Microbial Fundamentals of Biotechnology

Deutsche
Forschungsgemeinschaft
Microbial Fundamentals
of Biotechnology
Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft
Copyright 2001 WILEY-VCH Verlag GmbH, Weinheim
ISBN: 3-527-30615-3
015642 Titelei_Microbial 12.11.2004 11:16 Uhr Seite 1
Deutsche
Forschungsgemeinschaft
Microbial Fundamentals
of Biotechnology
Final report of the
collaborative research centre 323,
Mikrobielle Grundlagen der Biotechnologie:
Struktur, Biosynthese und Wirkung mikrobieller
Stoffe, 1986 1999
Edited by
Volkmar Braun and Friedrich Gtz
Collaborative Research Centres
015642 Titelei_Microbial 12.11.2004 11:16 Uhr Seite 2
Deutsche Forschungsgemeinschaft
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Cover:
Crystal structure of E.coli FhuA with bound rifamycin CGP 4832 as
determined by Ferguson et al. Structure 9:707-16 (2001)
Contents
Antibiotics and Other Biologically Active Microbial Metabolites
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Volkmar Braun and Friedrich Gtz
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1 Antibiotic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 The unique features of microbial iron transport . . . . . . . . . . . . . . . . 6
1.3 Transport of bacterial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Membrane components and membrane polarization . . . . . . . . . . . . 10
1.5 Chemistry of microbial peptides and proteins . . . . . . . . . . . . . . . . . 12
1.6 Summary of short-term projects of the collaborative research centre 14
2 Screening for New Secondary Metabolites from Microorganisms 16
Hans-Peter Fiedler and Hans Zhner
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2 Screening methods and novel compounds . . . . . . . . . . . . . . . . . . . . 18
2.3 Increasing structural diversity by directed fermentations . . . . . . . . . 41
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin . . . . . 52
Friedrich Gtz and Gnther Jung
3.1 History of lantibiotics and lantibiotic research in Tbingen . . . . . . . 52
3.2 Primary structure and proposed maturation of epidermin in
staphylococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 Genetic organization and regulation of the epidermin genes . . . . . 56
3.4 Isolation and characterization of genetically engineered gallidermin
and epidermin analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.5 Function of the epidermin immunity genes epiFEG . . . . . . . . . . . . . 66
3.6 Inactivation and characterization of the epidermin leader peptidase
EpiP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.7 The flavoenzyme EpiD and formation of peptidyl-amino-
enethiolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
V
Copyright #2001 WILEY-VCH Verlag GmbH, Weinheim
ISBN: 3-527-30615-3
3.8 Incorporation of d-alanine into S. aureus teichoic acids confers
resistance to defensins, protegrins, and other antimicrobial
peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4 Fermentation of Lantibiotics Epidermin and Gallidermin . . . . . . . 93
Uwe Theobald
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
4.2 Strains for gallidermin/epidermin production . . . . . . . . . . . . . . . . . . 94
4.3 Disadvantages during gallidermin process development . . . . . . . . . 94
4.4 Gallidermin a lantibiotic and its way towards industrial
production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5 Genetics of Nikkomycin Production in Streptomyces tendae T 901 102
Christiane Bormann
5.1 Introduction: nikkomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.2 Isolation of nikkomycin biosynthetic genes . . . . . . . . . . . . . . . . . . . 104
5.3 Isolation of the nikkomycin gene cluster and expression in
Streptomyces lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.4 Organization of the nikkomycin gene cluster . . . . . . . . . . . . . . . . . . 109
5.5 Roles of the nik genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.6 Transcriptional organization and regulation of the nik cluster . . . . . 120
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6 Glycosylated Antibiotics: Studies on Genes Involved in Deoxy-
sugar Formation, Modification and Attachment, and their Use in
Combinatorial Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Andreas Bechthold
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.2 Cloning of the avilamycin, landomycin, urdamycin,
and granaticin biosynthetic gene clusters . . . . . . . . . . . . . . . . . . . . . 127
6.3 Organization of avilamycin, landomycin, urdamycin, and granaticin
biosynthetic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
6.4 New genetically engineered natural compounds . . . . . . . . . . . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
7 Analysis of the Biosynthesis of Glycopeptide Antibiotics: Basis for
Creating New Structures by Combinatorial Biosynthesis . . . . . . . 139
Stefan Pelzer and Wolfgang Wohlleben
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
7.3 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Contents
VI
8 Homologous Recombination and the Induction of the
SOS-Response in Antibiotic Producing Streptomycetes . . . . . . . . . 151
Gnther Muth and Wolfgang Wohlleben
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
8.2 Mutational analysis of the S. lividans recA gene . . . . . . . . . . . . . . . 152
8.3 Regulation of RecA activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Membrane Processes
9 Regulated Transport and Signal Transfer Channels involved in
Bacterial Iron Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Volkmar Braun and Helmut Killmann
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
9.2 The Fhu proteins catalyze active transport of ferrichrome and the
antibiotic albomycin across the outer membrane and the cyto-
plasmic membrane of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
9.3 Transduction of energy from the cytoplasmic membrane into the
outer membrane for the activation of FhuA as a transporter and
phage receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
9.4 Transport of ferrichrome across the cytoplasmic membrane . . . . . . . 178
9.5 Ferric-carboxylate transport system of Morganella morganii
Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
9.6 Transport of ferric iron ions by the Sfu system of Serratia
marcescens
Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
10 Iron Transport in Gram-negative and Gram-positive Bacteria . . . . 188
Klaus Hantke
10.1 Ferric iron transport in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
10.2 Ferrous-iron transport systems (Feo) of E. coli . . . . . . . . . . . . . . . . . 194
10.3 Regulation of iron transport and metabolism . . . . . . . . . . . . . . . . . . 195
10.4 An [2Fe-2S] protein is involved in ferrioxamine B utilization . . . . . . 198
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
11 Regulation of the Ferric-Citrate Transport System by a Novel
Transmembrane Transcription Control . . . . . . . . . . . . . . . . . . . . . . 205
Volkmar Braun and Sabine Enz
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
11.2 Transport of Fe
3+
is mediated by citrate . . . . . . . . . . . . . . . . . . . . . . 205
11.3 Transcription initiation by a signaling cascade from the cell surface
into the cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
11.4 Iron regulation of fecIR and fecABCDE transcription . . . . . . . . . . . . 209
Contents
VII
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
12 Structure, Function, Import, and Immunity of Colicins . . . . . . . . . 212
Volkmar Braun and Helmut Pilsl
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
12.2 Colicin M inhibits murein biosynthesis and thus displays a unique
activity among the colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
12.3 Colicins 5 and 10 are taken up by a novel mechanism . . . . . . . . . . . 214
12.4 Colicins evolved by the exchange of DNA fragments which
precisely defined functional domains . . . . . . . . . . . . . . . . . . . . . . . . 215
12.5 Pore-forming colicins are inactivated by the cognate immunity
proteins shortly before the formation of the transmembrane pores . 217
12.6 Pesticin is a muramidase which is inactivated by the immunity
protein in the periplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
13 Structure, Activity, Activation, and Secretion of the Serratia
marcescens Hemolysin/Cytolysin . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Volkmar Braun and Ralf Hertle
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
13.2 Characterization of the S. marcescens hemolysin (ShlA) . . . . . . . . . 224
13.3 Pathogenicity of S. marcescens hemolysin/cytolysin . . . . . . . . . . . . 231
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
14 Staphylococcal Lipases: Molecular Characterization and Use as
an Expression and Secretion System . . . . . . . . . . . . . . . . . . . . . . . . 238
Friedrich Gtz and Ralf Rosenstein
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
14.2 Molecular organization of staphylococcal lipases . . . . . . . . . . . . . . . 239
14.3 Biochemical characterization of staphylococcal lipases . . . . . . . . . . 241
14.4 Role of the pro-peptide region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
14.5 The use of ShyL as expression and secretion system . . . . . . . . . . . . 244
14.6 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
15 A Multienzyme Complex Involved in Murein Synthesis of
Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Moritz von Rechenberg, Waldemar Vollmer, and
Joachim-Volker Hltje
15.1 The murein sacculus, a growing molecule . . . . . . . . . . . . . . . . . . 249
15.2 Murein growth is accompanied by massive turnover . . . . . . . . . . . . 252
15.3 Enlargement and division of a stress bearing structure . . . . . . . . . . 253
15.4 Interaction of murein hydrolases and synthases
as indicated by affinity chromatography . . . . . . . . . . . . . . . . . . . . . . 254
Contents
VIII
15.5 Dimerization of the bifunctional transpeptidase/transglycosylase
PBP1B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
15.6 Reconstitution of the core particle of a murein synthesizing
machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
15.7 Proposed structure of a hypothetical holoenzyme of murein
synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
15.8 Recent insights in the mechanism of growth of the murein sacculus
reveal novel targets for antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . 259
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
16 The Changing Path of Hopanoid Research: From Condensing
Lipids to New Membrane Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 263
Karl Poralla
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
16.2 The cyclization reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
16.3 Purification of squalene cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
16.4 Properties of purified cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
16.5 Cloning of squalene-hopene cyclases . . . . . . . . . . . . . . . . . . . . . . . . 270
16.6 Properties of SHC sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
16.7 The structure of squalene-hopene cyclase . . . . . . . . . . . . . . . . . . . . 272
16.8 Site directed mutagenesis of squalene-hopene cyclase . . . . . . . . . . 274
16.9 Hopanoid biosynthesis gene clusters . . . . . . . . . . . . . . . . . . . . . . . . 278
16.10 Miscellaneous results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
16.11 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
17 Genetic and Biochemical Analysis of the Biosynthesis of the
Orange Carotenoid Staphyloxanthin of Staphylococcus aureus . . 284
Friedrich Gtz
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
17.2 Cloning of the carotenoid biosynthetic genes from S. aureus
Newman in S. carnosus and E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . 285
17.3 Function of CrtM and CrtN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
17.4 Identification of carotenoids in S. carnosus (pOC21), E. coli (pUG1),
and S. aureus Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
17.5 Identification of dehydrosqualene in E. coli (pUG1) and E. coli
(UG9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
17.6 Squalene is very likely no substrate for CrtN, the proposed
dehydrosqualene desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
17.7 The crt operon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
17.8 Homology of CrtO, CrtP, and CrtQ . . . . . . . . . . . . . . . . . . . . . . . . . . 289
17.9 Construction of crtM mutants of S. aureus
strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
17.10 s
B
-regulated promoter of the crt operon from S. aureus strain
Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Contents
IX
17.11 The carotenoid biosynthesis genes . . . . . . . . . . . . . . . . . . . . . . . . . . 290
17.12 Function of the pigments in S. aureus strain Newman . . . . . . . . . . . 292
17.13 Distribution of pigment biosynthesis genes among staphylococcal
species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
18 Second Messenger Systems in Paramecium . . . . . . . . . . . . . . . . . . 295
Joachim E. Schultz and Jrgen Linder
18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
18.2 Identification and characterization of cGMP and cAMP second
messenger signaling systems in Paramecium . . . . . . . . . . . . . . . . . . 296
18.3 Biochemical properties of an adenylyl cyclase . . . . . . . . . . . . . . . . . 300
18.4 A guanylyl cyclase disguised as an adenylyl cyclase . . . . . . . . . . . . 302
18.5 On the way to an adenylyl cyclase with an intrinsic ion conduc-
tance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
18.6 Downstream of second messengers . . . . . . . . . . . . . . . . . . . . . . . . . 308
18.7 In vivo screening of bacterial secondary metabolites . . . . . . . . . . . . 311
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Chemical Synthesis and Structure Elucidation
19 Structure Elucidation and Chemical Synthesis of Microbial
Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Roderich D. Smuth, Jrg Metzger, and Gnther Jung
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
19.2 Development of methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
19.3 Structure elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
19.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Documentation
20 Documentation of the Collaborative Research Centre 323 . . . . . . . 345
20.1 List of institutes involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
20.2 List of supported project areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
20.3 Promotion of members of the collaborative research centre . . . . . . . 348
20.4 Recruitment of new project leaders . . . . . . . . . . . . . . . . . . . . . . . . . . 349
20.5 Alphabetical list of members and participants . . . . . . . . . . . . . . . . . 349
20.6 Support of young scientists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
20.7 Alphabetical list of guests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
20.8 International cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
20.9 International conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
20.10 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Contents
X
Preface
This book summarizes the scientific contributions of members of the collabora-
tive research centre 323. Tbingen is one of the very few academic places
worldwide where microbes have been systematically screened for biologically
active metabolites. This research started in 1964 when the first chair of micro-
biology was created at the University of Tbingen and was led by Hans Zhner.
Research on antibiotics in Tbingen continued in the 1980s, when most pharma-
ceutical companies had abandoned the development of new antibiotics as more
than 100 antibiotics were already available to treat seemingly all relevant micro-
bial infections. We now know that this decision was premature. Bacterial anti-
biotic resistance was already emerging and continued to progress at an increas-
ing pace, resulting in multi-resistant pathogens which now can only be con-
trolled by newly developed antibiotics.
The collaborative research centre 323 was an ideal instrument for bringing
together scientists of different disciplines and defining common interests. It con-
sisted of chairs and groups of Microbiology/Biotechnology, Microbiology/Mem-
brane Physiology, Microbial Genetics, Pharmaceutical Chemistry, Pharmaceuti-
cal Biology, and Organic Chemistry of the University and various groups of the
Max-Planck Institutes of Developmental Biology (Biochemistry Department),
and Infection Biology.
The members of the collaborative research centre met regularly in semi-
nars, which led to very successful co-operations in nearly all scientific projects.
Striking examples include the lantibiotic research, the identification of new
siderophores in pathogenic microorganisms, the screening/isolation/and subse-
quent structure elucidation of new antibiotics, and the synthesis of defined sub-
strates for iron transport analysis or hemolysin function. The extremely fruitful
cooperation between microbiologists and chemists is documented by co-author-
ship in numerous publications and is specified in more detail in the Research
Projects.
What is the secret of success of a collaborative research centre? There are
several reasons why the scientific outcome of a collaborative research centre is
usually more than the sum of the individual projects. First of all, the duration of
a collaborative research centre is long-term. Our collaborative research centre
323 was designed to run for 15 years, which allowed the realization of long-term
XI
ISBN: 3-527-30615-3
future-oriented projects. Research at universities is normally hampered by
short-term grants and fellowships. The collaborative research centre enabled
researchers to delve into scientific topics without the persistent fear of prema-
ture termination of the project, which would render the field open for competi-
tors to harvest the fruit.
Secondly, successful scientific cooperation is a matter of trust, competence,
and bilateral benefit. At a single university or research institution, it is not easy
to find a configuration that meets these prerequisites. A foundation of trust
takes time to develop and is largely dependent on individuals. The environment
of our collaborative research centre 323 facilitated and stimulated scientific co-
operation. The importance of the collaborative research centre for our place at
the frontier of science may be best illustrated by the recruitment of the scientific
staff to meet the requirements of the collaborative research centre 323.
It is no exaggeration to say that through the efforts of the members of the
collaborative research centre 323, fundamental and important results were
achieved in a number of areas, and many colleagues worldwide were influ-
enced and stimulated by the achievements of the collaborative research centre.
We are deeply indebted to the Deutsche Forschungsgemeinschaft for con-
tinuous support and to its reviewers for dedicated evaluation of the general con-
cept of the collaborative research centre and the individual research projects.
We would also like to thank the University of Tbingen and the Ministry for
Science of Baden-Wrttemberg for their understanding and financial support.
Volkmar Braun
XII
Preface
Antibiotics and Other Biologically
Active Microbial Metabolites
ISBN: 3-527-30615-3
1 Introduction
Volkmar Braun* and Friedrich Gtz**
Abstract
A summary of the major scientific achievements in the antibiotic, the mem-
brane-traffic, and chemistry projects of the collaborative research centre 323
from 1986 to 1999 will be presented in the Introduction, which is followed by a
more detailed description of the research projects of 1995 to 1999.
1.1 Antibiotic research
Because of the threatening spread of multi-resistant pathogenic microorganisms,
the search for and development of new antibiotics (anti-infective drugs) has be-
come more compelling than ever before. Vancomycin-resistant Staphylococcus
aureus strains have already been isolated in various countries, and one can fore-
see that we are facing an increased morbidity and mortality due to treatment
failure. Therefore, the search for new anti-infectives and lead compounds and
the development of new strategies will always be important.
Antibiotics are considered secondary metabolites, which, at least under la-
boratory conditions, do not participate in the primary metabolism essential for
microbial growth. Their role in the natural environment has always been an is-
sue in the collaborative research centre. Antibiotics inhibit microorganisms com-
* Mikrobiologie/Membranphysiologie, Universitt Tbingen, Auf der Morgenstelle 28,
D-72076 Tbingen
** Mikrobielle Genetik, Universitt Tbingen, Waldhuser Str. 70/8, D-72076 Tbingen
3
ISBN: 3-527-30615-3
peting for the same ecological niche. It was proposed that secondary metabo-
lism also forms the basis for the evolution of new metabolic pathways.
Through elucidation of the mode of action of various antibiotics, a large
number of distinct metabolic activities of both prokaryotic and eukaryotic organ-
isms were identified. Because of the beneficial activities of compounds related
to antibiotics, such as siderophores, which deliver iron to microorganisms, anti-
biotics can no longer be regarded as secondary metabolites. Antibiotics with
structures similar to those of iron complexes were found to be actively trans-
ported into target cells. This finding led to the concept of increasing the efficacy
of antibiotics by linking them to actively transported compounds.
1.1.1 Screening and fermentation
Antibiotic research in Tbingen involved the screening of many newly isolated
microorganisms for novel antibiotics and production of the antibiotics in
amounts sufficient for structure elucidation and utility testing. In the course of
the collaborative research centre, a state-of-the-art fermentation technology was
maintained, and new assay systems, and analytical and synthetic tools were de-
veloped. Metabolite production was increased by optimizing the fermentation
conditions, aided by a sophisticated on-line analysis of the products released
into the culture media. An extensive antibiotic database was created to differ-
entiate new compounds from known compounds at an early stage of the investi-
gation. The spectrum of products usually formed by the microorganisms was
modified by directed fermentation and more recently by genetic means, such as
mutation and combinatorial pathway recombination.
The concept was not a large-scale screening in the sense that thousands of
strains and compounds were tested per day. Rather, a selective screening was
employed using special growth conditions, novel test systems, and various
strains, some of which synthesized a number of different secondary metabolites.
The screening was also not target-oriented, and an unbiased selection proce-
dure was used. Some test systems were established within the collaborative re-
search centre, and others were used in cooperation with research groups outside
the collaborative research centre. Potential applications were focused not only
on antibacterial compounds, antifungal products and therapeutic compounds
were also considered.
Apart from the biotechnological purpose of searching for new microbial
metabolites, the wealth of compounds found demonstrated the extremely high
metabolic potential of microorganisms. For example, in the original nikkomycin-
producing Streptomyces strain, 14 nikkomycin variants were determined and
mutagenesis resulted in 24 additional derivatives. The metabolic diversity of
Streptomycetes along with their capability of differentiation is reflected by the
size of their genome, which is at the upper limit of bacterial genomes.
4
1 Introduction
1.1.2 Marketed compounds developed under the collaborative research
centre
Among the four commercially most interesting compounds developed under the
collaborative research centre, only gallidermin and avilamycin are antibacterial
compounds; the latter is employed as a food additive. Desferrioxamine B (trade
name Desferral
) is used to treat iron-overload diseases, phosphinothricin (trade

name BASTA
) is a herbicide, and the nikkomycins are potent inhibitors of

chitin synthases and thus kill fungi, insects, and acarids without toxic side ef-
fects on mammals. Nikkomycin Z is currently in the second stage of clinical
trial.
1.1.3 Lantibiotics
The lantibiotic era in Tbingen began in 1985 with the determination of the
structure of epidermin, a peptide antibiotic isolated from Staphylococcus epider-
midis. Four research teams in the collaborative research centre worked to reveal
this new class of natural products with a novel biosynthesis pathway. Tbingen,
where the name lantibiotic was coined, has been a stronghold in this research
area over the years. The functions of most of the proteins and enzymes involved
in biosynthesis were elucidated, although the mechanism of the key reaction,
lanthionine formation, is still unknown. Since chemical synthesis of large
amounts of epidermin and gallidermin is currently impossible, great efforts were
spent to improve the fermentation process, the scale up, and the downstream
processing.
1.1.4 Glycosyl antibiotics and regulation of biosynthesis
New derivatives of the glycosylated antibiotics avilamycin, landomycin, urda-
mycin, and granaticin were generated. The gene clusters involved in the bio-
synthesis of each of the antibiotics were analyzed, and the functions of most of
genes were identified. Especially those genes involved in deoxy-sugar forma-
tion, modification, and attachment were used to create novel natural products.
A series of new genetically engineered natural compounds were created by in-
activation or overexpression of certain genes.
Balhimycin is a glycopeptide with properties similar to those of vancomy-
cin, which is often used to combat bacterial infections when no other antibiotic
is effective. Balhimycin has the same heptapeptide core as vancomycin and dif-
5
1.1 Antibiotic research
fers in the glycosylation pattern. A cloning system for the producing strain was
developed, and a number of genes were identified by using DNA probes based
on consensus sequences of the typical biosynthetic enzymes, such as those en-
coding peptide synthetases and glycosyltransferases. Most of the balhimycin
biosynthesis gene cluster was sequenced, and the function of a number of
genes analyzed. The door is now open for the generation of hybrid antibiotics
using the combinatorial biosynthesis strategy.
Antibiotic production of mycelia-forming Streptomycetes is controlled by a
complex regulatory network that allows the cells to sense different growth con-
ditions and to react to these changes by producing antibiotics. Antibiotic pro-
duction of Streptomyces coelicolor was shown to be affected by cell density, nu-
tritional limitations, nutritional shiftdown, imbalance in metabolism, and differ-
ent kinds of stress. The key player of the SOS response in Streptomyces livi-
dans, RecA, was investigated.
1.1.5 Antibiotics and transport
Membrane studies were important for gaining knowledge on the entry of anti-
biotics into cells, resistance via permeability barriers, and active export systems,
and for identifying novel targets and altering the antibiotics to exert their detri-
mental function. One antibiotic studied is albomycin, which is actively taken up
by cells through an iron siderophore (ferrichrome) transport system. Inside the
target cell, the antibiotically active portion is cleaved off the carrier, which is re-
leased from the cells. Active transport reduces the minimal inhibitory concentra-
tion to the lowest value known for an antibiotic that kills Escherichia coli. An-
other example is phosphinothricyl-alanyl-alanine, which is transported into cells
by an oligopeptide system; the antibiotic is released from the peptide carrier by
intracellular proteases to generate phosphinothricin, which then inhibits gluta-
mine synthetase. These findings prompted research by the collaborative re-
search centre and by pharmaceutical companies aimed at developing antimicro-
bial compounds that are taken up by transporters.
1.2 The unique features of microbial iron transport
It was clear from the beginning that iron transport systems must have special
features not shared by transport systems of any other nutrient. Under oxic con-
ditions, iron occurs as the completely insoluble Fe
3+
. Since iron is an important
6
1 Introduction
cofactor of redox enzymes, iron shortage must be overcome by microorganisms;
they handle this by synthesizing iron-complexing compounds of low molecular
weight, designated siderophores (originally called sideramines, siderochromes).
The antibiotic-screening group of the collaborative research centre also had a
long-standing interest in microbial iron complexes because sideromycins, potent
antibiotics, belong to this class of compounds. Studies of the uptake of sidero-
mycins, the intracellular metabolism, and their mode of action were essential for
understanding antibiotic activity. In addition, it was clear that the iron supply
must be carefully balanced since iron overload is toxic due to iron-catalyzed ra-
dical formation, which results in the destruction of DNA, proteins, and mem-
brane lipids. Therefore, transport of iron-loaded siderophores and sideromycins
and regulation of siderophore synthesis and transport were the focus of the iron
projects.
1.2.1 Iron transport through the outer membrane of E. coli and
other pathogenic bacteria
Novel iron transport and regulatory mechanisms were expected and also found.
Transport of Fe
3+
-siderophores was unique in several respects. Transport across
the outer membrane of Gram-negative bacteria consumes energy, which is pro-
vided by the proton-motive force of the cytoplasmic membrane. Energy transfer
from the cytoplasmic membrane to the outer membrane became an important
research topic. A major breakthrough was the identification of the proteins in-
volved in energy transfer: TonB, ExbB, and ExbD (Ton system). The Ton system
was extensively characterized at molecular, biochemical, and structural levels.
Seven E. coli K-12 Fe
3+
-siderophore transport systems were identified, and
those of ferrichrome and ferric citrate were studied in detail. The receptors un-
dergo conformational changes upon substrate binding and through interaction
with the energized Ton system, as supported by the analysis of the crystal structure
of the FhuA transporter. Upon binding of ferrichrome to FhuA close to the cell sur-
face, a strong structural transition occurs. The long-range conformational change
takes place across most of the molecule and the width of the outer membrane.
The link to antibiotics is provided by FhuA, which serves as the active
transporter of two antibiotics: albomycin is structurally related to ferrichrome; ri-
famycin CGP 4832 is structurally unrelated to ferrichrome. Surprisingly, both oc-
cupy the same position as ferrichrome on FhuA.
Iron siderophores transporters homologous to the E. coli K-12 outer mem-
brane transporters were identified in Yersinia enterocolitica and Morganella
morganii. The ferrioxamine B transport system of the highly pathogenic Y. enter-
ocolitica O8 strain explains the occurrence of yersiniosis upon treatment of pa-
tients suffering from iron overload with Deferral
(mesylate salt of desferri-fer-

rioxamine B). In addition, a siderophore, designated yersiniabactin, was de-
tected in the culture supernatants of highly pathogenic strains. Yersiniabactin
7
1.2 The unique features of microbial iron transport
was isolated in amounts sufficient for determination of its novel structure.
Furthermore, the genes of the entire heme transport system of Y. enterocolitica
were cloned and sequenced, and functions were assigned to the encoded pro-
teins. This was the first characterized heme transport system.
Characterization of the iron transport systems of Serratia marcescens was
initiated by the finding that transcription of the hemolysin genes is iron-regu-
lated. These studies revealed a plethora of Fe
3+
-siderophore transport systems,
one of which transports Fe
3+
across the cytoplasmic membrane without involve-
ment of a siderophore. Other research groups later related this system to the up-
take of iron delivered by human transferrin to a variety of human pathogenic
bacteria.
1.2.2 Iron transport through the cytoplasmic membrane
Transport of Fe
3+
-siderophores, heme, and Fe
3+
across the cytoplasmic mem-
brane is catalyzed by ABC transporters, which consist of a periplasmic binding
protein, one or two integral membrane proteins, and a cytoplasmic ATPase. ABC
transporters represent the most frequently occurring transport systems in bac-
teria. Regions of interaction between the periplasmic binding protein and the cy-
toplasmic membrane transporter were shown for the first time with FhuB/D.
The same type of ferrichrome transport system was shown to occur in Ba-
cillus subtilis, a Gram-positive bacterium, which lacks a periplasm. Here, a pro-
tein similar to the periplasmic binding protein of Gram-negative bacteria is
linked by a lipid anchor of the murein lipoprotein type to the cytoplasmic mem-
brane.
1.2.3 Iron transport regulation
Fur was the first iron regulatory gene to be mapped, cloned, and sequenced.
Fur functions as an oligomer and binds when loaded with Fe
2+
to iron-regulated
promoters and inhibits transcription. An assay for the identification of Fur-regu-
lated promoters was developed.
The ferric citrate transport system displays the particular property that it is
not only repressed by iron, but is induced by ferric citrate. Ferric citrate binds to
the outer membrane FecA transport protein; this binding initiates a signal that
is transmitted by the FecR protein across the cytoplasmic membrane. In the cy-
toplasm, FecI is converted to an active sigma factor, which in turn transcribes
the fecABCDE transport genes. In this dual stepwise control, first iron limitation
is recognized and subsequently the transport system is synthesized only when
the cognate substrate is in the culture medium.
8
1 Introduction
Under anoxic conditions, bacteria may acquire Fe
2+
, which is much more
soluble than Fe
3+
and does not require chelating agents. Feo of E. coli, the only
Fe
2+
transport system characterized at the molecular level, is encoded by three
genes; one gene, feoB, is very likely involved in energizing the transport by nu-
cleotide triphosphate hydrolysis. Mutants in feo were shown to be attenuated in
the mouse gut.
1.2.4 Intracellular iron metabolism
Very little is known about the intracellular iron metabolism in bacteria. The aty-
pical [2Fe-2S] protein FhuF was characterized; the cysteine residues that bind
the iron-sulfur center were identified by amino acid replacement studies. FhuF
mutants no longer utilize ferrioxamine B as an iron source, which suggests that
FhuF may be involved in iron mobilization from ferrioxamine B. The two iron-
regulated genes sufS and sufD play a role in utilization of ferrioxamine B as an
iron source and possibly in intracellular iron metabolism. Sequence similarities
of SufS to NifS suggest that SufS is involved in the formation of the iron-sulfur
center of FhuF.
1.3 Transport of bacterial proteins
1.3.1 Transport of colicins and toxins
The activities, import, immunity, and evolution of bacterial protein toxins were
studied. The genes of eight colicins and pesticin were cloned and sequenced.
Cells that synthesize the toxins are protected by immunity proteins with a high
specificity for the cognate colicin. Most of the colicins are released from cells by
lysis proteins that are encoded downstream of the activity and immunity genes.
Colicins can be subdivided into the N-terminal translocation region, the
central receptor recognition region, and the C-terminal activity and immunity
regions. Comparison of amino acid sequences clearly demonstrated evolution of
the pore-forming colicins by exchange of DNA fragments that encode functional
domains.
How and when the immunity proteins in the cytoplasmic membrane inacti-
vate the pore-forming colicins was a major question. The transmembrane topol-
ogy of the immunity proteins and the regions of interaction with the colicins in-
9
1.3 Transport of bacterial proteins
dicate that the colicins are inactivated shortly before the pores are opened. In
contrast, colicin M, which inhibits murein and O-antigen biosynthesis by inter-
fering with C
55
-lipid carrier regeneration, and pesticin, which degrades murein
by a mechanism similar to that of lysozyme, are inactivated by their immunity
proteins in the periplasm before they reach their targets.
The hemolysin/cytolysin (ShlA) of Serratia marcescens is activated by a
single protein (ShlB) in the outer membrane through a novel mechanism during
secretion. The hemolysin is a large protein that remains in a non-hemolytic form
in the periplasm of cells that synthesize no ShlB protein. The N-terminal portion
of ShlA is important for activation and secretion, the central portion for binding
to erythrocytes, and the C-terminus for the formation of small pores in the mem-
brane of erythrocytes, leukocytes, and epithelial cells. ShlA represents one of
the very few cases where a major phospholipid of a biomembrane also serves as
a cofactor for activity. ShlB has the potential to form pores through which ShlA
might be exported.
1.3.2 Transport of staphylococcal (phospho)lipases
Five different staphylococcal lipase genes of S. aureus, S. epidermidis, and Sta-
phylococcus hyicus were cloned and sequenced. All corresponding proteins are
organized as pre-pro-enzymes in which the pro-region comprises between 207
and 267 amino acids. The pro-region acts as an intramolecular chaperone that
facilitates translocation of the native lipase; the pro-peptide can also translocate
a number of completely unrelated proteins fused to it. The pro-region protects
the proteins from proteolytic degradation. The lipase pro-peptide-based expres-
sion and secretion system is used by an increasing number of groups for produc-
tion of human proteins and peptides in Staphylococcus carnosus, a food-grade
microorganism for which a cloning system was developed.
1.4 Membrane components and membrane polarization
1.4.1 Biosynthesis of triterpenes in bacteria
There is a tremendous variety of triterpenes in the plant kingdom; a single
higher plant always contains several types of triterpenes. The triterpenoic hopa-
noids found in a large number of Gram-positive and Gram-negative bacteria
10
1 Introduction
show less structural variability. In some bacteria, hopanoid biosynthesis genes
are present, but are not expressed under laboratory conditions; therefore, an
even wider range of bacteria may synthesize hopanoids. The study of triterpene
biosynthesis and the structural variation of triterpenes in nature was ap-
proached by investigating the membrane-bound squalene-hopene cyclase of
Alicyclobacillus acidocaldarius, which proved to be easier to work with than
that of plants. The encoding gene was cloned, sequenced, and expressed, and
the gene product was purified and characterized. Comparisons of the amino
acid sequence with those of other triterpene cyclases revealed a conserved 16-
amino acid repeat. Interestingly, the highly purified A. acidocaldarius squalene-
hopene cyclase forms minor products of mostly tetracyclic structure; this finding
was important for a better understanding of the cyclase reaction mechanism.
The studies formed the basis for the determination of the crystal structure,
which, together with the crystal structures of two sesquiterpene cyclases, are
the first three-dimensional structures of terpene cyclases. The squalene-hopene
cyclase is a monotopic membrane-bound enzyme. Knowledge of the structure
allowed site-directed mutagenesis of specific residues in the catalytic cavity.
Some mutant squalene-hopene cyclases significantly increased the synthesis of
tetracyclic and bicyclic byproducts; a new cyclase in which a leucine in the
central cavity is replaced by lysine produced a bicyclic compound.
Additional genes involved in hopanoid biosynthesis were detected up-
stream of the squalene-hopene cyclase genes of Bradyrhizobium japonicum, Zy-
momonas mobilis, and Methylococcus capsulatus; the first bacterial gene found
to encode a squalene synthase is among them. In the aerial mycelium of Strep-
tomyces coelicolor, a differentiation-dependent formation of hopanoids was
found; hopanoids are not formed in substrate mycelium and when cultures are
grown in liquid.
1.4.2 Biosynthesis of staphyloxanthin
The yellow to orange colony color of S. aureus is one of the classical species
criteria. The main pigment is staphyloxanthin, a C
30
-carotenoid that is inte-
grated into the cytoplasmic membrane. The genes involved in the biosynthesis
of staphyloxanthin were identified and analyzed. Through the creation of dele-
tion mutants and the analysis of the intermediary compounds formed, a biosyn-
thetic pathway was postulated. The function of staphyloxanthin is still unclear;
however, the expression of its gene responds to the stress sigma factor, SigB,
which suggests that staphyloxanthin is necessary for survival under certain con-
ditions.
11
1.4 Membrane components and membrane polarization
1.4.3 Signal transduction by cAMP and cGMP
The initial steps in signal transduction in Paramecium involving the second
messengers cAMP and cGMP were characterized. Unlike in metazoans, where
hormones as first messengers elicit intracellular second messenger formation, in
the ciliate Paramecium abrupt changes in the cells membrane potential acti-
vated second messenger biosynthesis. Characterized behavioral mutants of the
ciliate with defined defects in electrogenesis showed that cAMP generation de-
pends on a K
+
-outward current, whereas cGMP formation is enhanced by a de-
polarizing Ca
2+
-inward current. Analysis of clones carrying genes of the respec-
tive protozoan nucleotide triphosphate cyclases demonstrated the presence of
an adenylyl cyclase embedded in a protein background that strongly resembles
a potassium ion channel. Most surprisingly, the guanylyl cyclase is disguised in
a membrane topology identical to that of canonical mammalian adenylyl cy-
clases and, in addition, carries an extended N-terminus that closely resembles a
P-type ATPase unit with a total of ten transmembrane-spanning helices. These
findings obtained with the ciliate Paramecium open new vistas on the structural
and functional evolution of nucleotide triphosphate cyclases and provide Rosetta
Stone sequences to decipher novel binding/regulating partnerships.
1.5 Chemistry of microbial peptides and proteins
During the course of the collaborative research centre, innovative analytical and
synthetic methods were introduced. These methods allowed high-level bio-
chemical investigations to solve microbiological research problems in interdisci-
plinary co-operations. Very often the analytical and synthetic work was even de-
cisive for the success of projects.
1.5.1 Structure determinations
The major contributions of the chemistry group were the structure determina-
tions of a large number of antibiotics, and the synthesis of precursors, which al-
lowed the elucidation of the activities of enzymes involved in biosynthesis. The
3-D structures of gallidermin and actagardine are the basis for the model of their
mode of action, which very recently became of increased interest due to the in-
hibitory activity on peptidoglycan biosynthesis. One of the most unusual and in-
12
1 Introduction
teresting peptide structures ever found is the 43-peptide antibiotic microcin
B17, elucidated by multidimensional NMR of the
13
C-,
15
N-labeled polypeptide
containing eight oxazole and thiazole rings in its backbone. This gyrase (topo-
isomerase II) inhibitor is ribosomally synthesized as a precursor peptide which is
post-translationally modified.
To elucidate such structures at that time, innovative instrumental methods,
such as greatly improved NMR methods, HPLC-ESI-MS, and the Edman se-
quencer coupled to an ESI-mass spectrometer, and novel chemical transforma-
tions had to be introduced. Unusual peptide structures can now be sequenced
using very small amounts of samples.
The number and complexity of the elucidated natural products increased
considerably, e. g. lipoglycopeptides and other unusual peptides, and new non-
peptidic metabolites, such as siderophores, macrolides, polyols, lactam antibio-
tics, and steroidal antibiotics. Recently, the complex structure of CDA (calcium-
dependent peptide antibiotic), a peptide pheromone carrying a thiolactone ring,
of intermediates in nikkomycin biosynthesis, and of the first linear glycopeptide
precursors in the biosynthesis of the antibiotic balhymicin were elucidated.
1.5.2 Peptide chemistry, peptide libraries, and mass spectrometric
analysis
The continuous improvements in parallel automated synthesis of peptides, pep-
tide mimetics, and peptide libraries contributed extraordinarily to structure ac-
tivity studies in various groups of the collaborative research centre. The out-
standing synthetic capabilities combined with novel achievements in library
analytics by ESI-MS, HPLC-ESI-MS, ICR-MS, and Edman pool sequencing sti-
mulated collaborations in microbiology and immunology, in and outside of T-
bingen.
The binding regions of the gating loop of FhuA were identified using syn-
thetic peptides. A number of enzyme activities (e. g. oxidative decarboxylase in
the epidermin biosynthesis) and binding domains of proteins to the cell wall
(e. g. autolysin) could only be studied with the aid of synthetic peptides and
peptide libraries. Furthermore, many new proteins were characterized by LC-
MS and Edman sequencing, circular dichroism, peptide mapping, and antipep-
tide antibodies, such as the novel antifungal protein from Streptomyces.
The recent introduction of a high-resolution, Fourier-transform, ion cyclo-
tron resonance mass spectrometer will provide powerful technologies for further
fruitful research between microbiologists and organic chemists.
13
1.5 Chemistry of microbial peptides and proteins
1.6 Summary of short-term projects of the collaborative
research centre
The collaborative research centre was designed to run for 15 years. During this
period, the details of the scientific program changed; however, the basic con-
cept was maintained.
Detailed descriptions of the results are contained in the research reports of
the collaborative research centre from 198687, 19881990, 19901993, and
19931995. This book contains the detailed reports of 19951999.
The following paragraphs describe the contributions made in short-term
projects by the groups of the listed project leaders.
Karl-Dieter Entian. Major contributions to the cloning and sequencing of
the epidermin and the pep5 lantibiotic biosynthesis gene clusters were made.
Heterologous proteins and peptides in yeast were synthesized.
Bernd Hamprecht. Intercellular communication in the human nerve system
was studied. A method was developed and successfully applied to the quantita-
tive determination of adenosine binding to neural adenosine receptors, which
paved the way for the isolation of adenosine receptors. The activities of glyco-
gen phosphorylase, creatine kinase, and sorbitol dehydrogenase were deter-
mined in an attempt to analyze metabolic processes regulated by cyclic AMP in
astroglia-enriched primary cultures. The group was further involved in the study
of the role carnosine plays in the brain, taurine transport, and the mode of action
of bradykinin. The neuronal cell cultures were used to analyze the activities of
products isolated in the microbial screening programs.
Thomas F. Meyer. Secretion of the IgA protease by Neisseria gonorrhoeae
was investigated, and a novel mechanism was discovered. The mode of action
of the translocating b-domain in the outer membrane was studied, and the do-
main was fused with heterologous proteins that became exposed at the cell sur-
face. The OmpT protease was identified as the enzyme that degrades the fused
proteins, which results in decreased yields. The formation of disulfide bonds by
oxidation in the periplasm was shown to prevent secretion by locking fusion
proteins in a secretion-incompetent conformation. The b-domain is therefore sui-
table for exposing antigens at the cell surface with the aim to produce antibo-
dies and to stimulate the human immune system.
Johannes Pohlner. The a-domain of the IgA protease of N. gonorrhoeae
was studied since it contains a sequence of basic amino acids found in proteins
that enter the nucleus of eukaryotic cells. The group also studied the post-trans-
lational processing of the IgA protease polypeptide to form the protease proper,
which is released into the culture medium along with the a-domain and the b-
domain that reside in the outer membrane.
Rainer Haas. The VacA cytotoxin of Helicobacter pylori was characterized.
The growth conditions for the production of the toxin were established, the
vacA structural gene was cloned and sequenced, the occurrence of vacA in var-
14
1 Introduction
ious Helicobacter strains was determined, vacA mutants were isolated and char-
acterized, and the secretion mechanism of VacA was studied. In addition, tools
for the genetic analysis of Helicobacter were developed.
Susanne Klumpp. The protein phosphatases type 1, 2A, and 2C of Parame-
cium were studied. The genes of the phosphatases 1 and 2C were cloned and
sequenced. The three phosphatases were purified to electrophoretic homogene-
ity, and functions were ascribed to protein domains. As part of a collaboration,
the biochemistry of sensory transduction in Paramecium was studied.
15
1.6 Summary of short-term projects of the collaborative research centre
2 Screening for New Secondary Metabolites
from Microorganisms
Hans-Peter Fiedler* and Hans Zhner
2.1 Introduction
Originally screening for secondary metabolites was focused on antibacterial
compounds. Later on the screening was extended to antifungal, antiviral and
antitumor activity and today it has expanded to human medicine, animal health,
and plant protection. The initial idea, using only natural products produced by
microorganisms has been replaced by the search for novel lead structures, ac-
companied by the development of novel targets in all application fields. Still,
the most prominent source for novel leads is found in nature and especially in
the secondary metabolism of microorganisms [1]. The new lead compounds can
be used for derivatisation programs or as platform for chemical synthesis. There-
fore, the screening for novel secondary metabolites received an increased inter-
est in the last 15 years.
More than hundred new test systems are described till today for applica-
tions in pharmaceutical and agricultural fields [2]. These in vitro-assays are
mostly based on key enzymes or receptors and differ from classical antibiotic as-
says by the following aspects:
. Proteases in microbial samples or extracts lead to false positive results by de-
gradation of assay enzymes or protein receptors.
. Numerous assays are sensitive to metabolites from the intermediary metabo-
lism, which are found in variable concentrations in all microbial cultures.
. The assays are sensitive to infections, osmotic conditions, and changes in the
pH value.
. The assays are selective for a distinct mode of action within a cascade and
do not take account to the whole cascade.
* Mikrobiologisches Institut, Universitt Tbingen, Auf der Morgenstelle 28, D-72076 T-
bingen
16
ISBN: 3-527-30615-3
. Not all new developed assays are suitable for automated robot screening.
. Quite a number of assays are sensitive to already known compounds.
. None of the target directed assays is suitable to detect chemical diversity in
microbial cultures or extracts.
Within the collaborative research centre 323 we developed screening stra-
tegies which aimed not only on classical antibacterial activity, but also on anti-
fungal activity, on activities which are involved in differentiation processes and
on detection of novel siderophores. However, our main screening strategy is
based on physico-chemical methods to detect a maximal number of novel sec-
ondary metabolites in freshly isolated Actinomycete strains. The so-called che-
mical screening which is based on thin-layer chromatography and staining re-
agents was first introduced by Hamao Umezawa [3] and continued a few years
later by Satoshi Omura, who detected staurosporin by this assay which his most
prominent compound found by chemical screening [4]. Hans Zhner modified
this method with respect to staining reagents, sample preparation, and variation
of the cultivation conditions of the isolated strains [5, 6]. The detection of a bio-
logical activity comes only second order.
A new dimension of insights into the chemical diversity of produced sec-
ondary metabolites was the coupling of high-performance liquid chromatogra-
phy with computer-assisted diode array detection (HPLC-DAD) and the con-
struction of a database of antibiotics and other natural products based on HPLC
and UV-visible absorbance spectral libraries (HPLC-UV-Vis Database) by Hans-
Peter Fiedler [7]. This efficient method allowed the identification of known com-
pounds in raw extracts at a very early stage of investigations or permitted a clas-
sification of the compound by comparing UV-visible spectra data. The efficiency
of HPLC-DAD screening technique was extended by HPLC-ESI-MS analysis in
co-operation with the members of the collaborative research centre Prof.
Gnther Jung and Prof. Jrg Metzger. The additional information of the molecu-
lar mass permitted a more accurate search in commercially available chemical
databases.
The goal of our strategy was to detect a novel compound which then was iso-
lated and broadly tested for its biological activity, considering that each secondary
metabolite will have an activity. A further advantage of chemical screening was
achieved by testing pure compounds in the assays. Quite a number of test systems
are not compatible with microbial cultures or crude extracts and need the applica-
tion of pure compounds. Nevertheless, we have not sufficient assay systems avail-
able and for many newcompounds we have so far not found any biological activity
despite intensive co-operation with various pharmaceutical and agrobiological
companies. We expect that the Naturstoffpool (sponsored since 1996 by BMBF
and German pharmaceutical companies) comprising a collection of compounds
will be tested by the end of 1999 by a large variety of molecular robot assays.
For the analysis of the structure-activity relationship of secondary metabo-
lites we increased the number of original compounds by directed fermenta-
tions and feeding, by modification of precursors during the production phase,
or, by mutasynthesis using blocked mutants.
17
2.1 Introduction
In the following subchapters all new secondary metabolites are described
which were detected in various screening programs during 1986 and 1999 in
the groups of Prof. Hans Zhner and Prof. Hans-Peter Fiedler within the colla-
borative research centre 323.
2.2 Screening methods and novel compounds
2.2.1 Classical screening for antimicrobial activity
The classical agar plate diffusion assay for the detection of antimicrobial agents
produced by Gram-positive and Gram-negative bacteria, yeasts or filamentous
fungi was applied only until 1988. By this assay system we found pyridazomycin
[8] and chlorotetain [9]. Both antibiotics show a selective antifungal activity. Pyr-
idazomycin is distinguished by the unusual pyridazine ring that was not de-
scribed before in microbial secondary metabolites, chlorotetain is a dipeptide
containing an unusual chlorinated amino acid.
A screening for growth inhibitors against Bacillus subtilis revealed a novel
peptide antibiotic named aborycin [10].
The structures of the novel antifungal antibiotics are shown in Fig. 2.1.
he
Phe
Trp Cys HO
S
S
Cys
Asn
Ile
Ser
Leu
Cys
Asp
Gly
S
P
Val Ala
Tyr
Val
Gly
Gly
Cys
S
Ala Gly
Aborycin
Streptomyces griseoflavus T 4072
Figure 2.1: Microbial secondary metabolites detected by classical screening for antimi-
crobial activity.
O
Cl
H H
2
C
CH COO
-
NH CO CH
CH
3
+
H
3
N
Chlorotetain
Bacillus subtilis ATCC 6633
N
N
COO
-
O
H
3
N
H
2
N
H
+
Cl
-
+
Pyridazomycin
Streptomyces violaceoniger sp. griseofuscus T 2557
18
2 Screening for New Secondary Metabolites from Microorganisms
2.2.2 Screening for antibiotics causing morphological changes of
hyphae of Botrytis cinerea
This assay is based on both, growth inhibition of Botrytis cinerea and morpholo-
gical changes of the hyphae, a so-called bulging effect. By this assay sub-
stances with antifungal action in the presence of polyene antibiotics were found.
Nikkomycin Z and X [11, review] were the most prominent antibiotics analysed
in the group of Prof. Hans Zhner during the collaborative research centre 76.
Nikkomycin Z is a potent inhibitor of chitin synthase and is non-toxic for hu-
mans. It was several years under intensive investigation as acaricide for agricul-
tural use at BAYER AG but cancelled in 1984 because of too high costs and its
too narrow application in plant protection. From 1994 till 1998 nikkomycin Z
was developed as an antimycotic agent for therapy of histoplasmosis, blastomy-
cosis and coccidoidomycosis in human medicine by Shaman Pharmaceuticals in
the USA and passed to the second clinical trial.
The Botrytis-assay was continued during the beginning of the collabora-
tive research centre 323 and resulted in the detection of galbonolides [12, 13],
four new members of antifungal macrolide antibiotics. Their structures are
shown in Fig. 2.2.
2.2.3 Screening for novel siderophores
With the exception of lactobacilli all other microorganisms are dependent on the
uptake of external iron ions to supply their iron-containing enzymes. Because of
the extreme water insolubility of Fe
3+
-ions all microorganisms have developed very
efficient iron-chelating compounds and specific iron-uptake systems. Iron-chelat-
ing compounds are for example trihydroxamates, catecholes, tricarboxylates, and
other compounds which are able to chelate iron. In the course of the collaborative
research centre 323 the following novel chelating metabolites were isolated:
HO
O
CH
3
CH
3
CH
2
O
CH
3
O
R
H
3
C
OH
OH
O
O
CH
3
CH
3
CH
2
CH
3
O
H
3
C
H
3
C
OH
OH
O
O
CH
3
CH
3
CH
2
H
3
CO CH
3
O
H
3
C
HO
A
B
C D
: R = OCH
3
: R = CH
3
Galbonolides A-D
Streptomyces galbus T 2253
Figure 2.2: Microbial secondary metabolites detected in the Botrytis assay.
19
Maduraferrin was isolated from strain Actinomadura madurae DSM 43067
after detection by an HPLC assay [14]. The complexing centres are a salicyl-
amide moiety, a hydroxamic acid group and an acid hydrazide group.
The highly hydrophilic carboxylate-type siderophores staphyloferrins A
and B were isolated from Staphylococcus hyicus DSM 20459 grown under
strong iron-restricted conditions [1518]. Both compounds are strictly iron-regu-
lated. Staphyloferrin A consists of two molecules citric acid, each linked to D-or-
nithine by an amino bond, whereas staphyloferrin B consists of 2,3-diaminopro-
pionic acid, citrate, ethylenediamine and 2-ketoglutaric acid.
From Bacillus sp. strain DSM 6940 was isolated besides schizokinen the
new dihydroxamate siderophore schizokinen B, in which citrate is replaced by
aconitate [19].
Rhizoferrin is a novel carboxylate-type siderophore which was isolated in
collaboration with the groups of Prof. Winkelmann and Prof. Jung from Rhizo-
pus microsporus and other fungi of the Mucorales [20, 21]. Rhizoferrin is similar
in structure to staphyloferrin A. In case of rhizoferrin, D-ornithine is replaced by
putrescin as bridge.
From a highly virulent Yersinia enterocolitica strain H1852a siderophore
named yersinibactin was isolated [22]. The novel compound contains a benzene
and a thiazolidine ring, as well as two thiazoline rings. It forms stable complexes
with trivalent cations such as iron and gallium.
While we investigated the fermentation of S,S-ethylenediamine disuccinic
acid (EDDS) with Amycolatopsis orientalis strain we found out that EDDS is not
an iron but a zinc chelator which opens new biotechnological applications. A
novel, not ferrioxamine-type siderophore named amycolachrome was isolated
that is similar in structure to the fungal ferrichrome-hexapeptides [23].
The structures of the isolated new siderophores are shown in Fig. 2.3.
2.2.4 Screening for secondary metabolites involved in differentiation
processes
Actinomycetes are characterised by complex differentiation processes. The
search for metabolites which influence these processes is of general importance
because they give insight in the tricky sequence and regulation of this dramatic
event in the live cycle of these organisms.
Prof. Heinz Wolf in the group of Prof. Zhner developed a screening system
that allows detection of compounds that stimulate formation of aerial mycelium in
Streptomycetes and he isolated hormaomycin from Streptomyces griseoflavus W-
384, a novel peptide-lactone antibiotic [24, 25]. Hormaomycin induces not only
aerial mycelium formation but also antibiotic production in these organisms.
Germicidins A and B were isolated from Streptomyces viridochromogenes
NRRL B-1551 [26]. Germicidin A is the first known autoregulative inhibitor of
spore germination in the genus Streptomyces.
Another novel peptide, streptofactin, was isolated from the nikkomycin
producer Streptomyces tendae T 901/8c in the group of Prof. Fiedler. This bio-
20
Figure 2.3: Novel iron-chelating compounds.
N
CH
3
N
NH
O
O
H
H
HO
OH
OH
O
HN
N
O N NH
O
O
H
H
Maduraferrin
Actinomadura madurae DSM 43067
N
N
COOH
OH
O
HO
HOOC
COOH
O
HOOC
H
H
Rhizoferrin
Cunninghamella elegans, Rhizopus microsporus
B
H
H
H
N
N
N
H
2
N COOH
COOH
O
O
O COOH
OH
O
A
H
HOOC
N
N
COOH
OH
O
HO
HOOC
COOH
O
COOH H
H
Staphyloferrins
Staphylococcus hyicus DSM 20459
N
O
N CH
3
O
OH
N
COOH
O
N CH
3
O
OH
H
H
Schizokinen B
Bacillus sp. DSM 6940
S
N
S
N
OH
H
3
C CH
3
OH
N
S
CH
3
COOH
Yersiniabactin
Yersinia enterocolitica
N
CH
2
CH
2
CH
2
CH
NH
C
O
CH
NH
C
O
CH
CH
2
CH
2
NH
C
O
CH
NH
C
O
CH
3
CH
2
OH OH
OH
C C O O
N CH
NH CH
C
O
H
2
C
CH
2
CH
2
H
2
C
N
C
OH
O
H
2
C
CH
2
N
H
HO
HO
H
3
C
Amycolachrome
H
Amycolatopsis orientalis
21
surfactant plays a structural role in aerial mycelium development of Streptomy-
cetes and supports the erection of aerial hyphae by lowering the surface tension
of water films enclosing the colonies. Mass spectrometry results and amino acid
analysis revealed the peptide sequence
H
2
N-Leu-Leu-Ala-Val-Ala-Leu-Lys-Thr
and a molecular mass of 1021 Daltons, including a further valine. The missing
small part with 94 Daltons of the molecule is bound to the N-terminal end of the
peptide [27]. Streptofactin is the first peptide described having structurally and
autoregulatory functions.
The structures of the isolated secondary metabolites involved in differen-
tiation processes are summarised in Fig. 2.4.
Figure 2.4: New secondary metabolites involved in differentiation processes.
C
CH N C
CH
3
N
C C
C C CH
2
O
O
N N
C
CH
3
C
O
O
C
C
CH
N
CH C
O NH
H
3
C
O
CH
CH
3
NO
2
HC CH
2
H
3
C
C
CH
NH
C O
CH
2
O
2
N
O
O
CH
3
H
H
H
H
H
N
HO
Cl
H
H
H
Hormaomycin
Streptomyces griseoflavus W-384
O
OH
O
CH
3
H
3
C
Germicidin A
O
OH
O
CH
3
H
3
C
H
3
C
Germicidin B
H
3
C
Streptomyces viridochromogenes NRRL B-1551
22
2.2.5 Chemical screening by TLC, monitoring coloured secondary
metabolites
Concentrated extracts of culture filtrates and mycelia of Streptomyces strains
were separated on silica gel TLC. Such strains were investigated whose extracts
showed coloured spots on TLC. The assay lead to the detection of various novel
anthraquinone, phenazine and polyene antibiotics.
Urdamycins AF were the most prominent secondary metabolites detected
by this method [2832]. These novel angucycline antibiotics, produced by Strep-
tomyces fradiae T 2717, are biologically active against Gram-positive bacteria
and show a strong cytotoxic activity against stem cells of murine L1210 leukae-
mia.
For the para-quinone metabolites cinnaquinone and di-cinnaquinone, which
were isolated from Streptomyces griseoflavus ssp. thermodiastaticus T 2484, no
biological activities have been detected so far [33, 34].
Two dark green substances, the esmeraldines A and B, were isolated from
Streptomyces antibioticus 2706 [35]. They formally derive by condensation of
two phenazine residues of the saphenic acid family. They dont have any anti-
bacterial activity, but esmeraldine B shows a cytotoxic activity against various
tumor cell lines.
From Streptomyces violaceus T 3556 the new naphthoquinone complex
naphthgeranines AD was isolated [36], from which naphthgeranines A and B
show a weak antibacterial and antifungal activity, whereas A, B and C have a
moderate cytocidal activity against various tumor cell lines. In addition, strain
T 3556 produced the new naphthoquinone compounds naphtherythrins DF.
The bright-yellow polyene carboxylic acid serpentene which shows an
antibacterial activity against Bacillus subtilis was isolated from Streptomyces sp.
T 3851 [37]. Remarkable regarding the structure is the benzene ring nearly in
the middle of the molecule.
The structures of the isolated secondary metabolites screened by this ap-
proach are summarised in Fig. 2.5.
2.2.6 Chemical screening by TLC, monitoring fluorescent secondary
metabolites
Two novel metabolites were detected regarding their blue fluorescence on TLC
plates by irradiation with UV light. Pyridindolol glycosides were isolated from
Streptomyces parvulus T 2480 [38]. No biological activity could be observed of
all three compounds.
Depsichlorins, isolated from Streptomyces antibioticus ssp. griseorubinosus
T 1661, represent a group of new cyclopeptide antibiotics which show biologi-
cal activity against Gram-positive and Gram-negative bacteria [39, 40].
The structures of pyridindolol glycosides and depsichlorins are sum-
marised in Fig. 2.6.
23
Figure 2.5: New secondary metabolites by chemical screening using TLC and monitor-
ing colored compounds.
O
O
HO
HO
O
O
O
OH O
O
O
CH
3
H
3
C
HO
R
OH
OH
O
OH
CH
3
CH
3
CH
3
A
E
: R = H
: R = SCH
3
O
Urdamycins
Streptomyces fradiae T 2717
O
O
HO
HO
CH
3
CH
3
B
O
O
O
H
3
C
HO
OH
CH
3
OH
O
O
O
O
O
HO
HO
O
OH
CH
3
CH
3
CH
3
C
O
O
O
O
CH
3
H
3
C
HO
OH
OH
O
O
O OH
O
R
D
: R =
: R =
HO
H
N
O
O
HO
HO
O
O
O
OH O
O
O
CH
3
H
3
C
HO
OH
OH
OH
O
O
OH
CH
3
CH
3
CH
3
F
24
Fig. 2.5 continued
O
COOH
NH
2
O
HO
OH
O
O
COOH
O
NH
2
O
HO
H
2
N
HOOC
Cinnaquinone
di-Cinnaquinone
Streptomyces griseoflavus ssp. thermodiastaticus T 2486
N
N N
COOH
CH
3
O
O R
N
HOOC
H
3
C
H HN
N N
COOH
CH
3
O
O
N
HOOC
H
3
C
OH
H
3
C
Esmeraldins
A
B
Streptomyces antibioticus T 2706
R = C
13
H
27
R = C
15
H
31
R = C
16
H
33
R = C
17
H
33
R = C
17
H
31
R = (CH
2
)
10
R =
R =
R =
(CH
2
)
12
CH
CH
3
CH
3
CH
CH
3
CH
2
CH
3
(CH
2
)
14
CH
CH
3
CH
3
(CH
2
)
13
CH
CH
3
CH
3
a
c
b
d
e
f
g
h
i
O
O
O
CH
2
R
1
R
2
H
H
R
3
CH
3
CH
3
HO
R
4
OH
Naphthgeranins
R
1
R
2
R
3
R
4
H H H H
H
H
H
H
H
H OH
OH
OH
OH
OH
O
O
O
CH
3
CH
3
HO
OH
CH
2
OH
OH
E
OH
Streptomyces violaceus T 3556
A
B
C
D
R
1
R
2
R
1
O
C
O
O
OH
CH
3
CH
3
R
2
NH CH
3
HOOC
Naphtherythrins D-F
D CHO H
E H H
F H OH
COOH
CH
3
Serpentene
Streptomyces sp. T 3851
25
N
N
OR
1
OR
2
H
OR
3
H
H T 2480 F
2
T 2480 F
3
T 2480 F
4
H
H
H
H
H
O
OH
OH
OH
HO
O
OH
OH
OH
HO
O
OH
OH
OH
HO
R
1
R
2
R
3
Pyridindolol glucosides
Streptomyces parvulus T 2480
CH C C
O
CH
3
Cl
Cl
HO
O
NH CH C
CH
O
C
CH
CH
2
O C CH
O
NH
NH C N C
CH
C
N
CH
O
O
O
O
H
3
C
OH
O
CH
2
CH
3
CH
3
CH
3
CH
3
O
HO
H
3
C
X Y
Depsichlorins
Streptomyces antibioticus ssp. griseorubinosus T 1661
X
Y
N
OAc
O
CH
3
N
OAc
O
CH
3
N
OAc
O
CH
3
CH
3
N
OAc
O
CH
3
CH
3
A
B
C
D
Leu
Homo-Ile
Leu
Homo-Ile
Figure 2.6: New secondary metabolites by chemical screening using TLC and monitor-
ing fluorescent compounds.
26
2.2.7 Chemical screening by TLC and Ehrlich reagent
Ehrlich reagent reacts mainly with primary amines and the products appear as
red-violet zones within few seconds on the TLC.
This reagent was successfully applied for detection of pyrrol-3-yl-2-prope-
noic acid and pyrrol-3-yl-2-propenamide, two further non-active secondary me-
tabolites isolated from Streptomyces parvulus T 2480, the producer of pyridin-
dolol glucosides [41].
The group of pyrrolams are four biosynthetically new pyrrolozidinones pro-
duced by Streptomyces olivaceus T 3082 [42]. They show no antibacterial and
antifungal activities, but a weak herbicidal activity against wheat and rice seed-
lings. Pyrrolam influences the embryonic development of the fish Brachydanio
rerio.
Obsurolides A
2
and A
3
produced by Streptomyces viridochromogenes
T 2580 represent a novel class of phosphodiesterase inhibitors [43]; they have
no growth inhibiting potency against bacteria, yeasts and filamentous fungi.
Two new phenylpentadienamides were detected in Streptomyces sp. T
3946 by orange spots on the TLC stained with Ehrlich reagent, 5-(4-aminophe-
nyl)penta-2,4-dienamide and N
2
-[5-(4-aminophenyl)penta-2,4-dienoyl]-L-gluta-
mine [44]. Both secondary metabolites show no antibacterial and antifungal ac-
tivities.
The structures of the novel secondary metabolites are summarised in Fig. 2.7.
2.2.8 Chemical screening by TLC and blue tetrazoliumstaining reagent
Blue tetrazolium is a relatively specific derivatisation reagent for steroids and re-
ducing compounds. Blue or violet coloured zones are formed on a light back-
ground on the TLC sheet.
From Streptomyces aurantiogriseus T 3149a compound was isolated
which revealed a yellow-orange colour by staining with blue tetrazolium. Be-
cause of its stimulation of aerial mycelium and spore formation of Streptomyces
glaucescens, the compound was named differolid [45]. No growth inhibiting ac-
tivity against bacteria, yeasts and filamentous fungi was observed.
A further blue tetrazolium positive compound, (2S,3R,4R,6R)-2,3,4-trihy-
droxy-6-methylcyclohexanone, was isolated from Streptomyces phaeochromo-
genes ssp. venezuelae T 3154 and Streptomyces albus T 3226 [46]. The com-
pound shows no biological activity to bacteria and fungi.
A new member of natural compounds having a thiotetronic acid structure
was isolated from Streptomyces olivaceus T 3010 [47]. The secondary metabo-
lite (2S)-4-ethyl-2,5-dihydro-3-hydroxy-2-[(1E)-2-methyl-1,3-butadienyl]-5-oxo-
2-thienylacetamide shows antibacterial activity especially against Streptomyces
strains.
From Streptomyces griseoflavus T 2880 the bright yellow colabomycins
AC were isolated which represent new members of the manumycin group [48,
49]. They react with blue tetrazolium as brown spots, with vanillin-sulphuric
27
acid as dark violet and with molybdatophosphoric acid as black spots, indicating
their reducing character. The main compound, colabomycin A, is active against
Gram-positive bacteria and shows a cytotoxic activity against stem cells of mur-
ine L1210 leukaemia.
In collaboration with Hoechst AG and Prof. Fiedler, seven musacin com-
pounds were detected in extracts of Streptomyces griseoviridis FH-S 1832 on
TLC plates. The compounds were detected by blue tetrazolium chloride (show-
ing a blue-violet colour), by anisaldehyde, orcinol, and Ehrlichs reagent, respec-
tively. The determination of their structure revealed that six of the seven com-
pounds were new [50]; musacin C shows an anthelmintic activity against Cae-
norhabditis elegans and Trichostrongylus colubriformis.
The structures of the isolated secondary metabolites are summarised in
Fig. 2.8.
Figure 2.7: New secondary metabolites by chemical screening using TLC and Ehrlich re-
agent.
N
COR
H
Pyrrol-3-yl-2-propenoic acid:
Pyrrol-3-yl-2-propenamide:
R = OH
R = NH
2
Streptomyces parvulus T 2480
N
H
O
N
R
O
A
B
C
: R = OH
: R = OCH
: R = O CH
CH
3
O CH
2
CH
3
3
Pyrrolam
Streptomyces olivaceus T 3082
Obscurolides
H
O
N
CH
3
HO
R
O
Streptomyces viridochromogenes T 2580
A
2
:: R = CHO
A
3
:: R = CH
2
OH
H
2
N
O
NH
2
H
2
N
O
NH
CONH
2
COOH
5-(4-Aminophenyl)penta-2,4-dienamide
N -(5-(4-aminophenyl)penta-2,4-dienoyl)-L-glutamine
2
Streptomyces sp. T 3946
28
Figure 2.8: New secondary metabolites by chemical screening using TLC and blue tetra-
zolium staining reagent.
O
O
H
H
O
O
Streptomyces aurantiogriseus T 3149
Differolid
H
3
C
OH
OH
OH O
(2S,3R,4R,6R)-2,3,4-trihydroxy-
6-methylcyclohexanone
Streptomyces phaechromogenes
ssp. venezuelae T 3154
O
O
N H
H
CH
3
OH
O
NH
O
H
O HO
Colabomycin A
S
OH
CH
3
CH
2
O
CH
2
CH
3
CH
2
H
2
N
O
C
Thiotetronic acid T 3010
HO O CH
3
OH
O
OH
OH
O CH
3
O
OH
OH
O
OH
O
O CH
3
O
OH
OH
OH
OCH
3
O
HO
O
O
H
3
C
OH
O
O
H
3
C
OH
HO
A
B
C
D
F
Musacins
Streptomyces griseoviridis FH-S 1832
29
2.2.9 Chemical screening by TLCand anisaldehyde and orcinol reagent
With anisaldehyde-sulphuric acid reagent sugars, steroids, and terpenes can be
detected. After heating the stained TLC sheets, a great variety of coloured spots
from violet, blue, grey to green were formed on a weakly ochre coloured back-
ground.
The same strain, Streptomyces griseoviridis FH-S 1832, that showed blue
violet musacin spots on the TLC plate when sprayed with blue tetrazolium
chloride, showed another pattern of spots with altered R
f
values and colour,
when the plate was sprayed with anisaldehyde and orcinol reagent, respec-
tively. Besides cineromycin B, three new members of the cineromycin group of
macrolide antibiotics were isolated [50]. The cineromycins showed weak activity
against Gram-positive bacteria; no further biological activities have yet been ob-
served.
The structures of the new cineromycins are summarised in Fig. 2.9.
2.2.10 Chemical screening by TLC and vanillin-sulphuric acid staining
reagent
Vanillin-sulphuric acid reacts relatively specific with higher alcohols, phenols
and steroids. Coloured zones are produced on a pale background on the TLC
sheet.
In the mycelium of the colabomycin producing strain Streptomyces griseo-
flavus T 2880, a further compound, called 2880-II, was detected by vanillin-sul-
phuric acid staining reagent, resulting in a dark brown spot on the TLC sheet.
The compound is related to ferulic acid and shows no antibacterial and antifun-
gal activity [51].
(3S,5R,6E,8E)-Deca-6,8-diene-1,3,5-triol and (3S,6E,8E)-1,3-dihydroxydeca-
6,8-diene-5-one were isolated from the (3S,8E)-1,3-dihydroxydec-8-en-5-one
Figure 2.9: New secondary metabolites by chemical screening using TLC and anisalde-
hyde or orcinol staining reagent.
Streptomyces griseoviridis FH-S 1832
2,3-Dihydrocineromycin B Oxycineromycin B Dehydrocineromycin B
O O
CH
3
CH
3
H
3
C
OH
H
3
C
HO H
O O
CH
3
CH
3
H
3
C
OH
HOH
2
C
HO H
O O
CH
3
CH
3
H
3
C
OH
H
3
C
O
30
producer Streptomyces fimbriatus T 2335. All compounds are inactive against
bacteria and fungi [52].
The structures of the isolated secondary metabolites are summarised in
Fig. 2.10.
2.2.11 Screening for new secondary metabolites by polystyrene resin
fermentation
Addition of the non-polar polystyrene resins Amberlite XAD-16 or XAD-1180 to
growing cultures of microorganisms, preferably at the end of the growth phase,
enables the absorption of unstable intermediate products or stimulates the pro-
ducing organism to an altered metabolite pattern.
The naphthgeranine and naphtherythrine producer Streptomyces viola-
ceus T 3556 (see Section 2.2.5) synthesised the novel series of naphtherythrins
AC, when Amberlite XAD-1180 was added to growing cultures after 36 hours
of incubation [53]. The main compounds, naphtherythrins A and B show a biolo-
gical activity against Gram-positive bacteria and a weak activity against fungi.
Under the same conditions Streptomyces exfoliates T 1424 produced a
group of three new naphthoquinone antibiotics named exfoliamycins [54, 55].
They inhibit growth of Gram-positive bacteria, whereas Gram-negative bacteria
and fungi are not sensitive against these antibiotics.
The structures of secondary metabolites produced during polystyrene resin
fermentations are summarised in Fig. 2.11.
Figure 2.10: New secondary metabolites by chemical screening using TLC and vanillin-
sulphuric acid staining reagent.
OH
OCH
3
NH O
O HO
2880-II
O OH
H
3
C OH
H
3
C OH
OH OH
(3S,5R,5E,8E)-Deca-6,8-diene-1,3,5-triol
(3S,6E,8E)-1,3-Dihydroxydeca-6,8-diene-5-one
Streptomyces fimbriatus T 2335
31
2.2.12 Screening for new secondary metabolites by HPLC and
photoconductivity detection
Photoconductivity detection has a complete different detection window than
UV-Vis spectroscopy and offers the detection of new secondary metabolites in
culture filtrates and extracts of microorganisms by HPLC analysis.
Streptomyces antibioticus T 99, who is known as a producer of chlorothri-
cin, juglomycins A and B, ketomycin, nikkomycins Z and J, as well as nocard-
amine, was reinvestigated using a HPLC photoconductivity screening system.
With this method we could detect four new butenolides [56]. The compounds,
which are summarised in Fig. 2.12, show a weak antibiotic activity against Pseu-
domonas aeruginosa and also a weak inhibition of the chitinase from Serratia
marcescens.
Figure 2.11: Newsecondary metabolites by screening using polystyrene resin fermentation.
C
A : R = CH
3
B : R = H
N
O
CH
3
H
3
C
O
O
OH
O
O
O
O
OH
HOOC
HOOC
O
O
O
N
O
CH
3
H
3
C
O
O
OH
O
CH
2
OH
R
Naphtherythrins A-C
H
O
CH
3
H
3
C
OR
OH O
O
O
HO OH
CH
2
OH O
CH
3
H
3
C
O
O
OH
O
HO OH
CH
2
OH
Exfoliamycines
Streptomyces exfoliatus T 1424
Exfoliamycin R = H
3-O-Methylexfoliamycin R = CH
3 Anhydroexfoliamycin
32
2.2.13 Screening for new secondary metabolites by HPLC and
diode-array detection
This method represents a modification of the classical chemical screening proce-
dure. TLC and staining reagents were replaced by the more efficient reversed-
phase HPLC technique coupled with computerised diode-array detection
(HPLC-DAD). Commercially available antibiotics and secondary metabolites
from our institute pool were analysed with identical standardised HPLC condi-
tions as culture filtrates and raw extracts from freshly isolated Actinomycete
strains. Retention times and UV-visible absorbance spectra of references and
biological samples were stored in libraries of a HPLC-UV-Vis-Database [7]. Till
today more than 600 secondary metabolites, mostly antibiotics, are stored in the
database. The technique was first used for detection of minor congeners and for
characterisation of blocked mutants and intermediate products of biosynthetic
pathways, and since 1990 as screening method for identification of new second-
ary metabolites in freshly isolated strains. All new secondary metabolites result-
ing from this screening strategy are summarised in Fig. 2.13.
In raw extracts from the elloramycin producer Streptomyces olivaceus T
2353 five minor congeners, elloramycins BF, were detected by HPLC-DAD and
determined in structure [57, 58]. All elloramycins are strongly active against
Streptomyces strains. As expected, the less methylated elloramycin B shows the
best activity against Gram-positive bacteria.
The new tetracenomycins B
3
and D
3
were detected in a blocked mutant of
the elloramycin producer, Streptomyces olivaceus T 2353-R [59]. The main
Figure 2.12: New secondary metabolites by HPLC-photoconductivity screening.
H
3
C
O
OH
H
3
C
O
H
HO
H
3
C
H
3
C
O
OH
O
H
HO
H
3
C
H
3
C
H
3
C
O
OH
O
H
3
C
H
3
C
H
3
C
O
OH
H
3
C
O
H
3
C
T 99 Butenolides
T 99-1 T 99-2
T 99-3 T 99-4
33
Figure 2.13: New secondary metabolites by HPLC-diode-array screening.
O
O
O OH O
O
O
OH
OH
O
CH
3
H
3
CO
OCH
3
CH
3
OCH
3
R
2
R
1
OR
4
O
O
O OH O
O
O
OH
O
CH
3
CH
3
OCH
3
H
3
CO
OCH
3
CH
3
OCH
3
OCH
3
O
OR
3
R
1
R
2
R
3
R
4
H
H
H
H H
H
H
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
OH
B
C
D
F
E
Elloramycins B-F
O
O OH CH
3
OH
COOH
OH
RO
Tetracenomycins B
3
and D
3
Streptomyces olivaceus T 2353-R
B
3
R = CH
3
D
3
R = H
N
N
COOH
O H
3
C
N
N
COOH
H
3
C OCH
3
N
N
COOH
H
3
C O R
O
6-Acetylphenazine-1-
carboxylic acid
Saphenic acid methyl ether
Saphenyl fatty acid esters
A
B
D
E
G
I
J
K
R = 12-methyltridecanoic acid
R = tetradecanoic acid
R = 12-methyltetradecanoic acid
R = 14-methylpentadecanoic acid
R = hexadecanoic acid
R = 14-methylhexadecanoic acid
R = unsaturated acid
R = 16- methylheptadecanoic acid
C
18
34
N
N
O
CHO
H
A =
N
N
O
O
H
B =
R
1
OH OH
O
CH CH CH C NH CH
C
R
2
O O
NH
2
R
3
OH
N
R
6
R
4
R
5
HN N
O
O
H
C =
R
1
OH OH
O
CH CH CH C NH CH
C
R
2
O O
NH
2
OH
N
CH
3
N
N
O
CHO
H
A =
N
N
O
O
H
B =
N
N
O
CHO
H
A =
N
N
O
O
H
B =
R
1
OH OH
O
CH C NH CH
C O
OH
CH
2
O
HO
NH
2
Nikkomycins
Streptomyces tendae T 901
Fig. 2.13 continued
35
Fig. 2.13 continued
O
O
OH
OH
C
OH
O
R
1
R
2
R
1
R
2
N
N
O
CHO
H
N
O
O
HN
N
N
O
CHO
H
N
O
O
HN
H
H
OH
OH
S
z
S
x
S
oz
S
ox
Nikkomycins
H
H
N
CH
3
HO
R
1
COOR
2
OH
B
2
: R = CHO
B
3
: R = CH
2
OH
B
4
: R = CH
2
OCH
3
A
1
: R = COOH
A
4
: R = CH
2
OCH
3
C : R
1
= CHO; R
2
= H D
2
C
2
methyl ester: R
1
= CHO; R
2
= CH
3
O
N
CH
3
OHC
O
O
H
O
N
CH
3
R
O
HO
H
O
N
CH
3
HO
R
O
Obscurolides
O
O
COOH
OH
H
HO
CH
3
Juglomycin Z
36
Fig. 2.13 continued
O
O
O
CH
3
HO
OH
CH
2
OH
CH
2
OH
3
5
Naphthgeranine F
N
O
OH
CH
2
OH
H
1-(3-Indolyl)-2,3-dihydroxypropan-1-one
H
H H
H
H
H H
H H
H
HO
H
H
Echinoserin
N
N
N
N
O
O CH
S
CH
2
C N
O
C C N
CH
3
O
C C N
O
C C
CH
3
CH
3
H
3
C
CH
N C C
CH
2
H
3
C CH
3
CH
C
CH
3
C
O
N C C
CH
3
N
O
C
CH
3
C N
O
C C N C
SCH
3
CH
3
HO
O
O
OH
CH
2
OH
O
O
NH
2
O
H OR
O
O
COOH
O OH
H
3
C
O
O
NH
2
O
H OR
Dioxolid A R = H
Dioxolid B R = COCH
3
Dioxolid D
Dehydrodioxolid A R = H
Dehydrodioxolid B R = COCH
3
Streptomyces ten a d e T 4042
O NH
2
OH
para-Hydroxybenzamide
OH
COOH
OCH
3
1-Hydroxy-4-methoxy-2-naphthoic acid
Streptosporangium cinnabarinum ATCC 31213
O
O
CH
3
O
CH
3
CH
3
OH OH
O
OH CH
3
CH
3
Spirofungin
Streptomyces violaceusniger T 4113
37
Fig. 2.13 continued
OH OH
H
3
C
OH
CH
3
O
O OH
O O
OH
OH
OH
CH
3
CH
3
O O
H
3
C
R
1
OH
HO
CH
3
OR
2
HO
1
5
10
15
20
25
30
35
40
1'
2'
3'
NH NH
2
O
NH NH
2
NH
2
+
NH NH
2
O
45
45
45
A
C
D
H
H
CH
3
Kanchanamycins
R
1
R
2
H
3
C
O
O
OH
(E)-4-oxonon-2-enoic acid
O
HO
O
HO
2E,4Z-decadienoic acid
2E,4Z,7Z-decatrienoic acid
Tigloside
Amycolatopsis sp.
CH
3
CH
3
CH
3
CH
3
O
O CH
3
CH
3
O
H
3
C
H
3
C
O
O
O
O
O
OH
OH
OH
OH
HO OH
HO OH
O
O
O
O
O
O
O
O
O
O
CH
3
CH
3
O
H
3
C
H
3
C
38
Fig. 2.13 continued
Simocyclinones
Streptomyces antibioticus Tu 6040
O
HO
OH OH
O
CH
3
OH O
O
H
3
C
O
R
2
R
1
HO
O
HO
OH OH
O
CH
3
OH O
O
H
3
C
O
O
R
2
O
O
OH
R
1
O
HO
OH OH
O
CH
3
OH O
O
H
3
C
O
O
O
O
N
O O
OH
OH
R
3
R
2
R
1
H
HO
OH OH
O
CH
3
OH O
O
R
Simocyclinones A
A1: R = H
A2: R = OH
Simocyclinones B
B1: R
1
= H
B3: R
1
= OH
B4: R
1
= OH
Simocyclinones C
C2: R
1
= OH
C3: R
1
= H
C4: R
1
= OH
Simocyclinones D
D2: R
1
= OH
D3: R
1
= H
D4: R
1
= OH
D6: R
1
= OH
D7: R
1
= H
D8: R
1
= OH
R
2
= H
R
2
= COCH
3
R
2
= COCH
3
R
2
= H
R
2
= H
R
2
= COCH
3
R
2
= H
R
2
= COCH
3
R
2
= COCH
3
R
2
= H
R
2
= COCH
3
R
2
= COCH
3
R
3
= H
R
3
= H
R
3
= H
R
3
= Cl
R
3
= Cl
R
3
= Cl
O
O
OH
OH OH N
O O
CH
3
CH
3
O
P
O OH
O
O
O
OH
C
13-16
H
27-33
O C
16-18
H
33-37
O
H
Kyanomycin
Nonomuria sp. NN22303
39
compound B
3
is antibiotically inactive against Gram-positive and Gram-negative
bacteria, but D
3
shows a moderate activity against Bacillus subtilis and Arthro-
bacter aurescens. The importance of the new compounds is based in their role as
key intermediates and in the elucidation of the biosynthetic pathway of ellora-
mycins and tetracenomycins.
Seven phenazine compounds were isolated from Streptomyces antibioticus
T 2706. Besides saphenamycin, saphenic acid and tubermycin B, three new
phenazines were detected by HPLC-DAD, 6-acetylphenazine-1-carboxylic acid,
saphenic acid methyl ether and a group of eight saphenyl fatty acid esters [60].
A great success of HPLC-DAD screening was the identification of new nik-
komycin compounds in mutants of Streptomyces tendae T 901. Twenty new
nikkomycins were detected by this method [6167] allowing intensive studies
on structure activity relationships [68] and getting new insights in the biosyn-
thetic pathway of nikkomycins. A further new compound from the juglomycin
family, juglomycin Z, was detected in the nikkomycin producing strain Strepto-
myces tendae T 901, when the organism was grown under modified nutrition
conditions [69]. The naphthoquinone antibiotic shows biological activity against
Gram-positive and Gram-negative bacteria and against yeasts.
A series of eight new obscurolides was detected besides the main com-
pounds A
2
and A
3
in Streptomyces viridochromogenes T 2580 [43, 70]. B
4
is
the most active obscurolide in the phosphodiesterase assay. All obscurolides re-
vealed no growth inhibiting potency against bacteria, yeasts and filamentous
fungi.
In the naphthgeranine producing strain Streptomyces violaceus T 3556
besides tubermycin B and 1-phenazinecarboxylate, a new minor congener
naphthgeranine F, as well as 1-(3-indolyl)-2,3-dihydroxypropan-1-one, which
was not previously described as a natural product, were detected [71]. Naphth-
geranine F showed a similar antibacterial activity against Gram-positive bac-
teria as naphthgeranine C, the main compound in fermentations of strain T
3556, whereas 1-(3-indolyl)-2,3-dihydroxypropan-1-one shows no biological ac-
tivity.
A new member of the quinoxaline group antibiotics, echinoserine, was de-
tected in strain Streptomyces tendae T 4031 [72]. The new compound is a non-
cyclic form of echinomycin, but is not a biosynthetic precursor. Echinoserine is
less antibiotically active than echinomycin.
Dioxolides, a novel class of secondary metabolites, were detected in the
culture filtrate of Streptomyces tendae T 4042 [73]. Besides dioxolides, which
consist of an unusual substituted dioxolane ring, para-hydroxybenzamide was
detected, which was not yet described as a natural product. All compounds
show no biological activity against Gram-positive and Gram-negative bacteria,
yeasts and filamentous fungi.
The kanchanamycins, a group of novel 36-membered polyol macrolide
antibiotics were detected in Streptomyces olivaceus T 4018 [74, 75]. The com-
pounds show antibacterial and antifungal activities, and are especially effective
against Pseudomonas. In the same strain the fatty acid (E)-4-oxonon-2-enoic
acid was detected, isolated and determined in structure [76]. This new second-
40
ary metabolite shows an antibacterial activity against various Gram-positive and
Gram-negative bacteria, especially against Staphylococcus aureus, but not
against yeasts and other fungi with the exception of Paecilomyces variotii.
Streptosporangium cinnabarinum ATCC 31213 was characterised as a pro-
ducer of the known antibiotics 43,334 and 43,596, but also as a producer of the
new naphthalene compound 1-hydroxy-4-methoxy-2-naphthoic acid, which
shows herbicidal activity in the Lemna minor assay [77].
In the culture filtrates and extracts of Streptomyces violaceusniger T 4113
the new secondary metabolite spirofungin was detected, a compound having a
polyketide-spiroketal structure that shows various antifungal activities, particu-
larly against yeasts [78].
Tigloside, a new tigloylated tetrasaccharide was detected in Amycolatopsis
sp. NN0 21702 [79]. This secondary metabolite shows unusual structural ele-
ments, which have never before been isolated from Actinomycete strains. Until
now, no biological activity could be observed.
Two fatty acids, (2E,4Z)-decadienoic acid and (2E,4Z,7Z)-decatrienoic acid,
the latter one being described for the first time as a natural product, were de-
tected in the culture filtrate of Streptomyces viridochromogenes T 6105 [80].
Both metabolites show strong herbicidal activities against Lemna minor and Le-
pidium sativum.
Simocyclinones, a novel group of angucyclinones, were detected during
the fermentation of Streptomyces antibioticus T 6040 [8184]. They are novel
natural hybrid polyketide antibiotics and consist of an unusual angucyclinone
ring with a tetraene side chain and a coumarin ring. Simocyclinones can be sub-
divided in A-, B-, C- and D-series regarding structures and UV-visible spectra.
Compounds of the D-series are distinguished by an activity against Gram-posi-
tive bacteria and against several tumor cell lines.
Kyanomycin, a blue-coloured secondary metabolite, was detected in the
mycelium extract of Nonomuria sp. by HPLC-DAD and HPLC-ESI-MS screen-
ing and determined as an unusual anthracycline-phosphatidylethanolamine hy-
brid that shows weak antibacterial activity [85].
2.3 Increasing structural diversity by directed fermentations
2.3.1 Biomodification of saphenamycin and esmeraldine
Streptomyces antibioticus T 2706 produces the antibacterial and antitumor ac-
tive saphenamycin, a phenazine compound, and the cytotoxic active esmeral-
dine B (see Fig. 2.5). Esmeraldine is the condensation product of one molecule
saphenic acid and one molecule saphenamycin. As saphenamycin is a methylsa-
licylic acid ester of saphenic acid, esmeraldine B shows the same methylsalicyc-
lic acid side-chain than saphenamycin. It was of interest to investigate whether
strain T 2706 modifies this side chain when derivatives of methylsalicylic acid
41
were fed during directed fermentations, and if modified saphenamycins and es-
meraldins show an altered antibacterial and antitumor spectrum.
Biomodification was achieved by feeding acetylsalicylic acid, 3-methylsa-
licylic acid, 4-methylsalicylic acid, 5-fluorosalicylic acid, 5-chlorosalicylic acid
and 5-bromosalicylic acid, resulting in six new saphenamycin and six new es-
meraldine compounds. No incorporation into the molecules was achieved by
feeding 5-iodosalicylic acid, 5-bromo-4-hydroxysalicylic acid, 3-hydroxysalicylic
acid, 3-methoxysalicylic acid, 3,5-dinitrosalicyclic acid, 4-aminosalicylic acid, 5-
sulfosalicylic acid, and thiosalicylic acid [86]. The structural modifications are
summarised in Fig. 2.14.
All saphenamycin derivatives show an antibacterial activity, however, only
4-methylsaphenamycin is as active as saphenamycin. The cytotoxic activity to-
wards an urinary bladder carcinoma cell line was lower in case of the deriva-
tives than with saphenamycin. In comparison to esmeraldine B, 4-methyl- and
5-fluoro-esmeraldine B show an altered antibacterial spectrum and an increased
antibacterial activity. 3-Methylesmeraldine B shows a higher cytotoxic activity
against the tested tumor cell line than the original esmeraldine B [86].
2.3.2 Biomodification of ferrioxamines
Streptomyces olivaceus T 2718 is naturally overproducing the iron-chelating
compound desferrioxamine E, also known as the antibiotically active nocard-
amine. Optimisation of the fermentation conditions yielded in amounts of more
than 10 g per litre desferrioxamine E. The siderophore consists of three mole
succinic acid and three mole L-lysine, forming a trihydroxamate ring. Desferriox-
amine E shows the strongest iron(III)-chelating complexing constant related to
desferrioxamines AI. The specificity for the incorporation of diamines contain-
ing two to six carbon atoms was investigated by feeding the following diamines:
1,2-diamine ethane, 1,3-diamine propane, 1,4-diamine butane (putrescine),
1,5-diamine pentane (cadaverine), 1,6-diamine hexane, 1,7-diamine heptane,
1,8-diamine octane, 1,5-diamine ethylether, S-2-aminoethyl cysteine, and N-gly-
cin-1,2-ethylenediamine. The incorporation was monitored by HPLC-DAD that
allowed the detection of all modified ferrioxamines [87]. Diamines with a space
of more than four carbon atoms between the amino groups were not incorpo-
rated into the molecule, such as diamines with more than six carbon atoms. All
other diamines were incorporated and led to the isolation and identification of
13 new ferrioxamines besides ferrioxamine D
2
and E [88], and were determined
in their differences of iron-complexation [89]. The new desferrioxamine struc-
tures are shown in Fig. 2.15.
42
Figure 2.14: Biomodifications of saphenamycin and esmeraldine B by directed fermenta-
tions with Streptomyces antibioticus T 2706.
N
N
O H
3
C
O
R
1
R
2
R
3
R
4
OR
5
COOH
N
N
O H
3
C
O
R
1
R
2
R
3
R
4
OR
5
COOH
N
N
HOOC
CH
3
H
43
Figure 2.15: Desferrioxamine structures produced by directed fermentations with strain
Streptomyces olivaceus T 2717.
N
R
4
O
C
O
C
O C (CH
2
)
2
C O
C
O
(CH
2
)
2
C
O
(CH
2
)
2
N HO R
2
NH
N
OH
NH
NH
R
1
R
3
44
2.3.3 Biomodification of rhizoferrins
Although rhizoferrin represents a fairly simple molecule that consists of two mo-
lecules of citric acid linked to 1,4-diaminobutane through two amide bonds (see
Fig. 2.3), it may have potential application in biotechnology due to its appreci-
able metal-binding properties and the ability to be easily degraded by various
microorganisms. The specificity of the biosynthetic enzymes of the rhizoferrin
producing fungus Cunninghamella elegans was investigated by modifying both,
the chain length of the diamine and the citric acid part of the molecule [21]. Var-
iations in chain length of the diamine backbone were very well tolerated.
Branching of functionalization in b-position to the amino group of the diamine
compounds was also accepted. However, it was not possible to introduce a-
amino acids. Therefore, the biosynthesis of rhizoferrin must be different to the
biosynthesis of staphyloferrin A, produced by staphylococci. Neither by feeding
of D-ornithine nor by application of inhibitors of ornithine decarboxylase, with
and without simultaneous addition of D-ornithine, it was possible to detect even
trace amounts of staphyloferrin A in Cunninghamella elegans. A higher degree
of enzyme specificity was involved in the formation of the activated citryl spe-
cies since analogues of citric acid are more difficult to be incorporated into rhi-
zoferrin analogues than diamines. The structures are summarised in Fig. 2.16
(series A are structures modified in the diamine part, series B are structures
modified in the citric acid part). All derivatives obtained by directed fermenta-
tions showed similar iron-chelating properties.
Acknowledgments
Following scientists from the Mikrobiologie/Antibiotika group contributed to
the success in search for novel secondary metabolites:
Post-docs: J. Bielecki, C. Bormann, Z. Chen, H. Decker, H. Drautz, U. Fauth,
M. Harder, T. Hrner, J. Mller, A. Plaga, O. Potterat, T. Schz, and U. Theobald.
Doctoral students: M. Alverado-Kirigin, N. Andres, S. Blum, M. Brandl,
D. Braun, K. Burkhardt, I. Cebulla, H.-J. Cullmann, H. Haag, U. Hartjen, H. Hoff,
W. Huhn, C. Isselhorst-Scharr, O. Jung,W. Katzer,W. Kuhn, J. Meiwes, F. Petersen,
C. Pfefferle, U. Pfefferle, P. Reuschenbach, M. Richter, J. Schimana, P. Schneider,
U. Schneider, A. Seiffert, J. Stmpfel, M. Tschierske, B. Wahl, and F. Walz.
The excellent collaboration with the groups of Walter Keller-Schierlein
(ETH Zrich), Axel Zeeck and Jrgen Rohr (Universitt Gttingen), Gnther
Jung and Jrg W. Metzger (Universitt Tbingen), Wilfried Knig (Universitt
Hamburg), Urs Squin (Universitt Basel), and Gerhard Bringmann (Universitt
Wrzburg), which were involved in structure elucidation of isolated secondary
metabolites, is gratefully acknowledged.
45
Acknowledgments
Figure 2.16: Rhizoferrin structures produced by directed fermentations with Cunningha-
mella elegans.
C
COO
-
OH
COO
-
N
O
C
HO
-
OOC
-
OOC
N
O
C
COO
-
COO
-
N
O
H
C
HO
-
OOC
-
OOC
N
O
C
COO
-
OH
COO
-
N
O
C
HO
-
OOC
-
OOC
N
O
C
COO
-
COO
-
N
O
H
C
-
OOC
-
OOC
N
O
H
C
COO
-
OH
COO
-
N
O
C
HO
-
OOC
-
OOC
N
O
O
C
COO
-
COO
-
N
O
CH
3
C
HO
-
OOC
-
OOC
N
O
C
COO
-
OH
COO
-
N
O
C
HO
-
OOC
-
OOC
N
O
CH
3
C
COO
-
COO
-
N
O
CH
3
C
-
OOC
-
OOC
N
O
H
3
C
C
COO
-
OH
COO
-
N
O
O
C
HO
-
OOC
-
OOC
N
O
H H
H H
H
H
H H
H H
Homorhizoferrin
Norrhizoferrin
Oxahomorhizoferrin
2-Methylhomorhizoferrin
2-Oxorhizoferrin
A-Series B-Series
H H
H H
H H
H H
Monodesoxyrhizoferrin
Didesoxyrhizoferrin
Monomethylmonodesoxyrhizoferrin
Dimethyldidesoxyrhizoferrin
46
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73. Blum, S., Groth, I., Rohr, J., and Fiedler, H.-P. (1996) Dioxolides, novel secondary me-
tabolites from Streptomyces tendae. J. Basic Microbiol. 36, 1925.
74. Fiedler, H.-P., Nega, M., Pfefferle, C., Groth, I., Kempter, C., Stephan, H., and Metz-
ger, J. W. (1996) Kanchanamycins, new polyol macrolide antibiotics produced by
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activities. J. Antibiot. 49, 101107.
75. Stephan, H., Kempter, C., Metzger, J. W., Jung, G., Potterat, O., Pfefferle, C., and Fie-
dler, H.-P. (1996) Kanchanamycins, new polyol macrolide antibiotics produced by
Streptomyces olivaceus T 4018. II. Structure elucidation. J. Antibiot. 49, 109113.
50
76. Pfefferle, C., Kempter, C., Metzger, J. W., and Fiedler, H.-P. (1996) (E)-4-oxonon-2-
enoic acid, an antibiotically active fatty acid produced by Streptomyces olivaceus T
4018. J. Antibiot. 49, 135137.
77. Pfefferle, C., Breinholt, J., Grtler, H., and Fiedler, H.-P. (1997) 1-Hydroxy-4-methoxy-
2-naphthoic acid, a herbicidal compound produced by Streptosporangium cinnabari-
num ATCC 31213. J. Antibiot. 50, 10671068.
78. Hltzel, A., Kempter, C., Metzger, J. W., Jung, G., Groth, I., Fritz, T., and Fiedler, H.-
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decatrienoic acid, two herbicidal metabolites from Streptomyces viridochromogenes
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81. Schimana, J., Fiedler, H.-P., Groth, I., Smuth, R., Beil, W., Walker, M., and Zeeck,
A. (2000) Simocyclinones, novel cytostatic angucyclinone antibiotics produced by
Streptomyces antibioticus T 6040. I. Taxonomy, fermentation, isolation and biological
activities. J. Antibiot. 53, 779787
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cytostatic angucyclinone antibiotics produced by Streptomyces antibioticus T 6040.
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New simocyclinones of the A-, B-, C- and D-series, novel angucyclinone antibiotics
from Streptomyces antibioticus T 6040. J. Antibiot., in press.
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sity of metabolites is dependent on fermentation conditions. J. Ind. Microbiol. Bio-
technol., in press.
85. Pfefferle, C., Breinholt, J., Olsen, C. E., Kroppenstedt, R. M., Grtler, H., and Fiedler,
H.-P. (2000) Kyanomycin, a complex of unusual anthracycline-phospholipid hybrid
from Nonomuria species. J. Nat. Prod. 63, 295298.
86. Kuhn, W. (1993) Untersuchungen zur Produktion der Esmeraldine und der Physiologie
von Streptomyces antibioticus. Doctoral Thesis, Universitt Tbingen.
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fication of new ferrioxamines by HPLC and diode array detection. J. Chromatogr. 513,
255262.
88. Meiwes, J., Fiedler, H.-P., Zhner, H., Konetschny-Rapp, S., and Jung, G. (1990) Pro-
duction of desferrioxamine E and new analogues by directed fermentation and feed-
ing fermentation. Appl. Microbiol. Biotechnol. 32, 505510.
89. Konetschny-Rapp, S., Jung, G., Raymond, K. N., Meiwes, J., and Zhner, H. (1992)
Solution thermodynamics of the ferric complexes of new desferrioxamine sidero-
phores obtained by directed fermentations. J. Am. Chem. Soc. 114, 22242230.
51
References
3 Biosynthesis of the Lantibiotics Epidermin
and Gallidermin
Friedrich Gtz* and Gnther Jung**
3.1 History of lantibiotics and lantibiotic research
in Tbingen
The first lantibiotic structure elucidated was that of nisin [23], and nisin was
probably the first lantibiotic identified [65]. Nisin is produced by the cheese star-
ter culture organism Lactococcus lactis subsp. lactis and has an antimicrobial ef-
fect on a broad variety of Gram-positive bacteria [23]. The pioneering research
of E. Gross and coworkers demonstrated that the peptide antibiotics nisin and
subtilin, the latter produced by Bacillus subtilis, actually contain lanthionine
(Lan) and 3-methyllanthionine (MeLan) as well as (Dha) and (Dhb), confirming
previous hypotheses [6, 21, 22].
The lantibiotic era in Tbingen began in 1985 with the publication of the
peptide sequence of epidermin isolated from Staphylococcus epidermidis. With
the elucidation of the structure of epidermin, it became clear that it is a hetero-
det tetracyclic 22-amino acid (2164 Da), amide peptide [1] that contains one re-
sidue each of Dhb and MeLan and two residues of Lan. The fourth cyclic struc-
ture results from the novel C-terminal mono-carboxy, di-amino acid, AviCys.
It was not clear at that time how these amino acid structures arose and
how the complex structures of nisin and epidermin are synthesized. Are they
non-ribosomally synthesized, as are gramidicin and valinomycin, which are ty-
pically synthesized by large multi-enzyme complexes in the cell and for which
no structural genes exists [27, 33], or are they synthesized ribosomally as a pre-
cursor peptide, which is subsequently posttranslationally modified?
In 1987, when Friedrich Gtz took over the chair in Mikrobielle Genetik in
Tbingen, he and his coworkers started to unravel the biosynthetic principles of
* Mikrobielle Genetik, Universitt Tbingen, Waldhuser Str. 70/8, D-72076 Tbingen
** Institut fr Organische Chemie, Universitt Tbingen, Auf der Morgenstelle 18,
D-72076 Tbingen
52
ISBN: 3-527-30615-3
epidermin. In co-operation with Karl-Dieter Entians group, a gene correspond-
ing to the epidermin amino acid sequence was identified on a 54-kb plasmid of
the epidermin-producing strain [72]. These pioneering results brought lantibio-
tic research a big step forward. For the first time it was shown that a lantibiotic
is ribosomally synthesized, and clues on the organization of epidermin as a pre-
cursor protein and the processing and post-translational modification steps were
found.
In the following years, the principles of the genetic organization and bio-
synthesis first found with epidermin were subsequently also found with other
lantibiotics all other lantibiotics studied later were also shown to be riboso-
mally synthesized. To date, antibiotics have been found in many Gram-positive
genera, such as Bacillus, Lactococcus, Pediococcus, Staphylococcus, Streptococ-
cus, and Streptomyces. Lantibiotics have not been found in a Gram-negative
species.
Subsequent studies of lantibiotics have revealed that they are post-transla-
tionally modified at specific positions to give rise to the large number of modi-
fied amino acids found in these peptides. In addition, the peptides are produced
with a leader peptide, which is removed during maturation, and are transported
by specific transport-related proteins out of the cell. Still other lantibiotic-speci-
fic proteins are involved in the genetic regulation of biosynthesis and generation
of the specific producer-cell self-protection mechanism(s) frequently observed.
The name lantibiotics was coined in Tbingen and refers to the rapidly
expanding group of antibiotic-like peptides that contain the non-protein amino
acids lanthionine and 3-methyllanthionine [72]. The discussion whether epider-
min and nisin should be regarded as antibiotics or as bacteriocins was intense.
The small size and compact structure of the compounds and the wide range of
antibacterial activity against most Gram-positive and some Gram-negative bac-
teria favors consideration as antibiotics bacteriocins are usually larger proteins
with a narrow range of activity, such as colicins. On the other hand, typical
amino-acid-derived antibiotics, such as penicillin and gramicidin, are non-ribo-
somally synthesized. With the knowledge available today, it is clear that lanti-
biotics fulfill criteria of both, antibiotics and bacteriocins and cannot be placed
in one or the other category. The name lantibiotic belies the full extent and com-
plexity of this class of bacterial peptides.
A strong and very fruitful collaboration was developed with Hans-Georg
Sahl (Bonn) and his coworkers. Since 1983, they had been studying the mode of
action of Pep5 and nisin, and later also of gallidermin and other lantibiotics. Ear-
lier studies suggested that nisin could interfere with the biosynthesis of the bac-
terial cell wall [46, 64]. This appears to be indeed true, as recent studies show
[13]. However, the binding to cell wall precursors does not explain the observed
bactericidal effect of lantibiotics. Type A lantibiotics form non-specific trans-
membrane pores in an energy-dependent fashion and allow efflux of preaccu-
mulated intracellular components [66, 69, 73].
In 1984, Friedrich Gtz, then at the Technical University in Mnchen, iso-
lated an antibiotic compound from a Staphylococcus gallinarum strain, which
belongs to a species previously described by Devriese et al. [17]. Preliminary
53
3.1 History of lantibiotics and lantibiotic research in Tbingen
studies showed that this antibiotic compound exerts a rather broad activity
against Gram-positive bacteria. However, it was not until he moved to Tbingen
that the compound was isolated in the group of Hans Zhner and the structure
was elucidated in the group of Gnther Jung [28]. The compound was named
gallidermin after the species name of the producing staphylococcus. Gallider-
min is a structural analogue of epidermin, but is slightly more active than epi-
dermin. Today, after long and laborious work to optimize the fermentation and
thus increase gallidermin production approximately hundred-fold [29], S. galli-
narum T 3928 is used for the biotechnological production of gallidermin by the
groups of Peter Fiedler (fermentation) and Rolf Werner (downstream processing;
Boehringer Ingelheim, Biberach).
To say that in the years following 1986, Tbingen became a stronghold in
lantibiotic research, by setting the pace of the research, inspiring many other
groups, and initiating quite a number of national and international co-opera-
tions, is no exaggeration. Three international workshops on lantibiotics have al-
ready taken place. Gnther Jung (Tbingen) initiated together with Hans-
Georg Sahl (Bonn) the first lantibiotic workshop, held in April 1991 in Bad Hon-
nef. The contributions were published under the title Nisin and novel lantibio-
tics with Jung and Sahl as editors (Leiden: Escom). The second workshop on
Lantibiotics: a unique group of antibiotic peptides was organized by Ruud
Konigs and Cees Hilbers (Nijmegen, The Netherlands) and held in Arnhem in
November 1994. The contributions were published in Antonie van Leeuwen-
hoek, vol. 69 in 1996. The third workshop on Lantibiotics and related modified
antibiotic peptides was organized by Friedrich Gtz, R. Jack, Gnther Jung,
and Hans-Georg Sahl and was held in Blaubeuren in April 1998. The ever pre-
sent international interest in the lantibiotic subject is best documented by the
increasing number of applicants and participants from workshop to workshop.
A number of potential applications have been found for the mature lanti-
biotics, including the use as an anti-infective in medical and veterinary areas, in
food, beverage and cosmetic preservation, and as regulators of both, human im-
mune function and blood pressure [26, 50, 62, 67]. Lantibiotics represent new
lead structures, but their chemical synthesis is too complicated and costly;
therefore, the only way to produce large enough quantities for marketing is
through biotechnology.
In the following sections, more detailed information on lantibiotic research
is presented. Since this is a final report on all achievements obtained in the fra-
mework of the collaborative research centre 323, we will focus on the results ob-
tained by the groups in Tbingen. However, the picture would be incomplete if
major achievements of other groups were not included. The topics on novel
structures, mechanism(s) of biosynthesis, genetic organization and regulation,
biological activities and mode of actions as well as potential applications for this
fascinating, novel class of bacterial-derived, biologically active peptides will be
addressed.
54
3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin
3.2 Primary structure and proposed maturation
of epidermin in staphylococci
The nucleotide sequence of the epidermin structural gene, epiA, revealed that
epidermin is part of a pre-peptide that consists of a 30-amino-acid N-terminal
leader peptide and a 22-amino-acid C-terminal propeptide [72]. A comparison of
the chemical structure of epidermin [2] with the peptide sequence deduced from
epiA indicates that this propeptide undergoes several modifications (Fig. 3.1)
before it is transported out of the cell. At each pair of positions where the mature
epidermin contains a lanthionine bridge, the precursor peptide contains one ser-
ine or threonine and one cysteine. Lanthionine is proposed to be formed in a
two-step process involving dehydration of serine and threonine and the sub-
sequent addition of a cysteine thiol group [10, 24].
Figure 3.1: Posttranslational modifications of the epidermin pre-peptide. (A) Model of
lanthionine synthesis: dehydration of serine (a) is followed by a nucleophilic addition reac-
tion of a thiol group, thereby forming a thioether bridge (b); the formation of meso-lanthio-
nine from threonine is analogous. (B) Biosynthesis of epidermin: dehydration (a), nucleo-
philic addition (b), oxidative decarboxylation (c), and removal of the leader peptide (d).
Abu, aminobutyric acid; Dhb, dehydrobutyrine.
55
3.2 Primary structure and proposed maturation of epidermin in staphylococci
3.3 Genetic organization and regulation
of the epidermin genes
3.3.1 Organization and function of epidermin genes
The gene cluster for biosynthesis of the lantibiotic epidermin is composed of
eleven genes in five transcription units. The genes are involved in 1) synthesis of
the epidermin precursor peptide, 2) modification of the precursor peptide, 3)
secretion of epidermin, 4) cleavage of the leader peptide, 5) immunity of the pro-
ducer to epidermin, and 6) regulation of the gene cluster. Four of the transcription
units are activated by the DNA-binding regulator EpiQ. The factors or conditions
leading to activation of the EpiQ protein or its expression remain unclear.
The gene cluster for epidermin biosynthesis, located on plasmid pT32 of
S. epidermidis T 3298 [72], contains genes for all activities proposed to be in-
volved in the biosynthetic pathway (Fig. 3.2). Like all lantibiotics, epidermin is
ribosomally synthesized and the gene cluster accordingly contains the structural
gene epiA, which encodes the precursor peptide [70, 72]. In the same operon,
the genes for modification reactions leading to the formation of thioether
bridges (epiB and epiC) [4, 41, 58] and to oxidative decarboxylation of the C-ter-
minus (epiD) [42, 43] are encoded. Between epiA and epiB, a weak terminator-
like structure reduces the expression of the subsequent genes [56, 70] whose
products are required only in catalytic amounts.
In a second transcriptional unit, the extracellular leader peptidase EpiP
[20] and the regulator protein EpiQ [56] are encoded. Upstream of epiA, the in-
dividually expressed genes epiT and epiH are located [60]. The deduced pro-
duct of epiT shares sequence similarity with an ABC-type transporter that is in-
volved in secretion of certain peptides and proteins [75]. The coding sequence
Figure 3.2: Organization of the epidermin gene cluster encoded on plasmid pT32. Epi-
dermin genes are shown as white arrows; flanking genes with putative functions in plas-
mid replication (rep) and maintenance (resolvase) are indicated in gray. Gene functions:
epiA, structural gene; epiB and epiC, dehydration and lanthionine formation; epiD, flavo-
protein (oxidative decarboxylation); epiQ, activator; epiP, pro-epidermin processing prote-
ase; epiH and epiT, translocation (export) of pro-epidermin; epiFEG, epidermin immunity.
Arrows indicate promoters that are activated by EpiQ. The epiT gene is incomplete due to
a deletion and frame-shift mutation, but its function can be complemented by the intact
gallidermin gene, gdmT.
56
is disrupted by two frame-shift deletions, and it is very questionable whether
epiT has a function in epidermin biosynthesis. Transcriptional reporter gene
fusions have demonstrated, however, that epiT is expressed (see below). The
homologous gene gdmT from the gene cluster of the closely related lantibiotic
gallidermin, has an intact sequence; it mediates an increase of epidermin pro-
duction in the heterologous host Staphylococcus carnosus [60] and thereby sub-
stantiates the proposed capacity of GdmT to secrete epidermin or gallidermin.
The gene adjacent to gdmT gdmH is also necessary for increased epidermin
production; GdmH may be an accessory factor for secretion. EpiH and GdmH
are hydrophobic proteins without conspicuous similarities to other proteins; they
furthermore exert a limited level of immunity to epidermin [57].
Upstream of epiH, three cotranscribed genes, epiF, epiE, and epiG, are en-
coded. They mediate resistance (immunity) to epidermin, and their products
very likely constitute the subunits of an ABC transporter that expels the harmful
epidermin molecules from the cytoplasmic membrane [54, 57]. The epidermin
genes are flanked by genes which apparently encode factors for replication and
stability of plasmid pT32 (Fig. 3.2). The res gene product has a high level of
identity to plasmid resolvases, whose function is the resolution of cointegrates
[58]. The open reading frame upstream of epiP shares significant similarity with
plasmid replication genes from various Gram-positive bacteria (M. Hille and
A. Peschel, unpublished). The epidermin gene cluster thus seems to be com-
plete; however, several chromosomally encoded genes are also very likely in-
volved in epidermin biosynthesis.
3.3.2 EpiQ is an unusual transcriptional regulator
EpiQ shares similarities with DNA-binding proteins of the response regulator fa-
mily [70] and binds to the epiABCD promoter region to activate expression [56].
Unlike conventional response regulators, EpiQ does not contain the conserved
aspartic acid residue that is phosphorylated by a corresponding sensor kinase to
activate the regulator [76]. Since a corresponding kinase gene is absent from
the epidermin gene cluster, it is very questionable whether EpiQ is phosphory-
lated at all. Several other lantibiotic gene clusters encode a conventional kinase
and regulator pair [75]; therefore, the regulator gene epiQ is unusual. The two-
component regulatory systems of nisin and subtilin activate the biosynthetic
genes in response to the extracellular concentration of the respective lantibiotic
[36]. Nisin and subtilin thus act as bacteriocin-like antimicrobial peptides and as
quorum-sensing peptide pheromones that control their own expression. Synth-
esis of epidermin seems to be regulated differently. Regulating agents or condi-
tions have not yet been identified, but EpiQ seems to be active under all labora-
tory conditions tested so far (A. Peschel, unpublished).
57
3.3 Genetic organization and regulation of the epidermin genes
3.3.3 Regulation of epidermin genes
Trans-activation experiments with transcriptional reporter gene fusions have de-
monstrated that EpiQ controls most of the transcriptional units in the epidermin
gene cluster; the promoters of the epiFEG, epiH, epiT, and epiABCD gene clus-
ters are activated [56, 58, 60] (Fig. 3.3). All these promoter regions have a palin-
dromic sequence motif (agAaAATTAC 6 bp GTAATTtTct) located immedi-
ately upstream of the only weakly conserved 35 regions. The motif upstream of
the epiABCD promoter is the EpiQ binding site [56]. The EpiQ operator is also
present in the gallidermin gene clusters at the same positions, and the promo-
ters are also sensitive to EpiQ.
The only transcriptional unit not controlled by EpiQ is the epiPQ operon
(Fig. 3.2); EpiQ therefore exerts no autoregulation. The epidermin gene cluster
contains additional promoters within the operon structures that are not con-
trolled by EpiQ, e. g. in front of epiC, epiD, and epiQ. The epiD promoter has a
particularly high activity (Peschel et al., unpublished).
The regulation of the epidermin genes remains elusive since no activating
agents have been found. Recent literature illustrates the importance of global reg-
ulatory relays in Gram-positive bacteria [31]. Quorum-sensing systems control the
production of antimicrobial substances in B. subtilis, and it is conceivable that a si-
milar, yet unknown system controls the epidermin genes. A further possibility is
the involvement of special sigma factors that couple epidermin production to cer-
tain growth phases or environmental conditions. In this respect, it is interesting to
note that epidermin production occurs mainly during the exponential growth
phase and is obviously switched off in the stationary phase [54]. Since EpiQ con-
Figure 3.3: Promoter activities of various epidermin genes. Extracellular lipase activities
of S. carnosus strains carrying a promoter test plasmid containing the lipase gene as a re-
porter under the control of the indicated epidermin gene promoters (Pepi). Lipase expres-
sion was determined in the presence and absence of the activator gene, epiQ, encoded on
a second plasmid.
1
10
100
1000
Pepi
FEG
Pepi
H
Pepi
T
Pepi
ABCD
Pepi
PQ
- EpiQ
+Epi Q
L
i
p
a
s
e
a
c
t
i
v
i
t
y
(
U
/
g
c
e
l
l
d
r
y
w
t
.
)
58
trols most of the epidermin genes, the promoters controlling its own expression are
likely targets for a chromosomally encoded regulatory system. Chromosomally en-
coded proteins may also be involved in secretion of epidermin, thereby substitut-
ing for the defective epiT, and may be involved in the modificationreactions.
3.4 Isolation and characterization of genetically engineered
gallidermin and epidermin analogues
Gallidermin (Gdm) and epidermin (Epi) are highly similar. To study the substrate
specificity of the modifying enzymes and to find variants of the lantibiotics with
altered or new biological activities, we exchanged certain amino acids of the gal-
lidermin and epidermin structural genes, gdmA and epiA, respectively, by site-
specific mutagenesis [53]. No epidermin/gallidermin analogues are found in the
supernatant when 1) the hydroxyamino acids involved in thioether amino acid
formation are substituted by non-hydroxyamino acids (S3N and S19A); 2) cys-
teine residues involved in thioether bridging are deleted (C21, C22 and C22); or
3) a ring amino acid is substituted by an amino acid with a completely different
character (G10E and Y20G). Production is greatly decreased when serine resi-
dues involved in thioether amino acid formation are exchanged by threonine resi-
dues (S16T, S19T). A number of conservative exchanges at positions 6, 12, or 14
of the gallidermin backbone are tolerated and lead to analogues with altered bio-
logical properties, such as an enhanced antimicrobial activity (L6V) or a remark-
able resistance to proteolytic degradation (A12L and Dhb14P). The T14S substi-
tution leads to the simultaneous production of two gallidermin species formed by
an incomplete posttranslational modification (dehydration) of the S14 residue.
The fully modified Dhb14Dha analogue has antimicrobial activity similar to galli-
dermin, whereas the Dhb14S analogue is less active. Both peptides are more sen-
sitive to tryptic cleavage than gallidermin. The construction and characterization
of the various analogues are described in more detail in the following sections.
3.4.1 Characterization of two Epi
mutants and development

of a host-vector system for expression of wild type and mutated
gdmA and epiA genes
We isolated a series of Epi
mutants of S. epidermidis T 3298 by ethyl-methane-

sulfonate(EMS) mutagenesis; two mutants (EMS5 and 6) carry mutations in the
epiA region [4]. Both mutants can be complemented to an Epi
+
phenotype by
59
transformation with a plasmid carrying epiA, which indicates that other epi bio-
synthetic genes are functional. Single point mutations in epiA result in an S3N
substitution (ring A) in EMS5 and a G10E substitution (ring B) in EMS6 (Fig. 3.1).
Analytical HPLC analyses indicated that no epidermin analogue or precursor
peptide is present in the culture supernatants of EMS5 and EMS6 [53].
We chose the mutant S. epidermidis EMS6 as an expression host for the
synthesis of epidermin and gallidermin analogues. To test this mutant for its
ability to synthesize the heterologous gallidermin, a 1.6-kb SalI/EcoRI subfrag-
ment (encoding gdmA and a part of gdmB and of gdmT) of the 4.8-kb EcoRI
chromosomal fragment of S. gallinarum T 3928 [71] was cloned into the poly-
linker region of the staphylococcal plasmid pT181mcs. S. carnosus TM300 was
transformed with the resultant plasmid, pTgdmA [53]. After isolation, the plas-
mid was transferred into S. epidermidis EMS6 by electroporation. This indirect
transformation method is necessary because S. epidermidis T 3298 and deriva-
tive strains are poorly transformed with ligation products; only ccc plasmids are
electroporated efficiently. EMS6 transformants carrying plasmid pTgdmA
formed halos on plates containing Micrococcus luteus, and analyses revealed
that only fully modified gallidermin is produced.
3.4.2 Characterization of gallidermin and epidermin analogues
generated by site-directed mutagenesis
The gallidermin and epidermin analogues were isolated from the culture super-
natant of various S. epidermidis EMS6 clones, purified, and analyzed by analytical
HPLC, electrospray mass spectrometry (ES-MS), and continuous Edman sequen-
cing. MIC values and tryptic sensitivity were determined for all gallidermin analo-
gues, except Gdm L6V/S16T and epidermin S19T because these peptides were
only produced in trace amounts. The results are summarized in Table 3.1 [53].
3.4.2.1 Mutations in ring A
Since Gdm (L6) is more active than Epi (I6) against various Gram-positive bac-
teria (23), we generated two more analogues of gallidermin with mutations at po-
sition 6: L6V and L6G. The production and modification of these two analogues
are not impaired. The L6G analogue is less active than gallidermin, regardless of
the indicator strain employed. The L6Vanalogue is twice as active as gallidermin
against Micrococcus luteus and Corynebacterium glutamicum, and just as active
against Arthrobacter cristallopoietes and B. subtilis. Antimicrobial activity
against S. aureus is reduced fivefold (Table 3.1). The L6G analogue is almost as
sensitive to trypsin as gallidermin, whereas the L6Vanalogue is more resistant.
60
3.4.2.2 Mutations in ring C and D
To determine the importance of the C-terminal bicyclic structure (intertwined
rings C and D) for epidermin/gallidermin biosynthesis, we exchanged various
amino acids in this region. Using the gdmA-L6V gene, we created the double
mutant Gdm L6V/S16T. The production of this analogue is only 8% of that ob-
served for Gdm L6V. Molecular mass determinations of Gdm L6V/S16Trevealed
that the T16 residue is dehydrated. However, it was not possible to verify the
formation of the 3-methyllanthionine in ring C by ES-MS. Gdm L6V/S16T has
antimicrobial activity; however, the MIC values were not determined because of
the very low production of this analogue.
The S19T codon of EpiA was replaced in order to create a posttranslation-
ally formed S-(2-aminovinyl)-2-methyl-d-cysteine residue as found in mersaci-
din. This analogue has antimicrobial activity and is also produced in very low
amounts. The production is only 0.4% of that observed for the gallidermin-pro-
ducing EMS6 (pTgdmA) clone, which produces approximately 12 mg Gdm/l.
ES-MS analysis revealed an average molecular mass of 2177 Da, indicating that
T19 is dehydrated and that the C-terminus is decarboxylated. It was also not
possible to verify the formation of the S-(2-aminovinyl)-2-methyl-d-cysteine resi-
due by ES-MS; however, thioether bridge formation is very likely because of
the observed chemical instability of the enethiol structure [42, 43].
To prevent formation of the S-(2-aminovinyl)-d-cysteine residue in ring D,
we generated an S19A (pTepiA-S19A) substitution and a C22 deletion (pTepiA-
DC22). These mutations could possibly lead to the formation of alternative
thioether bridges. However, the corresponding EMS6 clones form no halos on
Table 3.1: Antimicrobial activities of gallidermin and its analogues against various Gram-
positive indicator strains [53].
Gdm Minimal inhibitory concentration (lg/ml)
a
+
derivatives Micrococcus Arthrobacter Coryne- Bacillus Staphyloc.
luteus cristallo- bacterium subtilis aureus
poietes glutamicum Cowan I
Gdm 0.004 0.005 0.065 3.0 4.0
Gdm Dhb14Dha 0.004 0.005 0.065 3.0 5.0
Gdm Dhb14S 0.008 0.01 0.13 15.0 25.0
Gdm Dhb14A 0.004 0.02 0.065 >20.0 30.0
Gdm Dhb14P 0.12 0.08 0.52 >20.0 >30.0
Gdm A12L 0.15 0.02 0.13 10.0 12.0
Gdm L6G 0.008 0.04 0.13 8.0 30.0
Gdm L6V 0.002 0.005 0.032 3.0 20.0
a
MIC values obtained for M. luteus, A. cristallopoietes, and C. glutamicum are located
within the nanomolar range (0.0020.52 mg/ml; *1240 nM), whereas those observed
for B. subtilis and S. aureus are located within the micromolar range (3.0 to 630 mg/ml;
1.4 to 614 mM).
61
plates of M. luteus, and no epidermin analogue or precursor form was detected
by analytical HPLC. In addition no antimicrobial activity and no extracellular
product were detected when the last two C residues (pTepiA-DC21, C22) were
deleted.
An amino acid substitution not directly involved in thioether bridge for-
mation of ring D was created by replacing the bulky Y20 residue with a G
(pCUgdmA-Y20G). The corresponding EMS6 clone has no antimicrobial activity,
and no gallidermin analogue or precursor peptide was detected by analytical
HPLC.
3.4.2.3 Mutations in the flexible middle region (A12 to G15)
With the A12L substitution, a more bulky residue was introduced in the flexible
middle region. The production of this analogue is comparable to that of gallider-
min; however, antimicrobial activity against most of the test strains is reduced
threefold (Table 3.1). A striking feature of this analogue is its high resistance to
tryptic cleavage.
Further amino acid alterations mainly focused on the 2,3-unsaturated
Dhb14 residue to investigate whether the reactive C=C double bond is impor-
tant for biological activity, as suggested for the Dha5 residue of subtilin and ni-
sin [38, 48], or whether this residue plays a role in stabilizing a distinct structure
required for pore-forming activity, as reported for the P residue in the channel-
forming peptides alamethicin and melittin [78].
With the T14S substitution, two analogues were produced in nearly equi-
molar ratios, as judged from the respective peak areas. By ES-MS and continu-
ous Edman degradation, the two peptides were identified as gallidermin analo-
gues possessing either a dehydrated (Dha14) or an unmodified S14 residue (Fig.
3.1A, B). Antimicrobial activity of Gdm Dhb14Dha is similar to that of gallider-
min, whereas the Dhb14S analogue is less active, especially against B. subtilis
and S. aureus (Table 3.1). Both analogues were more sensitive to tryptic clea-
vage than gallidermin; the Dhb14S analogue was one of the most sensitive ana-
logues, being completely cleaved after 30 min.
To remove the reactive C=C double bond, a Dhb14A analogue was cre-
ated. Slightly more of this analogue is produced as compared to gallidermin.
Antimicrobial activity of Gdm Dhb14A is similar to that of gallidermin against
M. luteus and C. glutamicum and approximately seven-fold less against B. sub-
tilis and S. aureus (Table 3.1). Like Dhb14S, the Dhb14A analogue is more sensi-
tive to tryptic cleavage and is also fully degraded within 30 min.
The Dhb14P substitution was expected to cause the strongest conforma-
tional change of the middle region. Production of this analogue is comparable to
that of gallidermin. The antimicrobial activity is generally reduced 8- to 30-fold
(Table 3.1). This analogue has a pronounced resistance to tryptic cleavage.
62
3.4.3 Overviewof the characteristics of gallidermin and epidermin
analogues
We demonstrated that although epidermin and gallidermin are produced by two
different staphylococcal species, gallidermin can be synthesized successfully
using the heterologous S. epidermidis EMS6 as expression host and the cloned
gdmA gene. The gdmA promoter is controlled by the transcriptional activator
EpiQ with an efficiency similar to the epiA promoter [60], and the enzymes in-
volved in epidermin biosynthesis (including maturation and secretion) function
efficiently with the gdmA gene product.
Mutagenesis of amino acid positions that are directly involved in thioether
amino acid formation result in the loss of or a large decrease in production. Loss
of production (i. e. no HPLC-detectable extracellular product) is observed with
the analogues containing the S19A substitution, the deletion of the C22 residue
(ring D, Fig. 3.1), and the S3N substitution (ring A). Thus, it can be hypothesized
that biosynthesis and/or secretion of epidermin and gallidermin are severely im-
paired when formation of only one of the four thioether bridges is prevented.
There is also no production observed with the analogues containing the G10E
(ring B) and the Y20G (ring D) substitutions, which suggests that these mutations
interfere with thioether bridge formation even though the substituted residues
are not directly involved. The replacement of the G10 residue for a bulky and ne-
gatively charged E residue may impose steric hindrance on thioether bridge for-
mation at ring B. 2D-NMR studies of gallidermin [18] revealed that ring B, which
is identical to ring B of nisin and subtilin, adopts a typical b-turn type II conforma-
tion. This specific conformation would be disturbed by a G10E exchange and, as
a consequence, thioether bridge formation might be affected, regardless whether
this reaction is enzyme-catalyzed or occurs spontaneously in a Michael-addition-
like reaction [77]. Recent investigations of the substrate specificity of EpiD [43]
indicate that a precursor molecule possessing a G residue instead of the Y20 resi-
due is not a substrate of EpiD. This result suggests that at least oxidative decar-
boxylation of the last C residue of the mutant Y20G precursor peptide is pre-
vented. For all mutations that lead to the loss of production, further investigations
will determine whether only secretion or whether also other stages of biosynth-
esis are blocked. It also cannot entirely be ruled out that partially modified pre-
cursor peptides are rapidly degraded by the EMS6 host.
The only thioether amino acid mutations that result in the production of
antimicrobially active substances are the S19T (ring D) and the S16T (ring C)
substitutions. The extremely low production of Epi S19T and Gdm L6V/S16T
could be explained by assuming that dehydration of T16 and T19 is inefficient
and that only fully modified (dehydrated and subsequently thioether-bridged)
precursor peptides are efficiently secreted. The simultaneous production of
Gdm Dhb14S and Gdm Dhb14Dha is an indication of different efficiencies of S-
and T-dehydration.
For the applied use of peptide antibiotics, an advantageous trait would be
resistance to proteolytic degradation. Epidermin and gallidermin already natu-
63
rally have a rather high resistance. Among various peptidases tested (pronase,
trypsin, pepsin, thermolysin, collagenase, and carboxypeptidases A and B), only
pronase and trypsin cleave epidermin [2]. Proteolytic degradation by the other
peptidases is sterically hindered by the thioether bridges.
In one study, purified gallidermin and analogues were tested for their sen-
sitivity to tryptic cleavage in order to screen for analogues with an increased re-
sistance to proteolytic degradation and, as a consequence, a prolonged period
of action. All gallidermin analogues are cleaved by trypsin only in the central
part of the molecule between K13 and the residue at position 14, which is in
agreement with earlier reports on gallidermin [28] and epidermin [2]. Tryptic
cleavage at the second putative tryptic cleavage site, the K4-F5 bond (ring A), is
sterically hindered, at least under the conditions employed.
2D-NMR studies of the gallidermin molecule [18, 19] revealed that the ri-
gid rings A/B and C/D are connected by a flexible middle region (A12 to G15).
The conformation (e. g. flexibility of the peptide backbone) of this region ap-
pears to be important for antimicrobial activity and resistance to tryptic clea-
vage. Gallidermin analogues with a supposed decreased flexibility of the middle
region, such as Gdm Dhb14P and Gdm A12L, are highly resistant to tryptic clea-
vage, whereas their antimicrobial activity against various Gram-positive indica-
tor bacteria is greatly reduced (Table 3.1).
According to modeling studies of the gallidermin/trypsin interaction [26],
the gallidermin molecule must adopt a bent structure in order to fit into the cat-
alytic cleft of the trypsin molecule. This bent structure is supported by the flex-
ibility of the peptide backbone within the middle region. Thus, the high resis-
tance to tryptic cleavage observed for Gdm Dhb14P and Gdm A12L may reflect
a decreased flexibility of the middle region, impeding the formation of the bent
structure.
A P residue following a K residue usually has a strong negative influence
on trypsin action. This also may explain the high resistance to tryptic cleavage
observed for Gdm Dhb14P (tryptic cleavage site: K13P14). However, a negative
influence of a preceding L residue is not known [28], which suggests that the
large increase in resistance to tryptic cleavage observed for Gdm A12L (tryptic
cleavage site identical to that of gallidermin) is mainly caused by a restricted
flexibility of the middle region.
The K13Dhb14 bond of gallidermin is cleaved by trypsin with a strikingly
lower efficiency than bonds of K to normal protein amino acids [28]. Thus, an in-
creased substrate affinity of the trypsin molecule and an increased flexibility of
the peptide backbone within the middle region, which may lead to a greater ac-
cessibility of the tryptic cleavage site, may be responsible for the greatly re-
duced resistance to tryptic cleavage observed for Gdm Dhb14A and Gdm
Dhb14S (tryptic cleavage sites: K13A14 and K13S14, respectively). This effect
was strikingly less enhanced for Gdm Dhb14Dha (tryptic cleavage site: K13
Dha14). Interestingly, the tryptic sensitivity of Gdm L6V and Gdm L6G differ,
although these gallidermin analogues are not altered in the vicinity of the tryp-
tic cleavage site; this observation possibly indicates a slight overall change in
conformation of the L6V and the L6G analogue.
64
Of all the gallidermin derivatives altered within the middle region, only
the Dhb14Dha analogue has antimicrobial activity similar to that of gallidermin
(Table 3.1). This may indicate the importance of the 2,3-didehydroamino acids
for maintaining a structure required for efficient pore formation. A critical role
of the polypeptide backbone flexibility for biological activity has also been sug-
gested for the channel-forming peptides alamethicin and melittin [78]. With
Gdm Dhb14A, a decrease in antimicrobial activity is only observed for three of
five indicator strains (Table 3.1). Thus, one can speculate that gallidermin can
exert its antimicrobial activity by different mechanisms: a Dhb/Dha-dependent
and a Dhb/Dha-independent mechanism. Subtilin exerts its antimicrobial activ-
ity against Bacillus by two different mechanisms. The inhibitory effect on Bacil-
lus spore germination is dependent on the Dha5 residue and is most likely
caused by a Michael-type reaction of the didehydro residue with nucleophilic
membrane sulfhydryl groups [48]. The ability to lyse vegetative Bacillus cells,
however, is independent of the Dha5 residue [47].
Thus, our results [53] and the results obtained previously by mutagenesis
of other type A lantibiotics strongly indicate that the molecular mechanisms by
which these peptides exert their antimicrobial activity may differ and that a gen-
eral function of the 2,3-didehydroamino acids for biological activity is not yet
known.
The various test strains varied greatly in their susceptibility to gallidermin
and its analogues (Table 3.1), possibly because of differences in the membrane
phospholipid composition [25, 35] or different membrane potentials [34, 68].
Both factors are crucial for the formation of voltage-dependent transmembrane
pores. Furthermore, the composition of the bacterial cell wall might influence
the kinetics of pore formation. Cationic lantibiotics interact with polyanions
(e. g. teichoic acids) of the cell wall [11] and with membrane-bound cell wall
precursor molecules [64]. Binding of lantibiotics to these components may influ-
ence the kinetics of pore formation, and may also be responsible for secondary
killing mechanisms observed for Pep5 and nisin, such as activation of autolytic
enzymes and inhibition of murein synthesis [64]. Recently it was indeed demon-
strated that an increased negatively charged cell wall leads to an increased sen-
sitivity to gallidermin and other positively charged antimicrobial peptides [59].
Since B. subtilis and S. aureus produce a variety of exoproteases, the strikingly
lower susceptibility of these test strains (Table 3.1) might be partially due to pro-
teolytic degradation of gallidermin and its analogues.
In summary, we successfully engineered gallidermin and epidermin analo-
gues with increased antimicrobial activities and/or resistance to proteolytic de-
gradation. Furthermore, we obtained valuable information about the structural
elements required for proper biosynthesis of epidermin and gallidermin. NMR
analyses of the generated analogues and black lipid membrane studies may
provide further information on the structure/activity relationship of these lanti-
biotics and may pave the way for target-oriented peptide engineering.
65
3.5 Function of the epidermin immunity genes epiFEG
Epidermin, gallidermin, Pep5, subtilin, and nisin are the best-studied members
of type A group of lantibiotics, which are all synthesized by Gram-positive bac-
teria. Type A lantibiotics act by forming membrane-potential-dependent pores
in the cytoplasmic membrane of bacteria [5, 68, 69]. Because of this activity,
self-protection against the lantibiotic is of vital importance to the producing or-
ganism. Several genes responsible for producer self-protection have been found
in the gene clusters of the respective systems: 1) nisI and spaI, which code for li-
poproteins [32, 37]; 2) epiF, epiE and epiG [57] which are homologous to nisF,
nisE, and nisG of the nisin system [74] and to spaF and spaG of the subtilin sys-
tem [32], all of which code for ABC transporters; and 3) pepI, which does not
share any sequence similarity to any known genes [63]. Analysis of the amino
acid sequences suggests that the EpiFEG-type transporters and the lipoproteins
NisI and SpaI are membrane-located; this has been experimentally shown for
PepI [63]. The mechanism of action has not been elucidated for any of the gene
products. The sequence similarity of the EpiFEG proteins to that of ABC trans-
porters led to the proposal that the EpiFEG-type proteins act by transporting the
lantibiotic out of the cytoplasmic membrane, thus keeping its concentration be-
low a critical level and preventing pore formation. Two conceivable directions of
transport have been discussed: import into the cell for inactivation by proteolytic
cleavage and export into the surrounding medium.
As shown by heterologous expression experiments in S. carnosus, the self-
protection (immunity) of the epidermin-producing strain S. epidermidis T 3298
against the pore-forming lantibiotic epidermin is mediated by an ABC transpor-
ter composed of the EpiF, EpiE, and EpiG proteins. We developed a sensitive as-
say based on HPLC analysis of the substrate gallidermin in cell supernatants to
investigate the mechanism of the EpiFEG transporter. Our results indicate that
the EpiFEG transporter works by expelling the lantibiotic from the cytoplasmic
membrane into the surrounding medium with a high substrate specificity. Thus,
the EpiFEG transporter functions according to the hydrophobic vacuum clea-
ner mechanism [12]. Furthermore, we showed that the gallidermin derivative
L6G has an EpiE-dependent enhanced activity. These results will be covered in
more detail in the following sections.
To our knowledge, the EpiFEG transporter is the first of its kind to be in-
vestigated that has a pore-forming peptide as substrate. Future research on the
EpiFEG transporter will focus on the question whether the EpiFEG transporter
can substitute in S. epidermidis T 3298for the non-functional EpiT transporter,
whose task is to export completely modified pre-epidermin (with the leader pep-
tide still present) out of the producer cell.
66
3.5.1 The epiFEG genes confer immunity to the epidermin producer
We characterized a DNA region located upstream of the structural gene epiA
that mediates immunity and increased epidermin production. The sequence of a
2.6-kb DNA fragment revealed three ORFs (Fig. 3.2), epiF, E, and G, which form
an operon [57]. In the cloning host S. carnosus, the three genes mediate an in-
creased tolerance to epidermin, and the highest level of immunity (sevenfold) is
achieved with S. carnosus carrying epiFEG and epiQ (Fig. 3.4). The promoter of
the first gene epiF responds to the activator protein EpiQ and contains a palindro-
mic sequence similar to the EpiQ-binding site of the epiA promoter, which is also
activated by EpiQ. Inactivation of epiF, E, or G results in the complete loss of the
immunity phenotype. An epidermin-sensitive S. epidermidis T 3298 mutant is
complemented by a DNA fragment containing all three genes. When the epiFEG
genes are cloned together with plasmid pTepi14, which contains the biosynthetic
genes epiABCDQP, the production of epidermin is approximately fivefold higher.
The deduced amino acid sequences of EpiF, E, and G are similar in se-
quence and proposed structure to the components of various ABC transporter
systems. EpiF is a hydrophilic protein with conserved ATP-binding sites, while
EpiE and G have six alternating hydrophobic regions and very likely constitute
the integral membrane domains. When EpiF is overproduced in S. carnosus, it is
at least partially associated with the cytoplasmic membrane.
Figure 3.4: The epiFEG genes confer immunity against gallidermin in the heterologous
host S. carnosus. MIC values were determined with S. carnosus TM300 harboring all or
only two of the epiFEG genes (in all possible combinations) on plasmid pRB473. Black
bars represent MIC values of strains harboring in addition the positive regulator of the
epidermin biosynthetic system, epiQ, on plasmid pTepiQ10.
67
3.5.2 Transport mechanism
In most cases, it has been assumed that ABC transporters reported to be in-
volved in resistance to non-pore-forming antibiotics have to export the antibiotic
from the cytoplasm to the surrounding medium. For an antibiotic whose target
is located in the cytoplasm, this mechanism seems self-evident, whereas for a
pore former whose target is the cytoplasmic membrane, import into the cell as
well as export to the surrounding medium are both conceivable mechanisms for
removing the antibiotic from its target (Fig. 3.5).
To investigate the direction of transport mediated by the EpiFEG transpor-
ter, it was not possible to use membrane vesicle systems because the cytoplas-
mic orientation of the ATPase site in the EpiF part of the transporter constitutes
the only means to discriminate between right-side-out and inside-out vesicles
and because the substrate gallidermin is known to cause efflux of small mole-
cules such as ATP out of the cell. This would, unfortunately, lead to an equal
Figure 3.5: Two alternative models for the mechanism of the EpiFEG transporter. The
model shows the EpiE and EpiG proteins situated within the cytoplasmic membrane and
an EpiF dimer bound to the complex at the cytoplasmic side of the membrane. EpiF har-
bors the ATPase binding site, as predicted by hydropathy plots and sequence alignments
(Peschel and Gtz 1996). Black arrows illustrate the mechanism model 1: the export of gal-
lidermin into the surrounding medium. Every exported molecule in model 1 is likely to be
able to reintegrate into the membrane, thereby causing an incessant expulsion process.
Gray arrows illustrate model 2: the import of gallidermin into the cytoplasm, where one
would expect proteolytic degradation of the lantibiotic.
68
distribution of ATP in the internal and external fluid. We therefore developed an
assay in which the amount of the substrate gallidermin remaining in the super-
natant of whole cells incubated with gallidermin was determined. In this trans-
porter assay, a concentration of gallidermin in the supernatant of cells expres-
sing the epiFEG genes higher than that of a control strain would suggest that
the EpiFEG transporter works by export; a lower concentration in the superna-
tant would suggest that the transporter works by import. All experiments were
performed with S. carnosus TM300 as a host for heterologous expression of all
or some of the epiFEG genes. Gallidermin was quantified by HPLC detection.
Our results indicate that model 1 shown in Fig. 3.5 represents the mechan-
ism used by the EpiFEG transporter. It is not known whether the transporter re-
cognizes the substrate in its monomeric or oligomeric form; both possibilities are
illustrated in Fig. 3.5.
Optimal results were observed with rather low gallidermin concentrations,
and HPLC quantification was optimized and resulted in a detection limit of
about 20 ng at the very specific wavelength of 266 nm (absorption of C-terminal
aminovinylcysteine in gallidermin and all derivatives). At 2 mg gallidermin/ml,
the extracellular gallidermin content is about fourfold higher for the epiFEG-ex-
pressing strain than for the control strain. At higher and lower concentrations of
applied gallidermin, the difference is less pronounced.
To exclude the possibility that the observed difference in extracellular gal-
lidermin concentration is caused by non-specific interaction of gallidermin with
one of the EpiFEG proteins and not by the functional transporter, the epiFEG-
expressing strain was compared with strains harboring the genes encoding only
two of the three transporter components (epiF, epiE and epiG) in all three possi-
ble combinations. The gallidermin concentration in the assay supernatant of
each of the three strains showed a similar basal level, whereas in the epiFEG-
expressing strain, this concentration was about fourfold higher, demonstrating
that the observed effect is not caused by non-specific interaction with one of the
protein components of the EpiFEG transporter (Fig. 3.6).
3.5.2.1 Energy dependence of transport
Energy dependence of the EpiFEG transporter could not be directly studied
using ATPase inhibitors because destruction of the membrane potential would
also result in inhibition of gallidermin activity. Instead, dependence of the Epi-
FEG-mediated transport on the glucose concentration was determined. Glucose
was the only energy source in the incubation buffer. In the absence of glucose,
transport is strongly reduced. Increases in the concentration of glucose up to
1% result in increases in the transport efficiency; at concentrations of glucose
higher than 1%, transport efficiency does not increase further. These results are
in accordance with the expected energy dependence of the ATP-consuming
EpiFEG transporter.
69
3.5.2.2 Specificity of the transporter
Eight gallidermin derivatives with single amino acid substitutions were used to
determine the specificity of the EpiFEG transporter in the transporter assay. In
addition to epidermin (gallidermin L6I), seven other derivatives originating from
site-specific mutagenesis of the gallidermin structural gene gdmA [53] were in-
vestigated. Transporter efficacy was defined as the value obtained with the epi-
FEG-expressing strain divided by the value obtained with the control strain.
This factor is in the range of 3 to 5for each substance, with the exception of gal-
lidermin Dhb14P and gallidermin L6G, where there is almost no detectable ef-
fect. No differences among the test strains were detected with nisin, which sug-
gests that nisin is not a substrate of the EpiFEG transporter. The most prominent
feature of gallidermin Dhb14P is that the flexibility of the so-called hinge region
is strongly decreased; gallidermin L6G was the most hydrophilic derivative in-
vestigated (as concluded from reversed-phase HPLC retention).
The three-step model for the mode of action of nisin and type A lantibiotics
in general includes adhesion to the outer surface of the cell membrane mediated
by electrostatic interaction between the cationic peptide and anionic charges on
the surface of the cytoplasmic membrane, integration into the membrane with a
DC or DpH present where hydrophobicity and flexibility are considered to be
important, and formation of oligomeric pores [51, 52]. Thus, gallidermin Dhb14P
and gallidermin L6G appear to be impaired in the integration into the mem-
Figure 3.6: Comparison of the effect of the entire EpiFEG transporter with that of the
transporter protein components. The transporter assay was carried out with S. carnosus
strains harboring plasmid pTepiQ10, which contains the positive regulator of the epider-
min system, and two (FE, EG, FG), none of (473), or all of (FEG) the epiF, epiE
and epiG genes on plasmid pRB473. Gallidermin was used as substrate at a concentration
of 2 mg/ml. The values of the determined extracellular gallidermin content from five sam-
ples for each strain were averaged.
70
brane, and this is presumably also the cause for their low bactericidal activity.
Adhesion to the cytoplasmic membrane is not likely to be affected in any of the
gallidermin derivatives tested because the charge of the molecule is not chan-
ged. Since specifically gallidermin Dhb14P and gallidermin L6G are by far the
poorest substrates among all gallidermin derivatives investigated, we assume
that integration of the substrate into the membrane is important for EpiFEG ac-
tivity. This suggests that the substrate binding site of the EpiFEG transporter is
within the membrane-spanning part of the protein complex, an assumption that
is further supported by the interaction of gallidermin L6G with the internal
membrane protein EpiE (see below).
MIC values of gallidermin, epidermin, gallidermin derivatives, and nisin of
S. carnosus (pRBepiFEG/pTepiQ10) and S. carnosus (pRB473/pTepiQ10) were
also determined. No difference in the MIC values of nisin among the test strains
was detected. Gallidermin, epidermin, and the gallidermin derivatives Dhb14S,
Dhb14Dha, and A12L are more active against the control strain, whereas the ac-
tivity of gallidermin Dhb14P is only slightly influenced by the presence of the
epiFEG genes in the test strain. In contrast, the MICs of gallidermin derivatives
L6V and Dhb14A are not influenced by the presence of the epiFEG genes in the
strain; however, they are good substrates of the EpiFEG transporter, as shown
in the transporter assay. While conditions in the transporter assay were selected
to optimize EpiFEG transporter efficacy, this was not the case in the complex
medium used for MIC determinations. Conditions in the complex medium
seemed to suppress the interaction of EpiFEG with some derivatives.
3.5.2.3 Interaction of EpiE with gallidermin L6G
Paradoxically, the activity of gallidermin L6G is clearly higher in the presence of
the epiFEG genes in the test strain. Further experiments were designed to in-
vestigate this phenomenon. The MIC of gallidermin L6G of S. carnosus (pRBe-
piFE/pTepiQ10), S. carnosus (pRBepiEG/pTepiQ10), S. carnosus (pRBepiFG/
pTepiQ10), and the strains used before was determined (Table 3.2). The activity
of gallidermin L6G is higher whenever the epiE gene is expressed in the strain;
the expression of the other two genes (epiF and epiG) is not necessary for this
effect. The activity of gallidermin L6G is also higher against S. carnosus (pTXe-
piE), in which the epiE gene is under the regulation of a xylose-inducible pro-
moter, than against the control strain S. carnosus (pTX16). The integration of
gallidermin L6G into the cytoplasmic membrane is usually impaired because of
its comparatively low hydrophobicity, yet the activity of gallidermin L6G against
strains that expressed the epiE gene was always relatively high (Table 3.2). As
an explanation for this phenomenon, we propose a direct protein-protein inter-
action of gallidermin L6G with EpiE; this interaction would also suggest a parti-
cipation of EpiE in substrate binding in general. Gallidermin L6G probably
binds to the substrate binding site on EpiE and remains bound there, forming a
nucleus to which other gallidermin L6G molecules adhere to form a pore. With-
out this interaction, gallidermin L6G would be too hydrophilic to form a pore.
71
Table 3.2: Activity of gallidermin L6G [53].
Strain MIC
a
(mg/ml)
S. carnosus (pRB473/pTepiQ10) 1.0
S. carnosus (pRBepiFEG/pTepiQ10) 0.1
S. carnosus (pRBepiFE/pTepiQ10) 0.1
S. carnosus (pRBepiEG/pTepiQ10) 0.1
S. carnosus (pRBepiFG/pTepiQ10) 1.0
S. carnosus pTX16 1.0
S. carnosus pTXepiE 0.2
a
MIC values of gallidermin L6G (Ottenwlder et al. 1995) of various strains were deter-
mined as described in the experimental procedures to investigate the gallidermin L6G-
EpiE interaction. Strains S. carnosus pTXepiE, in which epiE is under the control of a xy-
lose-inducible promoter, and S. carnosus pTX16 as a control were grown in basic med-
ium without glucose and with 0.5% xylose.
3.6 Inactivation and characterization of the epidermin
leader peptidase EpiP
The sequences of lantibiotic leader peptides differ from Sec-dependent protein
export signal sequences. Type A lantibiotics have a negatively charged, amphi-
philic a-helix that is removed at the characteristic processing-site P
2
Q/R
1
;X
+1
(Fig. 3.7). In nearly all lantibiotic gene clusters, a serine protease is encoded,
which is proposed to mediate precursor processing. These proteases are usually
located in the cytoplasm. In contrast, NisP, the nisin leader peptidase, and EpiP
contain a signal sequence, indicating that they act extracellularly. Therefore, the
location of the processing of the various lantibiotics may differ [75].
The serine protease EpiP from S. epidermidis T 3298 catalyzes the extra-
cellular processing of the epidermin precursor peptide, as shown in experiments
where epiP in the xylose-regulated expression vector pCX15 in S. carnosus is
Figure 3.7: The processing site of modified pre-epidermin. The arrow indicates the pro-
cessing site of the secreted EpiP protease.
72
overexpressed. The cleavage of the unmodified EpiA precursor peptide to lea-
der peptide and pro-epidermin by EpiP-containing culture filtrate of S. carnosus
(pCX15epiP) was followed by reversed-phase chromatography and subsequent
electrospray mass spectrometry [20]. The epidermin leader peptidase gene epiP
was inactivated so that the final intermediates of epidermin biosynthesis could
be isolated to characterize the mechanism of precursor processing. The isolated
precursor peptides may also serve as natural substrates for EpiP in future ex-
periments.
3.6.1 epiP gene replacement in S. epidermidis T 3298
To construct a plasmid for the replacement of epiP, 1 kb upstream and 1 kb
downstream of the gene were amplified by PCR and sequenced. The two ampli-
fied fragments were ligated into pBT2, a temperature-sensitive shuttle vector
[14], flanking an erythromycin resistance cassette. The resulting plasmid was in-
troduced into S. epidermidis T 3298 by electroporation. A homologous recom-
bination event deleted the entire epiP gene, leaving the epiP promoter intact to
ensure expression of the regulator epiQ [56] downstream of epiP (Fig. 3.2); there
is no terminator structure in the erythromycin resistance cassette that could inhi-
bit transcription of epiQ. The antimicrobial activity of S. epidermidis
T 3298DepiP mutant strains on agar plates containing the epidermin-sensitive
M. luteus was strongly decreased. By transforming the mutant with an epiP-ex-
pressing plasmid, epidermin production could be reconstituted (Kies and Gtz,
unpublished).
3.6.2 Detection and isolation of epidermin precursor peptides
Epidermin and epidermin precursor peptides were purified by reversed-phase
chromatography of the supernatant from S. epidermidis T 3298DepiP grown in
synthetic medium. A silver-stained nitrocellulose blot of an SDS-polyacrylamide
gel with fractions from the reversed-phase chromatography showed peptides
with an apparent molecular weight similar to that of mature epidermin as well
as peptides with an apparent molecular weight similar to that of the N-termin-
ally cleaved precursor peptides isolated from the S. epidermidis T 3298 wild
type strain grown in defined medium. These preliminary results indicate that in
S. epidermidis T 3298DepiP, the same or at least a similar processing at the N-
terminus occurs as in the S. epidermidis T 3298 wild type strain. To test this hy-
pothesis, the precursor peptides isolated from S. epidermidis T 3298DepiP were
analyzed by mass spectrometry and Edman degradation.
73
3.6 Inactivation and characterization of the epidermin leader peptidase EpiP
From S. carnosus (pTepiABCDQ) grown in synthetic medium, completely
modified epidermin precursors could be isolated that were N-terminally pro-
cessed at different positions within the leader peptide, i. e. before amino acids
17, 19, or 20 (Fig. 3.7). Mature epidermin was also produced. The peptides were
characterized by Edman degradation and mass spectrometry. The same peptide
mixture was detected in S. epidermidis T 3298 (wild type strain) when grown
in synthetic medium, where EpiP shows a significant reduction in activity be-
cause of the low buffer capacity of this medium, which becomes rather acidic.
The precursor peptides isolated are not antimicrobially active against the epi-
dermin-sensitive M. luteus. Treatment with endoprotease ArgC, a protease that
simulates the EpiP reaction in vitro, leads to antimicrobially active, mature epi-
dermin, as shown by mass spectrometry and in the M. luteus bioassay.
In conclusion, the serine protease EpiP catalyzes the last modification step
in epidermin biosynthesis the processing of the epidermin precursor peptide.
Due to the inactivation of epiP in S. epidermidis T 3298DepiP, it was possible to
detect the final intermediate of epidermin biosynthesis, i. e. the completely mod-
ified precursor peptide. The precursor had undergone proteolytic cleavage
within the leader peptide. Similar results have been observed for the S. epider-
midis T 3298 wild type strain in synthetic medium, where EpiP shows de-
creased activity, and for S. carnosus (pTepiABCDQ), which indicate a two-step
processing of the epidermin precursor peptide.
3.7 The flavoenzyme EpiD and formation of peptidyl-
aminoenethiolates
One of the goals of lantibiotic research is the analysis of the enzymatic mechan-
isms involved in the modification process from pro-epidermin to pre-epidermin.
The key function is certainly associated with the EpiB and EpiC enzymes, which
are apparently involved in dehydration of serine and threonine residues and in
the thioether bridge formation. Homologous protein sequences that occur in all
other lantibiotic genes are referred to as LanB and LanC. Several groups are
studying the enzymatic mechanism of these two enzymes; however, to date, no
clean in vitro reaction has been achieved. It was speculated that the enzymatic
reaction is strictly oxygen sensitive and/or that unusual co-factors are involved.
Epidermin and gallidermin modification involves a third enzyme, EpiD, whose
enzymatic function was extensively studied by Thomas Kupke (see below). An
epiD homologue has so far only been found in the mersacidin biosynthesis gene
cluster [9], but not the nisin, pep5, or subtilisin gene clusters.
74
3.7.1 Preparation of precursor peptide EpiA
To investigate the enzymes involved in epidermin biosynthesis, it is necessary to
produce sufficient amounts of pre-epidermin (EpiA) as a substrate and to design
methods to detect EpiA. EpiA was therefore expressed in Escherichia coli using
a malE-epiA fusion. The malE gene encodes the maltose-binding protein MBP.
The fusion protein MBP-EpiA was expressed from pIH902-epiA and purified in
one step by amylose affinity chromatography. EpiA was cleaved from MBP-EpiA
by factor Xa, and the identity of purified EpiA was confirmed by ES-MS and
amino acid sequencing. Upon prolonged incubation, factor Xa not only cleaves
EpiA from the fusion protein, but also less efficiently cleaves EpiA internally be-
tween R
1
and I
+1
. The internal factor Xa cleavage site of EpiA was masked by
altering the sequence -A
4
-E-P-R
1
- to -A
4
-E-P-Q
1
- by site-directed mutagen-
esis. Since the mutant peptide EpiA R
1
Q) has properties different than EpiA
(the molecular mass is 28 Da and the isoelectric point is approximately 1.5 pH
units lower), it is used as a control peptide in incubation experiments. Anti-EpiA
antisera were raised to detect EpiA [44].
3.7.2 Construction and purification of MBP-EpiD fusion proteins
According to the nucleotide sequence, epiD encodes a 181-amino acid protein.
In order to obtain more information on the function and biochemical properties
of EpiD, epiD was expressed in E. coli using the maltose-binding protein (MBP)
fusion system. The soluble 67-kDa MBP-EpiD fusion protein was purified in one
step by amylose affinity chromatography and was used to raise polyclonal anti-
bodies directed against EpiD. The MBP-EpiD fusion protein was yellow and
identified as a flavoprotein by its absorption spectrum with maxima at 277, 378,
and 449 nm (compare Fig. 3.8). The coenzyme is very tightly, but not covalently
attached. It could only be removed by TCA extraction of the fusion protein and
was identified by thin-layer chromatography (TLC) as FMN.
S. epidermidis T 3298/EMS11 [4] carries a mutation within epiD, desig-
nated epiD*, which was PCR-amplified using purified pT32from the mutant as
a template. This DNA fragment was inserted in the StuI site of the pIH902 poly-
linker. Plasmids were isolated from four E. coli TB1 (pIH902-epiD*) clones, and
the entire epiD* region was sequenced. The epiD* start codon immediately fol-
lows the factor Xa cleavage sequence and all four isolated plasmids have a
point mutation in codon 93, substituting the wild type GGT (Gly) with GAT
(Asp) in epiD*. This GA transition concurs with the use of EMS as the muta-
genizing agent.
MBP-EpiD* is not yellow and does not exhibit the characteristic absorption
maxima of MBP-EpiD when similar concentrations of fusion proteins are used; it
was necessary to dilute MBP-EpiD because of the low amount of soluble MBP-
75
3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates
EpiD*. MBP-EpiD* does not bind FMN. Since MBP-EpiD* differs from MBP-
EpiD only in the substitution of Gly
93
by Asp, Gly
93
may be important for FMN
binding [45].
3.7.3 Characterization of purified EpiD
EpiD was expressed using the T7 RNA polymerase promoter system in E. coli
and was purified to homogeneity in three column chromatography steps:
DEAE-Sepharose Fast Flow, Mono-Q, and Phenylsuperose chromatography. The
first 18 N-terminal amino acids of purified EpiD were determined by Edman de-
gradation. The amino acid sequence correlates exactly with the amino acids
(Met-Tyr-Gly-Lys-) deduced from the epiD sequence [70]. Purified EpiD is yel-
low. The absorption spectrum exhibits maxima at 274, 382 and 453 nm, which
are characteristic for flavoproteins in the oxidized state (Fig. 3.8). The liberated
flavin component was analyzed by TLC and ES-MS. The mass of the flavin
coenzyme was determined to be 455.5 Da (457 Da in a second experiment),
which closely agrees with the theoretical value of 456.3 Da and confirms that
the flavin component is FMN and not FAD (theoretical mass 785.6 Da). The
average molecular mass of EpiD was determined to be 20,827 +/ 5 Da, which is
in close agreement with the theoretical value of 20,825 Da calculated for the
amino acid sequence derived from the nucleotide sequence, indicating that
EpiD is not covalently modified.
Figure 3.8: Absorption spectrum of purified EpiD.
76
3.7.4 Model of oxidative decarboxylation of peptides
Since flavin coenzymes are normally involved in oxidation-reduction reactions,
EpiD belongs to the class of oxidoreductases. There is only one obvious reaction
involved in epidermin biosynthesis that requires an oxidoreductase activity: The
synthesis of S-((Z)-2-aminovinyl)-D-cysteine [1] includes the removal of two re-
ducing equivalents from a -CC- group to form a -C=C- group. Regarding the
proposed order of the modification reactions [72], it was assumed that the four
lanthionine rings are formed first, followed by the oxidation of the C-terminal
lanthionine by EpiD and the subsequent decarboxylation reaction. In the course
of the oxidation, EpiD-FMN becomes reduced. According to this first published
hypothesis, EpiD catalyzes the final step in the modification of pre-epidermin,
and its reaction product is then processed by the leader peptidase, forming ma-
ture epidermin [45].
Thioether formation is only possible if dehydrated serine and threonine re-
sidues are present, but in principle, oxidative decarboxylation could be inde-
pendent of the other modification reactions. Therefore, the following alternative
model of the C-terminal oxidative decarboxylation of peptides has been sug-
gested (Fig. 3.9): in the first reaction, EpiD catalyzes the removal of two redu-
cing equivalents from the C-terminal cysteine residue of unmodified EpiA. A
double bond is formed, and FMN is reduced to FMNH
2
. The C-terminal car-
boxyl group is then removed, catalyzed either by EpiD or occurring sponta-
neously [42]. The occurrence of a reaction between EpiD and the unmodified
precursor peptide EpiA was investigated.
3.7.5 Interaction between EpiD and EpiA
EpiA or EpiAR
1
Q was coupled to an NHS-activated HiTrap column in order to
purify the enzymes involved in epidermin biosynthesis by affinity chromatogra-
phy. Binding studies were carried out with purified EpiD or extracts of induced E.
coli K38 (pGP12, pT75epiD) cells. In both cases, the migration of EpiD is re-
Figure 3.9: Model of the C-terminal oxidative decarboxylation of unmodified precursor
peptide EpiA and the role of flavoprotein EpiD. The C-terminal cysteine residue of the
precursor peptide EpiA is shown. After oxidation and decarboxylation, the peptidyl-ami-
noenethiols are formed.
HN CHC
CH
2
OH
O
SH
EpiD-FMN
EpiD-FMNH
2
HN C C
CH
OH
O
SH
HN C
CH
SH
H
CO
2
77
tarded only under reducing conditions. The applied EpiD focuses as a sharp yel-
low band on the EpiA-HiTrap column. Other proteins of the cell extract are not re-
tarded, which indicates the specificity of the interaction. Even under reducing
conditions, the interaction is weak, and EpiD already elutes with the loading buf-
fer containing no salt. In a control experiment, a column without any coupled pep-
tide was used, and as expected, the migration of EpiD was not retarded [40, 43].
3.7.6 Reaction of EpiD with EpiA and pro-epidermin
Under reducing conditions, purified EpiD reacts with unmodified precursor pep-
tide EpiA. Several products were identified after separation of the reaction mix-
ture by reversed-phase chromatography and ES-MS analysis. The product
formed depends on the incubation time. The first product formed, product 3
(the products were designated according to increasing hydrophobicity), is more
hydrophobic than the unmodified peptide EpiA and has a molecular mass of
5,5795,585 Da (values obtained from several experiments), i. e. 4548 Da less
than EpiA. In order to determine the mass difference between EpiA and product
3 with greater accuracy, the peptides were measured together, giving a mass
difference of 46.5 Da. Furthermore, product 3 has an absorbance at 260 and
280 nm in 0.1% TFA/H
2
O/acetonitrile, higher than that of EpiA [42].
This enzyme reaction was also detected using crude cell extracts of S. car-
nosus (pTepi14) and EpiA [40]. This clone has been used for heterologous ex-
pression of epidermin [3, 70].
With longer incubation times, two additional, less hydrophobic peptides
with molecular masses of 5,5245,528 Da (product 1; 102105 Da less than
EpiA) and 5,5795,585 Da (product 2; 4548 Da less than EpiA) were identified.
Product 3 (oxidatively decarboxylated peptide) is unstable and is non-enzymati-
cally converted to products 1 and 2. Product 2 has the same mass as product 3,
but its absorbance at 260 and 280 nm is comparable to that of unmodified pep-
tide EpiA. Based on its molecular mass, product 1 is either the peptide EpiA-
(M
1
-C
51
) (lacking the last cysteine residue; 103.2 Da less) or the peptide EpiA-
(M
1
-C
51
)-NH
2
(104.1 Da less) [42].
All known lantibiotics are synthesized as pre-peptides with an N-terminal
leader peptide. It has been proposed that all the processing signals are in the lea-
der region of the pre-peptides [15]. Thus, the pre-peptides would be recognized
as substrates by binding of the leader peptides to the enzymes involved in post-
translational modifications. To test this hypothesis, unmodified pro-epidermin
(obtained by factor Xa cleavage of EpiA) was used as a substrate for the flavopro-
tein EpiD. Even with this substrate, the reaction occurs. Hence, a leader peptide
is not required for substrate recognition by EpiD. The primary reaction product
was analyzed by Edman degradation to detect unmodified amino acid residues.
A pattern of pmol amounts of the PTH-amino acids Ile
+1
to Tyr
+20
similar to that of
unmodified pro-epidermin was obtained, which indicates that amino acids 1 to
78
20 are unmodified and that one of the last two cysteine residues (Cys
+21
, Cys
+22
)
is modified [42]. Recently, a more detailed analysis of the reaction product has be-
come possible using tandem mass spectrometry and NMR methods.
3.7.7 In vivo reaction of affinity-tag-labeled epidermin precursor
peptide with the flavoenzyme EpiD
The genes encoding the His-tag-labeled epidermin precursor peptide EpiA and
the flavoenzyme EpiD or the mutant protein EpiD-G
93
D, which lacks the coen-
zyme, were co-expressed and the proteins were synthesized in vivo in E. coli.
Only in the presence of EpiD was the precursor peptide converted to a reaction
product with a decrease in mass of 4446 Da. This result confirms the in vitro
experiments carried out with purified EpiA and purified EpiD from S. epidermi-
dis. In the presence of EpiD, the amount of purified (modified) peptide EpiA
was several-fold higher than in the presence of EpiD-G
93
D, indicating that the
stabilization of EpiA against proteolysis is due to an interaction with EpiD or to
the presence of the C-terminal modification [39].
3.7.8 Determination of the substrate specificity of EpiD using mutant
precursor peptides and chemically synthesized peptides
The precursor peptide EpiAC
52
S, which is altered by gene mutation and con-
tains a C-terminal serine, was used to test the production of enols by reaction
with EpiD [31]. No reaction occurred, which indicates the necessity for a C-
terminal cysteine residue. However, not all peptides with a C-terminal cysteine
residue, e. g. the peptide EpiA M
1
-C
51
, were a substrate of EpiD. EpiAS
49
A was
a substrate for oxidative decarboxylation, providing the first hint that EpiD has
no absolute substrate specificity.
The leader peptide of the precursor peptide EpiA has no significant influ-
ence on the reaction with EpiD. Synthetic peptides with increasingly larger dele-
tions of the amino-terminus were used to determine the minimal size of the sub-
strate. Aweak reaction was even observed for the tetrapeptide SYCC. The hepta-
peptide SFNSYCC was used to study the substrate specificity of EpiD further
(Table 3.3). The serine and cysteine residues of this heptapeptide form the C-
terminal bicyclic structure of mature epidermin, and it could not be excluded that
amino acid exchanges in this peptide have an influence on the reaction with
EpiD [31]. Interestingly, the penultimate cysteine residue can be exchanged with
at least a serine or threonine residue. Modification or exchange of the C-terminal
cysteine residue results in loss of the reaction with EpiD. The peptides SFNSYCS,
SFNSYCM, and SFNSYC were no substrates of EpiD, which confirms the results
79
obtained with the mutant precursor peptides. The analogue with a C-terminal
ethyl-thioether structure [SFNSYCC(Et)], the amide SFNSYCC-NH
2
, and the
peptide SFNSYCHcy (C-terminal homocysteine residue) were not substrates of
EpiD.
Mersacidin is another lantibiotic containing a C-terminal -NHCH=CHS-
group [61], probably formed by modification of a C-terminal cysteine residue.
The C-terminal peptide TLTSECIC of the mersacidin precursor peptide is not a
substrate of EpiD [43].
Table 3.3: Determination of the substrate specificity of the flavoprotein EpiD [43].
Peptide Reaction
with EpiD
EpiA, EpiAR-1Q, K-EpiA +
EpiAS+19A +
EpiAC+22S
EpiAdesC
+22
proepidermin +
synthetic proepidermin +
AKTGSFNSYCC +
SFNSYCC +
FNSYCC +
NSYCC +
SYCC +
YCC (?)
AFNSYCC +
SANSYCC +
SFASYCC +
SFNAYCC +
SFNSACC
SFNSYGC
SFNSYCS
SFNSYC
SFNSYCC-NH
2

SFNSYCC(Et)
SFNSYCHCy
SFNSYCM
SFNSYSC +
SFNSYTC +
SFNSFCC +
SFNSWCC +
SFNYYCC +
SYNSYCC +
SWNSYCC +
TLTSECIC
80
3.7.9 Tandem mass spectra of modified SFNSYTC and SFNSYSC
The sequence and structure of peptides can be analyzed with tandem mass
spectrometry (collision-induced dissociation). In collision-induced dissociation
experiments, peptides are preferentially cleaved at the peptide bonds between
NH and CO, resulting in amino-terminal B
n
fragments and C-terminal Y
n
frag-
ments [7, 8]. To verify that the C-terminal cysteine residue of the reaction pro-
ducts is modified, product ion scans of SFNSYTC and SFNSYSC and the corre-
sponding reaction products were recorded. These peptides were used to ex-
clude intramolecular disulfide bridge formation in the peptide SFNSYCC. The
B
1
B
6
fragments of the peptide and its reaction product are identical, proving
that the C-terminal amino acid residue is modified. SFNSYTC and SFNSYSC
and their reaction products differ from each other by 14 mass units in their B
6
fragments, showing the S/T exchange. The mass difference between the modi-
fied peptide and its B
6
fragment is 75 mass units; the mass difference between
the unmodified peptide and its B
6
fragment is 121 mass units. It was, therefore,
possible to identify the reaction products by neutral loss mass spectrometry [43].
3.7.10 The application of neutral loss mass spectrometry to determine
the substrate specificity of EpiD
Tandem mass spectrometry methods have already been used to determine the
composition of synthetic multicomponent peptide mixtures. For example, O-tert-
butylated by-products of peptide libraries were identified by neutral loss scans
[49]. In the constant neutral loss scan, the first and second analyzer of the mass
spectrometer are scanned together such that there is a constant mass difference
between the ions transmitted by the two analyzers. Under these conditions, only
ions that lose a neutral fragment with a mass corresponding to the chosen mass
difference will be detected.
Heptapeptide sublibraries with one variable amino acid residue at posi-
tions 1 to 7 of the peptide substrate S
1
FNSYCC
7
were synthesized and incu-
bated with EpiD. Peptides were identified by their masses using product ion
scans and neutral loss scans, and by comparison of the mass spectra obtained
after various incubation times. The heptapeptides with a single amino acid sub-
stitution at positions 1 to 4 are substrates of EpiD. Not all amino acid residue
substitutions at positions 5 to 7 of the peptide led to active substrates; therefore,
the last three amino acids determine the substrate specificity for EpiD. For the
sublibraries SFNSX
5
CC and SFNSYX
6
C, the reaction products were identified
by neutral loss mass spectrometry of the peptide mixtures. The tyrosine residue
at position 5 can be replaced by the hydrophobic amino acid residues V, I/L,
(M), F, and W; it is not possible to differentiate between Ile and Leu by ES-MS.
The cysteine residue at position 6 can be replaced by A, S, V, (I/L), and T. Pep-
81
tides containing amino acid residues with acidic or basic side chains at position
5 or 6 are not substrates of EpiD. In position 7 of the peptide substrates, only cys-
teine is accepted [43].
3.7.11 Structure analysis of the reaction products by two-dimensional
NMR methods
Due to the complexity of the NMR spectra, the introduction of a
13
C isotope in
the C-terminal cysteine residues was necessary to obtain the interesting signals
using heteronuclear correlation experiments. Hence, the peptide KKSFNSYTC
was synthesized by solid-phase synthesis using cysteine labeled with
13
C at the
b-carbon atom. The two lysine residues at the N-terminus were introduced to in-
crease the solubility of the substrate in water.
Two crosspeaks occur in the HSQC spectrum of the educt KKSFNSYTC.
The first signal is due to the
13
C-labeled b-carbon, while the second is due to
the methyl groups in Tris[tris(hydroxymethyl)aminomethane]. The NMR data,
the UV-VIS spectra, the MS-MS experiments, the observed reaction product
with Ellmans reagent, and the mass difference between educt and product of
46 Da indicate that the product contains a C-terminal thioenol [30].
3.7.12 The pK
a
value of the enethiol side chain of the reaction products
is lower than that of the thiol side chain of peptides
The UV-VIS spectra of the reaction products of EpiD are pH-dependent, indicat-
ing that the enethiol side chain is converted to an enethiolate anion. The pK
a
value of the enethiol group was determined to be 6.0 and is substantially lower
than the pK
a
value of the thiol side chain of cysteine residues. This increased
acid strength of the enethiol side chain compared to that of the thiol group is at-
tributed to the resonance stabilization of the negative charge of the anion [39].
3.7.13 Overviewof the function of EpiD
In conclusion, the formation of the lantibiotic epidermin from the precursor pep-
tide EpiA includes the oxidative decarboxylation of the C-terminal cysteine resi-
due of EpiA to a (Z)-enethiol catalyzed by the FMN-containing enzyme EpiD.
This oxidative decarboxylation reaction was the first analyzed posttranslational
82
modification reaction involved in lantibiotic biosynthesis. Two reducing equiva-
lents from the C-terminal cysteine residue of EpiA are removed, a double bond
is formed, and the coenzyme FMN is reduced to FMNH
2
. The decarboxylation
occurs spontaneously or is catalyzed by EpiD. The in vivo conversion of (His)
6
-
tag-labeled precursor peptide EpiA by EpiD was demonstrated. The (Z)-enethiol
derivative is the intermediate in the formation of the C-terminal S-((Z)-2-amino-
vinyl)-D-cysteine residue of epidermin. The enethiol structure has been con-
firmed by UV-VIS spectroscopy, mass spectrometry, tandem mass spectrometry,
two-dimensional NMR spectroscopy, and conversion of the enethiol to a mixed
disulfide with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellmans reagent). At physiolo-
gical pH, the dominant form of the reaction product is the enethiolate anion
(peptidyl-aminoenethiolates), and the pK
a
of the enethiol group is 6.0. EpiD
has a low substrate specificity, and most of the peptides with the sequence (V/I/
L/(M)/F/Y/W)-(A/S/V/T/C/(I/L))-C at the carboxy terminus are substrates of
EpiD, as elucidated by analysis of the reaction of EpiD with single peptides and
peptide libraries.
Amino acid residues of EpiD involved in FMN binding or substrate bind-
ing, or important for the catalytic action are currently identified by analyzing
analogues generated by site-directed mutagenesis. In addition, the structure of
EpiD has been determined by X-ray crystallography (Kupke et al. unpublished).
The overall aim is the detailed molecular analysis of the flavoenzyme EpiD and
related enzymes, and a more detailed investigation of the role of EpiD in epider-
min biosynthesis. It is still not known how reduced EpiD is re-oxidized in vivo
and whether EpiD forms a complex with the enzymes EpiB and EpiC. The
chemistry of the peptidyl-aminoenethiolate reaction products has to be studied
in order to develop these peptides as enzyme inhibitors.
3.8 Incorporation of d-alanine into S. aureus teichoic acids
confers resistance to defensins, protegrins, and other
antimicrobial peptides
Why some staphylococcal strains are more tolerant to gallidermin than other
strains has always been of interest. In order to answer this question, Andreas
Peschel [59] isolated S. aureus and S. xylosus transposon-insertion mutants that
were hypersensitive to gallidermin, and found that genes hit by the transposon
are involved in teichoic acid modification.
83
3.8 Incorporation of d-alanine into S. aureus teichoic acids confers resistance
3.8.1 Identification and sequence analysis of the S. aureus
and S. xylosus dlt operon
S. aureus Sa113 and the coagulase-negative S. xylosus C2a have high innate
tolerances towards several antimicrobial peptides. To investigate the resistance
mechanism, S. xylosus C2a was mutagenized with Tn917, and the resulting
transposon-insertion mutants were analyzed for reduced growth on agar plates
containing the antimicrobial peptide gallidermin. The nucleotide sequence up-
stream and downstream of the transposon from seven clones whose growth is
specifically reduced in the presence of gallidermin was determined. In all se-
ven mutants, the transposon had integrated into the same determinant of 4.6
kb, which encodes four open reading frames arranged in an operon-like struc-
ture, followed by a typical terminator (Fig. 3.10). The open reading frames
showed sequence similarity to the Lactobacillus casei and B. subtilis dltABCD
operons, which are responsible for esterification of teichoic acids with d-ala-
nine [16, 55]. The growth rates in the absence of gallidermin and the micro-
scopic appearance of the mutants were indistinguishable from those of the
wild type strain.
3.8.2 Disruption of the S. aureus dlt operon and analysis
of the d-alanine content in lipoteichoic acids (LTA)
and wall teichoic acids (WTA)
The dltA gene of S. aureus Sa113 was replaced by a spectinomycin resistance
gene (spc) by homologous recombination, thereby producing the gallidermin-
sensitive strain AG1. WTA and LTA of S. aureus and S. xylosus wild type strains
and mutants were isolated, and the molar ratios of d-alanine to phosphorus
were determined (Table 3.1). In the wild type strains, 75% (S. aureus) and 95%
(S. xylosus) of the alditol phosphate residues in LTA are esterified with d-ala-
Figure 3.10: Genetic organization and proposed function of the dlt operon in staphylo-
cocci.
84
nine, while only 51% (S. aureus) and 15% (S. xylosus) are esterified in WTA. In
the dlt mutants, no d-alanine is detected in LTA or WTA, indicating that the
pathway for d-alanine incorporation is inactivated by the spectinomycin resis-
tance gene and transposon insertions. When the mutant strains are complemen-
ted with plasmid pRBdlt1, which carries the dlt operon, normal or slightly in-
creased amounts of d-alanine are found in LTA and WTA. Transformation of the
wild type strains with pRBdlt1 results in an increase of d-alanine in LTA and
WTA by 518% [59].
3.8.3 Sensitivity towards antimicrobial peptides
The minimal inhibitory concentrations of gallidermin and of several other mem-
brane-damaging antimicrobial peptides were determined for the S. aureus and
S. xylosus wild type and mutant strains. The mutants are sensitive to a variety
of antimicrobial peptides that bear a positive net charge [59]. The sensitivity of
the S. aureus mutant to defensin from human neutrophils and to protegrins 3
and 5from porcine leukocytes is at least 10- to 23-fold higher. Factors of 712
were determined with tachyplesin 1 and 3from hemocytes of the horseshoe crab
and to a variant of magainin II from the skin of the clawed frog. The tolerance
towards the lanthionine-containing bacterial peptides gallidermin from S. galli-
narum and nisin from L. lactis is 850-fold lower; very similar results were ob-
tained with the S. xylosus strains.
The increased sensitivity of dlt mutants seems to be restricted to cationic
peptides since no considerable differences are observed in the inhibitory con-
centrations of the neutral peptide gramicidin D from Bacillus brevis. Further-
more, the mutants are not sensitive to cationic polylysine, indicating that catio-
nic properties are not sufficient for activity of a peptide against staphylococci
lacking d-alanine esters in their teichoic acids [59].
The results indicate that the general surface charge of staphylococci can
be severely influenced by the degree of esterification with d-alanine. Loss of d-
alanine esterification is not deleterious to the bacterium, but has other pleiotro-
pic effects. One such effect is that mutants become hypersensitive to a large
variety of cationic peptides. Other effects are currently being studied. One can
speculate whether the D-alanination of teichoic acids is a means whereby S. aur-
eus overcomes the human defensin attack to which they are confronted during
the first defense regimen.
85
3.8 Incorporation of d-alanine into S. aureus teichoic acids confers resistance
3.9 Conclusions
The collaborative research centre 323 allowed the lantibiotic research to boom,
particularly in Tbingen. The fermentation group (H. Zhner and P. Fiedler), the
organic chemistry group (G. Jung) and microbial genetics group (F. Gtz) com-
plemented each other ideally. The fermentation group optimized the fermenta-
tion process of the producing strains and provided sufficient amounts of the
compounds to the chemists for structure determination and mass analysis, and
the molecular biologists studied the biosynthetic genes and the functions of the
corresponding enzymes together with the organic chemists. This concerted ac-
tion was the secret of success. Quite a few of the Tbingen achievements can
be regarded as milestones in lantibiotic research, such as:
. Determination of the chemical structure of epidermin (Allgaier et al. 1986)
. First finding of an antibiotic substance (later named gallidermin) in Staphylo-
coccus gallinarum (F. Gtz 1984)
. First identification of the structural gene (epiA) encoding the lantibiotic pre-
cursor for epidermin; and the first proof that epidermin, and as we now know,
all lantibiotics, are ribosomally synthesized (Schnell et al. 1989)
. Determination of the NMR structure of gallidermin (Freund et al. 1991)
. First cloning and nucleotide sequence of lantibiotic (epidermin) biosynthesis
genes: epiB, C, D, P, and Q (Schnell et al. 1992, Augustin et al. 1992)
. First characterization of a novel flavin-mononucleotide-dependent decarbox-
ylase encoded by the epiD gene (Kupke et al. 1992)
. Regulation of epidermin biosynthesis by the activator EpiQ (Peschel et al.
1993)
. Isolation and characterization of genetically engineered gallidermin and epi-
dermin analogues (Ottenwlder et al. 1995)
. Further optimization of gallidermin production leading to a productivity of
300 mg/liter culture supernatant (Kempf et al. 1997)
. Secretion mechanism of the lantibiotics epidermin and gallidermin (Peschel
et al. 1997)
. Unique producer self-protection against the lantibiotic epidermin by the
ABC transporter EpiFEG (Peschel et al. 1996; Otto et al. 1998)
. Nearly complete X-ray structure of the lantibiotic biosynthesis enzyme, the
flavoprotein EpiD (Kupke et al., in progress)
In Fig. 3.11 an overview of the biosynthetic pathway of epidermin (galli-
dermin) is presented. There are still many open questions that deserve atten-
tion. For example, the underlying enzyme mechanisms for the key reaction, the
lanthionine formation, is still unsolved and it is not known which cofactors are
involved. The environmental conditions under which the activator EpiQ is func-
tional are unknown; there are no indications for a two-component system like
that of nisin or subtilisin. Finally, the question remains why Gram-positive bac-
86
teria produce such lantibiotics what is their real function? It is unlikely that
the antibiotic activity is the only function since it only becomes obvious when
larger amounts of the lantibiotic are produced. Many isolates produce only min-
ute amounts, which might, however, be sufficient for communication with other
bacteria. Most of the lantibiotics interact with the cytoplasmic membrane; some,
such as mersacidin and gallidermin, bind to the cell wall precursor lipid I and,
as recently shown, interact with the teichoic acids to an extent that depends on
the degree of negative charges. Perhaps they represent a measure for the fine
tuning of cell wall structures.
Since genes nearly identical to the epidermin biosynthesis genes are also
present in S. aureus, one can ask whether they have an influence on infection
and survival in the host. Where the lantibiotic biosynthesis genes originate is
not known. The nucleotide sequences of most lantibiotic genes suggest that the
genes are either plasmid encoded (e. g. epidermin) or part of a transposon (e. g.
nisin). They might be relicts of bacteriophages that used lantibiotics for a better
penetration of their genome into the target cells; in this respect, a pore-forming
activity could be helpful. Whether any of the lantibiotics were once used as anti-
biotics or not, the study of these unique peptides sheds light on various areas of
microbiology.
Figure 3.11: Pathway of epidermin biosynthesis.
87
3.9 Conclusions
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92
4 Fermentation of Lantibiotics Epidermin
and Gallidermin
Uwe Theobald*
4.1 Introduction
In recent years the research of the lantibiotics epidermin and gallidermin fo-
cused on two topics, mainly the more genetically based work on the biosynth-
esis of those antimicrobial peptides [13] and their production in bioreactors, re-
spectively. Since antibiotics serve a vital role in modern medicine, fermentation
technology represents a commercially significant part [4, 5]. Additionally, the
need for an industrial production of new bioactive compounds become more
and more important due to the increasing problem of multi-resistence in bac-
teria [6]. Both lantibiotics, gallidermin produced by Staphylococcus gallinarum
T 3928 [7] and epidermin produced by Staphylococcus epidermidis T 3298 [8]
exhibit strong activity against Gram-positive bacteria, particularly against pro-
pioni bacteria which are involved in the acne disease. Other favourable proper-
ties for a large-scale production are the comparable biological activity to re-
nowned antibiotics in current clinical practice like erythromycin or fusidin be-
cause of the bactericidal impressiveness of both compounds against actively
growing and non-dividing bacteria, the advantage for treatment of endocarditis,
abscesses or skin infections. Finally, gallidermin or epidermin are eligible alter-
natives to vancomycin, so that these compounds gained pharmaceutical interest.
The remarkable pharmacological properties led to extensive investigations dur-
ing the last decade to optimise the production process of these drugs for indus-
trial purpose [912]. However, each biotechnological production process is an
unique operation. So, every step of this process needs optimisation for a promis-
ing conversion into an industrial standard. Major crucial points and optimisation
steps known in general are the variation of media components or the design of
special media compositions [13], the development of a suitable and reproducible
process [14], the scale-up problems including oxygen transfer and mixing in the
* LSMW GmbH, Robachstrae 38, D-70499 Stuttgart
93
ISBN: 3-527-30615-3
bioreactor [15], and the implementation of down stream processing steps to the
process [16].
Hence, a complete summary of data concerning the fermentative produc-
tion of gallidermin or epidermin has to reflect all optimisation steps of a fermen-
tation from strain cultivation to large scale production. Therefore, this chapter
will focus on several different topics of an entire antibiotic production process
that enabled efficient metabolite production.
4.2 Strains for gallidermin/epidermin production
The fermentative production of epidermin or gallidermin was possible with dif-
ferent microbial systems. Besides the mentioned wild type strains epidermin
could be produced by a recombinant strain of Staphylococcus carnosus. In con-
trast to those wild type strains, Staphylococcus carnosus is a food-grade strain
which is applied in starter cultures. Beyond that the strain Staphylococcus car-
nosus TM 300 was successfully used for heterologous expression of epidermin
in a two-plasmid system. With the recombinant strain Staphylococcus carnosus
TM 300 (pTepiMA+, pRBgepSI), containing the biosynthesis genes for epider-
min production and immunity against its own antibiotic, nearly 70 % of the wild
type production yield were observed. A problem of the two-plasmid system was
the insufficient plasmid stability, so that most work on process development and
optimisation was carried out with the wild type strains S. gallinarum T 3928
and S. epidermidis T 3298. According to the somewhat higher activity against
living and non-dividing Gram-positive bacteria, this review will focus on the
antibiotic gallidermin solely, although a lot of successful work was done for epi-
dermin, too [1721].
4.3 Disadvantages during gallidermin process
development
Regarding the development of a gallidermin production process over a couple
of years a few problems were generally noticed. First of all, a exceedingly strain
instability concerning product excretion was observed, though biomass forma-
tion remained still stable. This fluctuating lantibiotic production yield was con-
tradictory to a reproducible and standardised process. A second crucial point
94
4 Fermentation of Lantibiotics Epidermin and Gallidermin
was the composition of the production medium. Several investigations dealt
with the search for a defined and synthetic medium for staphylococci, but the
biomass and product concentrations gained with the developed medium [22]
could not compete with any of the tested complex media. Best production yield
could be observed with a medium based upon meat extract. Meat extract was
supposed to be the only suitable complex medium component that led to appro-
priate product yields [9] but is first of high costs for production or downstream
processing and second carried a possible risk of prion infections such as bovine
spongiform encephalopathy or Creutzfeld-Jakob-disease [2325].
4.4 Gallidermin a lantibiotic and its way towards
industrial production
The basis for a microbial process is the stock culture for storage of the talented
strain. This first step of the production could be identified as prime cause for the
problem of the mentioned instability in product formation during fermentation
[26]. A new mathematical parameter (hs-value) based upon the commonly ana-
lysed parameters biomass and product concentration was introduced to facilitate
the optimisation of the media used for this cultivation of stock cultures on agar
slants. The hs-value (high and stable product concentration) reduced the
amount of data generated in optimisation experiments to one single value for
each medium composition and allowed an assessment of any medium formula-
tion with regard to reproducibility and product formation [26]. Figure 4.1 illus-
trates the time course of the product concentration over several passages found
in the corresponding liquid cultures after 24 hours of incubation (A) and the cor-
responding hs-values (B) obtained exemplary from four different stock culture
media (out of 53 different formulations tested) during several passages.
Another indispensable optimisation step was the design of a new medium
composition. An analysis of different complex compounds [13] suggested the
possibility for a successful use of yeast extract (without risk of prion infection).
In contrast to earlier results [9, 27] the investigations revealed that a special
yeast extract could efficiently replace meat extract in the production medium of
S. gallinarum T 3928. In addition to that the special yeast extract (Ohly KAT)
applied in a three-fold lower concentration led to approximately 20 % higher
product yields compared to the old medium composition [28]. Figure 4.2 shows
a comparison of the gallidermin concentrations obtained with these media in
batch fermentations.
Since the Staphylococcus species are very halotolerant in general, the ef-
fect of salt (cation) is very important for growth and production [911, 27]. To
optimise this parameter, different cations in various concentrations were
95
4.4 Gallidermin a lantibiotic and its way towards industrial production
tested [12, 2830]. Best results were gained with CaCl
2
in very high concen-
trations of 30 to 40 g/l whereas the other cations like NaCl showed only slight
influence. Figure 4.3 indicated, that there is an optimum in CaCl
2
concentra-
tion whereas the NaCl concentration is only of slight influence upon product
formation.
However, Peschel and co-workers demonstrated that cationic teichonic
acids at the staphylococcal cell surface are involved in gallidermin resistance
[31]. This may be due to an ionic interaction between the teichonic acids and
the gallidermin molecule that also has a positive net charge (3-fold). Although
the mechanism of resistance is not yet clear in detail [1] it is possible that Ca
2+
Figure 4.1: Gallidermin concentrations (A) and corresponding hs-values (B) obtained
from a suitability test shown for four different stock culture media during several pas-
sages.
96
Figure 4.2: Comparison of gallidermin formation by Staphylococcus gallinarum T 3928
grown in the previously used medium (open symbols) and the new developed production
medium (full symbols). Both cultures were inoculated from the same agar plate.
Figure 4.3: Maximal gallidermin concentrations in the culture broth of Staphylococcus
gallinarum T 3928 in dependency on different NaCl/CaCl
2
proportions in the production
medium.
97
4.4 Gallidermin a lantibiotic and its way towards industrial production
probably may be involved in the immunity as well. Hence, the components of
the new medium were roughly (qualitatively) adjusted. Final optimisation were
carried out with a computer-aided procedure. Genetic algorithms are able to
optimise simultaneously a multi-parameter system like a medium composition
(quantitatively optimisation) [32].
Several fermentation modes (batch, fed-batch, continuous) were tested.
Preliminary experiments in continuous cultivations resulted in lower gallidermin
yields compared to those gained in batch or fed-batch cultures. Best results
were determined in batch processes or fed-batch processes with pulses of sev-
eral amino acids or maltose during the antibiotic production phase [33]. A com-
parison of different types of bioreactor (dialysis fermenter, airlift reactor, stirred
tank and loop reactor) showed no significant differences in growth and produc-
tion. The effect of dissolved oxygen tension seemed to be more critical [11] than
the type of reactor. The dialysis reactor consisted of two chambers which are se-
parated by a dialysis membrane with an exclusion limit of 10 000 Dalton [10].
This reactor has the advantage of on-line product separation during the produc-
tion phase but has the crucial drawback that no scale-up procedure is possible.
Hence, a transfer from laboratory scale to industrial scale seemed to be out of
question at that time. Scale-up investigations were focused on the batch and
fed-batch processes in stirred tank reactors, which enabled a reliable scale-up
procedure from 0.2 litre to 200 litre scale [34]. Best results in large scale fermen-
tations were achieved by addition of maltose during the late production phase
as shown in Fig. 4.4.
Figure 4.4: Concentrations of gallidermin (full symbols) and cell dry weight (open sym-
bols) observed during a scale-up from 20 to 200 litres bioreactor. The arrow marks the
transition from 20 to 200 litres and the time when maltose is pulsed into the 200 litres reac-
tor; the grey area shows the range of the maximal gallidermin concentration (including er-
rors) expected in a 200 litres bioreactor without maltose addition.
98
The downstream processing (product recovery from the fermentation
broth) was successfully optimised by product adsorption onto resin (Amberlite
XAD-1180) [35]. An integration of the product separation step was performed
by direct addition of resin into the bioreactor during fermentation or addition of
an cross-flow filtration step between bioreactor and adsorber column. This re-
sulted in a cell retention by the cross-flow module during the production phase.
Product containing filtrate was harvested afterwards onto an adsorber column
with XAD resin. So, the overall production time could be prolonged.
4.5 Conclusion
Extensive investigations led to a new method for strain storage and cultivation,
the design of a new production medium, process development and the ability for
scale-up into technical scale. An integration of a filtration step during production
permitted an on-line product harvesting. Finally, it should be emphasised that
higher production yields were obtained (more than 300 mg/l) and costs for med-
ium components were drastically reduced more than 15-fold from 72 Euro per
gram gallidermin to approximately 4.6 Euro per gram. Figure 4.5 summarised the
reduction of medium costs and the increase in gallidermin concentration during
the three optimisation steps (replacement of ingredients, rough adjustment of salt
concentrations and final optimisation using a computer program).
Figure 4.5: Evolution of medium costs (columns) and product yields during three optimi-
sation steps (see text).
99
4.5 Conclusion
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101
References
5 Genetics of Nikkomycin Production
in Streptomyces tendae T 901
Christiane Bormann*
5.1 Introduction: nikkomycins
Nikkomycins are peptidyl nucleoside antibiotics that are potent and specific in-
hibitors of chitin synthetases. They are structurally similar to UDP-N-acetylglu-
cosamine, the natural substrate of these enzymes, and inhibition occurs in a
competitive manner [1, 2]. Structures of nikkomycins are shown in Fig. 5.1. Nik-
komycins X and Z, the main compounds produced by Streptomyces tendae
T 901, exhibit high antifungal, insecticidal, and acaricidal activity [3]. Since
their toxicity towards mammals and bees is very low or not detectable, and since
nikkomycins are easily degraded in nature, they are potentially useful in agri-
culture or as therapeutic antifungal agents in humans. Nikkomycin Z shows sig-
nificant activity towards the highly chitinous, pathogenic, dimorphic fungi Coc-
cidioides immitis and Blastomyces dermatitidis [4]. Polyoxins, antibiotics related
to nikkomycins, are commercially produced by Streptomyces cacaoi and applied
as agricultural fungicides in Japan (for a review, see [5]).
Nikkomycins X and Z are composed of the unusual amino acid hydroxy-
pyridylhomothreonine (HPHT; nikkomycin D) and a peptidically linked nucleo-
side moiety. The nucleoside moiety comprises an aminohexuronic acid N-glyco-
sidically linked to 4-formyl-4-imidazolin-2-one, forming nikkomycin C
x
, or to ur-
acil, forming nikkomycin C
z
. Minor components of the culture filtrate of S. ten-
dae T 901 are nikkomycins I and J, which are structures analogous to nikkomy-
cins X and Z that contain glutamic acid peptidically bound to the 6'-carboxyl
group of the aminohexuronic acid. Nikkomycins C
x
, C
z
, and D are also detected
in culture filtrates and arise by hydrolytic cleavage of nikkomycins X and Z.
These three compounds neither act as chitin synthetase inhibitors nor display
biological activity. Nikkomycins K
x
, K
z
, O
x
, and O
z
(Fig. 5.1), which will be dis-
* Mikrobiologie/Biotechnologie, Universitt Tbingen, Auf der Morgenstelle 15, D-72076
Tbingen
102
ISBN: 3-527-30615-3
O
OH OH
R
1
CH
C O
R
3
NH R
2
A B C
R
1
N
N
CHO
O
H
N
HN
O
O
R
2
N
CH CH
OH
CH
3
CH C
NH
2
HO
O
H
N
CH CH
2
OH
CH C
NH
2
O
D
R
2
N
CH CH
2
OH
CH C
NH
2
HO
O
nikkomycin R
1
R
2
R
3
X A A OH
Z B A OH
I A A Glu
J B A Glu
C
x
A B OH
C
z
B B OH
K
x
A C OH
K
z
B C OH
O
x
A D OH
O
z
B D OH
D
N
CH CH
OH
CH
3
CH C
NH
2
HO
O
OH
Figure 5.1: Structures of nikkomycins.
103
5.1 Introduction: nikkomycins
cussed in a later section, are synthesized by mutants derived from S. tendae
T 901/8c by UV treatment and chemical mutagenesis [6].
Biosynthesis of nikkomycins can be divided into two parts: the nucleoside
and peptidyl moieties are synthesized in separate pathways and then are linked
by peptide bonds [6]. Figure 5.2 summarizes the steps of the nikkomycin bio-
synthetic pathway based on isotopic labeling and chemical characterization of
pathway intermediates and shunt products. It also includes data on the bio-
synthesis of the polyoxin nucleoside, which contains an aminohexuronic acid
moiety identical to that of nikkomycin nucleosides. Isono and coworkers [7]
have shown that the polyoxin nucleoside arises from uridine and carbon-3 of
phosphoenolpyruvate, and they proposed the reaction mechanism as a conden-
sation of uridine and phosphoenolpyruvate to octofuranuloseuronic acid as the
intermediate, followed by oxidative elimination of carbon-7' and carbon-8' and
introduction of an amino group on carbon-5'. Isolation of octosyl acids, shunt
metabolites derived from the postulated intermediate, supports this hypothesis.
Analogues of octosyl acids, nikkomycins S
x
and S
z
, have been isolated from nik-
komycin-producing S. tendae, and therefore the same biosynthetic pathway has
been suggested for the nikkomycin nucleosides nikkomycins C
x
and C
z
[8]. His-
tidine is the precursor of the imidazolone base [9]; l-lysine is incorporated via
picolinic acid into the pyridyl moiety and the attached carbon atom of nikkomy-
cin D [10, 11]. Pyridylhomothreonine (PHT, nikkomycin E) and 4-pyridyl-2-oxo-
4-hydroxy-isovalerate (POHIV), which have been isolated from S. tendae culture
filtrate [12, 13], are potential biosynthetic precursors of HPHT.
5.2 Isolation of nikkomycin biosynthetic genes
A number of approaches have been successfully used to isolate genes of anti-
biotic biosynthetic pathways from several Streptomyces strains. The strategy of
cloning antibiotic-resistance genes, which are usually clustered with antibiotic
biosynthetic genes, is not applicable to nikkomycin genes because the produ-
cing strain lacks the nikkomycin target site. Complementation of S. tendae mu-
tants blocked in nikkomycin synthesis has led to the isolation of a 9.4-kb frag-
ment that complements a non-producing mutant to nikkomycin C
x
, C
z
, and K
x
synthesis [14]. However, structural genes of the nikkomycin pathway have not
been identified. Therefore, the nikkomycin genes were cloned by identifying
gene products involved in nikkomycin synthesis, microsequencing the N-ter-
mini to design oligonucleotide probes, and cloning the corresponding genes
using these probes [15].
Nikkomycin biosynthetic enzymes were identified in gene expression stu-
dies using two-dimensional gel electrophoresis to separate cellular proteins. In-
itially, gene expression was analyzed in S. tendae wild type and mutant strains
104
5 Genetics of Nikkomycin Production in Streptomyces tendae T 901
Figure 5.2: Nikkomycin biosynthetic pathway based on the incorporation of labeled pre-
cursors and chemical characterization of pathway intermediates and shunt products; in-
cluded are data on the biosynthesis of the polyoxin nucleoside (for references, see text).
POHIV, 4-pyridyl-2-oxo-4-hydroxyisovalerate; PHT, pyridylhomothreonine.
105
blocked in nikkomycin biosynthesis [6]. Mutants which were able to synthesize
nikkomycins but which differed from the wild type in the spectrum of nikkomy-
cin structures formed had a protein pattern identical to that of the wild type. In
contrast, protein profiles of mutants which did not synthesize any known nikko-
mycin structure differed from the wild type pattern as well as from each other.
Selection of protein spots that were present in the producing strains and absent
in each non-producing mutant led to the identification of ten gene products
(P1P10) (Fig. 5.3).
As nikkomycin biosynthesis is regulated by the growth phase, the kinetics
of the appearance of proteins P1P10 was investigated during growth of S. ten-
dae T 901/8c in production medium (Fig. 5.4). Growth of S. tendae is charac-
terized by a biphasic growth curve with two phases of rapid growth separated
by a 1.5 to 2 h period in which growth slows down. Nikkomycin production be-
gins in the transition to the stationary phase, after approximately 27 h after in-
oculation. Proteins P1P6 and P10 were detected in extracts of mycelia har-
vested in the second exponential growth phase after 22.5 h of inoculation, and
the amount of all of proteins P1P10 increased to maximum levels at the early
stationary phase and remained constant throughout the stationary phase. Synth-
esis of proteins P1P10 preceded nikkomycin production, as would be expected
for nikkomycin biosynthetic enzymes.
N-terminal amino acid sequences were obtained for six of the ten identi-
fied proteins. Oligonucleotide probes designed from the N-terminal sequences
of proteins P4, P5, and P8 gave positive signals of similar intensities with more
Figure 5.3: Two dimensional gels of silver-stained cell extracts prepared from wild type
T 901/8c (A) and nikkomycin non-producing mutant T 901/NP13 (B). Mycelia were har-
vested in the stationary phase after 27.5 h of incubation. Proteins P1P10 observed in the
T 901/8c extract (A) and their corresponding positions in the T 901/NP13 extract (B) are
indicated by arrows. These proteins were identified on the basis of three experiments per-
formed with each producing and non-producing S. tendae strain. Positions of protein mo-
lecular mass standards are shown on the left. The acidic end of the gel is on the left, and
the basic on the right.
106
than ten bands of digested genomic DNA and therefore were unsuitable for iso-
lating the corresponding genes. A mixture of oligonucleotide probes designed
from proteins P1 and P2, which have identical N-terminal sequences except that
protein P1 has three additional amino acid residues at its N-terminus, and
probes designed from protein P6 led to the isolation of genomic DNA fragments
containing the binding sites for the probes used. The isolated fragments, an
8-kb BamHI fragment and a 6.5-kb PvuII fragment, appeared to overlap by
1.5-kb, and the former contained the binding sites for both oligonucleotide
probes used. DNA sequence analysis revealed that the 8-kb BamHI fragment
contains two open reading frames (ORFs) whose deduced N-terminal amino
acid sequences are identical to that of proteins P1/P2 and that of protein P6. As
gene-disruption mutants in the ORF encoding proteins P1/P2 (designated nikJ)
and encoding protein P6 (designated nikI) failed to synthesize nikkomycins, it
was evident that nikkomycin biosynthesis genes had been isolated.
Figure 5.4: Time course of nikkomycin production (A) and gene expression (B) in S. ten-
dae T 901/8c cultivated in production medium. Nikkomycin Z plus X (g) in the culture
filtrate compared to growth as determined by dry weight (&). Arrows indicate time points
at which mycelia were harvested to prepare protein extracts for 2-D gel electrophoresis.
(B) Enlarged sections of silver-stained 2-D gels of extracts of S. tendae T 901/8c har-
vested in the exponential phase (17.5 h), the late exponential phase (22.5 h), and the sta-
tionary phase (27.5 h). The position of proteins P1, P2, P3, P4, P7, P8, and P9 are marked
by arrows.
107
5.2 Isolation of nikkomycin biosynthetic genes
5.3 Isolation of the nikkomycin gene cluster and expression
in Streptomyces lividans
In bacteria, antibiotic biosynthetic genes are usually clustered. In order to isolate
the entire nikkomycin gene cluster, a genomic library of S. tendae T 901/8c was
constructed in the Escherichia coli/Streptomyces shuttle cosmid pKC505 [16] and
screened with the 8-kb BamHI fragment containing the nikI and nikJ genes. Hy-
bridizing cosmids containing inserts of approximately 30 kb were mapped with
restriction enzymes. Cosmids p24/32 and p9/43 carried the recognition sites for
oligonucleotide probes designed from the N-terminal amino acid sequences of
proteins P1/P2, P4, P5, P6, and P8, and of proteins P1/P2, P5, P6, and P8, respec-
tively. S. lividans TK23, which does not produce nikkomycins, was used as a host
to express the cloned nikkomycin biosynthetic genes. The nik cluster was not iso-
lated on a single plasmid, as shown by the lack of synthesis of nikkomycins I, J,
X, and Z by S. lividans TK23 transformants containing one of the recombinant
plasmids. The nik cluster was cloned on two plasmids, p24/32 and p9/43, which
carry inserts of about 31 and 27 kb, respectively, 15 kb of which overlap (Fig. 5.5).
To facilitate selection for both plasmids, the apramycin resistance gene of p9/43
was removed by restriction with XhoI, which did not cut within the insert, and re-
placed with the aphII gene from Tn5. One of the apramycin- and neomycin-resis-
tant S. lividans TK23 transformants synthesized relatively large amounts of nik-
komycins I, J, X, and Z, approximately 50% of that synthesized by S. tendae
T 901/8c. This transformant contained a large (>100-kb) plasmid that had
formed by recombination between homologous regions of the two plasmids,
either the vector pKC505 regions or the homologous regions of the inserts [17].
orfR nikV U T S R Q P2 P1 A B C D E F G I J K L M N O
B B BB B B B
VM4 VM5 VM8 VM6 VM1/2
S S S S B
genome
S S S B B
p24/32
S
probe
p9/43
27 kb
31 kb
8 kb
Figure 5.5: Organization of the nik gene cluster and restriction map of the cloned chro-
mosomal region containing the nik cluster. The structural genes (nik) and the regulatory
gene (orfR) of the nik cluster are indicated by arrows. Wavy arrows indicate transcripts.
The boxes below the genome map indicate the recognition sites for oligonucleotide probes
VM1/2, VM4, VM5, VM6, and VM8, designed from proteins P1/2, P4, P5, P6, and P8. The
hybridization probe used to screen the S. tendae gene library and the inserts of cosmid
p24/32 and p9/43 are also indicated. B, BamHI; S, SacI.
108
5.4 Organization of the nikkomycin gene cluster
The nucleotide sequence of an approximately 36-kb genomic region containing
the nik cluster has been determined, and a series of 23 ORFs comprising a re-
gion of 29 kb identified. The translational start points of the ORFs have been
tentatively located using the following criteria: (a) the G+C content in the third
position of codons, (b) the location of a potential ribosome binding site at a suita-
ble distance from the putative translation start [18], and (c) the observed simila-
rities of the deduced amino acid sequence with proteins in databases. Each of
the deduced proteins of the nikJ, nikS, nikA, nikI, and nikC genes had an
N-terminal amino acid sequence identical to that determined for proteins P1/P2,
P4, P5, P6, and P8, respectively. The most relevant features of the nik cluster
and the encoded proteins deduced from the nucleotide sequence are summar-
ized in Table 5.1, and the organization of the nik cluster is shown in Fig. 5.5. The
ORFs of the nik cluster are arranged in three sets of adjacent genes with an in-
tergenic spacing of <92 bp, and the stop codon of several ORFs overlaps with
Table 5.1: Relevant features of the nik cluster and the encoded proteins deduced from
the DNA sequence.
ORF G+C [mol%] Residues/MW Predicted/known function
nikA 69.3 296/31,429 Aldehyde dehydrogenase
nikB 72.2 357/37,146 Synthetase
nikC 72.3 426/47,072 Aminotransferase
nikD 71.0 389/43,055 Oxidase
nikE 74.6 561/60080 Ligase
nikF 71.5 410/45,908 Cytochrome P450
nikG 74.5 64/6,565 Ferredoxin
nikI 68.4 218/24,489
nikJ 70.1 453/50,422
nikK 73.9 374/40,164 Aminotransferase
nikL 74.7 240/25,895
nikM 70.4 213/24,086
nikN 75.8 550/57,238 Membrane transport
protein
nikO 73.4 471/50,345 Enoylpyruvate transferase
nikP1 73.2 677/73,061 Peptide synthetase
nikP2 75.5 272/29,514 Thioesterase
nikQ 73.1 396/44,479 Cytochrome P450
nikR 76.7 226/24,991 Phosphoribosyltransferase
nikS 70.8 424/46,873 Ligase
nikT 73.6 440/46,227 Acyl carrier protein and
aminotransferase
nikU 63.8 155/16,549 Mutase, small subunit
nikV 69.2 423/46,077 Mutase, large subunit
orfR 74.0 1062/114,701 Regulatory protein
109
5.4 Organization of the nikkomycin gene cluster
the start codon of the subsequent gene, implicating translational coupling. The
nikA-nikG gene cluster and the nikI-nikO gene clusters are separated by 944 bp
and are oriented in the same direction, while the nikP1-nikV gene cluster is di-
vergently transcribed; the intergenic region between nikP1 and nikA is 273 bp.
The orfR gene, which encodes a pathway-specific activator protein for the nik
biosynthesis genes is located at the left end of the nik cluster and is divergently
oriented relative to the nikP1-nikV genes, with a 447-bp intergenic spacing.
5.5 Roles of the nik genes
The likely roles of the nik genes in nikkomycin biosynthesis as discussed below
have been established from: (a) the nikkomycins structures synthesized by
strains with inactivated nik genes, (b) sequence comparisons, and (c) enzymatic
activities of nik gene products. Except for the nikG and nikU genes, whose roles
seemed to be obvious, each gene of the nik cluster was inactivated by inserting
a kanamycin resistance cassette via double cross-over homologous recombina-
tion. Novel nikkomycin structures accumulated by nik::aphII mutants were iso-
lated from culture broth and their chemical structure (Fig. 5.6) was elucidated.
Figure 5.6: Novel nikkomycin structures and biosynthetic intermediates synthesized by
nik gene insertion mutants. Nikkomycins L
x
(1) and L
z
(2) are synthesized by nikF::aphII;
4-formyl-4-imidazolin-2-one (3) by nikK::aphII and fluorouracil-resistant nikR::aphII; and
ribofuranosyl-4-formyl-4-imidazolin-2-one (4) by nikO::aphII.
O
O
N
HN HN
N
CHO
O
CH
O
OH OH
NH CH
CH
3
CH C
NH
2
O
CH
OH
N
COOH
R
R:
(1) (2)
HN
N
CHO
O
O
OH OH
CH
2
HO
(4)
HN
N
CHO
O
H
(3)
110
Table 5.2 summarizes the nikkomycin structures synthesized by gene-insertion
mutants. Except for the nikJ::aphII and nikP1::aphII mutants, all mutants were
able to synthesize the wild type nikkomycin spectrum by genetic complementa-
tion or by feeding suitable biosynthetic intermediates, which excluded the possi-
bility that the mutant phenotype was caused by a polar effect of the integrated
kanamycin cassette on downstream genes. Table 5.1 summarizes the predicted
function of the deduced Nik proteins based on results of database searches. The
nikC, nikT, and nikR genes have been expressed in E. coli, and the gene pro-
ducts catalyze the reactions shown in Fig. 5.7.
5.5.1 Biosynthetic pathway of the peptidyl moiety
Figure 5.8 shows the proposed biosynthetic pathway for the peptidyl moiety
HPHT [19]. The genes needed for its synthesis are all the genes of the nikA-
nikG operon and the clustered nikT-nikV genes of the nikP1-nikV operon (Fig.
5.5). Except for the nikF::aphII mutant, all mutants inactivated in one of these
genes fail to synthesize biologically active nikkomycins, but accumulate the nu-
cleoside structures nikkomycins C
x
and C
z
.
The initial reaction in the HPHT biosynthetic pathway is catalyzed by
NikC, which has been characterized as l-lysine-2-aminotransferase [20]. NikC
Table 5.2: Nikkomycin structures synthesized by mutants containing an inactivated nik
gene.
Mutant Nikkomycin structures Mutant Nikkomycin structures
nikA::aphII C
x
, C
z
nikP1::aphII C
x
*, C
z
nikB::aphII C
x
, C
z
nikP2::aphII I*, J, X*, Z
nikC::aphII C
x
, C
z
nikQ::aphII RT 2.7, J, Z
nikD::aphII C
x
, C
z
nikR::aphII I*, J, X*, Z
nikE::aphII C
x
, C
z
nikS::aphII C
x
, C
z
nikF::aphII L
x
, L
z
nikT::aphII C
x
, C
z
nikI::aphII RT 2.7 nikV::aphII C
x
, C
z
, K
x
, K
z
, O
x
, O
z
nikJ::aphII RT 2.7
nikK::aphII S
x
, S
z
, (3) orfR::aphII none
nikL::aphII RT 0.75, S
x
, S
z
nikM::aphII S
x
, S
z
nikN::aphII S
x
, S
z
nikO::aphII (4), RT 2.7
Structures of nikkomycins, intermediates, and shunt products are shown in Figs. 5.1, 5.2,
and 5.6. Numbers in parentheses refer to structures shown in Fig. 5.6. RT 0.75 and RT 2.7
indicate unknown compounds that might comprise nikkomycin structures; RTrefers to re-
tention time in HPLC analyses.
* reduced amount; approximately 60% of wild type nikkomycin X production.
111
removes the a-amino group from lysine to create piperideine-2-carboxylic acid,
the cyclic and dehydrated form of 2-oxo-6-aminohexanoic acid (Fig. 5.7, reac-
tion (1)). NikC belongs to a family of pyridoxamine/pyridoxal-phosphate-depen-
dent enzymes, such as dehydrases involved in the formation of dideoxyhexoses
of the Gram-negative cell wall and aminotransferases involved in deoxyamino
sugar biosynthesis in secondary metabolism [21, 22]. NikC is a secondary meta-
bolite enzyme that is specific for nikkomycin biosynthesis and is not required
for l-lysine catabolism in S. tendae.
The next step, the oxidation of piperideine-2-carboxylic acid to picolinic
acid, is predicted to be catalyzed by the nikD-encoded protein, as feeding of pi-
colinic acid to nikD mutants restores the ability to synthesize nikkomycins I, J,
X, and Z. The NikD protein belongs to the family of monomeric sarcosine oxi-
dases that contain FAD as the coenzyme. Members of this family metabolize sar-
cosine, l-pipecolic acid, and l-proline [23]. These latter two substrates are oxi-
dized to D-piperideine-6-carboxylic acid and D-pyrrolidine-5-carboxylic acid, re-
spectively. Piperideine-2-carboxylic acid is structurally similar to these com-
pounds. A reaction mechanism similar to that of sarcosine oxidases is proposed
Figure 5.7: Reactions catalyzed in vitro by recombinant nik gene products. Reactions (1),
(2), and (3) are catalyzed by NikC, NikT, and NikR, respectively. PLP, pyridoxalphosphate.
N N
N
H H
H
2 2
2
O
O
OH
NH
2
O
O
O
O
OH
OH
OH
NikC
PLP
R R
H O
2
(1)
(2)
P
P P
O
CH
2
OH OH
O
O
+
H
N
HN
O
O
O
CH
2
OH OH
O P
N
HN
O
O
PP
i
NikR
(3)
N
CH CH
OH
CH3
CH C
O
OH
NH2
N
CH CH
OH
CH3
C C
O
OH
O
PLP
phenylalanine
NikT
phenylpyruvate
C
O
OH
N
112
N
C
H
C
O
H
C
H
3
C
H
C
O
O
H
H
N
H
2
C
O
O
H
N
N
i
k
E
A
T
P
C
o
A
-
S
H
A
M
P
+
P
P
i
H
O
2
N
N
N
H
H
H
2
2
2
O
O
H
O
O
O
H
N
i
k
C
L
-
l
y
s
i
n
e
p
i
p
e
r
i
d
e
i
n
e
-
2
-
c
a
r
b
o
x
y
l
i
c
a
c
i
d
R
H
O
2
N
i
k
D
C
O
O
H
S
C
o
A
C
N
O
4
[
H
]
p
i
c
o
l
i
n
i
c
a
c
i
d
p
i
c
o
l
i
n
i
c
a
c
i
d
-
C
o
A
N
C
H
C
O
H
C
H
3
C
H
C
O
O
H
H
N
H
2
H
O
O
+
2
H
2
+
H
O
2
N
i
k
F
,
N
i
k
G
H
P
H
T
N
C
H
CC
H
3
C
C
O
O
H
H
P
O
H
I
V
N
P
H
T
C
C
C
O
H
H
O
O
C
C
O
O
H
H
2
2
N
i
k
T
N
i
k
U
,
N
i
k
V
N
H
2
O
O
H
R
O
O
O
H
R
2
-
o
x
o
g
l
u
t
a
m
i
c
a
c
i
d
2
-
o
x
o
-
3
-
m
e
t
h
y
l
-
s
u
c
c
i
n
i
c
a
c
i
d
N
i
k
B
O
O
H
R
N
A
D
H
+
H
H
C
N
O
p
y
r
i
d
i
n
e
-
2
-
c
a
r
b
a
l
d
e
h
y
d
e
N
i
k
A N
A
D
+
C
o
A
-
S
H
+
C
O
O
H
C
C
O
H
H
O
O
CC
H
3
C
O
2
O
H
O
F
i
g
u
r
e
5
.
8
:
P
r
o
p
o
s
e
d
b
i
o
s
y
n
t
h
e
t
i
c
p
a
t
h
w
a
y
f
o
r
t
h
e
p
e
p
t
i
d
y
l
m
o
i
e
t
y
H
P
H
T
.
2
-
o
x
o
g
l
u
t
a
r
i
c
a
c
i
d
113
for NikD, except that oxidation of piperideine-2-carboxylic acid to picolinic acid
requires the removal of two electron pairs. The NikD protein contains an ADP-
binding, bab-fold fingerprint sequence at its N-terminus, with an aspartate at
position 1, which has been found in several FAD-containing enzymes and seems
to be important for the interaction with FAD [24]. This finding implies that NikD
might use FAD as a coenzyme.
The presumed role of the nikE gene product is the activation of picolinic
acid to form picolinic acid-coenzyme A, the predicted substrate for NikA. This
proposed reaction rests on the similarity of NikE to adenylate-forming enzymes.
NikE is most similar to enzymes that adenylate aromatic acids, such as SnbA,
which activates 3-hydroxy-picolinic acid in the pristinamycin biosynthetic path-
way [25], and 2,3-dihydroxybenzoate-AMP ligases of enterobactin biosynthesis
[26] and other EntE homologs. NikE contains the conserved core sequences
needed for ATP-binding and hydrolysis and for adenylate formation [27]. As
feeding of HPHT to nikD mutants restores the ability to synthesize nikkomycins
I, J, X, and Z, it is clear that NikE functions in the nikkomycin biosynthetic
pathway and is not responsible for the formation of peptide bonds.
The role of the nikA- and nikB-encoded proteins is deduced from their sig-
nificant sequence similarity to aldehyde dehydrogenases (acylating) (ADAs) and
4-hydroxy-2-oxovalerate aldolases (HOAs), respectively, of the meta-cleavage
pathway for the metabolism of aromatic hydrocarbons. The aldolase releases pyr-
uvate and acetaldehyde, and the acylating dehydrogenase converts acetalde-
hyde to acetyl-CoA using NAD
+
and CoA as cofactors. The mechanistically re-
verse reactions are postulated for NikA and NikB to form the carbon-carbon
bond, thereby yielding the entire skeleton of HPHT. The proposed role of NikA is
to catalyze the reduction of picolinic acid-CoA to pyridine 2-carbaldehyde with
the release of coenzyme A. The NikA protein has an ADP-binding, bab-fold fin-
gerprint sequence at its N-terminus [28], indicating that NAD(P)H might function
as a hydrogen donor. NikB is proposed to catalyze a Claisen condensation of pyri-
dine 2-carbaldehyde and a 2-oxo acid compound that will be discussed below.
Two regions of NikB also exhibit similarity with regions of NifV homocitrate
synthetases, which catalyze condensation of acetyl-CoA with 2-oxoglutarate to
form homocitrate. The reaction mechanism of carbon-carbon bond formation by
NifV homologs, also suggested for NikB, is proposed to be similar to that of car-
bon-carbon-cleavage by HOAs [29]. Interestingly, the order of the nikA and nikB
genes in the nikA-nikG operon is the same as that of genes encoding ADA and
HOA in the meta-pathway gene clusters. The order of these genes is strictly con-
served; the ADA-encoding gene is located directly upstream of the HOA-encod-
ing gene. The two proteins are suggested to form a pre-adapted catabolic mod-
ule, which is thought to be assembled with other modules of the meta-cleavage
operons in Pseudomonas [30]. NikA and NikB represent the first proteins shown
to have significant sequence similarities along their entire length with ADAs and
HOAs, respectively, that are not involved in meta degradation of aromatic hydro-
carbons. These features indicate a common evolutionary origin.
The presumed role of nikU and nikV is to catalyze the isomerization of 2-
oxoglutaric acid to yield 2-oxo-3-methylsuccinic acid, which is the predicted 2-
114
oxo acid substrate in the NikB-mediated condensation reaction that yields the
skeleton for HPHT with the release of carbon dioxide [31]. This hypothesis is
based on the significant sequence similarity of NikU and NikV to methylaspar-
tate mutase S and E chains, respectively, from Corynebacterium. The coenzyme
B
12
-dependent mutase from Corynebacterium is composed of two components
(S and E) and converts L-glutamate to threo-b-methyl-L-aspartate [32]. The ana-
logous reaction is proposed for the putative mutase composed of NikV and
NikU. The phenotype of nikV mutants is compatible with the proposed role for
NikV, as the nikV mutants accumulate nikkomycins K
x
, K
z
, O
x
, and O
z
(Fig. 5.1)
in addition to the nucleoside moieties nikkomycins C
x
and C
z
. Compared to
HPHT, the peptidyl moiety of these nikkomycins lacks the methyl group at car-
bon-3. The phenotype of the nikV mutants can be rationalized by assuming that
the lack of the nikV gene leads to an increased intracellular concentration of 2-
oxalacetate or pyruvate, which might be substrates for NikB, yielding the pepti-
dyl moiety of nikkomycins K
x
, K
z
, O
x
, and O
z
.
The nikT gene encodes a protein which consists of two domains [31]. The
N-terminal region (approximately 70 amino acid residues) exhibits 60% se-
quence similarity to acyl carrier proteins and contains a putative 4'-phosphopan-
tetheine binding sequence. This domain of NikT is predicted to be involved in
peptide bond formation (Section 5.5.3). The other part of NikT (amino acid 113
to 440) reveals significant sequence similarity to aminotransferase class-II and
contains a putative pyridoxal-phosphate attachment site. As recombinant NikT
transaminates POHIV in vitro to yield PHT (Fig. 5.7, reaction (2); shown in colla-
boration with K. Liebetrau, University of Mnster), the role proposed for NikT is
to catalyze this transamination reaction.
The final reaction in the HPHT biosynthetic pathway is presumed to be
catalyzed by the nikF-encoded cytochrome P450. The NikF protein is responsi-
ble for introducing the hydroxy group into the pyridyl ring since disruption of
the nikF gene abolishes nikkomycin I, J, X, and Z synthesis, but leads to the for-
mation of nikkomycin L
x
and L
z
, homologs of nikkomycins X and Z that contain
an unhydroxylated pyridyl residue ((1), (2) in Fig. 5.6). PHT is assumed to be the
substrate for NikF since PHT has been detected in culture filtrates of S. tendae
[12]. NikF is a member of the Class I cytochrome P450 monooxygenases, which
are reduced via an iron-sulfur protein by a flavin-containing reductase that ac-
cepts electrons from NAD(P)H. The ferredoxin-encoding gene nikG is located
immediately downstream of the nikF gene, implying that NikG might be the in
vivo electron transport protein for NikF.
5.5.2 Biosynthetic pathway for the nucleoside moiety
Figure 5.9 summarizes the information on the nucleoside biosynthetic pathway.
The genes nikQ and nikR, arranged in this order within the nikP1-nikV operon,
are predicted to be responsible for the formation of 5'-phosphoribofuranosyl-4-
115
Figure 5.9: Proposed biosynthetic pathway for the nucleoside moieties nikkomycins C
x
and C
z.
formyl-4-imidazolin-2-one, the precursor for nikkomycin C
x
[33]. The genes
nikO, nikI, nikJ, nikL, nikM, and nikK, which are located in a single operon, are
proposed to be responsible for the formation of the aminohexuronic acid portion
of the nucleoside skeletons originating from 5'-phosphoribofuranosyl-4-formyl-
4-imidazolin-2-one, uridine-monophosphate, and phosphoenolpyruvate [34].
The presumed role of the nikQ-encoded cytochrome P450 monooxygenase,
the conversion of L-histidine to 4-formyl-4-imidazolin-2-one, has been deduced
from the following findings. The nikQ mutants fail to produce nikkomycins X
and I, which contain the imidazolone base, but synthesize nikkomycins Z and J,
which contain uracil as the base. Feeding of 4-formyl-4-imidazolin-2-one ((3) in
Fig. 5.6) isolated from culture filtrate of a nikK mutant restores the ability of
nikQ mutants to synthesize nikkomycins X and J.
The nikR gene product is thought to catalyze the transfer of 4-formyl-4-
imidazolin-2-one to 5-phosphoribosyl-1-pyrophosphate with the release of pyro-
phosphate, yielding 5'-phospho-ribofuranosyl-4-formyl-4-imidazolin-2-one. The
NikR protein has significant sequence similarity to uracil-phosphoribosyl trans-
ferases (UPRTases), which catalyze the conversion of uracil and 5-phosphoribo-
syl-1-pyrophosphate to uridine-monophosphate in the uracil salvage pathway.
This reaction is also catalyzed in vitro by recombinant NikR (Fig. 5.7, reaction
(3)). The nikR mutants still synthesize nikkomycins containing the imidazolone
base (X, I), but at levels of approximately 60% of wild type levels. Fluorouracil-
resistant nikR mutants that have no detectable UPRTase activity fail to produce
nikkomycins X and I, but synthesize wild type levels of the uracil-containing
nikkomycins Z and J. In addition, 4-formyl-4-imidazolin-2-one (Fig. 5.6, struc-
ture (3)) is accumulated in culture filtrate. This indicates that the primary meta-
bolism enzyme UPRTase can partly substitute for NikR in nikkomycin biosynth-
esis in nikR mutants.
The proposed role of the nikO gene product is the transfer of phosphoenol-
pyruvate to the oxygen atom of carbon-5' of the riboside structures, yielding in
further steps octofuranuloseuronic acids. NikO has significant sequence similar-
ity to UDP-N-acetylglucosamine enolpyruvyl transferases, which catalyze the
first committed step in murein biosynthesis for the bacterial cell wall, and to a
minor extent to 5-enol-pyruvylshikimate-3-phosphate synthases of the shiki-
mate pathway. These enzymes catalyze the transfer of phosphoenolpyruvate to
a substrate. The nikO mutants accumulate ribofuranosyl-4-formyl-4-imidazolin-
2-one ((4) in Fig. 5.6), compatible with the role proposed for NikO [35].
The gene products of nikL, nikM, and nikK are involved in the nucleoside
biosynthetic pathway after the nikO gene product, as all mutants inactivated in
one of these genes accumulate nikkomycins S
x
and S
z
, which derive from the
predicted octofuranuloseuronic acids (Fig. 5.2). While the amino acid sequence
of NikL and NikM gave no clue to their role, NikK has significant sequence si-
milarity to histidinol-phosphate aminotransferases. Based on this finding, NikK
is proposed to introduce the amino group to 5'-oxohexuronic acid compounds,
which are assumed to be the direct precursors for nikkomycins C
x
and C
z
.
Less information is available on the role and order of the nikI and nikJ
gene products in the nucleoside biosynthetic pathway. The nikI and nikJ mu-
117
tants do not accumulate nikkomycins S
x
and S
z
, but accumulate the unknown
compound RT 2.7, which might be a biosynthetic intermediate. Since the nikJ
mutants could not be complemented genetically, a polar effect of the inserted
kanamycin cassette on the downstream located genes cannot be excluded.
However, this seems to be unlikely since the nikJ phenotype differs from that of
the nikK and nikL mutants. The nikJ gene product displays limited sequence si-
milarity with methyltransferases involved in the biosynthesis of bialaphos and
fortimycin, and with magnesium-protoporphyrin monomethylester oxidative
cyclases [36]. The former enzymes use methylcobalamin as the methyl donor.
Methylcobalamin and protoporphyrin are structurally similar, and the conserved
amino acids might participate in binding or recognizing these cofactors. This im-
plies that NikJ might contain cobalamin as a cofactor and might be responsible
for the removal of carbon-7' and/or carbon-8' from the predicted octofuranu-
loseuronic acid intermediates.
5.5.3 Peptide bond formation
The genes nikS, nikT, nikP1, and nikP2 are proposed to catalyze the peptide
bond formation between the peptidyl moiety HPHT and the nucleoside struc-
tures nikkomycins C
x
and C
z
, yielding nikkomycin X and Z, respectively (Fig.
5.10) [31]. The NikS protein belongs to a superfamily of enzymes that is charac-
terized by a specific ATP-binding structure. Members of this superfamily reveal
strong structural similarity, but limited sequence similarity and catalyze carbox-
ylate-amine/thiol ligation reactions. NikS is proposed to transfer POHIV through
Figure 5.10: Predicted reactions for peptide bond formation. The nikT and nikP1 gene
products are represented as boxes, and their domains are indicated by bars. Substrates
are attached to the 4'-phosphopantetheine prosthetic group by a thioester bond.
R, 4-formyl-4-imidazolin-2-on, uracil, nikX, Z, nikkomycins X, Z.
118
the phosphate to the 4'-phosphopantetheinyl group attached to the acyl carrier
domain of NikT. Aminotransferase reaction and hydroxylation reaction (Fig. 5.8)
could involve substrates bound to NikT. nikT and nikS mutants accumulate nik-
komycins C
x
and C
z
and were not complemented to nikkomycin X/Z formation
by feeding HPHT to the production medium. In addition, the finding that HPHT
has never been found to be accumulated as a biosynthetic intermediate in any
of the S. tendae T 901 mutants, is compatible with the roles proposed for NikS
and NikT.
The nikP1 gene encodes a peptide synthetase containing all the core se-
quences necessary for ATP-binding and adenylate-formation, and also displays
the conserved binding site for the 4'-phosphopantetheinyl prosthetic group [27].
The nikP1 mutants accumulate nikkomycins C
x
and C
z
; HPHT is not detected in
culture filtrates. NikP1 is predicted to activate the nucleoside moieties, nikko-
mycin C
x
and C
z
, and catalyze peptide bond formation between them and
HPHT attached to the acyl carrier domain of NikT. The nikP2 gene product re-
sembles thioesterases required for the termination step in non-ribosomal pep-
tide synthesis, the cyclization reaction in the biosynthesis of cyclic peptides, or
the removal of the synthesized peptide from the peptide synthetase. The nikP2
mutants do not have a noteworthy phenotype; they synthesize the entire wild
type nikkomycin spectrum. This finding indicates that another cytoplasmic
thioesterase might substitute for NikP2 in nikkomycin synthesis.
The mechanism of the formation of the second peptide bond between car-
bon-6' and glutamic acid in the tripeptidyl nikkomycins J and I is still a mystery
since there are no more genes available within the nik cluster to encode for a
peptide synthetase.
5.5.4 Export
The protein deduced from the nikN gene displays limited sequence similarity to
some putative membrane transport proteins. The finding that NikN is predicted
to have six transmembrane helices and a binding site for ATP/GTP suggest that
the nikN gene might be involved in the export of di- and tripeptidyl nikkomy-
cins.
119
5.6 Transcriptional organization and regulation
of the nik cluster
The 29-kb nik cluster contains three polycistronic operons and the monocistroni-
cally transcribed regulatory gene orfR (Fig. 5.5) [19, 31, 34, 37]. Transcriptional
analyses have revealed that transcripts of the nikA-nikG genes, the nikI-nikO
genes, and the nikP2-nikV genes first become evident at the beginning of the
second rapid growth phase (Fig. 5.4), about five hours before nikkomycins be-
come detectable in the culture broth, while transcription of the orfR gene is in-
itiated approximately two hours earlier. Levels of all transcripts increase mark-
edly during the transition to the stationary phase, and high levels are main-
tained during the stationary phase. Transcription of the nikA-nikG, the nikI-
nikO, and the nikP1-nikV operons is activated by the orfR-encoded protein, as
insertion of the kanamycin cassette in the 5'-half of the gene abolishes transcrip-
tion of the three operons in these mutants. Heptameric repeat sequences de-
tected in the promoter regions of these operons provide potential binding sites
for OrfR.
OrfR has a molecular mass of 114,701 Da (1062 amino acids), deduced
from the nucleotide sequence and determined by SDS-PAGE of recombinant
protein expressed in E. coli [37]. OrfR has a potential ATP/GTP-binding site at
position 344 to 351, and its N-terminal half (amino acid residues 1 to 275) shows
a high level of sequence similarity to members of the family of Streptomyces
antibiotic regulatory proteins (SARPs), which are characterized by an OmpR-
like DNA-binding fold at their N-terminus [38]. This family includes ActII-Orf4
(225 amino acids) from S. coelicolor A3(2) and DnrI (272 amino acids) from
S. peucetius, pathway-specific regulatory proteins that activate transcription of
actinorhodin and daunorubicin biosynthesis genes, respectively. The AfsR pro-
tein (993 amino acids), a pleiotropic regulatory protein from S. coelicolor A3(2)
that effects several biosynthetic pathways, is the only member of the SARPs that
has a large size similar to that of OrfR. In addition, AfsR contains a P-loop and
has been shown to be phosphorylated by the afsK gene product, a serine/threo-
nine protein kinase.
The open question is which signal triggers expression of the orfR gene. In
S. tendae T 901/8c, the switch of metabolism to nikkomycin biosynthesis ap-
pears to take place during the short phase of reduced growth between the two
exponential growth phases.
120
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123
6 Glycosylated Antibiotics: Studies on Genes
Involved in Deoxysugar Formation, Modification
and Attachment, and their Use in Combinatorial
Biosynthesis
Andreas Bechthold*
6.1 Introduction
6.1.1 Combinatorial biosynthesis
Advances in recombinant DNA technology have enabled the cloning and expres-
sion of many biosynthetic gene clusters from different biological sources. Gene
clusters encoding proteins catalyzing the biosynthesis of different natural bioac-
tive products have been isolated and characterized from plants, fungi and bac-
teria. From the first isolation of antibiotic biosynthetic genes from a Streptomy-
cete [13] and the cloning and expression of entire biosynthetic pathways of anti-
biotic producing Streptomycetes [4, 5], a lot of progress has been achieved. Func-
tions have been assigned to many gene products in the biosynthesis of different
bioactive compounds. More recently, researchers in this area including molecular
biologists and bioorganic chemists have started to use these different genes to al-
ter the structure of natural compounds by genetic engineering or to combine
genes from different biosynthetic pathways. This new technology named combi-
natorial biosynthesis [6, 7] resulted in the formation of novel natural products.
6.1.2 Glycosylated antibiotics
Deoxysugars are an important class of carbohydrates occurring in plants, fungi and
microorganisms. In microorganisms deoxygenated sugars can be found as elements
of lipopolysaccharides (LPS), extracellular polysaccharides (EPS) and antibiotics.
* Pharmazeutische Biologie, Universitt Freiburg, Stefan-Meier-Str. 9, D-79104 Freiburg
124
ISBN: 3-527-30615-3
Traditionally, the sugar moieties of antibiotics have been viewed as mole-
cular elements that control the pharmacokinetics of a drug. This view is begin-
ning to change. Recent results clearly show that deoxygenated sugars actively
contribute as recognition elements to the mechanism of action of the respective
drug [8, 9].
In 1994 the author of this manuscript initiated investigations on the genetic
analysis of avilamycin, landomycin and urdamycin biosynthesis and continued a
cooperation on granaticin biosynthesis. Our research was motivated by the be-
lieve that genes, especially glycosyltransferase genes, are very important tools
for combinatorial biosynthesis.
6.1.2.1 Avilamycin
The avilamycins which are produced by Streptomyces viridochromogenes T 57
(S. viridochromogenes T 57) are oligosaccharide antibiotics and belong to the
orthosomycin group of antibiotics [10, 11]. Avilamycins as well as other impor-
tant members of the orthosomycins contain a dichloroisoeverninic acid moiety,
as well as one or more orthoester linkages which are associated with carbohy-
drate residues. Components of the saccharide side chain are D-olivose, 2-deoxy-
D-evalose, 4-O-methyl-D-fucose, 2,6-Di-O-methyl-D-mannose, L-lyxose and
eurekanic acid. Main product of S. viridochromogenes T 57 is avilamycin A
(Fig. 6.1). The compound SCH27899, another orthosomycin, shows excellent ac-
tivity against Gram-positive bacteria and is presently being tested for its possi-
ble use against human infectious diseases [12, 13].
6.1.2.2 Landomycin
S. cyanogenus S136 is the producer of landomycins, consisting of a benz[a]an-
thraquinone-type aglycone moiety and a varying phenol-glycosidically linked
oligosaccharide chain [14]. Landomycin A, the principal metabolite of S. cyano-
genus, is the largest angucycline so far described, and the most active landomy-
cin [15]. Its deoxysugar moiety is an unusual hexasaccharide consisting of four
D-olivose and two L-rhodinose residues (Fig. 6.1). Besides landomycin A S. cya-
nogenus S136 produces the related congeners landomycin B and landomycin D.
They differ from landomycin A in the length of the sugar side chain. In various
tests landomycin A showed interesting antitumor activities, in particular against
prostate cancer cell lines. This antitumor activity was discussed to result from in-
teractions with DNA, for which the hexasaccharide chain plays an important
role.
125
6.1 Introduction
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126
6.1.2.3 Urdamycin
Urdamycins are produced by S. fradiae. Like landomycins they are polyketides
belonging to the angucyline type of antibiotics [16]. In difference to landomy-
cins urdamycins mostly differ in the structure of the polyketide moiety. Main
product of S. fradiae is urdamycin A (Fig. 6.1), minor compounds are urdamycin
B, C, D, E, F, G and H. Urdamycin A consists of aquayamycin which contains a
C-glycosidically linked D-olivose and three additional O-glycosidically linked
deoxysugars: two L-rhodinoses, and another D-olivose. Urdamycin A shows only
some anticancer and antimicrobial activity.
6.1.2.4 Granaticin
Granaticin made by S. violaceoruber T 22 is a member of a class of polyketide
antibiotics known as benzoisochromanequinones with activity against Gram-po-
sitive bacteria and P-388 mouse leukemia [17]. Granaticin contains a D-olivose
moiety attached to the polyketide moiety via two carbon-carbon bonds at C-9
and C-10 (Fig. 6.1). A second sugar, L-rhodinose, is attached to D-olivose by a
conventional glycosidic bond.
6.2 Cloning of the avilamycin, landomycin, urdamycin,
and granaticin biosynthetic gene clusters
6.2.1 Development of a PCR method to clone dNDP-hexose
4,6-dehydratase genes
In the past the cloning of biosynthetic gene clusters started with the shotgun
cloning of random fragments of DNA from a Streptomyces strain, producing a
given antibiotic. Mutants of the strain blocked at a step in the biosynthesis of
the antibiotic were transformed with these fragments. Fragments restoring anti-
biotic production were used for further investigations. In another approach re-
sistance genes of antibiotic producers were cloned in a heterologous host. This
led to the identification of linked biosynthetic genes. More recently genes with
known functions were used as probes in Southern hybridization to identify
homologous genes in other organisms. The most popular gene probes were actI
and actIII from S. coelicolor and strD from S. griseus [18, 19].
In our projects a different approach to clone the biosynthetic gene clusters
of avilamycin, landomycin, urdamycin and granaticin was performed. The com-
127
6.2 Cloning of the avilamycin, landomycin, urdamycin
parison of the sequence of known dTDP-glucose 4,6-dehydratases revealed sev-
eral regions of high similarity. Based on these sequences two oligonucleotide pri-
mers were synthesized taking into account the codon usage of Actinomycetes.
These primers were used to amplify DNA fragments from our Actinomycetes.
PCR fragments obtained were subcloned and sequenced. The deduced amino
acid sequences of the isolated fragments revealed similarity to each other and to
the dNDP-glucose dehydratase (StrE) isolated fromS. griseus N-2-3-11 [20].
6.2.2 Preparation and screening of cosmid libraries
For the construction of gene libraries from our producer strains, DNA fragments of
2540 kb were ligated into pOJ446. DNA was packed into phages by using the
Gigapack Packaging Extract Gold
TM
fromStratagene (Heidelberg, Germany). The
phages were used to transduce E. coli XL1-Blue-MRF'. For screening of the cosmid
libraries, DNAfragments obtained by PCR amplification were used as probes. Sev-
eral cosmid clones hybridizing to the probes were isolatedin each case [21].
6.2.3 Identification of the biosynthetic gene clusters by gene
inactivation and expression experiments
Gene inactivation has been used to show that we in fact had cloned the urda-
mycin- and avilamycin biosynthetic gene cluster [22, 23], and expression of
genes in heterologous strains was used to show that cosmid clones isolated from
S. violaceoruber T 22 and S. cyanogenus S136 contained the entire granaticin-
and landomycin biosynthetic gene cluster, respectively [24, 25].
6.3 Organization of avilamycin, landomycin, urdamycin,
and granaticin biosynthetic genes
6.3.1 Genes involved in avilamycin biosynthesis
More than 42 kb of the avilamycin biosynthetic gene cluster have been se-
quenced (Fig. 6.2, Table 6.1a) [26]. So far eight deoxysugar biosynthetic genes
(aviQ1 aviQ2, aviQ3, aviD, aviE1, aviE2, aviE3, and aviJ), four glycosyltransfer-
128
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.
ase genes (aviGT1, aviGT2, aviGT3, and aviGT4) and four putative sugar
methyltransferase genes (aviG1, aviG2, aviG3, and aviG5) were detected. AviD
and AviE1 are probably involved in the biosynthesis of the important key inter-
mediate NDP-4-keto-6-deoxy-D-glucose, which can be converted to the 2,6-di-
deoxysugar components of avilamycin A, namely ring B (D-olivose), C (D-oli-
vose) and D (D-evalose). AviQ1, AviQ2 and AviQ3 are similar to epimerases
(AviQ1, after expression in E. coli, showed UDP-glucose 4-epimerase activity),
AviE2 and AviE3 to NDP-D-glucose 4,6-dehydratases and AviJ to oxidoreduc-
tases. AviM is an orsellinic acid synthase and AviH seems to be involved in ha-
logenation. AviG4 might code for a methyltransferase involved in the biosynth-
esis of the dichloroisoeverninic acid moiety. AviO1 and AviO2 resemble oxyge-
nases and might catalyze the formation of the orthoesters. Further genes, aviB1,
aviB2, aviRa, aviRb, aviGTP1, aviReg1, aviReg2, aviT, aviN, aviABC1, and
aviABC2 are either involved in resistance, transport or regulation. The function
of many other genes remains unknown [26, 27].
6.3.2 Genes involved in landomycin A biosynthesis
The landomycin gene cluster from S. cyanogenus S136 contained several genes
involved in deoxysugar- and polyketide biosynthesis (Fig. 6.2, Table 6.1b). The
formation of dTDP-D-glucose from glucose-1-phosphate is catalyzed by LanG
which was shown after expression of lanG in E. coli. dTDP-D-glucose might
then be converted to TDP-4-keto-6-deoxy-D-glucose by LanH. Deoxygenation
at C-2 is probably catalyzed by LanS leading to a hypothetical intermediate
which can be reduced by LanT. TDP-4-keto-2,6-dideoxy-D-glucose might be a
Table 6.1a: Putative functions of genes of the avilamycin biosynthetic gene cluster.
Gene Deduced function
aviM orsellinic acid synthase (PKS)
aviH halogenase (tailoring)
aviO1, aviO2 oxygenase (tailoring)
aviG1, aviG2, aviG3, aviG4, aviG5 methyltransferase (tailoring)
aviB1, aviB2 acetoin-dehydrogenase (tailoring)
aviD dNDP-hexose synthase (sugar)
aviE1, aviE2, aviE3 dNDP-hexose-4,6-dehydratase (sugar)
aviQ1, aviQ2, aviQ3 dNDP-hexose-4-epimerase (sugar)
aviJ1 oxidoreductase (sugar)
aviGT1, aviGT2, aviGT3, aviGT4 glycosyltransferase
aviRb, aviRa resistance protein
aviGTP, aviReg1, aviReg2 regulatory protein
aviT, aviN, aviABC1+aviABC2 transporter
? unknown function
130
central intermediate in the biosynthesis of both TDP-L-rhodinose and TDP-D-
olivose. A reduction step at C-4 (LanR) is required to complete the biosynthesis
of TDP-D-olivose. 3,5-Epimerization (LanZ1), dehydroxylation at C-3 (LanQ) and
reduction at C-4 (LanZ3) would lead to the formation of TDP-L-rhodinose. So far
four genes (lanGT1lanGT4) coding for putative glycosyltransferases have been
detected in the biosynthetic gene cluster. Genes involved in the biosynthesis of
the polyketide moiety are lanA, lanB, lanC, lanD, lanF, lanL and lanP. Further
genes are either involved in tailoring reactions (lanE, lanM, lanZ5, lanN, lanO,
lanV, lanZ4) or can be viewed as regulatory (lanK) or transporter (lanJ) genes
(Fig. 6.2) [25, 28].
Table 6.1b: Putative functions of genes of the landomycin, urdamycin and granaticin bio-
synthetic gene cluster.
Gene Deduced function
lanA, urdA, graORF-1 b-ketoacyl-ACP synthase (PKS)
lanB, urdB, graORF-2 chain length factor (PKS)
lanC, urdC, graORF-3 acyl carrier protein (PKS)
lan F, lanL, urdF, urdL, cyclase (PKS)
graORF-18, graORF-4, graORF-33
lanD, lanN, lanO, lanV, lanZ4, reductase (PKS)
urdD, urdN, urdO,
graORF-5, graORF-6, graORF-34
lanE, lanM, lanZ5, urdE, urdM, oxygenase (tailoring)
graORF-21, graORF-29
lanP decarboxylase (PKS)
lanG, lanZ2, urdG, graORF-16 dNDP-hexose-synthetase (sugar)
lanH, urdH, graORF-17 dNDP-hexose-4,6-dehydratase (sugar)
lanQ, urdQ, graORF-23 dNDP-4-keto-6-deoxy-hexose-3-dehydratase
(sugar)
lanR, lanZ3, urdR, graORF-22 dNDP-hexose-4-ketoreductase (sugar)
lanS, urdS, graORF-27 dNDP-4-keto-6-deoxy-hexose-2,3-dehydratase
(sugar)
lanT, urdT, graORF-26 oxidoreductase (involved in C-2 deoxygenation)
(sugar)
lanZ1, urdZ1, graORF-25 dNDP-hexose-3,5-epimerase (sugar)
lanGT1, lanGT2, lanGT3, lanGT4, glycosyltransferase
urdGT1a, urdGT1b, urdGT1c, urdGT2,
graORF-14
lanK, urdK, graORF-7, graORF-9, regulator
graORF-10, graORF-11, graORF-19,
graORF-20, graORF-37
lanJ, urdJ, urdJ2, graORF-15 transporter
lanU, lanZ6, lanX, graORF-8, graORF-12, ? (unknown)
graORF35, graORF-36
131
6.3 Organization of avilamycin, landomycin, urdamycin
6.3.3 Genes involved in urdamycin A biosynthesis
So far eight genes (urdZ1, urdG, urdH, urdZ3, urdQ, urdR, urdS, urdT) probably
involved in deoxysugar formation have been identified in the urdamycin biosyn-
thetic gene cluster (Fig. 6.2, Table 6.1b). The deduced amino acid sequences of
these genes are very similar to the deduced amino acid sequences of genes
from the landomycin cluster indicating similar functions. Four putative glycosyl-
transferase genes (urdGT1a, urdGT1b, urdGT1c, and urdGT2), which have
been detected in the cluster, are involved in the formation and attachment of
the trisaccharide chain as well as the attachment of L-rhodinose at C12b. Genes
involved in the biosynthesis of the polyketide moiety or in tailoring reactions
have also been detected in the urdamycin cluster. Products of these genes
(urdA, urdB, urdC, urdD, urdF, urdL, urdE, urdM, urdO) are again very similar
to the deduced amino acid sequences of genes from the landomycin cluster, and
UrdJ and UrdJ2 resemble antibiotic transporters [26, 27, 2931].
6.3.4 Genes involved in granaticin biosynthesis
As in the landomycin and urdamycin cluster several genes involved in the for-
mation of the polyketide and sugar moieties have been sequenced (Fig. 6.2,
Table 6.1b). The deduced amino acid sequences are very similar to proteins
from the landomycin and urdamycin cluster [17, 24].
6.4 New genetically engineered natural compounds
Gene inactivation experiments by introducing in-frame deletions or frame-shift
mutations into the genes and combinatorial biosynthetic approaches were car-
ried out. New molecules were created and functions of genes could be deter-
mined.
132
6.4.1 Avilamycin
6.4.1.1 New avilamycin derivatives through gene inactivation experiments
To improve the water solubility of avilamycin A we started a project to inactivate
methyltransferase genes of the avilamycin cluster. So far aviG1 and aviG4 have
been deleted. Extracts of mutants were analyzed by HPLC-UV and by HPLC-
MS and were also analyzed for antibiotic activity (bioassay). Experimentally ob-
tained data indicate that aviG1 is encoding a C-methyltransferase and that inac-
tivation of aviG1 is leading to a less antibiotically active derivative. Inactivation
of aviG4, which is probably involved in methylation of ring A of avilamycin A
was leading to the formation of a more hydrophilic compound which showed
high antimicrobial activity.
6.4.1.2 Production of orsellinic acid by expression of aviM
The expression of one gene of the avilamycin biosynthetic gene cluster (aviM)
in S. lividans was leading to the production of orsellinic acid (Fig. 6.3), a struc-
tural component of avilamycin A [22].
6.4.2 Landomycin and urdamycin
6.4.2.1 Accumulation of intermediates of the urdamycin biosynthesis
after expression of a cosmid containing genes of the landomycin cluster
Cosmid H2-26, containing genes of the landomycin cluster was transformed into
the urdamycin producer S. fradiae. Transformants containing the cosmid were
accumulating aquayamycin and compound 100-2. The structures of both com-
pounds indicated that both compounds were not produced through combinator-
ial biosynthesis. Both compounds are likely to be intermediates of urdamycin A
biosynthesis and a competition between glycosyltransferases of the urdamycin-
and landomycin cluster might be responsible for the accumulation of both com-
pounds (Fig. 6.3) [25].
6.4.2.2 New compounds by inactivation of biosynthetic genes
Several genes of the urdamycin cluster have been deleted by gene inactivationex-
periments. Mutants were accumulating new compounds instead of urdamycin A.
(Table 6.2, Fig. 6.3) [2931]. From these experiments the function to several genes
can be assigned.
133
6.4 New genetically engineered natural compounds
UrdGT2 must catalyze the earliest glycosyltransfer step in the urdamycin
biosynthetic pathway which is the C-glycosyltransfer of an activated D-olivose.
UrdGT1a is involved in the glycosylation of aquayamycin during urdamycin A
biosynthesis and UrdM is an oxygenase involved in oxygenation at position 12b
of urdamycin A.
Table 6.2: Production of new antibiotics by gene inactivation- and gene expression ex-
periments.
Deleted gene Gene expressed Products
in the corresponding
mutant
urdGT2 urdamycin I, urdamycin J, urdamycin K
urdGT2 urdGT2 urdamycin A
urdGT1a 12b-desrhodinosyl-urdamycin C,
12b-desrhodinosyl-urdamycin D,
12b-desrhodinosyl-urdamycin F,
urdamycinon D, urdamycin B
urdGT1a urdGT1a urdamycin A
urdGT1b, urdInt
and urdGT1c 100-2
urdGT1b, urdInt
and urdGT1c urdGT1c urdamycin G, urdamycin A
urdGT1a, urdGT1b,
urdInt and urdGT1c aquayamycin
urdGT1a, urdGT1b,
urdInt and urdGT1c urdGT1c 12b-desrhodinosyl-urdamycin G
urdGT1a, urdGT1b,
urdInt and urdGT1c urdGT1b and urdGT1c 12b-desrhodinosyl-urdamycin G,
12b-desrhodinosyl-urdamycin A
urdGT1a, urdGT1b,
urdInt and urdGT1c lanGT3 12b-desrhodinosyl-urdamycin G
urdGT1a, urdGT1b,
urdInt, urdGT1c
and urdGT2 urdamycin I, urdamycin J
urdM rabelomycin
urdR aquayamycin
urdZ3 urdamycin I, urdamycin J
urdS (no product)
urdZ1 aquayamycin
urdQ aquayamycin
134
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i
g
u
r
e
6
.
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:
S
t
r
u
c
t
u
r
e
o
f
c
o
m
p
o
u
n
d
s
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e
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i
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s
t
u
d
y
.
135
6.4.2.3 New compounds by expression of single genes
All glycosyltransferase genes of the urdamycin and landomycin biosynthetic
gene cluster have been expressed in glycosyltransferase mutants of S. fradiae.
The expression of urdGT1c and lanGT4 (independent experiments) in a mutant,
in which urdGT1a, urdGT1b, and urdGT1c had been deleted and which was
accumulating compound 100-2, resulted in the production of 12b-desrhodino-
syl-urdamycin G. After coexpression of urdGT1c and urdGT1b the accumula-
tion of 12b-desrhodinosyl-urdamycin A was detected (Table 6.2, Fig. 6.3). Thus
UrdGT1c is catalyzing the transfer of an activated L-rhodinose to 1002 during
urdamycin biosynthesis and UrdGT1b is attaching the final sugar [31]. LanGT4
seems to catalyze the attachment of L-rhodinose and might act twice during
landomycin biosynthesis.
References
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11. Mertz, J. L., Peloso, J. S., Barker, B. J., Babbitt, G. E., Occolowitz, J. L., Simson, V. L.,
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(1996) Comparative in-vitro activity of SCH 27899, a novel everninomycin, and vanco-
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Streptomyces violaceoruber T22 genes involved in the biosynthesis of granaticin.
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407.
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Chem. Rev. 97, 24652497.
20. Decker, H., Gaisser, S., Pelzer, S., Schneider, P., Westrich, L., Wohlleben, W., and
Bechthold, A. (1996) A general approach for cloning and characterization of dNDP-
glucose dehydratase genes from actinomycetes. FEMS Microbiol. Lett. 141, 195201.
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22. Gaisser, S., Trefzer, A., Stockert, S., Kirschning, A., and Bechthold, A. (1997) Cloning
of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes
T57. J. Bacteriol. 179, 62716278.
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the angucycline antibiotic urdamycin A and a gene probably involved in its oxygena-
tion. J. Bacteriol. 177, 61266136.
24. Ichinose, K., Bedford, D. J., Tornus, D., Bechthold, A., Bibb, M. J., Revill, W. P., Floss,
H. G., and Hopwood, D. A. (1998) The granaticin biosynthetic gene cluster of Strepto-
myces violaceoruber T22: sequence analysis and expression in a heterologous host.
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25. Westrich, L., Domann, S., Faust, B., Bedford, D., Hopwood, D. A., and Bechthold, A.
(1999) Cloning and characterization of the landomycin biosynthetic gene cluster of
Streptomyces cyanogenus S136. FEMS Microbiol. Lett. 170, 381387.
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nauer, G., and Westrich, L. (1999) Glycosidierte Naturstoffe, Perspektiven fr die
Kombinatorische Biosynthese. Chemotherapie Journal 4, 130135.
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natorial biosynthesis. In: New aspects in Bioorganic Chemistry (Diederichsen, U.,
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Rohr, J. (1999) The urdGt2 gene encodes a glycosyltransferase responsible for the C-
glycosyltransfer of activated D-olivose, the earliest glycosyl transfer step in the urda-
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H., Knzel, E., Rohr, J., and Bechthold, A. (1999) Two new tailoring enzymes, a glyco-
syltransferase and an oxygenase, involved in biosynthesis of the angucycline antibio-
tic urdamycin A in Streptomyces fradiae. Microbiology, accepted for publication.
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genes involved in the biosynthesis of urdamycin A. Chemistry & Biology 7, 13142.
138
7 Analysis of the Biosynthesis of Glycopeptide
Antibiotics: Basis for Creating New Structures
by Combinatorial Biosynthesis
Stefan Pelzer and Wolfgang Wohlleben*
7.1 Introduction
Together with gentamicin the glycopeptide antibiotic vancomycin (Fig. 7.1) and
the structurally related lipoglycopeptide antibiotic teicoplanin are considered to
be the last lines of defence against a variety of serious infections caused by
Gram-positive organisms, such as enterococci, methicillin-resistant Staphylococ-
cus aureus (MRSA), and Clostridium difficile [1]. Their annual turnover amounts
to about one billion US$ [2], which demonstrates their high social and economic
value.
Glycopeptide antibiotics inhibit the synthesis of the cell wall of Gram-posi-
tive organisms. Their primary molecular target is the D-alanyl-D-alanine (D-Ala-
D-Ala) terminus of growing peptidoglycan. Dimers of vancomycin bind specifi-
cally to D-Ala-D-Ala inhibiting simultaneously the two key-steps of the peptido-
glycan biosynthesis, the transglycosylase and the transpeptidase reaction [2].
In the last ten years numerous vancomycin resistant enterococci (VRE)
emerged in clinical isolates [3] and spread rapidly. These organisms avoid their
cell deaths by modifying the drugs target, specifically by modifying it to the
depsipeptide D-alanyl-D-lactat (D-Ala-D-Lac). The affinity of vancomycin to D-
Ala-D-Lac is reduced significantly (factor 1000).
The fear that this high-level resistance may eventually spread to methicil-
lin-resistant Staphylococcus aureus (MRSA) has prompted the search for new or
modified glycopeptide antibiotics.
One way to generate new glycopeptide antibiotics is the manipulation of
biosynthetic genes by combinatorial biosynthesis. In order to employ this ap-
proach successfully, corresponding gene clusters have to be identified and ana-
lysed. Until now only putative genes of the vancomycin producer A. orientalis
Tbingen
139
ISBN: 3-527-30615-3
Figure 7.1: Chemical structures of the glycopeptide antibiotics balhimycin, vancomycin
and chloroeremomycin [17].
140
7 Analysis of the Biosynthesis of Glycopeptide Antibiotics
C329.4 [4] and the chloroeremomycin producer A. orientalis A82846 were de-
scribed [5]. However, the role of the sequenced genes was not elucidated.
A glycopeptide with similar properties as vancomycin is balhimycin, which
is produced by Amycolatopsis mediterranei DSM5908 [6]. Balhimycin shares the
heptapeptide core of vancomycin and differs only in the glycosylation pattern
(Fig. 7.1). In the following we present the establishment of a transformation pro-
tocol and the construction of vectors for manipulation of the balhimycin produ-
cer, the isolation of the balhimycin gene cluster, and the functional analyses of
different biosynthetic genes.
7.2 Results
7.2.1 Establishment of a gene cloning system for the balhimycin
producer Amycolatopsis mediterranei DSM5908
Although transformation techniques were described for the vancomycin produ-
cer A. orientalis ATCC 19795 [7] and the producer of the teicoplanin-like anti-
biotic A47934, S. toyocaensis [8], the application for generating glycopeptide
antibiotic mutants was never reported.
For the establishment of a gene cloning system for the balhimycin produ-
cer A. mediterranei DSM5908 a transformation method was developed first. In
order to monitor successful transformation events, DNA of the Amycolatopsis
phage W2 [9], which was able to infect the balhimycin producer was employed.
Using W2-DNA in transfection experiments, the three different methods de-
scribed for the transformation of Amycolatopsis strains were tested for their ap-
plicability in A. mediterranei DSM5908. Neither various modifications of the
PEG induced protoplast transformation method, which was reported as an effi-
cient method for A. orientalis [8], nor electroporation methods described for
transformation of A. mediterranei and A. orientalis [10] enabled introduction of
W2-phage DNA into A. mediterranei DSM5908.
Only after modifying the direct transformation method [11], which was pre-
viously described for the rifamycin producer A. mediterranei [12] and for
A. methanolica [13], transformants of the balhimycin producer were obtained.
Using the optimised protocol it was not possible to establish any of the de-
scribed Amycolatopsis vectors or broad host range Streptomyces vectors [11].
Therefore, novel non-replicative vectors for integration mutagenesis were devel-
oped. Since the balhimycin producer showed a naturally high resistance against
the customary actinomycetes resistance markers thiostrepton and kanamycin,
the erythromycin-resistance gene ermE [14] and the chloramphenicol-resistance
gene cat [15] were used to construct the vectors pSP1 and pSP2 [11].
141
7.2 Results
The application of the vectors in gene disruption, gene replacement and
in-frame deletion experiments revealed, that the integration frequencies were
influenced by several parameters [11]: Highest integration frequencies were ob-
served when the DNA was isolated from the dam/dcm E. coli strain JM110 and
PEG 1000 from NBS Biologicals was employed. The efficiency of integration de-
pended directly on the size of the cloned insert. Plasmids with fragments smaller
than 1 kb were difficult to integrate. In gene replacement experiments a high
double crossover rate (31%) was demonstrated.
7.2.2 Identification of biosynthetic genes by reverse genetic approaches
The application of standard strategies for isolation of antibiotic biosynthetic
genes like cloning of resistance genes or complementation of non-producing
mutants was not possible. Therefore, different reverse genetic approaches were
chosen considering characteristic features of glycopeptide antibiotics like the
heptapeptide backbone or the sugar residues.
7.2.2.1 Isolation of different peptide synthetase genes using conserved
oligonucleotide probes
Since the central core heptapeptide of balhimycin includes five non-proteino-
genic amino acids, a non-ribosomal synthesis by peptide synthetases is most
likely.
Peptide synthetases of bacterial and fungal origins are highly conserved
and show a modular organisation. Each module activates the cognate amino
acid and incorporates it in the peptide product by a stepwise elongation [16].
Comparison of the amino acid sequences of peptide synthetase modules from
various microorganisms showed five strongly conserved regions [11]. These con-
served motives were used to design oligonucleotide probes, which were adapted
to the Streptomyces codon usage.
Using these gene probes in plaque hybridisation experiments we identi-
fied two different clusters of peptide synthetase genes in a l library of A. medi-
terranei. Gene disruption experiments revealed, that both different peptide
synthetase gene clusters are not involved in balhimycin production [8]. Today
we know that genes encoding peptide synthetases are common in Actinomy-
cetes and that the balhimycin producer carries at least four different peptide
synthetase gene clusters, two of them with unknown function.
142
7.2.2.2 Identification of a glycosyltransferase gene fragment involved
in balhimycin biosynthesis
Another reverse genetic approach considered, that balhimycin includes two su-
gar groups, a glucose and the rare aminosugar dehydrovancosamine (Fig. 7.1).
Such sugars are normally attached to the aglycon by the action of glycosyltrans-
ferases.
To design PCR primers for the amplification of balhimycin-specific glyco-
syltransferase gene fragments, sequences of glycosyltransferases probably in-
volved in the biosynthesis of vancomycin and chloroeremomycin [4] were com-
pared and two conserved regions were chosen.
Using conserved oligonucleotides we were able to amplify a 900 bp DNA
fragment (bgtfB) from genomic DNA of the balhimycin producer in an opti-
mised PCR-protocol [17]. DNA sequencing and comparison of the deduced
gene product showed significant similarities to various glycosyltransferases.
To prove that the glycosyltransferase gene is involved in balhimycin bio-
synthesis, a gene disruption experiment was performed. An internal fragment of
the bgtfB gene was cloned into the gene disruption vector pSP1. This vector
was used to inactivate the chromosomal bgtfB gene by homologous recombina-
tion (Fig. 7.2). In bioassays, the mutant (HD1) was able to synthesise a sub-
stance which was still antibiotically active. HPLC-MS and MS-MS analyses
showed that HD1 produced at least four different compounds, which were not
glycosylated anymore [17, 18].
Thus the first glycopeptide biosynthetic mutant was created by a targeted
approach and the role of bgtfB in balhimycin biosynthesis was proved.
7.2.3 Analysis of the balhimycin biosynthetic gene cluster
Since antibiotic biosynthetic genes including resistance and regulatory genes
are normally physically linked in the genome of Streptomycetes [19] isolation of
the whole balhimycin gene cluster was intended by using a gene fragment of
the glycosyltransferase bgtfB as probe.
7.2.3.1 Isolation and sequencing of the major part of the balhimycin
biosynthetic gene cluster
Hybridisation experiments with the glycosyltransferase PCR fragment (bgtfB)
led to the identification of twelve cosmids in an A. mediterranei cosmid library.
By DNA sequence, one cosmid (16.1) was characterised in detail (manuscript in
preparation). An ORF analysis revealed, that a region of 46 kb carries 26 com-
plete and 1 incomplete putative ORFs (Fig. 7.3). Computer-assisted homology
searches with the sequences of the deduced gene products showed significant
143
7.2 Results
similarities to many gene products, which may participate in balhimycin bio-
synthesis like peptide synthetases, glycosyltransferases, cytochrome P450
mono-oxygenases, sugar biosynthetic enzymes, and an enzyme similar to chal-
cone/stilbene synthases of plants.
The organisation of the major part of the balhimycin gene cluster is very si-
milar to the organisation described for the putative chloroeremomycin gene
cluster [5]. Differences between the two clusters are on one hand the presence
Figure 7.2: Inactivation of the glycosyltransferase gene bgtfB by gene disruption result-
ing in mutant strain HD1. HPLC and MS-MS analyses showed that HD1 produced four
different balhimycin derivatives which are all not glycosylated [17]. The chemical struc-
ture of one derivative (HD-1142) is shown in detail.
144
of an additional ORF (ORF 7, Fig. 7.3) in the balhimycin cluster showing simila-
rities to Na/H antiporter. On the other hand, ORF 9 in the balhimycin cluster
(Fig. 7.3), showing significant similarities to NDP-hexose-4-ketoreductases car-
ries an in-frame deletion of 226 amino acids in comparison with the correspond-
ing intact ORF of the chloroeremomycin cluster (manuscript in preparation).
This corresponds to the occurrence of the keto-sugar residue dehydrovancosa-
mine in balhimycin and the reduced 4-epi-vancosamine in chloroeremomycin,
respectively (Fig. 7.1).
Figure 7.3: Organisation of the major part of the balhimycin biosynthetic gene cluster
and the proposed function of the encoding ORFs.
145
7.2 Results
7.2.3.2 Characterisation of the function of different balhimycin biosynthetic
genes by gene inactivation experiments
Cytochrome P450 mono-oxygenases are involved in the coupling of the aromatic
side chains of the balhimycin heptapeptide
In the major part of the balhimycin cluster four ORFs (OxyAD, Fig. 7.3) with
remarkable similarities to cytochrome P450 mono-oxygenase were identified.
Integration of a plasmid between oxyA and oxyB resulted in the balhimycin
mutant SP11, which was not able to synthesise an antibiotically active com-
pound [17]. Structural analyses by HPLC-MS, fragmentation studies, and
amino acid analyses demonstrated, that SP11 produced two balhimycin pre-
cursors (SP969 and SP1134; Fig. 7.4), which were linear (unbridged) and not
glycosylated [18, 20].
These results showed clearly that oxygenases are involved in the coupling
of the aromatic side chains, which are essential to hold the peptide backbone in
a rigid, cup-shaped form. This structure is important for the antibiotic activity,
because only this form interacts with the D-Ala-D-Ala terminus of the cell wall
precursor [2].
The halogenase BhaA is responsible for chlorination of balhimycin
The product of the bhaA gene (Fig. 7.3) showed significant similarities (42%) to
PrnC, a new type of non-heme halogenase of Pseudomonas fluorescens which is
involved in chlorination of pyrrolnitrin [21]. Since balhimycin carries two chlor-
ine atoms at the aromatic side chains of amino acid two and six, the participa-
tion of BhaA in chlorination of balhimycin is plausible.
To prove this, a bhaA in-frame deletion-mutant (PH4) was constructed
using a two-step strategy [22]. HPLC-MS analyses and the analysis of the isoto-
pic pattern clearly showed, that the mutant PH4 was not able to synthesise
chlorinated balhimycin-derivatives (Fig. 7.5). Thus bhaA is responsible for the
incorporation of both chlorine atoms into the glycopeptide antibiotic. Bioassays
revealed that dechlorobalhimycin is still antibiotically active (manuscript in pre-
paration).
The non-ribosomal biosynthesis of the balhimycin backbone: Eight peptide
synthetase modules are involved in the biosynthesis of a heptapeptide
For the non-ribosomal biosynthesis of a heptapeptide seven modules were ex-
pected. Until now, three peptide synthetase genes (bpsBD) encoding five pep-
tide synthetase modules were characterised in the balhimycin biosynthetic gene
cluster (manuscript in preparation, Fig. 7.3). From comparison with the putative
chloroeremomycin biosynthetic gene cluster [5], we conclude that a fourth pep-
tide synthetase gene (bpsA) encoding three further modules is located in front
of the peptide synthetase bpsB. Surprisingly, altogether eight peptide synthe-
tase modules are expected to be located within the balhimycin biosynthetic
gene cluster.
146
The function of three peptide synthetase genes (bpsBD) was investigated
by gene inactivation experiments and by analysing the enzymatic activities of
overexpressed peptide synthetase modules (manuscript in preparation). All mu-
tants were unable to synthesise an antibiotically active compound. The success-
ful complementation of the bpsD replacement-mutant, encoding the eighth
module, with a copy of the native bpsD gene showed that polar effects of the
gene replacement could be excluded.
Figure 7.4: Construction of mutant strain SP11 by integration mutagenesis between the
cytochrome P450 mono-oxygenase genes oxyA and oxyB. HPLC-MS and MS-MS revealed
that the mutant produced a linear balhimycin precursor (SP-1134) which is antibiotically
inactive showing that oxygenases are involved in coupling of the aromatic rings [17, 18].
147
7.2 Results
The results of the mutational analyses were confirmed by biochemical ex-
periments showing that the modules 4, 5 and 7 activated the expected amino
acid (manuscript in preparation). Moreover, these experiments demonstrated
clearly that also the eighth module is involved in the biosynthesis of the hepta-
peptide core.
Figure 7.5: Construction of the mutant PH4 which carries an in-frame deletion in the ha-
logenase gene bhaA. HPLC-MS analysis and determination of the isotopic pattern re-
vealed that the mutant produces the antibiotically active compound dechloro-balhimycin
indicating that bhaA is responsible for the chlorinating steps [21].
148
7.3 Outlook
The balhimycin biosynthesis includes further interesting biosynthetic steps. First
experiments showed that the css gene, encoding a gene product similar to plant
chalcone/stilbene synthases, is presumably involved in the biosynthesis of the
seventh amino acid 3,5-dihydroxyphenyl-glycine.
Furthermore, the balhimycin producer and the identified genes are suita-
ble tools for the generation of hybrid antibiotics by rational combinatorial bio-
synthesis.
References
1. Cunha, B. A. (1995) Vancomycin. Med. Clin. N. Am. 79, 817831.
2. Williams, D. H. and Bardsley, B. (1999) Die Vancomycin-Antibiotika und der Kampf
gegen resistente Bakterien. Angew. Chem. 111, 12641286.
3. Arthur, M., Depardieu, F., Reynolds, P., and Courvalin, P. (1996) Quantitative analysis
of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-
resistant enterococci. Mol. Microbiol. 21, 3344.
4. Solenberg, P. J., Matsushima, P., Stack, D. R., Wilkie, S. C., Thompson, R. C., and
Baltz, R. H. (1997) Production of hybrid glycopeptide antibiotics in vitro and in Strep-
tomyces toyocaensis. Chem. Biol. 4, 195202.
5. Van Wageningen, A. M. A., Kirkpatrick, P. N., Williams, D. H., Harris, B. R., Kershaw,
J. K., Lennard, N. J., Jones, M., Jones, S. J. M., and Solenberg, P. J. (1998) Sequen-
cing and analysis of genes involved in the biosynthesis of a vancomycin group anti-
biotic. Chem. Biol. 5, 155162.
6. Nadkarni, S. R., Patel, M. V., Chaterjee, S., Vijayakumar, E. K. S., Desikan, K. R.,
Blumbach, J., and Ganguli, B. N. (1994) Balhimycin, a new glycopeptide antibiotic
produced by Amycolatopsis sp. Y-86,21022. J. Antibiot. 47, 334341.
7. Matsushima, P., McHenney, M., and Baltz, R. H. (1987) Efficient transformation of
Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids.
J. Bacteriol. 169, 22982300.
8. Matsushima, P. and Baltz, R. H. (1996) A gene cloning system for Streptomyces toyo-
caensis. Microbiology 142, 261267.
9. Lechevalier, M. P., Prauser, H., Labeda, D. P., and Ruan, J.-S. (1986) Two new genera
of nocardioform actinomycetes: Amycolata gen. nov. and Amycolatopsis gen. nov. Int.
J. Syst. Bacteriol. 36, 2937.
10. Lal, R., Lal, S., Grund, E., and Eichenlaub, R. (1991) Construction of a hybrid plasmid
capable of replication in Amycolatopsis mediterranei. Appl. Environ. Microbiol. 57,
665671.
11. Pelzer, S., Reichert, W., Huppert, M., Heckmann, D., and Wohlleben, W. (1997) Clon-
ing and analysis of a peptide synthetase gene of the balhimycin producer Amycola-
topsis mediterranei DSM5908 and development of a gene disruption/replacement sys-
tem. J. Biotechnol. 56, 115128.
149
References
12. Madon, J. and Htter, R. (1991) Transformation system for Amycolatopsis (Nocardia)
mediterranei: Direct transformation of mycelium with plasmid DNA. J. Bacteriol. 173,
63256331.
13. Vrijbloed, J. W., Madon, J., and Dijkhuizen, L. (1995) Transformation of the methylo-
trophic actinomycete Amycolatopsis methanolica with plasmid DNA: Stimulatory ef-
fect of a pMEA300-encoded gene. Plasmid 34, 96104.
14. Uchiyama, H. and Weisblum, B. (1985) N-methyl transferase of Streptomyces ery-
thraeus that confers resistance to the macrolide-lincosamide-streptogramin B antibio-
tics: Amino acid sequence and its homology to cognate R-factor enzymes from patho-
genic bacilli and cocci. Gene 38, 103110.
15. Gil, J. A., Kieser, H. M., and Hopwood, D. A. (1985) Cloning of a chloramphenicol
acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces
and Escherichia coli. Gene 38, 18.
16. Konz, D. and Marahiel, M. A. (1999) How do peptide synthetases generate structural
diversity? Chem. Biol. 6, R39R48.
17. Pelzer, S., Smuth, R., Heckmann, D., Recktenwald, J., Huber, P., Jung, G., and
Wohlleben, W. (1999) Identification and analysis of the balhimycin biosynthetic gene
cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis medi-
terranei DSM5908. Antimicrob. Agents Chemother. 43, 15651573.
18. Smuth, R., Metzger, J., and Jung, G., Chapter 19 this edition.
19. Chater, K. F. (1990) The improving prospects for yield increase by genetic engineer-
ing in antibiotic-producing streptomycetes. Biotechnology 8, 115121.
20. Smuth, R., Pelzer, S., Nicholson, G., Walk, T., Wohlleben, W., and Jung, G. (1999).
New advances in the biosynthesis of glycopeptide antibiotics of the vancomycin type
from Amycolatopsis mediterranei. Angew. Chem. Int. Ed. Engl. 38, 19761979.
21. Kirner, S., Hammer, P. E., Hill, D. S., Altmann, A., Fischer, I., Weislo, L. J., Lanahan,
M., van Pe, K.-H., and Ligon, J. M. (1998) Functions encoded by pyrrolnitrin biosyn-
thetic genes from Pseudomonas fluorescens. J. Bacteriol. 180, 19391943.
22. Pelzer, S., Huber, P., Smuth, R., Recktenwald, J., Heckmann, D., and Wohlleben, W.
(1999) Nucleic acid fragment and vector comprising a halogenase, and a process for
halogenating chemical compounds. US patent application 19926770.7
150
8 Homologous Recombination and the Induction
of the SOS-Response in Antibiotic Producing
Streptomycetes
Gnther Muth and Wolfgang Wohlleben*
8.1 Introduction
Antibiotic production of mycel forming streptomycetes is controlled by a com-
plex regulatory network allowing Streptomyces to sense different growth condi-
tions and to react to changes in the environment by the production of antibiotics
[2]. Antibiotic production of S. coelicolor was shown to be affected by cell den-
sity, nutritional limitations, nutritional shiftdown, imbalance in metabolism and
by different kinds of stress [2]. To sense and to respond to these environmental
conditions Streptomyces evolved a set of pleiotropic regulatory factors such as
catabolite repression, sigma factor heterogeneity, two component systems, and
pleiotropic regulatory proteins which bind small diffusable butyrolacton autore-
gulators. These regulatory systems were shown to crosstalk to each other, and
they might be also linked to the basic cellular stress response mechanisms as
stringent response (ppGpp level), N-limitation (NTR) or SOS-response to trigger
antibiotic production.
On the other site antibiotic production is also affected by the genetic in-
stability of Streptomyces. Genome rearrangements and deletion of chromosomal
ends were shown to result in the deletion of antibiotic pathways [8]. Recently it
was demonstrated that the amplification process [14] and the deletion of chro-
mosomal ends [4] depended on the RecA protein.
RecA is the key enzyme in homologous recombination and in the induc-
tion of the SOS-response by supporting autocleavage of the LexA repressor
(reviewed in [15], [7]). The recA genes of a variety of bacteria have been char-
acterized. An alignment of the Streptomyces RecA proteins to RecA sequences
from other bacteria revealed a high conservation [6]. Only the C-terminus of
the Streptomyces RecA protein was about 20 amino acids longer and showed
Tbingen
151
ISBN: 3-527-30615-3
no similarity to the C-termini of other RecA proteins [8]. RecA seems to play a
particular role in Streptomyces. Overexpression of recA is detrimental to Strep-
tomyces. It was only possible to reintroduce the recA gene into S. lividans on a
single-copy plasmid, but not when located on a multi-copy plasmid. If recA
was placed under control of an inducible promoter, induction resulted in
growth impairment (unpublished data). In contrast to other bacteria, where
recA could be inactivated without major problems, the recA gene of Strepto-
myces seemed to be essential for viability. Extensive attempts to generate recA
mutants either by UV-mutagenesis [5] or by gene disruption experiments [9, 1]
only allowed the isolation of mutants with residual activity. The gene disrup-
tion mutant FrecD3 which lacked the very last 86 amino acids was UV-sensi-
tive, its recombination activity was drastically reduced and genetic instability
was enhanced [9]. The recA mutant FRECD3 lost the chromosomal end con-
taining the chloramphenicol (CM) resistance gene 70 times more frequently
than the S. lividans wild type [14]. Thus it was concluded that the recombina-
tion activity of RecA might be required for the repair of single-stranded breaks
occurring during the replication of the Streptomyces chromosome. Due to the
linearity of the Streptomyces chromosome a single-stranded break would cause
the collapse of the replication fork and result in the deletion of the respective
chromosomal end.
To analyze the role of RecA in Streptomyces we characterized the S. livi-
dans recA gene by site directed mutagenesis and studied the modulation of
RecA activity by the LexA repressor and by the RecX protein.
8.2 Mutational analysis of the S. lividans recA gene
8.2.1 Site directed mutagenesis of recA to discriminate the different
biochemical activities of RecA
A great variety of different E. coli recA mutants have been characterized in
great detail. Certain amino acid exchanges were shown to differentially affect
the distinct biochemical activities of the RecA protein (reviewed in [7]). Some of
these amino acid residues were found to be highly conserved in other RecA
proteins [3]. To identify which activity of RecA, recombination or coprotease ac-
tivity was responsible for the toxic effects of recA overexpression/deficiency,
several amino acid residues of the S. lividans RecA protein were exchanged
(Pro
67
?Arg, Gly
157
?Asp, Arg
169
?His, Arg
243
?His) by site directed mutagen-
esis in analogy to well characterized E. coli recA mutants (manuscript in pre-
paration).
152
8 Homologous Recombination and the Induction of the SOS-Response
Figure 8.1: 3D-model of the S. lividans RecA protein. The structure was modeled in ana-
logy to the crystal structure of the E. coli RecA protein [11] using the Swissprod DB viewer
and insight II (MSI). (A) Binding of a RecA hexamer to the DNA. (B) Location of the amino
acid residues that have been mutated by site directed mutagenesis within the DNA bind-
ing domain of a RecA hexamer.
A
B
153
Since the S. lividans recA gene was fully able to complement an E. coli
recA deletion mutant, the effect of the different amino acid exchanges was in-
vestigated in E. coli and subsequently in the S. lividans recA mutant SV64 (see
below). The mutated recA genes were placed under control of the lacZ promoter
and introduced into the E. coli recA deletion strain DK1. UV-resistance was ana-
lyzed to indicate proficiency for induction of the SOS-response and recombina-
tional repair. The ability to perform homologous recombination was studied in
HFR mating assaying the mobilization of a chromosomal tetracycline resistance
marker.
The mutated S. lividans RecA proteins conferred only in part the expected
phenotype. Whereas the E. coli strain containing Pro
67/Arg
was UV-sensitive, the
respective S. lividans strain was UV-resistant as the wild type. Obviously the
mutant protein was defective in supporting autocleavage of the E. coli LexA re-
pressor, but proficient to interact with the S. lividans LexA homologue.
Exchange of the Gly
157/Asp
and Arg
243/His
in the S. lividans RecA protein
showed a very similar phenotype as reported for the respective E. coli mutants
(constitutive coprotease, reduced recombination activity). Whereas the Arg
169/
His
substitution in the E. coli RecA protein still allowed homologous recombina-
tion at a reduced ratio, the Streptomyces RecA-Arg
169/His
protein was comple-
tely defective in homologous recombination.
Thus, the amino acid exchanges in the S. lividans RecA protein had only
in part the effects of the respective aa substitutions of the E. coli RecA. This
shows that despite the high evolutionary conservation of RecA proteins the en-
zymatic activity is differentially affected by analogous amino acid substitutions.
8.2.2 3D-modeling of the mutated RecA proteins
The X-ray structure of the E. coli RecA protein was elucidated [10]. Due to the
high similarity of the S. lividans RecA protein to the E. coli RecA protein it was
possible to model the 3D-structure of the S. lividans protein and to simulate the
effect of the above described amino acid exchanges on the 3D-structure of the
RecA filaments. Computer modeling was done using insight II (MSI) and the
SWISSPROD DB viewer3.5b3.
Surprisingly we found that all the mutated residues located at the inner
site of RecA filaments (manuscript in preparation), probably interacting with
DNA (Fig. 8.1, s. p. 153). This suggests, that small differences in the DNA-bind-
ing activity of the RecA filaments are responsible for the selective inactivation of
the different biochemical functions, as LexA cleavage, recombinational repair
and homologous recombination. This also explains why analogous amino acid
exchanges in the S. lividans RecA protein conferred only in part the same phe-
notype as observed for the respective E. coli recA mutants.
154
8.2.3 Construction of a recA replacement mutant
Since extensive attempts to inactivate recA by gene disruption or gene replace-
ment failed [9], we intended to replace the chromosomal recA gene while pro-
viding a second copy of recA (Fig. 8.2). The plasmid-encoded copy of recA was
placed under control of the tipA promoter to allow inducible recA expression.
Following the replacement of the chromosomal recA gene by the aphII casette,
the recA expression vector (pEXrecA) was eliminated by plasmid incompatibil-
Figure 8.2: Construction of the S. lividans recA gene replacement mutant SV64. After
elimination of the temperature-sensitive replacement plasmid and selection for the repla-
cement of the chromosomal recA gene by the aphII cassette, the expression plasmid pEX-
recA was removed using the incompatible plasmid pIJ920.
155
ity: After transformation of the recA mutant with plasmid pIJ920, which carried
the same replicon as pEXrecA, several colonies could be selected that had lost
pEXrecA. By PCR analysis and Southern hybridization the lack of recA se-
quences was proved. Thus we were able to delete the chromosomal copy of
recA, previously thought to be indispensable [13].
There are two possible explanations why we were able to generate a com-
pletely defective recA mutant by this procedure whereas it was not possible by
the classical protocol:
1) Due to the lack of chi sequences or other unknown reasons the recA con-
taining DNA-fragment is only poorly recombinogenic. Overexpression of the
RecA protein, however, could result in an enhanced recombination activity that
allows the recombination of poor substrates.
2) The recA mutant had acquired an additional, suppressing mutation which
overcomes the RecA deficiency. Since the plasmid-encoded copy of recA which
is under control of the tipA promoter might be just sufficient to override the
toxic effect of recA deficiency, but might not be able to complement recA with
wild type efficiency, a selection pressure could exist to select for such suppres-
sing mutations. The recA mutant SV64 represents a so-called whi mutant, that
is able to erect aerial mycelial, but is deficient in sporulation. Even after comple-
mentation with the wild type recA this sporulation defect is still present,
whereas UV-sensitivity and recombination activity are fully complemented. This
might be a clear indication that SV64 had acquired an additional mutation that
could suppress the toxic effects of recA deficiency.
8.3 Regulation of RecA activity
8.3.1 Transcriptional regulation of recA by the LexA repressor
In all bacteria recA is regulated by the SOS-repressor LexA. LexA binds to so
called SOS Boxes (Cheo Box) in the promoter region of the genes of the SOS-re-
sponse. In the putative promoter region of recA there is a sequence GAACATC-
CATTC which resembles the B. subtilis SOS Box GAACNNNNGTTC/T. To ana-
lyze transcriptional regulation of recA by LexA, we expressed the S. coelicolor
lexA gene in E. coli as a His
6
-fusionprotein and purified LexA by Ni
2+
-NTA chro-
matography. A 109 bp fragment containing the putative SOS Box of the S. lividans
recA gene was amplified by PCR and 3'-labeled with digoxigenin. After incuba-
tion with LexA the reaction mixture was separated on a 4% Tris-Borat-PAGE and
blotted onto a nylon membrane and visualized with Anti-Dig-antibody-conjugate.
The retardation of the SOS box containing fragment demonstrated that the Strep-
tomyces LexAis able to bind the proposed SOS box [13].
156
8.3.2 Interaction of RecAwith RecX
8.3.2.1 Construction of a recX mutant
Downstream of the S. lividans recA gene lies an orf with significant similarity to
bacterial recX genes. The function of RecX which is in many bacteria transla-
tionally coupled to recA is unknown. Since in P. aeruginosa overexpression of
recA was lethal if recX was not coexpressed a regulatory role on RecA activity
was suggested.
To analyze the function of RecX, a recX mutant of S. lividans was con-
structed. A cloned recX gene was disrupted by the insertion of a tsr cassette
into the single BclI site. Subsequently, the chromosomal copy of recX was re-
placed by the inactivated recX copy. The resulting mutant (SVX1) represents
the first bacterial recX mutant that is well defined and had no additional defects
[12]. SVX1 displayed the following phenotype:
Homologous recombination was not affected. UV-resistance and genetic
instability (determined by the percentage of CM sensitive segregants) were
identical to that of S. lividans WT. However, the recX mutant showed a clearly
reduced colony size (Fig. 8.3).
An even more drastic phenotype was observed after overexpression of
recA. In a S. lividans TK64 culture carrying the recA expression plasmid pEX-
recA the colony titer under inducing conditions was about 60% of that of unin-
duced cultures, indicating the toxic effect of recA overexpression. In the recX
mutant however, no single colony could grow on thiostrepton-containing med-
ium [12]. This showed that induction of the tipA promoter resulting in the over-
expression of recA was lethal in the absence of RecX in S. lividans.
Figure 8.3: Morphology of the recX mutant SVX1. The recX gene of S. lividans was dis-
rupted by a thiostrepton resistance cassette. The colony size of the resulting mutant SVX1
was approximately 30% (area) of that of the wild type strain.
157
To confirm that the overexpression of recA is responsible for the toxic ef-
fect in the SVX1 mutant, we expressed an inactive RecA protein (Arg
169-His
) in
the mutant. The mutated recA gene was not able to complement an E. coli recA
deficient mutant with regard to recombination efficiency and UV-resistance,
whereas the wild type RecA protein could. The inactive recA gene was inserted
into the expression plasmid under control of the tipA promoter and transferred
into the mutant SVX1. After induction with thiostrepton 95% growth in compar-
ison to non-induced conditions was observed. Obviously, overexpression of an
inactive RecA protein was tolerated in the recX mutant [12]. This demonstrated
that the toxic effect of recA expression is caused by the enzymatic activity of the
RecA protein and not by unspecific effects of protein overexpression.
8.3.2.2 Transcriptional analysis of the recX gene
In contrast to the situation in other bacteria, where recX overlaps or lies immedi-
ately downstream of recA, the recX gene of S. lividans is separated from recA
by a fragment of 220bp. The distance and a potential termination structure (DE
26.2 kJ) 65 bp downstream of recA suggested that these two genes were not
cotranscribed. To assess whether both genes were expressed from a single tran-
script a reverse transcription polymerase chain reaction (RT-PCR) analysis was
performed. Since recA is regulated by the SOS-repressor LexA, RNA was iso-
lated at different intervals after induction with the DNA damaging methyl
methanesulfonate (MMS, 25 g ml
1
). The RTreaction was performed using ran-
dom nonamer oligonucleotides. Primers for subsequent PCR were designed for
the specific amplification of recA, recX or recA-recX transcripts.
The suitability of the primers chosen for RT-PCR was demonstrated by
PCR on genomic DNA as template (Fig. 8.4, lanes DNA). To confirm the ab-
sence of contaminating DNA, a control PCR was performed using RNA as tem-
plate. From uninduced cultures no recX transcript and only a weak band indi-
cating basal expression of the recA gene were detected (Fig. 8.4, panel A and
C, lane 0' ). 20 minutes after induction the intensity of the recA-specific band in-
creased, demonstrating induction of the recA gene during the SOS-response.
Transcription of the recX gene, however, was not detectable even 20 minutes
after induction. After 40 minutes and 60 minutes, expression of recA reached its
maximum. Concomitantly a fragment corresponding to the recA-recX cotran-
script was amplified. The recA-recX cotranscript, however, was less abundant
(~10%) than the recA transcript alone [12].
The terminator downstream of recA is probably responsible in ensuring
the different transcript levels within the recAX operon.
8.3.2.3 Putative role of the RecX protein in modulating RecA activity
The phenotype of the recX mutant SVX1 indicated, that RecX is required to
overcome the toxic effects of recA overexpression. To analyze whether RecX
158
functions by regulating down recA transcription, RT-PCR analysis was per-
formed with RNA isolated from SVX1. However, recA transcription was not sig-
nificantly affected in the recX mutant [12], and an induction pattern very similar
to that of the wild type was observed following DNA damage (Fig. 8.4 panel D).
Because RecX did not seem to influence transcription of recA we propose an in-
teraction of RecX with the RecA protein. Since all of the biochemical functions
of RecA are directly affected by the DNA binding an alteration of the binding
characteristics of RecA filaments by the interaction with RecX might efficiently
interfere with the specific activity of RecA.
Figure 8.4: RT-PCR analysis of the recAX operon. Following induction with MMS, RNA
was isolated from S. lividans TK64 (panel A, B, C) and SVX1 (panel D) at 0', 20', 40' and
60' minutes and converted to DNA using AMV reverse transcriptase. Subsequently, PCR
was performed using different primer combinations to detect the specific transcripts.
Panel A and D, internal recA primers; panel B, recA-recX primers; panel C, internal recX
primers; lane DNA: control PCR using genomic DNA as template.
159
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Streptomyces lividans recA gene suggests that only mutants with residual activity re-
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160
Membrane Processes
ISBN: 3-527-30615-3
9 Regulated Transport and Signal Transfer
Channels involved in Bacterial Iron Supply
Volkmar Braun* and Helmut Killmann
9.1 Introduction
During studies on the structure and function of the phage receptor TonA (for
phage T1, T one), we discovered in 1975 that the receptor participates in the
transport of the iron complex ferrichrome [1]; TonA was renamed FhuA for ferric
hydroxamate (ferrichrome) uptake. At that time it was known that the product
of an additional gene, termed tonB, is required for the FhuA-dependent infec-
tion of Escherichia coli by certain phages, and we were soon able to show that
the electrochemical potential of the cytoplasmic membrane is somehow trans-
mitted by TonB to the outer membrane protein FhuA and serves to activate
FhuA [2]. The study of energy-consuming active import not only of iron com-
plexes, but also of bacterial protein toxins (colicins), to gain insight into the
function of protein transporters and the transfer of energy from one membrane
to an adjacent membrane, became a major topic of our laboratory.
The Fe
2+
/Fe
3+
pair displays a wide range of redox potentials from 300 to
+700 mV, depending on the iron ligands and the protein environment. All or-
ganisms, with the exception of certain lactobacilli, take advantage of this unu-
sually wide range of electron transport capacity. However, despite its high abun-
dance in nature, iron is difficult to acquire by most organisms. Under oxic condi-
tions and at the physiological pH of 7.0, the concentration of free ferric ions in
equilibrium with the ferric hydroxide polymer is in the order of 10
12
M. The
growth-promoting concentration of iron for microbes is approximately 10
1
M.
Bacteria contain about 10
5
iron ions per cell. Since bacteria usually reach densi-
ties of 10
9
cells per ml, they require 10
14
iron ions per generation, which sharply
contrasts the available 10
3
free ions. Bacteria have solved the iron supply pro-
blem by synthesizing iron-chelating compounds, called siderophores, which
Tbingen
163
ISBN: 3-527-30615-3
they secrete and take up again after loading with Fe
3+
. The bacteria employ
highly specific and efficient transport systems for ferric siderophore uptake. In
addition to the siderophores they synthesize themselves, bacteria can use side-
rophores synthesized and secreted by other bacteria and fungi or iron sources of
their hosts (Fig. 9.1). In the human body, iron is bound to proteins: transferrin in
the serum, lactoferrin in secretory fluids and in granules of polymorphonuclear
leukocytes, and intracellular ferritin. Bacteria take up the free iron in equili-
brium with transferrin and lactoferrin via siderophores, or they take up free
heme, heme from hemoglobin and hemopexin, and iron from transferrin and
lactoferrin without the involvement of siderophores. Heme and iron is released
from the host proteins at the bacterial cell surface.
Figure 9.1: Iron transport systems of Gram-negative bacteria. Transport proteins located
in the outer membrane (OM), periplasm (PP), and in the cytoplasmic membrane (CC) are
symbolized by filled barrels. Siderophores are produced by some species and released as
indicated by the dashed arrow. Acquisition of ferric iron from lactoferrin, transferrin,
heme, hemoglobin, hemopexin, and ferric siderophores, or of ferrous iron is indicated by
solid arrows. The Ton complex composed of TonB, ExbB, ExbD is indicated by empty bar-
rels. Fe
2+
is transported by the Feo system consisting of a large and two small transport
proteins.
164
9 Regulated Transport and Signal Transfer Channels
E. coli K-12, the most widely used laboratory strain, synthesizes six distinct
siderophore-mediated Fe
3+
transport systems, each of which recognizes a single
type of ferric siderophore, and one Fe
2+
transport system. However, E. coli K-12
synthesizes only one siderophore, enterobactin (cyclic trimer of 2,3-dihydroxy-
benzoylserine, also designated enterochelin). Two of the transport systems take
up the biosynthetic precursors of enterobactin, 2,3-dihydroxybenzoic acid and
2,3-dihydroxybenzoylserine [3]. One transport system is specific for ferric citrate,
although the concentration of citrate released from E. coli K-12 into the growth
medium under laboratory conditions (not more than 15 M) is not sufficient to
induce the system (threshold concentration: 100 M citrate). Citrate is an exo-
genous siderophore, as are ferrichrome and coprogen of fungal origin, all of
which are also used as siderophores by E. coli. Pathogenic E. coli strains fre-
quently synthesize aerobactin and a ferric-aerobactin-specific transport protein,
whose genes are encoded on plasmids. The many iron transport systems in E.
coli and in other bacteria probably reflect the iron sources to which the bacteria
are exposed in their various environments to which they have to be able to
adapt.
In the following, the systems of iron transport via ferrichrome in E. coli and
Bacillus subtilis, citrate in E. coli, and heme in Yersinia enterocolitica will be de-
scribed as they represent the first systems studied and those studied in most de-
tail and are typical for Fe
3+
-siderophore transport systems in these organisms
and in other bacteria. The reader is referred to recent references for more com-
prehensive reviews [414].
9.2 The Fhu proteins catalyze active transport of ferri-
chrome and the antibiotic albomycin across the outer
membrane and the cytoplasmic membrane of E. coli
9.2.1 Transport across the outer membrane the need for receptor
proteins
Gram-negative bacteria such as E. coli are surrounded by an outer membrane,
the inner cytoplasmic membrane, and the periplasm between the two mem-
branes (Fig. 9.1). Most substrates diffuse across the outer membrane through
the water-filled channels of the porins, which in E. coli have an exclusion limit
of approximately 600 Da [15]. In contrast, the Fe
3+
-siderophores, heme, and vita-
min B
12
are actively transported across the outer membrane by proteins
(Fig. 9.1). The Fe
3+
-siderophores with molecular masses of 7001000 Da are too
large to diffuse through the porins. The concentration of Fe
3+
-siderophores is
165
9.2 The Fhu proteins catalyze active transport of ferrichrome
also too low for a sufficient iron supply if one considers the strong dilution of the
exported siderophores in the environment of the cells. Competition of the side-
rophores for iron with other strong iron-chelating compounds, such as sidero-
phores produced by bacteria and fungi that cannot be used by a particular
strain, or proteins produced by the infected hosts, e. g. transferrin, lactoferrin,
and ferritins, further lowers the Fe
3+
-siderophore concentration. Fe
3+
-sidero-
phores that make contact with bacterial cells are captured by their cognate re-
ceptor proteins. By this means, the Fe
3+
-siderophores are extracted from the
medium and concentrated at the bacterial cell surface. The low K
D
of the recep-
tors (below 0.1 M) favors binding of the Fe
3+
-siderophores. A function similar
to that of the receptor proteins at the cell surface of Gram-negative bacteria is
displayed by the binding proteins, which are anchored by a lipid moiety of the
murein-lipoprotein type to the outer face of the cytoplasmic membrane of
Gram-positive bacteria, as demonstrated for the ferrichrome transport in B. sub-
tilis [16]. In Gram-positive bacteria, the Fe
3+
-siderophores diffuse through the
multi-layered murein and teichoic acids and gain direct access to the Fe
3+
-side-
rophore transport systems in the cytoplasmic membrane.
9.2.2 The FhuA transport protein forms a regulated channel in the outer
membrane of E. coli
FhuA was originally chosen and continuously studied since it is a multifunc-
tional protein, and the mechanisms of energy-dependent phage infection, active
transport of ferric siderophores and the antibiotics albomycin and rifamycin
CGP 4832, and the import of the protein toxin colicin M and of the peptide toxin
microcin J25 by FhuA can be studied. At the beginning of the studies, it was
known that a function encoded by the tonA gene, later designated as the fhuA
gene [17], is required for infection by the phages T1, T5, and f80 and for the
sensitivity of cells to colicin M. In 1975, we and the Neilands group indepen-
dently showed that FhuA is involved in ferrichrome transport [1, 18] and that
mutants resistant to albomycin are mostly impaired in albomycin and ferri-
chrome transport. This provided a convenient way to characterize the transport
across the outer membrane and the cytoplasmic membrane. Transport genes
and their products were identified, and the transport proteins were localized.
Evidence for a channel in the FhuA outer membrane protein involved in
ferrichrome transport has been obtained by excision of DNA fragments from the
fhuA structural gene. Deletion of residues 322355 (FhuAD322355) results in a
stable protein that is integrated into the outer membrane and contains an open
channel through which ferrichrome enters cells at a rate proportional to the ex-
ternal ferrichrome concentration, without showing saturation at higher ferri-
chrome concentrations. Transport across the cytoplasmic membrane is not rate
limiting. At a low ferrichrome concentration (1 M), FhuAD322355 supports
iron uptake much less than the FhuA wild type. At higher ferrichrome concen-
166
trations (above 7 M), iron uptake through FhuAD322355 exceeds uptake via
the FhuA wild type, as one would expect for a diffusion-controlled process.
FhuAD322355 virtually does not bind ferrichrome. FhuAD322355 incorporated
into artificial lipid bilayers (black lipid membranes) forms open channels with a
single channel conductance more than three times as high as that of E. coli por-
ins [19]. Cells that synthesize FhuAD322355 can grow on maltotetraose and
maltopentaose in the absence of the LamB protein through which maltodextrins
diffuse across the outer membrane, and they are sensitive to SDS and bacitracin
[19] because the maltodextrins and noxious agents diffuse through the outer
membranes via the open FhuA channels. Segment 322355 of FhuA is exposed
to the cell surface, as revealed by proteolysis of FhuA by added proteases after
insertion of a tetrapeptide or a hexadecapeptide after residue 321, which results
in ferrichrome-transport-active FhuA derivatives [20].
FhuA contains four cysteine residues that form disulfide bridges [21]. After
cleavage of the disulfide bridge with mercaptoethanol between the two cys-
teines at positions 318 and 329, FhuA in living cells reacts weakly with biotin-
maleimide; however, a newly introduced cysteine at position 336 is highly reac-
tive, which demonstrates its exposure near the cell surface [21]. Deletion of a
single amino acid, Asp 348, inactivates ferrichrome transport activity of FhuA
and therefore is in a region important for FhuA activity [22].
9.2.3 FhuA as phage receptor
FhuA not only catalyzes transport of ferrichrome and of the structurally related
antibiotic albomycin, but also serves as a receptor for the infection of a number
of phages and for killing by toxic proteins (Fig. 9.2). The large variety of ligands
that bind to the multifunctional FhuA protein makes FhuA a particularly attrac-
tive receptor for studying structure-function relationships. Cells that synthesize
FhuAD322355 and FhuAD335355 are resistant to the phages T1, T5, and f80
(UC-1 was not tested) and to colicin M (microcin 25 was not tested). This shows
that this segment is involved in binding of the phages and in binding and up-
take of colicin M.
The binding sites of the phages in the region 316355 were demonstrated
by competitive peptide mapping [23]. This method avoids long-range conforma-
tional changes, caused by mutations in the gating loop, at regions outside the
gating loop that may be involved in phage binding. Synthetic hexapeptides that
span the entire region were employed, and those identical to sequences of three
segments inhibited infection by phages T1, T5, and f80. Similar results were ob-
tained with pentapeptides comprising residues 316320, 332337, 348351, and
with selected tetrapeptides (Killmann, H., unpublished results). The hexapep-
tides not only interfere with phage binding, but inactivate the phages by trig-
gering DNA release from the phage head [23]. The peptides mimic binding of
phage T5 to the entire isolated FhuA protein, which causes DNA release from
167
168
the phage as shown by electron microscopy. The peptides also trigger release of
DNA from phage f80, in contrast to the entire FhuA molecule, which does not
cause DNA release from f80 [23, 24] or f80 inactivation [23]. DNA release
caused by the peptides requires input of thermal energy since, under the condi-
tions used, all phages are inactivated at 378C and half of them remain intact at
208C. After the release of DNA caused by FhuA, phage T5 largely maintains its
shape. In contrast, the peptides cause a collapse of the phage head and release
of the phage tail. The stronger effect of the peptides compared to that of FhuA
can be explained by their flexible conformation, which enables them to adapt to
the surface of the phage tail region with which the phages bind to FhuA. One
of the peptide conformations is the one that the FhuA segment assumes when
energized by the proton-motive force across the cytoplasmic membrane via the
Ton complex (see Section 9.3). This energized conformation is required for bind-
ing or uptake of all FhuA ligands except phage T5. In contrast, the entire FhuA
protein assumes a defined conformation with less flexibility.
9.2.4 Sequence comparison of the FhuA proteins of various Entero-
bacteriaceae reveal conserved sites important for FhuA activity
Comparison of the ferrichrome transporters of E. coli K-12 [25, 26], Salmonella
paratyphi, Salmonella typhimurium, and Pantoea agglomerans (formerly Erwinia
herbicola) [27] support the division of the E. coli FhuA segment 322355 into
two regions. Mutant FhuAD322336 still transports ferrichrome, but is resistant
to phage T1, and FhuAD335355 does not transport ferrichrome and is resistant
to the phages T1, T5 and f80. FhuA of S. typhimurium lacks residues 321337
(E. coli FhuA numbering) and P. agglomerans lacks residues 318331 and 338
341 [27]. Both strains are resistant to the E. coli phages, but transport ferri-
chrome. S. paratyphi lacks none of the amino acids, transports ferrichrome, and
is sensitive to the E. coli phages. The entire segment 322355 is therefore re-
quired for infection by the phages, but residues 322336 are dispensable for fer-
richrome transport.
3 Figure 9.2: Upper panel (A): Crystal structure of the FhuA protein in the unloaded and
albomycin-loaded form [31]. Note the large structural change that occurs at the periplas-
mic side of FhuA when loaded with albomycin. Lower panel (B): Ligands of the multifunc-
tional FhuA protein in the outer membrane (OM) and transmembrane topology of the
TonB, ExbB, and ExbD proteins anchored to the cytoplasmic membrane (CM) and extend-
ing into the periplasm (PP). A predicted gating loop that controls the permeability of FhuA
is indicated in the cork domain. Interaction of TonB with FhuA is proposed to occur via a
region in which residue 160 resides. N designates the N-terminus, and C designates the
C-terminus of the proteins. T5, T1, f80, and UC-1 designate phages that use FhuA as a re-
ceptor to infect E. coli cells.
169
Deletion of fragment 236248 of the E. coli FhuA [27], which is identical in
all four species, abolishes ferrichrome binding and transport. As will be shown
below, this region contains a ferrichrome binding site.
9.2.5 Crystal structure of the FhuA transporter in free form and loaded
with ferrichrome
Initial attempts of the laboratory of Prof. W. Welte of the University of Konstanz to
crystallize our cloned FhuA protein yielded only crystals with a diffraction of 8 .
Later, Andrew Ferguson of the same laboratory obtained crystals with a diffraction
of 2.6 of a FhuAderivative with a His-tag at residue 405 [28, 29]. Locher et al. [30]
succeeded in the determination of the crystal structure of our cloned FhuA protein
purified without a His-tag [30]. According to the results of these two groups, FhuA
assumes a novel structure (Fig. 9.2, left panel) in which residues 160714 form a b-
barrel composed of 22 antiparallel b-strands. The b-barrel is closed by residues 19
159, which form a globular structure and enter the b-barrel from the periplasmic
side. Since this portion entirely closes the b-barrel, it has been designated the cork
or plug. Residues 118 are not seen and presumably are flexible. The crystal struc-
ture reveals that the above-described segment 322336 indeed forms a loop (L4),
which is the most prominent loop at the cell surface, consistent with the finding that
L4 serves as a binding site for the phages and colicin M.
In the crystal structure, ferrichrome is bound at a site located outside the
outer membrane bilayer (Fig. 9.2, right panel shown with albomycin instead of
ferrichrome). Four amino acid residues of the cork and six residues of the b-bar-
rel come close enough to ferrichrome to form hydrogen bonds and van der
Waals contacts (Table 9.1). The aromatic residues lining the inner walls of the
external pocket of FhuA probably extract ferrichrome from the external med-
ium. Upon binding of ferrichrome, a portion of the cork moves by 1.7 towards
ferrichrome, accompanied by a large structural transition at the periplasmic side
where a short a-helix of the cork is unwound, and Glu19 moves 17.3 away
from the position it occupies in ferrichrome-unloaded FhuA. Despite this large
structural change across the entire FhuA molecule and the thickness of the
outer membrane, the b-barrel is not converted to an open channel.
9.2.6 FhuA-albomycin is the first example of an antibiotic-protein-
transporter crystal structure
Most antibiotics diffuse into bacteria. Their efficiency, as measured by the mini-
mal inhibitory concentration (MIC), is determined by the diffusion rate and the
activity at the target sites. Gram-negative bacteria are usually less sensitive to
170
antibiotics than Gram-positive bacteria because they contain an outer mem-
brane that functions as a permeability barrier. However, if antibiotics are ac-
tively transported across the outer membrane, their MIC may be lower in Gram-
negative bacteria than in Gram-positive bacteria because the antibiotic is accu-
mulated in the periplasm and forms a steep concentration gradient across the
cytoplasmic membrane into the cytoplasm, which enhances the diffusion rate, or
it may even be actively transported across the cytoplasmic membrane. Both up-
take routes have been investigated.
The crystal structure of FhuA loaded with albomycin (Fig. 9.2, right panel)
reveals that the Fe
3+
-hydroxamate portion of albomycin occupies the same site
on FhuA and is bound by the same amino acid chains as ferrichrome [31]
(Table 9.1). In Table 9.1, ferricrocin is shown instead of ferrichrome; ferricrocin
has the same structure as ferrichrome except that one of the glycine residues is
replaced by a serine residue. Ferricrocin is produced by Aspergillae, functions
as an Fe
3+
-ligand, and is transported as well as ferrichrome. The predominant
binding sites of albomycin on FhuA are by far aromatic residues (69%). Binding
of the thioribosyl pyrimidine moiety occurs in the external pocket and involves
residues Phe 115, Lys 344, Tyr 393, Tyr 423, and Gln 505. These additional bind-
Table 9.1: Interactions of FhuA with its ligands
a
.
Ligand
Ferricrocin Albomycin
b
Albomycin
c
CGP 4832
Residue Arg 81 Arg 81 Arg 81
Glu 98
Gly 99 Gly 99 Gly 99 Gly 99
Gln 100 Gln 100 Gln 100 Gln 100
Ser 101
Phe 115 Phe 115
Tyr 116 Tyr 116 Tyr 116 Tyr 116
Tyr 244 Tyr 244 Tyr 244 Tyr 244
Trp 246 Trp 246 Trp 246 Trp 246
Tyr 313 Tyr 313 Tyr 313 Tyr 313
Tyr 315 Tyr 315 Tyr 315
Lys 344 Lys 344
Phe 391 Phe 391 Phe 391 Phe 391
Gly 392
Tyr 393 Tyr 393
Tyr 423 Tyr 423
Gln 505 Gln 505
Phe 557
Phe 558
Phe 693 Phe 693 Phe 693 Phe 693
Tyr 696
a
Distance within 4 forming hydrogen bonds and van der Waals contacts;
b
Extended conformation;
c
Compact conformation.
171
ing sites of albomycin as compared to those of ferricrocin do not prevent release
of albomycin from FhuA and transport through FhuA. The albomycin transport
rate is half the transport rate of ferrichrome, but it is not clear whether the lower
rate is caused by transport across the outer membrane or across the cytoplasmic
membrane.
The structure of the FhuA-albomycin co-crystal also reveals the hitherto
unknown conformation of albomycin and the conformation in the transport-com-
petent form. The most unexpected result is the existence of two albomycin con-
formations in the crystal an extended and a compact conformation. Both con-
formations fit into the external cavity of FhuA and occupy different amino acid
ligands (Table 9.1). The solvent-exposed external cavity of FhuA is sufficiently
large to accommodate the voluminous side chain bound to the Fe
3+
-hydroxa-
mate moiety of albomycin.
With the modular composition of albomycin, in which the iron carrier is
linked by a peptide linker to the antibiotically active thioribosyl pyrimidine, nat-
ure provides a clue of how to design highly efficient antibiotics that are actively
transported into bacteria. Such antibiotics could be synthetically assembled
from Fe
3+
-hydroxamates, which fit into the active center of the transporters, and
from an antibiotic that diffuses too slowly into cells to be useful itself as a drug.
The FhuA-albomycin structure demonstrates that the water-filled cavities in
transporters can tolerate rather large antibiotics that are structurally unrelated
to the carrier. The tolerance to the antibiotic structure is not confined to FhuA;
albomycin is also transported very well across the cytoplasmic membrane and
during this process is recognized by the FhuD and the FhuB proteins.
9.2.7 Crystal structure of FhuAwith bound rifamycin CGP 4832
In 1987, a group at Ciba-Geigy reported on a semisynthetic rifamycin derivative,
CGP 4832, with an activity against many Gram-negative bacteria 200-fold
higher than unmodified rifamycin [32]. It was then shown with our mutants [33]
that CGP 4832 is transported by FhuA across the outer membrane of E. coli and
that TonB activity is required [34]. Mutants in the fhuBCD genes, which encode
the proteins required for active transport of ferrichrome across the cytoplasmic
membrane, display unaltered CGP 4832 sensitivity. Our attempts to find addi-
tional transport mutants only revealed mutations in fhuA and tonB exbB exbD,
which suggests that CGP 4832 crosses the cytoplasmic membrane by diffusion
rather than by transport [35]. The use of FhuA as transporter for CGP 4832 is
surprising since CGP 4832 does not contain iron and has no structural resem-
blance to ferrichrome. Therefore, it was particularly of interest to determine the
crystal structure of FhuA loaded with CGP 4832 [35].
Analysis of the X-ray diffraction data reveals that CGP 4832 largely occu-
pies the site in FhuA that is also used by ferrichrome (Table 9.1). Interestingly,
the amino acid residues Gly 99, Tyr 116, and Tyr 244, which also bind ferri-
172
chrome, recognize those side chains in which CGP 4832 differs from unmodified
rifamycin.
In contrast to ferrichrome and albomycin, CGP 4832 in the crystal does not
cause the large structural change in the periplasmically oriented pocket of
FhuA. This does not seem to be caused by restriction of FhuA movements in the
crystal since binding of CGP 4832 to FhuA in solution does not result in intrinsic
FhuA tryptophan fluorescence quenching [35], as is observed when FhuA binds
ferrichrome [35, 36]. This finding has an impact on the concept of how FhuA in-
teracts with TonB since, as discussed above, the large structural transition is
thought to facilitate interaction of FhuA with TonB. Since transport of CGP 4932
depends on TonB, interaction of TonB with FhuA may occur in the absence of
the structural change. Analysis of a FhuA deletion derivative described below
supports this conclusion.
9.2.8 The b-barrel domain of FhuAD5160 is sufficient
for TonB-dependent FhuA activities
The FhuA crystal structure reveals that residues 160 to 714 of the mature protein
form a b-barrel that is closed from the periplasmic side by the globular N-proxi-
mal fragment, residues 1 to 159 designate the cork. We deleted the cork with
the idea that the resulting FhuAD5160 protein might form an open channel
[37]. To be active, the remaining b-barrel domain has to be stable and exported
across the cytoplasmic membrane into the outer membrane. If the b-barrel lack-
ing the cork does not collapse, one could expect that it would form a perma-
nently open channel larger than the channels of the previously described
FhuAD322355 and FhuAD335355 deletion derivatives [19, 38]. Such a channel
would allow diffusion, but no active transport of ferrichrome, and would confer
sensitivity to SDS and antibiotics that are too large to diffuse through the porin
channels. One could anticipate that FhuAD5160 might still function as a recep-
tor for the TonB-independent infection by phage T5, provided loop 4 retains its
conformation. In fact, deletion of the cork results in a stable protein, FhuAD5
160, that is incorporated into the outer membrane. Cells that synthesize
FhuAD5160 display a higher sensitivity to large antibiotics such as erythromy-
cin, rifamycin, bacitracin, and vancomycin, and grow on maltotetraose and mal-
topentaose in the absence of LamB. High concentrations of ferrichrome support
growth of a tonB mutant that synthesizes FhuAD5160. These results demon-
strate non-specific diffusion of compounds across the outer membrane of cells
that synthesize FhuAD5160 [37].
However, growth of a FhuAD5160 tonB
+
strain occurs at low ferrichrome
concentrations, and ferrichrome is transported at about 45% of the FhuA wild
type rate, despite the lack of ferrichrome binding sites provided by the cork.
FhuAD5160 confers sensitivity to phages T1 and f80 and the uptake of colicin
M at levels as high or nearly as high through wild type FhuA; FhuAD5160 also
173
confers sensitivity to albomycin and rifamycin CGP 4832. These data confirm the
high activity of FhuAD5160 and its strong dependence on TonB [37], despite the
lack of the TonB box (residues 7 to 11) previously implicated in the interaction of
FhuA with TonB. FhuAD5160 still functions as a specific transporter and as a
phage receptor, and sites in addition to the TonB box are involved in the TonB-
mediated response of FhuA to the proton gradient of the cytoplasmic membrane.
It is proposed that TonB interacts with the TonB box of FhuA and with the b-barrel
to release ferrichrome from the FhuA binding sites and to open the channel in
FhuA. For transport of ferrichrome through the open channel of FhuAD5160, in-
teraction of TonB with the b-barrel is sufficient to release ferrichrome from the re-
sidual binding sites at the b-barrel and to induce the active conformation of the
L4 loop at the cell surface for infection by the TonB-dependent phages T1 and
f80. For the infection by phages T1 and f80, induction of the infection-compe-
tent conformation of loop 4 by TonB cannot be mediated by the cork domain, but
must be transmitted by the b-barrel to the ferrichrome binding pocket close to the
cell surface. This implies that the interaction of FhuA with TonB is propagated
from the periplasm through the b-barrel up to loop 4 [37].
The activity of FhuAD5160 sheds light on the mode of action of wild type
FhuA. There is more than a single interaction site between FhuA and TonB. In-
teraction of TonB with the TonB box of FhuA might impose the structural change
in the cork that is required to open a channel; without this interaction, the cork
tightly closes the channel lumen of FhuA. This structural change may contribute
to the release of ferrichrome from its FhuA binding site. However, the structural
change in the cork might only alter the orientation of the cork amino acids that
contribute to ferrichrome binding and not affect the amino acids at the b-barrel
that bind ferrichrome. For the complete release of ferrichrome, the b-barrel also
changes its structure in response to TonB. As our results demonstrate, TonB can
activate FhuAD5160 exclusively through the b-barrel, and we propose that this
occurs by a conformational transition that changes the orientation of the aro-
matic residues such that they no longer bind ferrichrome. These findings imply
that the b-barrel does not form such a rigid structure that it cannot change the
conformation to alter its activity.
Export of FhuAD5160 across the cytoplasmic membrane and insertion into
the outer membrane may also shed light on the assembly of wild type FhuA.
Obviously, the FhuA barrel can be assembled in the absence of the cork; this
suggests that the barrel is constructed first and then the cork is introduced into
the pre-formed barrel. This conclusion is supported by the stability of the
FhuAD5160 b-barrel to intracellular and added proteases as high as the stabi-
lity of wild type FhuA. FhuA, like many other outer membrane proteins, con-
tains at its C-terminus a phenylalanine; in the outer membrane protein PhoE,
this amino acid is important for the formation of an assembly-competent folded
monomer. In FhuA, the C-terminal Phe residue is part of the barrel, which may
fold in the periplasm and insert into the outer membrane prior to the insertion of
the cork.
174
9.3 Transduction of energy from the cytoplasmic membrane
into the outer membrane for the activation of FhuA as a
transporter and phage receptor
Resistance of cells that synthesize wild type FhuA to SDS and large antibiotics
and the failure of the cells to grow on maltodextrins when LamB is lacking indi-
cate that FhuA does not form a permanently open channel with an inner dia-
meter larger than that of the porins. Cells that synthesize the FhuAD5160,
FhuAD322355, or FhuAD335355 deletion protein have an increased antibiotic
sensitivity, allow diffusion of ferrichrome, and grow on maltodextrins in the ab-
sence of LamB, which indicate that the FhuA channels are open. The question
arises how the FhuA wild type channel is opened physiologically. The first indi-
cation how this may happen and still the best evidence obtained comes from
studies on the infection of phage T1 [2]. Phage-resistant cells were mutated in
two genes, designated tonA and tonB (ton from T one). tonA was later renamed
fhuA [17] to indicate its physiological activity in ferric hydroxamate (ferri-
chrome) uptake. Early studies at the time when phage genetics opened a field
now known as molecular biology revealed that phage T1 adsorbs to tonB mu-
tants reversibly and that cellular energy is required for irreversible adsorption
accompanied by infection. Phage T1 does not bind to tonA (fhuA) mutants, sug-
gesting that FhuA determines the primary binding site and TonB defines a later
step in infection (cited in [2]). The nature of the energy required for irreversible
adsorption of phage T1 (and f80) was then shown to be the electrochemical po-
tential of the cytoplasmic membrane [2]. Correlation of the energy and TonB re-
quirement suggests that TonB is somehow involved in coupling the energy con-
served in the transmembrane potential of the cytoplasmic membrane to irrever-
sible adsorption of phage T1 to the outer membrane. Later, the fhuA gene pro-
duct was shown to be a protein in the outer membrane [39] and that phage T5,
which does not require energy and TonB for productive adsorption (infection), is
inactivated by the isolated protein, in contrast to phages T1 and f80, which do
require TonB in energized cells [40]. Phage T5 inactivation is prevented by coli-
cin M, which suggests a common binding site for phage T5 and colicin M [1].
This has been proven with point mutations and deletions in the region 316355
and by competitive peptide mapping [19, 22, 23, 38]. The relationship between
the adsorption step and the requirements for energy and the TonB activity was
shown by isolating phage T1 host mutants that infected tonB mutants. Infection
by these mutants demonstrates that transfer of DNA across the outer membrane
does not require TonB activity. The energized cytoplasmic membrane induces
an infection-competent conformation in FhuA through the activity of TonB. This
conformation is recognized by phages T1 and f80, while phage T5 recognizes
the unenergized conformation. Evidence for two FhuA conformations has also
been obtained by competition studies between phage T5 and ferrichrome at
FhuA. Ferrichrome inhibits phage T5 adsorption to FhuA of unenergized cells
175
(tonB mutants or energy-deprived tonB
+
cells) more strongly than adsorption to
FhuA of energized cells [41]. It is likely that all Ton-dependent receptors un-
dergo similar conformational changes upon energization, which results in trans-
location of the ferric siderophores across the outer membrane.
Transport of ferrichrome across the outer membrane into the periplasm of
E. coli was determined with a fhuB mutant devoid of ferrichrome transport
across the cytoplasmic membrane. The periplasmic FhuD binding protein had to
be overproduced to measure accumulation of radioactive [
55
Fe
3+
]ferrichrome in
the periplasm [42]. About 8000 ferrichrome molecules per cell bind to FhuD and
can be chased with non-radioactive ferrichrome. Under the same conditions,
100 000 ferrichrome molecules are taken up into the cytoplasm of a fhuB
+
strain
and can no longer be chased because iron is released from ferrichrome and in-
corporated into heme and non-heme iron proteins and into the undefined iron
pool of the cell.
For energization of outer membrane transport, the electrochemical poten-
tial (proton-motive force) of the cytoplasmic membrane is required. Three pro-
teins are known to be involved in the transduction of energy from the cytoplas-
mic membrane into the outer membrane, where FhuA is activated to transport
ferrichrome, albomycin, rifamycin CGP 4832, colicin M, and microcin J25, and
to serve in the energized state as receptor for the infection by phages T1 and
f80 (Fig. 9.2). The proteins are TonB, ExbB, and ExbD. tonB mutants are devoid
of all FhuA activities, except infection by phage T5, while exbB and exbD mu-
tants display residual activities. For this reason, an accessory role was ascribed
to the ExbB and ExbD proteins until it was shown that two other proteins, TolQ
and TolR, can partially substitute for ExbB and ExbD, respectively [4345]. exbB
tolQ and exbD tolR double mutants are both completely inactive, as are tonB
deletion mutants. tonB mutants do not accumulate ferrichrome in the periplasm
of a fhuB mutant [42], and uptake in cells that synthesize FhuAD322355 does
not require TonB, which suggests that TonB is required only for active transport
across the outer membrane.
The transmembrane topology of TonB (Fig. 9.2) further supports its role as
an energy transducer between the cytoplasmic and the outer membranes. TonB
is anchored by the N-terminal end to the cytoplasmic membrane and extends
into the periplasm [46]. Interaction of TonB with outer membrane receptors has
been demonstrated by mutations close to the N-terminal end of receptors, in the
so-called TonB box, which are suppressed by mutations at residue 160 of TonB
[4749]. Furthermore, overproduced FhuA prevents proteolytic degradation of
overproduced TonB in cells only when both proteins functionally interact with
each other [50]. Evidence for a structural complex between TonB, ExbB, and
ExbD is based on the inhibition by ExbB of TonB and ExbD degradation by cel-
lular proteases [46, 51]. A fragment of TonB, consisting of only 44 N-terminal re-
sidues, is still stabilized by ExbB [52]. Since this fragment consists mainly of the
portion of TonB that spans the cytoplasmic membrane, it is likely that interaction
between the two proteins occurs within or close to the cytoplasmic membrane.
Weak suppression of a TonB derivative lacking Val-17 by an Ala-39-to-Glu mu-
tation in the first transmembrane region of ExbB further indicates functional in-
176
teraction of the two proteins in the cytoplasmic membrane [53]. Furthermore,
TonB and ExbB can be chemically cross-linked to each other in cells [53]. In vi-
tro evidence for interaction of the three proteins has been obtained by binding
isolated ExbB which carried a His-tag at the C-terminal end to a nickel-nitrilo-
triacetate agarose column. His-tag ExbB specifically retains ExbD and TonB on
the column [54].
Although the N-terminal transmembrane segment of TonB and the region
around residue 160 are important functional regions of TonB, they are certainly
not the only regions that determine TonB activity. Mutations in the C-terminal
region beyond residue 160 (E. coli TonB consists of 239 residues) strongly affect
uptake of ferrichrome, sensitivity to certain colicins and phages [52, 55], and the
activity of certain mutated FhuA proteins with phage T5 [56].
ExbD is, like TonB, anchored by the N-terminal region in the cytoplasmic
membrane (Fig. 9.2) and extends into the periplasm [57]. Replacement of Asp
25, the only charged amino acid in the transmembrane region, by Asn results in
an inactive ExbD [54]. It is conceivable that Asp 25 interacts with His 20 of
TonB, and when replaced by Asn, largely inactivates TonB [52]. Substitution of
Leu 132 by Asn also completely inactivates ExbD [54]. Like TonB, ExbD con-
tains important functional sites in the cytoplasmic membrane and in the peri-
plasm.
In contrast to TonB and ExbD, ExbB spans the cytoplasmic membrane
three times (Fig. 9.2) and most of the protein is located in the cytoplasm [58].
The ExbB protein may be the key of the device that senses the electrochemical
potential of the cytoplasmic membrane by the TonB-ExbB-ExbD protein com-
plex.
Although infection by phage T5 does not require TonB and phage T5 mul-
tiplies in tonB deletion mutants as well as in tonB
+
cells, certain fhuA point mu-
tants display a strongly altered sensitivity to phage T5 when combined with cer-
tain tonB point mutants. For example, the sensitivity of cells that synthesize
FhuA(L106P, DD348) to phage T5 is 100-fold less than the sensitivity of cells
that synthesize wild type FhuA. Sensitivity is fully restored by replacing wild
type TonB by TonB(R204H). By contrast, FhuA(L106P) confers full phage T5 sen-
sitivity when combined with wild type TonB and is reduced three orders of mag-
nitude when combined with TonB(G174R,V178I). These data are consistent with
the proposal that various TonB derivatives impose distinct conformations on the
FhuA variants that are differently suitable for phage T5 infection [56]. TonB mu-
tant proteins and TonB proteins of different strains confer various sensitivities to
TonB-dependent ligands. For example, TonB of Serratia marcescens [59] confers
sensitivity of E. coli to colicins B and M, but not to colicin Ia [52, 55].
Transfer of energy from the cytoplasmic membrane into the outer mem-
brane via the TonB-ExbB-ExbD protein complex can be envisioned to occur by
an allosteric mechanism. TonB assumes an energized conformation through the
action of ExbB and ExbD induced by the proton-motive force of the cytoplasmic
membrane. Interaction of energized TonB with outer membrane receptor in-
duces a conformational change that opens the receptor channel and lowers the
affinity of the receptor for ferrichrome, albomycin, CGP 4832, colicin M, and mi-
177
crocin J25. The FhuA ligand is released from the receptor and diffuses through
the channel into the periplasm, where ferrichrome and albomycin bind to the
FhuD binding protein. It is not known whether diffusion through the open
FhuA channel takes place vectorially, or whether the FhuA ligand can also be
released into the culture medium, from where it has to adsorb again to FhuA to
be transported across the outer membrane. At low concentrations, the rate of
energy-dependent ferrichrome transport is much higher than the diffusion rate
through the permanently open channels of the FhuA deletion derivatives. This
could mean that the channel opens only towards the periplasm and prevents es-
cape of the ferric siderophore into the culture medium. The higher rate could
also be caused by binding of the ferric siderophore to wild type FhuA, which
does not occur with the deletion derivatives. Binding of the ferrichrome to FhuD
imposes a concentration gradient from the outside to the inside that facilitates
diffusion from the cell surface into the periplasm.
Binding of ferrichrome induces a conformational change in FhuA, as
shown in vitro by the crystal structures of FhuA in the ferrichrome-loaded and
unloaded form, in vivo by a change in the pattern of proteolytic FhuA degrada-
tion products [60], by prevention of trypsin cleavage at Lys-67 of isolated FhuA,
and by inhibition of binding of certain monoclonal antibodies to FhuA [61]. It is
conceivable that ferrichrome binding triggers energization via the Ton system.
However, it is questionable whether this is a requirement for FhuA energization
because all the other Ton-dependent FhuA ligands would also have to induce
the same or a very similar FhuA conformation capable of interacting with the
Ton system.
The concept of how FhuA might be activated by the proton-motive force
through the action of the TonB-ExbB-ExbD protein complex probably applies to
all Ton-dependent active transport processes across the outer membrane de-
picted in Fig. 9.1.
9.4 Transport of ferrichrome across the cytoplasmic
membrane
Three types of systems for transport of iron across the cytoplasmic membrane
are found in bacteria. They mediate the import of ferrous iron, of ferric iron in
the ionic form, and of ferric iron coupled to siderophores or heme. The latter two
systems are members of the periplasmic-binding-protein-dependent transport
(ABC transporter or traffic ATPase) composed of a periplasmic binding protein,
one or two different integral membrane proteins, and one or two different
ATPases that face the cytoplasm and supply the systems with energy. The al-
most identical design suggests a common origin of all ABC transporters.
178
The FhuD binding protein of E. coli accepts a number of structurally differ-
ent siderophores of the hydroxamate type (e. g. ferrichrome, coprogen, aerobac-
tin, ferrioxamine B, shizokinen, rhodotorulic acid) and the antibiotic albomycin
[62, 63]. FhuD is synthesized as a precursor with a typical signal sequence and
then is processed and exported into the periplasmic space [6467]. Binding of ir-
on(III) hydroxamates to the mature FhuD protein has been shown by three types
of experiments. First, accumulation of [
55
Fe
3+
]-ferrichrome in the periplasm of
intact cells was shown in an FhuD-overproducing strain, which, due to a muta-
tion in the integral membrane protein FhuB, is unable to translocate the sub-
strate into the cytoplasm. In a second assay, radiolabeled FhuD from the peri-
plasm is protected against proteolytic degradation by proteinase K and trypsin.
This protection is exclusively observed in the presence of those ferric hydroxa-
mates that support growth of the bacterial cells under iron-limiting conditions
[42]. In a third approach, FhuD was isolated and purified as a His-tag-labeled
derivative on a Ni-chelate resin. The dissociation constants of ferric hydroxa-
mates were estimated from the concentration-dependent decrease in the intrin-
sic fluorescence intensity of His-tag-FhuD: 0.4 M for ferric aerobactin,
1.0 M for ferrichrome, 0.3 M for ferric coprogen, and 5.4 M for the antibiotic
albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are taken
up poorly via the Fhu system, display dissociation constants of 79, 36 and 42
M, respectively [63].
FhuD delivers the ferric hydroxamates to the FhuB transport protein in the
cytoplasmic membrane. Three experimental approaches have indicated a physi-
cal interaction of FhuD with FhuB: 1) in spheroplasts, FhuD protects radioac-
tively labeled overproduced FhuB from being degraded by trypsin and protein-
ase K [62], 2) His-tag-FhuD added to spheroplasts is chemically cross-linked
to overproduced radiolabeled FhuB [63], and 3) peptides of 10 and 20 amino
acid residues identical in sequence to two transmembrane regions, two periplas-
mic loops and two cytoplasmic loops (Fig. 9.3) bind specifically to FhuD and in-
hibit ferrichrome transport [68]. The competitive peptide binding experiments
represent a novel approach in this field and were only possible after construc-
tion of FhuAD322355 [19], which rendered the outer membrane permeable to
the added peptides. These data also imply a model of the FhuB structure with
an open cylinder at the periplasmic side and a closed configuration at the cyto-
plasmic side. FhuD inserts into the cylinder and delivers its substrates to FhuB.
FhuD comes very close to the proposed binding side of FhuC (Fig. 9.3) and may
trigger in the substrate-loaded form ATP hydrolysis by FhuC. Induction of ATP
hydrolysis by substrate-loaded binding protein has been demonstrated with re-
constituted maltose and histidine transport systems [69, 70]. However, the model
derived from these studies proposes a transmembrane-signaling across the en-
tire thickness of the cytoplasmic membrane to accommodate the activation of
the cytoplasmic ATPase by the periplasmic binding protein. In our model, FhuD
approaches FhuC in the FhuB transmembrane channel so closely that the pro-
teins can interact directly or through a short FhuB peptide region.
The transmembrane topology model of FhuB as shown in Fig. 9.3 is de-
rived from genetically constructed FhuB-b-lactamase hybrid proteins in which
179
9.4 Transport of ferrichrome across the cytoplasmic membrane
increasing N-proximal segments of FhuB were fused to the BlaM b-lactamase
devoid of its signal sequence. Cells become resistant to ampicillin when the fu-
sion site is located in a periplasmic site of FhuB. In addition, a multiple se-
quence alignment of 33 transmembrane ABC transporter proteins was used to
construct the model [71].
FhuB is unique among the integral membrane proteins in that it is about
twice the size (70 kDa) of integral membrane proteins of other ABC transporters.
It consists of two domains that display significant sequence similarity to each
other. The FhuB halves are connected by a linker region of a few amino acid re-
sidues. The linker can be cleaved and the two halves assemble to form an active
transporter [72]. Both halves, FhuB(N) and FhuB(C), are essential for transport;
no activity is observed with one individually expressed domain.
Several areas of striking sequence similarity are found in the primary
structures of hydrophobic components of ABC transporters. The so-called con-
served region (CR), located in the last third of the polypeptide chains, includes
an invariant glycine residue at a distance of about 100 amino acids from the C-
terminus [73, 74]. The conserved Gly is identical with (or corresponds to) a con-
served Gly, contained in the E A A - - - G - - - - - - - - - I - L P motif defined
by Dassa and Hofnung [75]. Point mutations at two corresponding glycine resi-
dues located within FhuB[N] at position 226 and FhuB[C] at position 595 de-
crease transport activity.
The FhuC amino acid sequence contains two highly conserved sequences
typical for ATPases, the so-called Walker A and Walker B consensus motifs.
Figure 9.3: Proposed transmembrane topology of the FhuB Fe
3+
-hydroxamate transporter
in the cytoplasmic membrane [71]. The sites of interaction with the FhuD protein, as re-
vealed by binding of synthetic FhuB peptides to isolated FhuD protein and competition of
ferrichrome transport by the peptides [68], are indicated by black bars in the cytoplasmic
membrane (CM), and by asterisks in periplasmic (PP) turns and in cytoplasmic (CP) turns.
180
A total loss of function in all these FhuC derivatives suggests that FhuC indeed
acts as an ATP-hydrolase, thereby energizing the transport process, most likely
by inducing conformational changes in the components of the permease com-
plex that might open a channel [76]. Interaction of FhuC with FhuB has been
demonstrated by dominant negative effects on the transport of FhuC derivatives
with single amino acid replacements in the putative ATP-binding domains [77].
9.5 Ferric-carboxylate transport system of
Morganella morganii
Volkmar Braun
M. morganii cells are able to take up the fungal siderophore rhizoferrin. Two
genes essential for the utilization of this polyhydroxycarboxylate siderophore
have been identified. They encode an outer membrane protein and a periplas-
mic protein named RumA and RumB, respectively (rhizoferrin uptake into Mor-
ganella). RumA displays striking sequence similarities to FecA from E. coli and
to other TonB-dependent receptors; RumB shows similarity to binding proteins
of the siderophore family. rumA and rumB have the same transcription polarity
and are probably cotranscribed from an iron-regulated promoter upstream of
rumA. A predicted Fur regulatory sequence upstream of rumA has been con-
firmed by using the Fur titration assay. Analysis of a 10-kb sequence flanking
rumA and rumB upstream and downstream has revealed seven additional open
reading frames for which no role in ferric rhizoferrin uptake can be discerned.
Thus, rumA and rumB form an isolated operon, and additional genes required
for the uptake of ferric rhizoferrin across the cytoplasmic membrane must map
at chromosomal sites distinct from rumA and rumB [78].
181
9.5 Ferric-carboxylate transport system of Morganella morganii
9.6 Transport of ferric iron ions by the Sfu system of Serratia
marcescens
Volkmar Braun
The first ferric iron transport system was characterized in Serratia marcescens
(Sfu) [79]. In iron-depleted medium, the plasmid-encoded sfuABC genes confer
growth to an E. coli K-12 strain that does not synthesize enterobactin, the only
siderophore formed by wild type E. coli K-12, and to E. coli mutants unable to
transport ferric enterobactin [80]. The Sfu system also stimulates growth of tonB
and exbB mutants, and no genes encoding outer membrane proteins could be
identified in the S. marcescens genome, thereby excluding an active ferric side-
rophore transport system across the outer membrane. Ferric iron solubilized in
phosphate buffer or oxalate buffer is transported equally well, which indicates
that there is no requirement for a specific ligand. At very low iron concentra-
tions, ferric citrate stimulates iron uptake via the Sfu system in E. coli, provided
that the FecA outer membrane protein and the Ton system are active. Under
these conditions, ferric citrate is actively transported across the outer membrane
by the ferric citrate transport system, and iron is released from citrate and is
further transported by the Sfu system.
Sequence analysis of a 4.8-kb fragment of the S. marcescens chromosome
revealed three genes, sfuA, sfuB, and sfuC, which are arranged in an operon
[79]. The sfuC start codon overlaps the sfuB stop codon, indicating a regulation
of protein synthesis by translational coupling. Upstream of the sfu operon, a pu-
tative Fur box overlaps a putative promoter region. The Sfu proteins constitute a
typical ABC transporter of which the SfuA protein is localized in the periplasm
and the very hydrophobic SfuB protein is located in the cytoplasmic membrane;
the SfuC protein is degraded too fast to be located, but contains the two Walker
nucleotide binding motifs of bacterial traffic ATPases.
Through sequence analysis, homologous genes organized in operons have
been found in Neisseria gonorrhoeae (fbp) [81], Haemophilus influenzae (hit)
[82], Yersinia enterocolitica (yfu) [83], and Actinobacillus pleuropneumoniae
(afu) [84]. These systems transport ferric iron. In Neisseria meningitidis, DNase I
footprinting experiments suggest the binding of two Fur repressor dimers up-
stream of fbpA at two putative Fur consensus sequences that overlap the poten-
tial 35 region [85].
182
Acknowledgments
The authors thank the many coworkers listed in the reference list who made im-
portant contributions to the results described in these chapters. In particular we
acknowledge the original work of Wolfgang Kster on transport across the cyto-
plasmic membrane. The financial support of the Deutsche Forschungsge-
meinschaft and the Fonds der Chemischen Industrie made this work possible.
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coli K-12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic
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51. Fischer, E., Gnter, K., and Braun, V. (1989) Involvement of ExbB and TonB in trans-
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partate25 in the transmembrane region are important for ExbD activity. J. Bacteriol.
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membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities
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coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, f80
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57. Kampfenkel, K. and Braun, V. (1992) Membrane topology of the Escherichia coli ExbD
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187
References
10 Iron Transport in Gram-negative and
Gram-positive Bacteria
Klaus Hantke*
10.1 Ferric iron transport in bacteria
10.1.1 Siderophore-iron uptake in Escherichia coli
In the 1970s it was observed that six proteins in the outer membrane of E. coli
K-12 in the range of 74 to 83 kDa were overproduced when the cells were
grown under low-iron stress conditions. All these proteins, as we now know, are
receptors for siderophores (iron carriers), and some are also receptors for phages
and/or colicins: FhuA for ferrichrome, phage T1, and colicin M (see Chapter 9);
FepA for enterochelin (enterobactin) and colicin B; FecA for ferric citrate (see
Chapter 11); and Cir for colicin I [1]. Later, in a systematic study of iron-regu-
lated genes, FhuE, the receptor protein for the fungal siderophore coprogen,
and Fiu, the receptor for catecholates and colicins G and H, were identified [2].
Analysis of the nucleotide sequence of the E. coli genome has revealed a
seventh siderophore receptor gene, whose gene product possibly corresponds to
a minor protein band observed between FhuE and FhuA [2]. This protein has
not been characterized. In a BLAST search, the closest relative of this putative
receptor was shown to be FyuA, the receptor for yersiniabactin, a siderophore of
Yersinia pestis (see Section 10.1.2).
When these studies first began, it was a surprise to find so many different
iron-siderophore transport systems in one organism. Since then, a similar multi-
tude of ferric iron transport systems has been found in most other bacteria that
live under oxic conditions. More than 110 siderophore type receptors are now
found in the NCBI database of non-redundant protein sequences another indi-
cation of the ubiquity of this protein type in Gram-negative bacteria.
D-72076 Tbingen, Germany
188
ISBN: 3-527-30615-3
10.1.1.1 FhuE is a receptor for ferri-coprogen
The FhuE protein, a receptor for coprogen, has been characterized by sequen-
cing and transport studies [3, 4]. It is similar to FhuA and other hydroxamate
and pyochelin siderophore receptors. Comparison of the amino acid sequence of
FhuE with that of other receptors led to the discovery of an important binding
site, the TonB box. The TonB protein interacts with all siderophore receptors in
the outer membrane to transduce energy from the cytoplasmic membrane to the
receptor to allow release of the bound siderophore into the periplasm. A site for
the TonB interaction, the TonB box, has been identified in the N-terminal end of
the receptors (see Chapter 9).
A mutation,V8P, in the TonB box of FhuE leads to the inability of the cells to
grow on coprogen or ferrioxamine B. Different suppressor mutations were isolated
in vivo by selecting for growth on coprogen as sole iron source. Interestingly, the
suppressor mutations isolated are found only in FhuE (V8L, V8Q, and V8R), and
not in TonB, in contrast to the case with FhuA and BtuB (see Chapter 9).
10.1.1.2 Ferric-catecholate transport systems
Most E. coli strains and many Enterobacteriaceae produce the catecholate-type
siderophore enterochelin (enterobactin), which is a cyclic trimer composed of 2,3-
dihydroxy-N-benzoyl-serine. Enterochelin transport has mainly been studied by
groups in the USA [5]. We have investigated the functions of the two iron-regu-
lated outer membrane proteins, Cir and Fiu, which were originally defined as co-
licin receptors of E. coli [6, 2]. Their in vivo function is the uptake of ferric cate-
cholates and ferric dihydroxybenzoyl serine, a degradation product of enteroche-
lin [7]. Both receptors recognize an astonishingly broad spectrum of catecholate
derivatives, including catecholate-containing cephalosporin derivatives that are
transported into the periplasm, the location of their action. Transport of the cate-
cholate-cephalosporins as opposed to diffusion of the cephalosporins reduces the
minimal inhibitory concentration about 100-fold [4]. Receptors with a similar sub-
strate specificity are present in Yersinia (see Section 10.1.2) and many Gram-ne-
gative bacteria. In Pseudomonas aeruginosa these receptors make the cephalos-
porin-resistant strains highly sensitive to catecholate-cephalosporins [8].
10.1.2 Iron uptake in Yersinia enterocolitica
Y. enterocolitica is a close, but less virulent relative of Yersinia pestis, the causa-
tive agent of bubonic plague. Highly virulent serotypes (O8 and O13) and less
virulent serotypes (O3 and O9) of Y. enterocolitica have been distinguished.
One reason for these differences in virulence is the ability to produce a sidero-
phore, which we have called yersiniabactin.
189
The existence of this siderophore has been a matter of debate. In 1975,
Wake et al. [9] claimed that Y. pestis produces a siderophore related to viru-
lence. However, this was questioned by a study where it was not possible to iso-
late a siderophore with biological activity. Later it was shown in a plate test that
siderophore production of Y. enterocolitica is correlated to virulence [10]. The
characterization of the siderophore yersiniabactin specifically produced by viru-
lent Yersinia is described below.
10.1.2.1 Siderophore-mediated iron uptake
10.1.2.1.1 Yersiniabactin
The siderophore produced by Y. enterocolitica WA-C O8 is unable to crossfeed
siderophore-dependent E. coli K-12 strains. In collaboration with H. Zhner and
P. Fiedler (see Chapter 2), H. Haag isolated and purified the siderophore yersi-
niabactin, which was shown to be relatively labile. We isolated a fur mutant
using the manganese enrichment technique [11] described in Section 10.3.1.
Fur is the iron(II)-dependent repressor of iron uptake systems and also regulates
siderophore biosynthesis. As expected, the fur mutant of Y. enterocolitica consti-
tutively produced yersiniabactin. For wild type strains, it is difficult to maintain
low-iron growth conditions for optimal siderophore production without retarding
growth. In contrast, the fur mutant allowed siderophore production under iron-
rich growth conditions, which was helpful for the large-scale productions neces-
sary for the structural elucidation of yersiniabactin. The structure of yersiniabac-
tin (Fig. 10.1) was determined in close collaboration and is described in Chap-
ters 2 and 19. The purified siderophore was used to prove that 1) yersiniabactin
is a siderophore of Y. enterocolitica, and 2) the receptor FyuA is a 65-kDa pro-
tein [12].
Pesticin is a colicin-like protein, and its production had been used in the
early 1950s as a characteristic marker of Y. pestis. It was observed that virulent
Yersinia were attenuated when they became pesticin resistant. This is now ex-
plained by the genetic instability of the virulence island encoding yersiniabactin
synthesis and transport. Rare strains of E. coli are sensitive to pesticin. A tonB
mutation renders such a strain insensitive to pesticin, which provided a hint that
the receptor was a siderophore receptor. Using pesticin-resistant mutants of Y.
enterocolitica, it was possible to identify the yersiniabactin siderophore receptor
FyuA as the pesticin receptor [12, 13]. Pesticin, with its mode of action, belongs
to the rare bacteriocins with a muramidase activity [14]. In line with this, the
pesticin immunity protein, which protects the producing cells from the toxic ac-
tion of pesticin, is found in the periplasm [15]. The pesticin plasmid is a small,
colicin-E1-like plasmid that also encodes the plasminogen activator that may be
responsible for the highly invasive fulminate character of Y. pestis infections.
One hypothesis is that the bacteriocin stabilizes the presence of the plasmid in
the population.
190
10 Iron Transport in Gram-negative and Gram-positive Bacteria
10.1.2.1.2 Ferrichrome
Like many other bacteria, Y. enterocolitica is able to utilize ferrichrome (Fig.
10.1) as an iron source. Using the antibiotic albomycin, a structural analogue of
ferrichrome, it was possible to isolate a mutant unable to utilize ferrichrome.
The mutation is in the fcuA gene, and the FcuA protein (approximately 70 kDa)
is found in the outer membrane. The ferrichrome receptors of the Enterobacter-
iaceae Salmonella typhimurium, Salmonella paratyphi, Pantoea agglomerans
(formerly Enterobacter agglomerans), and E. coli have been compared and are
very similar [16]. The sequence analysis of the receptor FcuA of Y. enterocolitica
provided surprising results. The closest relative of FcuA in the siderophore re-
ceptor protein family was AngR, the receptor for anguibactin in Vibrio anguil-
larum. Anguibactin is a siderophore with similarities to yersiniabactin (Fig.
10.1), which is not structurally related to ferrichrome [17]. This is an example
where a few changes in the receptor may lead to a new receptor specificity, il-
lustrating that it is not always possible to predict the substrate specificity based
on sequence similarities.
Figure 10.1: Structures of the siderophores yersiniabactin from Y. enterocolitica and Y.
pestis, anguibactin from V. anguillarum, and the fungal siderophore desferri-ferrichrome.
191
10.1.2.1.3 Catecholate
Y. enterocolitica does not produce enterochelin, the characteristic siderophore
found in many enterobacteria. Recent studies [19] have indicated that Y. entero-
colitica WA contains only a truncated fep-ent operon, which may allow transport
of catecholes but not synthesis of enterochelin. This explains the fact that Y. en-
terocolitica is able to utilize several catecholes. A catecholate-specific receptor
mutant with a mutation in the gene cccA has been isolated using a catecholate-
cephalosporin. The receptor protein is in the outer membrane and has an appar-
ent molecular mass of 65 kDa [18]. In the Sanger sequencing project of Y. pestis
an open reading frame with similarity to Cir, a catecholate receptor of E. coli, is
found which may be the cccA gene.
10.1.2.1.4 Ferrioxamine
Ferrioxamines are siderophores produced by many Streptomyces strains and
also by some Gram-negative species, such as Pseudomonas putida and the En-
terobacteriaceae Pantoea agglomerans and Hafnia alvei. Ferrioxamine B en-
hances the virulence of the Y. enterocolitica serotypes O3 and O9 in a mouse in-
fection model, but has no effect on the pathogenicity of highly virulent strains.
This has been interpreted to mean that these highly virulent strains gain en-
ough iron through their yersiniabactin transport system for growth in their host.
In humans, Desferal (mesylate salt of desferri-ferrioxamine B) is used ther-
apeutically to chelate iron in iron-overload disease. Such patients are prone to
Yersinia infections, which may be the result of two synergistic effects of ferrioxa-
mine B: 1) the stimulation of growth of Y. enterocolitica by increasing the iron
supply via ferrioxamine B and 2) the immunosuppressing nature of Desferal,
which weakens the host defense [20].
The ferrioxamine receptor gene (foxA) of Y. enterocolitica has been cloned
and sequenced, and FoxA has been found to be similar to FhuA, the ferri-
chrome receptor of E. coli. The sequence similarities to porins and some rules
derived from the porin structural studies have been used to propose the first b-
barrel model of a siderophore receptor [21], which now has to be revised in the
light of the FhuA and FepA crystal structures (see Chapter 9).
10.1.2.2 Heme-iron uptake in Y. enterocolitica
In vertebrates, heme is the most abundant iron carrier because of the high he-
moglobin content of erythrocytes. Heme is released by tissue damage and des-
quamation of epithelial cells, which is important for bacteria colonizing the mu-
cosa. Heme can be used as an iron source by several bacteria [22]. The uptake
systems of mainly pathogenic bacteria have been studied and have revealed an
astonishing multitude of substrates and ways how bacteria extract heme from
different sources. An overview is given in [23].
We characterized the first heme uptake system of Y. enterocolitica at the
molecular level. This system strongly resembles a typical siderophore uptake
192
system [24, 25]. The six genes hemPRSTUV seem to constitute an operon. HemP
is a small protein that may have regulatory functions, and HemR is the outer
membrane receptor for heme. The sequence of HemR shows similarities to the
TonB-dependent siderophore receptors. In addition, it has been shown with a
Y. enterocolitica tonB mutant that the heme uptake is TonB dependent [24].
HemS most likely helps to degrade heme and to liberate the iron inside the cell.
The three genes hemTUV code for a binding-protein-dependent transport sys-
tem across the cytoplasmic membrane. HemT is the periplasmic binding protein,
HemU is the integral membrane protein, and HemV provides the energy as an
ATPase for the transport process [25].
Heme transport systems from E. coli O157:H7 [27], Shigella dysenteriae
serotype 1 [26] and Vibrio cholerae [28], which are similar to the heme transport
system of Y. enterocolitica have been cloned and sequenced.
10.1.3 Hydroxamate-iron transport in Bacillus subtilis
Gram-positive bacteria such as B. subtilis do not contain an outer membrane
and therefore also have no periplasm between an outer membrane and a cyto-
plasmic membrane. The question has arisen whether they contain iron trans-
port systems of the ABC transporter type, which include a periplasmic binding
protein in Gram-negative bacteria. The genes of the B. subtilis fhu operon,
which encode the ferrichrome transport system, are organized in two divergent
transcription units with overlapping promoter regions that are regulated by iron
and Fur. fhuD encodes a binding protein with a signal sequence and a cysteine
residue at the N-terminus of the mature protein, which are typical for lipopro-
teins of the murein lipoprotein type. It is therefore assumed that the FhuD pro-
tein functions as a binding protein and is anchored to the cell surface by a
covalently linked lipid. fhuD is oriented in the direction opposite to that of
fhuB, fhuG, and fhuC, which most likely form an operon. Unlike transport via
FhuB in E. coli, transport across the cytoplasmic membrane of B. subtilis is
mediated by two integral membrane proteins, FhuB and FhuG, each of which
is half the size of the E. coli FhuB. fhuC, the last gene in the operon, encodes
an ATPase. A second putative siderophore binding protein has been detected
by sequence analysis. The high sequence similarity to FhuD suggests a func-
tion as a binding protein for an iron(III) hydroxamate, most likely ferrioxamine
B, or possibly schizokinen.
193
10.2 Ferrous-iron transport systems (Feo) of E. coli
Enterobacteriaceae are facultatively anaerobic bacteria. Under anoxic condi-
tions, ferrous iron is stable, and many bacteria reduce ferric iron to ferrous iron
under anoxic conditions. Fe
2+
is more soluble than Fe
3+
and this allows its trans-
port without being complexed by ligands. A ferrous-iron transport system has
been found in E. coli, but the encoding genes could not be cloned on a plasmid,
most likely due to the high toxicity of Fe
2+
under oxic conditions. Three genes,
feoABC, have been identified [29]. feoA and feoC encode two small proteins of
unknown function, each with a molecular mass <10 kDa. feoB encodes an
84-kDa protein located in the cytoplasmic membrane. Analysis of the FeoB
sequence shows a typical nucleotide-binding motif at the N-terminal end of the
protein, which led to the assumption that ferrous iron uptake is driven by ATP
hydrolysis. More recent comparisons of this domain with proteins in the data-
bases revealed similarities to the Era/Obg/Ras protein family, some members of
which have been shown to be GTP-binding proteins. If GTP is recognized by
FeoB, it will be interesting to determine whether this domain has an energizing
or, more likely, a regulatory function.
Mutants in feoA or feoB take up ferrous iron at a greatly reduced rate. In
addition, feo mutants are derepressed for many Fur-regulated genes. These re-
sults indicate that ferrous iron transport contributes to the iron supply of the
cells also under oxic conditions [29]. In a mouse model, it has been shown that
feo helps E. coli to colonize the gut [30]. feo genes have also been identified in
S. typhimurium [31] and in some other Gram-negative bacteria, and genes simi-
lar to feoB are found in Methanococcus janaschii [32] and other anaerobic ar-
chaea.
Mg
2+
is taken up by the constitutively expressed CorA protein, which is lo-
cated in the cytoplasmic membrane and which has been studied in E. coli and
in S. typhimurium [33]. As the name of the gene corA (cobalt resistance) indi-
cates, cobalt can be used for the selection of mutations in this gene. This reflects
that also Co
2+
, Mn
2+
, and Ni
2+
can be taken up by this transport system; this
may be toxic for the cells when high concentrations of these metals are in the
medium. Ferrous iron is also a substrate for this transporter, which may be the
often-mentioned low affinity iron uptake system of E. coli [34].
194
10.3 Regulation of iron transport and metabolism
Empirically, it has long been known that the iron supply of bacteria may have a
strong influence on the metabolism of bacterial cells. One famous example is
the observation in the 1930s that low iron concentrations stimulate the produc-
tion of diphtheria toxin by Corynebacterium diphtheriae [35]. This type of regu-
lation was later also observed for many other toxins and virulence factors of
pathogenic bacteria [36]. Another early observation was that under iron-limiting
conditions, many microorganisms secrete chelating substances that produce red
to brown colored iron complexes. When the function of these substances as
Fe
3+
-carriers was recognized, they were termed siderophores [37]. How the cells
sense their iron needs became clear with the introduction of molecular biology
techniques. The first mutation in the iron regulatory gene fur (ferric iron uptake
regulation) was isolated in 1978 in S. typhimurium [38]. Later we isolated a simi-
lar mutant in E. coli [39], which allowed the cloning and sequencing of the fur
gene [40].
The development of the reporter gene technology by Casadaban [41] was
important for the study of iron regulation. The expression of the lac gene is put
under the control of the promoter of the gene of interest. The regulation of this
reporter gene under different growth conditions, for instance +/ iron, can be
seen directly on MacConkey lactose plates or with X-gal as an indicator of b-ga-
lactosidase activity. A promoter of an iron uptake gene responds to low-iron
growth conditions by greatly increasing expression the colonies of the clone
are red on MacConkey lactose plates due to the increased expression of the lac
gene under the control of the promoter; with high-iron conditions, the colonies
are white [42]. Mutants that produce red colonies on high-iron MacConkey
plates contain mutations either in the regulatory gene fur or in the feo genes,
which encode a ferrous-iron transport system.
10.3.1 Regulation of gene transcription by Fe
2+
-Fur
The Fur protein of E. coli is a repressor of 148 amino acid residues [40], 12 of
which are histidines. The high content of histidines allows the isolation of the
Fur protein by chelate-affinity chromatography [43, 44]. The histidines and car-
boxylate groups of aspartate or glutamate mainly in the C-terminal domain of
Fur probably bind the corepressor Fe
2+
[45]. However, manganese resistant mu-
tants in E. coli were often found to be mutated in the fur gene. We assume that
Mn
2+
at high concentrations binds to the Fur repressor instead of Fe
2+
. This inhi-
bits iron uptake systems although the cell may be in need for iron [45a]. Mu-
tants with a defect Fur protein have constitutively derepressed iron uptake sys-
tems and may grow better than their parents in the presence of Mn
2+
. This
195
method has also been used with success in other bacteria to isolate fur mutants.
1
H-NMR studies with the isolated Fur protein have indicated that metal binding
depends on both, metal ion concentration and proton concentration [46]. This
may be a link to the observed influence of Fur on the acid tolerance response,
which has been shown to be defective in Fur mutants of S. typhimurium [47].
The function of the four cysteines in the C-terminal domain of Fur is not clear;
however, they seem not to bind Fe
2+
directly [48, 49]. Two cysteines (C93 and
C95) have been shown to bind zinc [50].
Footprinting experiments using the promoter region of the aerobactin bio-
synthesis genes have shown that the binding of Fur to DNA is mediated by di-
valent ions, such as Co
2+
and Mn
2+
[51]. Because of the instability of Fe
2+
under
oxic conditions, only a few experiments have been conducted with this natural
partner of Fur. The protected region on the DNA is a 19-bp degenerate palin-
drome. A similar sequence is found in the promoter region of fur, and autoregu-
lation has been demonstrated [52]. Similar sequences are found in the promoter
regions of many other iron-regulated genes and are assumed to be binding sites
for Fur; this site is termed the Fur box or the iron box. An overview on Fur-
mediated regulation of gene transcription is given in [53]. When high-copy-
number plasmids containing cloned Fur binding sites are introduced into the
cells, Fur is removed from its chromosomal binding sites, and derepression of a
reporter gene under the control of an iron-regulated promoter is observed. We
developed this Fur titration assay and used it to clone new iron-regulated genes
that have a Fur box in the promoter region. In addition, we observed that the
presence of genes coding for iron-binding proteins could lead to derepression of
the reporter gene by lowering the regulatory concentration of iron in the cell
[53]. Since Fur boxes from different bacteria are similar to the Fur box of E. coli,
this system has also been used to clone iron-regulated genes from other bac-
teria.
Regulation by Fur has been studied by isolating mutants with mutations in
the fur gene that are able to inhibit repression of iron-regulated genes by the
chromosomally encoded Fur protein. Studies with such mutants indicated for the
first time that Fur acts as an oligomer. Defective Fur protein can dimerize with
wild type Fur protein encoded on the chromosome, which leads to an inactive di-
mer (negative complementation) unable to bind to the Fur box on the DNA [44].
Further proof for the binding of a Fur oligomer to DNA has been obtained by
electron microscopy and atomic force microscopy of Fur-DNA complexes. At
higher concentrations, Fur polymerizes to a multimer along the DNA [54], which
is in accordance with the results of DNA-footprinting experiments [51].
Many regulatory proteins have a typical helix-turn-helix motif at their
DNA binding site. Such a motif for Fur has not been identified using structure
prediction programs. Studies with Fur-lambda cI hybrid proteins have shown
that the N-terminal domain of Fur contains the DNA binding site [55]. This re-
sult agrees with the predicted structural similarities of the N-terminal domain of
Fur to the LexA helix-turn-helix DNA-binding motif [56].
Fur-like proteins have been found in many other bacteria. A recent BLAST
search has identified about 65 homologs; a Fur-like function has been demon-
196
strated for about 20 of these proteins. These results show that this principle of
iron regulation is widely distributed among bacteria. In the low-GC Gram-posi-
tive bacteria, such as in Bacillus subtilis, three Fur-like genes have been identi-
fied in the genome sequencing project. In addition, the promoter structure of
several iron-related genes has led to the postulation that a Fur-like regulator
should also exist in B. subtilis [57]. Helman and coworkers [58] then showed
that one such regulator has a Fur-like activity, one is a zinc-dependent repressor
called Zur, and one, PerR, regulates the oxidative response. Gram-positive bac-
teria with a high GC content, such as Corynebacteriae and Streptomyces, seem
to be the only exception. In these genera, Fur-like proteins have been identified
that seem to regulate only the oxidative-stress response genes and not iron-re-
lated genes. In these organisms, DtxR-like proteins, another type of iron-regula-
tory proteins, fulfill the function of Fur in regulating iron uptake and the bio-
synthesis of siderophores.
Proteins of the Fur family recognize different divalent cations and have ful-
filled very different functions during evolution: regulation of iron transport and
metabolism, regulation of Zn
2+
transport and metabolism, and regulation of the
oxidative-stress response in connection with cations such as iron or manganese.
These different activities of Fur-like proteins makes it difficult to predict from
sequence similarities alone the in vivo activities of the proteins. A sequence
comparison of most of the better-known Fur proteins is shown as an unrooted
phylogenetic tree in Fig. 10.2. Fur proteins that act as iron-transport regulators
in pseudomonads and enteric bacteria form a defined subgroup, while the other
Fur-like proteins seem to be very diverse. It is apparent that the oxidative stress
responsive proteins FurA from Mycobacterium marinum, FurS from Strepto-
myces reticulum, and PerR from B. subtilis are not closely related. The observed
regulation of heme biosynthesis by Irr in Bradyrhizobium japonicum may also
be related to the oxidative-response regulation since catalases and the respira-
tory chain contain heme. Also the two identified Zur proteins for zinc regulation
from B. subtilis and E. coli are not closely related. However, further characteriza-
tion of these regulons is necessary to determine which metals act as corepressor
and which genes are regulated by these proteins.
The two cysteines which bind zinc in E. coli Fur are well conserved in all
Fur proteins with exception of Fur from some pseudomonads. Further research
has to find out if the Fur protein has been a zinc regulator which in evolution
got the function of an iron regulator.
197
10.4 An [2Fe-2S] protein is involved in ferrioxamine B
utilization
Very little is known about the mobilization of iron from siderophores inside the
cell. It is generally assumed that the iron is reduced to ferrous iron and then
bound by an unknown component of the cytoplasm. We assume that the FhuF
protein is somehow involved in these processes. In E. coli, a fhuF-lacZ operon
fusion has been used as a reporter to study iron regulation since this fusion re-
acts very sensitively to slight changes in the iron concentration of the medium.
The only phenotype observed for fhuF mutations is a diminished ability of the
cells to use ferrioxamine B as an iron source. This siderophore is a poor iron
source for E. coli K-12 [3] because this strain lacks a specific receptor for ferriox-
amine B in the outer membrane; this receptor is found in Yersinia and other en-
terobacteria (see Section 10.1.2.1.4).
Figure 10.2: Unrooted tree of the Fur protein family. Only some of the corresponding pro-
teins have been shown to regulate iron transport. Fur-like proteins with a demonstrated
different function are indicated: in some Gram-positive bacteria, Fur-like proteins seem to
regulate mainly oxidative response genes2and Zur from E. coli and B. subtilis regu-
late zinc uptake&.
198
In an attempt to understand the function of FhuF, the protein has been
purified and characterized [59]. The fhuF gene has been identified as open
reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12.
The FhuF protein was labeled with a His-tag and purified. Based on sulfur de-
terminations and Mssbauer and EPR spectroscopy, FhuF has been identified as
an [2Fe-2S] protein. The g values (g
x
= 1.886, g
y
= 1.961, g
z
= 1.994) and some
of the Mssbauer parameters of FhuF obtained (oxidized protein as isolated:
DE
Q,4.2K
= 0.474 mm s
1
; Fe
3+
(reduced protein): DE
Q
= 0.978 mm s
1
) are not ty-
pical for common [2Fe-2S] proteins and indicate that FhuF has unusual struc-
tural properties. The amino acid sequence of FhuF does not show any similari-
ties to known [2Fe-2S] proteins. By site-directed mutagenesis, each of the six cy-
steines of FhuF was replaced by serine. EPR of the six reduced mutant proteins
revealed that the terminal cysteine residues 244, 245, 256, and 259 form the
[2Fe-2S]Cys
4
cluster. Mutants having the Cys-to-Ser replacement at positions
244, 245, 256, or 259 do not complement a fhuF mutant. The motif Cys-Cys-
Xaa
10
-Cys-Xaa
2
-Cys in FhuF differs considerably from the motif Cys-Xaa
2
-Cys-
Xaa
915
-Cys-Xaa
2
-Cys found in other [2Fe-2S] proteins. The unusual Cys-Cys
terminal group of the cluster may explain the atypical EPR and Mssbauer spec-
tra of the FhuF protein; possibly the tetrahedral symmetry at the ferric ion site is
distorted. The phenotype of fhuF mutants and the structural features of the
FhuF protein suggest that FhuF is involved in the reduction of ferric iron in cy-
toplasmic ferrioxamine B.
Mutants with an FhuF
-like phenotype were selected with the aim of

further defining the function of FhuF in ferrioxamine B uptake. Two mutants,
sufS::MudI and sufD::MudI, have the same phenotype as a fhuF mutant,
namely the inability to use ferrioxamine B as an iron source in the plate assay.
The sufS gene and the sufD gene were shown to be regulated by the iron-de-
pendent Fur repressor. Sequence analysis has revealed that the sufS open read-
ing frame corresponds to orf_f406. The protein SufS belongs to the family of
NifS-like proteins, which supply sulfur for [Fe-S] centers. The NifS protein is re-
quired for the assembly of the [Fe-S] cluster of the nitrogenase in Azotobacter
vinelandii. Three open reading frames coding for proteins with similarities to
NifS have been identified in the E. coli K-12 genome (Fig. 10.3). One is the
NifS-like protein, now called IscS, which has been purified and described as a
pyridoxal-phosphate-dependent cysteine desulfurase by Flint [60] and is en-
coded at 57 min on the genetic map of E. coli. A second E. coli protein of this fa-
mily has been characterized as a selenocysteine lyase (called Csd) with cysteine
sulfinate desulfinase activity. The corresponding gene has been cloned and
mapped at 63 min on the E. coli genetic map. The similarity of NifS-like proteins
has been discussed extensively by Mihara et al. [61], who pointed out that Csd
and NifS are members of two subfamilies. SufS and Csd show 43% amino acid
identity, and SufS and IscS have 22% identity, which reflects that SufS also be-
longs to the Csd group of the NifS-like proteins.
Examination of the genes in the neighborhood of sufS have revealed an op-
eron structure. The product of the first gene sufA is similar to IscA from E. coli
and Azotobacter vinelandii; IscA is encoded in the iscSUA gene cluster (Fig.
199
10.4 An [2Fe-2S] protein is involved in ferrioxamine B utilization
10.3). Genes encoding counterparts of IscSUA have been found near nifS in A. vi-
nelandii and also in different bacteria and eukaryotes. The function of IscA and
IscU is not known, but it is assumed that the proteins from these operons synthe-
size iron-sulfur centers of various proteins. An obvious counterpart for IscU or
NifU is missing in the SufABCDSE cluster. However, SufE and YgdK from E. coli
have 35% amino acid identity. ygdK is an open reading frame directly down-
stream of csd, which again points to a relationship between Csd and SufS.
Since the insertion of MudI into sufS and sufD only had an effect on fer-
rioxamine B utilization, no other vital functions seem to depend solely on these
genes. In this early stage of the investigation, it is not known whether the sufD
product is directly involved in ferrioxamine utilization or whether the Mu inser-
tion has a polar effect on the downstream genes, sufSE. However, it is interest-
ing to note that these genes are regulated by Fur and iron, as observed for fhuF.
Since the FhuF protein contains an [2Fe-2S] center, we assume that SufS
may be responsible for the assembly of this iron-sulfur center. A mutation in the
upstream sufD gene (orf_f423) causes the same phenotype. The T7 expression
system and a His-tag allow the isolation of the FhuF protein from a wild type
strain in good yield. In contrast, overproduction of the protein in a DsufD strain
has failed. Radioactive labeling of N-His-FhuF with [
35
S]methionine has shown
that the protein is unstable in the DsufD mutant [62]. The instability of FhuF
could have various reasons. FhuF may be part of the Suf complex and is un-
stable as a single protein. Another interpretation, which we prefer, is that the
Suf complex is necessary for the building of the distorted [2Fe-2S] center in
FhuF and that the apo-FhuF protein is unstable in the cell. However, this in-
stability must be very peculiar since the various FhuF Cys?Ser mutant proteins
which did not contain a [2Fe-2S] center have been isolated in relatively large
amounts [59].
Figure 10.3: Gene clusters with similarities to the E. coli sufABCDSE genes. Similar
genes are indicated with the same shading: sufA is similar to iscA; sufS is similar to csd,
iscS, and nifS; sufE is similar to ygdK; and part of nifU is similar to iscU.
200
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uptake regulator protein (Fur). Mol. Gen. Genet. 247, 199205.
56. Holm, L., Sander, C., Rterjans, H., Schnarr, M., Fogh, R., Boelens, R., and Kaptein,
R. (1994) LexA repressor and iron uptake regulator from Escherichia coli: new mem-
bers of the CAP-like DNA binding domain superfamily. Protein Eng. 7, 14491453.
57. Schneider, R. and Hantke, K. (1993) Iron-hydroxamate uptake systems in Bacillus
subtilis: identification of a lipoprotein as a part of a binding protein dependent trans-
port system. Mol. Microbiol. 8, 111121.
58. Gaballa, A. and Helmann, J. D. (1998) Identification of a zinc-specific metalloregula-
tory protein, Zur, controlling zinc transport operons in Bacillus subtilis. J. Bacteriol.
180, 58155821.
59. Muller, K., Matzanke, B. F., Schunemann, V., Trautwein, A. X., and Hantke, K. (1998)
FhuF, an iron-regulated protein of Escherichia coli with a new type of [2Fe-2S] center.
Eur. J. Biochem. 258, 10011008.
60. Flint, D. H. (1996) Escherichia coli contains a protein that is homologous in function
and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vi-
nelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid
dehydratase. J. Biol. Chem. 271, 1606816074.
61. Mihara, H., Kurihara, T., Yoshimura, T., Soda, K., and Esaki, N. (1997) Cysteine sulfi-
nate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase
and cysteine desulfurase activities. Gene cloning, purification, and characterization of
a novel pyridoxal enzyme. J. Biol. Chem. 272, 2241722424.
62. Patzer, S. I. and Hantke, K. (1999) SufS is a NifS-like protein, and SufD is necessary
for stability of the [2Fe-2S] FhuF protein in Escherichia coli. J. Bacteriol. 181, 3307
3309.
204
11 Regulation of the Ferric-Citrate Transport
System by a Novel Transmembrane
Transcription Control
Volkmar Braun* and Sabine Enz
11.1 Introduction
Citrate does not serve as a carbon source for Escherichia coli K-12 since it is not
taken up by the cells; however, Fe
3+
delivered as a citrate complex is actively
taken up by E. coli. Our investigation of the transport of radiolabeled [
55
Fe
3+
]
[
14
C]citrate has revealed uptake of iron and only residual uptake of citrate, indi-
cating that only iron and not the iron complex enters the cytoplasm. Yet ferric
citrate induces transcription of ferric-siderophore transport genes and is the
only ferric siderophore known to do so in E. coli. The question arose how an in-
ducer initiates transcription of transport genes in the cytoplasm when it does
not enter the cytoplasm.
11.2 Transport of Fe
3+
is mediated by citrate
A ferric-citrate transport system has been characterized only from E. coli (Fig.
11.1). Since a 20-fold surplus of citrate over iron is required to obtain a predomi-
nantly low molecular weight form of ferric citrate, the transport-active composi-
tion of ferric citrate is not certain, but most likely consists of ferric dicitrate [1, 2].
Ferric citrate is transported across the outer membrane via the FecA pro-
tein at the expense of the electrochemical potential of the cytoplasmic mem-
brane, mediated by the Ton system. Once in the periplasm, ferric citrate binds
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to the FecB protein. Only iron seems to be transported further across the cyto-
plasmic membrane since ten times more radioactive iron than radioactive citrate
is firmly associated with the cells, and iron transport, but not citrate uptake, is
inducible. It is not known where iron dissociates from citrate. This could occur
at the FecB protein because FecB binds iron in the absence of citrate. Iron is
transported across the cytoplasmic membrane with the help of two integral
membrane proteins, FecC and FecD, together with the FecE protein, which is
presumably bound to the inside surface of the cytoplasmic membrane. FecE
contains two Walker motifs of nucleotide-binding proteins and can be photoaffi-
nity-labeled with [
32
P]8-azido-ATP [3]. These results indicate that iron delivered
as ferric citrate is transported across the cytoplasmic membrane by a mechanism
that is typical for ABC transporters. FecB delivers ferric iron to the FecC/FecD
Figure 11.1: (A) Arrangement of the fec operon at 97.3 minutes on the E. coli chromo-
some. P
fec
denotes the promoter induced by ferric citrate, P
fur
denotes the promoters re-
pressed by the Fe
2+
-loaded Fur protein. (B) Location of the signaling and transport pro-
teins and the proposed transport and signal pathways. N, N-terminus of FecA; OM, outer
membrane; PP, periplasm; CM, cytoplasmic membrane; CP, cytoplasm; RNAP, RNA poly-
merase; aa, amino acids.
206
11 Regulation of the Ferric-Citrate Transport System
transport proteins in the cytoplasmic membrane, and subsequent translocation
of iron across the cytoplasmic membrane is energized by ATP hydrolysis cata-
lyzed by FecE.
11.3 Transcription initiation by a signaling cascade from
the cell surface into the cytoplasm
Not intracellular ferric citrate, but extracellular ferric citrate serves as an inducer
of the ferric-citrate transport system. fecBCDE mutants impaired in the transport
of iron across the cytoplasmic membrane are fully inducible, but fecA, tonB,
exbB, or exbD mutants (the latter in combination with tolQ or tolR mutations),
which are devoid of ferric-citrate transport across the outer membrane, are not
inducible [4, 5]. The obvious conclusion that entry of ferric citrate into the peri-
plasm is required for induction has been ruled out by supplying to a fecA null
mutant growth-promoting concentrations of ferric dicitrate (molecular mass 434
Da) that enter the periplasm by diffusion through the porins. No induction of fec
transport genes is observed [6], and the transport genes encoding cytoplasmic
membrane transport activities have to be constitutively overexpressed from a
multicopy plasmid to provide sufficient amounts of transport proteins. A direct
involvement of FecA in induction has been shown with fecA missense mutants,
which induce fec transcription, but do not transport ferric citrate, and which ex-
press fec genes constitutively in the absence of ferric citrate [6].
In contrast to other E. coli ferric siderophore receptors, the FecA protein
contains an N-terminal extension. When this extra peptide (residues 47101;
numbering includes the signal peptide) is removed by genetic means, induction
is abolished but FecA fully retains transport activity. Overexpressed N-terminal
FecA(1100) and FecA(1127) fragments inhibit induction, but not transport [5].
This proves that the N-terminus of mature FecA (741 amino acids; signal pep-
tide comprises 33 residues) extending from residue 1 to approximately 120 parti-
cipates in signal transduction. The N-proximal portion of FecA is localized in
the periplasm [5] and most likely interacts with the FecR regulatory protein, of
which residues 101317 are contained in the periplasm, residues 86100 span
the cytoplasmic membrane, and the N-terminal portion is in the cytoplasm [7, 8].
The transmembrane topology of FecR suggests that it transmits the signal, eli-
cited by ferric citrate on FecA, across the cytoplasmic membrane into the cyto-
plasm. Cytoplasmic N-terminal fragments of FecR, the smallest of which consists
of 59 residues, induce fec transport gene transcription constitutively [7], show-
ing that the cytoplasmic segment participates in transcription regulation and the
periplasmic region responds to the signal. Signal transduction is inhibited by
carbonylcyanide-m-chlorophenylhydrazone (CCCP), which dissipates the elec-
207
11.3 Transcription initiation by a signaling cascade
trochemical potential of the cytoplasmic membrane [5]. The energized cytoplas-
mic membrane is required for signal transduction across the outer membrane,
but not for signal transduction across the cytoplasmic membrane, as shown by
the lack of effect of CCCP on the transcription of the fec transport genes in the
absence of ferric citrate and TonB in the fecA4 mutant which constitutively tran-
scribes the fec transport genes [5]. The TonB-ExbB-ExbD complex transmits the
energy from the cytoplasmic membrane to the outer membrane for signal trans-
duction, as for ferric citrate transport across the outer membrane. The molecular
mechanisms underlying the two metabolic processes do not have to be the same
since the fecA4 mutant signals but does not transport.
FecR does not directly act on the promoter upstream of the fecA gene that
regulates transcription of fecA and of the fecBCDE genes downstream of fecA
(Fig. 11.1). The fecI gene upstream of fecR encodes a sigma factor with an
amino acid sequence only slightly similar to that of s
70
factors. The sigma factors
of FecI type are widespread in different genera and are designated as ECF (ex-
tracytoplasmic functions) factors because they all seem to be involved in extra-
cytoplasmic metabolic activities [9]. However, no other ECF regulatory system
has been studied to the extent as that of the ferric citrate regulation. Purified
FecI mediates specific binding of the RNA polymerase core enzyme to the pro-
moter region upstream of fecA, as revealed by DNA-mobility band-shift experi-
ments, and promotes fecA transcription in vitro [10]. Band shifting of fecA-pro-
moter DNA caused by cell lysates requires synthesis of FecA, FecI, and FecR
and growth of cells with ferric citrate [10], which implies that activated FecI re-
mains active during disruption of the cells and the band-shift assay.
Interaction between the FecA, FecR, and FecI signaling proteins has been
demonstrated by utilizing two methods. In in vitro binding assays, FecA is re-
tained by FecR His-tagged at the N-terminus [(His)
10
-FecR] and bound to a Ni-
NTA agarose column and is co-eluted with [(His)
10
-FecR]; FecI is retained by
FecR His-tagged at the C-terminus [FecR-(His)
6
] and is co-eluted with [FecR-
(His)
6
] from the column. An N-terminally truncated, induction-negative but
transport-active FecA protein does not bind to (His)
10
-FecR. In the in vivo assay,
the FecA-, FecR-, and FecI-interacting domains have been determined using
the bacterial two-hybrid Lex-based system. FecA
179
interacts with FecR
101317
,
and FecR
185
interacts with FecI
1173
[11]. Moreover, mutations in the cytoplas-
mic region of FecR are suppressed by FecI(Ser15?Ala) and FecI(His20?Glu)
mutations (A. Stiefel, unpublished results). These data clearly support a model
that proposes interaction of the periplasmic N-terminus of FecA with the peri-
plasmic C-terminal portion of FecR, and interaction of the cytoplasmic N-termi-
nus of FecR with the N-terminus of FecI, which results in FecI activation.
208
11.4 Iron regulation of fecIR and fecABCDE transcription
fecIR and fecABCDE form separate transcripts [12]. Transcription of fecIR is re-
pressed by iron and the Fur protein, but is uninfluenced by ferric citrate, while
fecABCDE transcription is regulated by iron and Fur and by ferric citrate via
FecI and FecR [13]. FecI and FecR regulate fec transport genes transcription, but
they display no autoregulation [14]. The iron transport genes are regulated by
the internal iron concentration and by external ferric citrate. This is a very eco-
nomical way of using ferric citrate as an iron source. When iron is not needed or
when ferric citrate is not present, the transport system can be almost totally shut
off by cytoplasmic iron. When the iron concentration in the cytoplasm falls be-
low a certain limit, the fecIR genes are transcribed. To turn on the ferric citrate
transport system, the carrier has to be in the culture medium. The FecA and
FecR proteins do not only have regulatory functions, they also have vectorial ac-
tivities in that they transmit information through three cell compartments (Fig.
11.2). Binding of ferric citrate to FecA induces a signal that is transferred from
the cell surface into the periplasm and across the cytoplasmic membrane into
the cytoplasm. It is likely that the information flux involves coupled conforma-
tional changes of FecA and FecR. The conceptual question that remains to be
answered is how FecI is activated by FecR. No phosphorylation or proteolytic
processing of FecI has been found.
Transmembrane transcription control of the E. coli Fec type is not confined
to the regulation of ferric-citrate transport. A regulatory system most likely simi-
lar to the fec transcription control has been found in Pseudomonas putida. P. pu-
tida WCS358 transports ferric pseudobactin BN8. An outer membrane protein,
PupB, transports the ferric pseudobactins BN7 and BN8, and synthesis of PupB
is induced by BN7 and BN8. It has also been shown that the N-terminus of
PupB is involved in induction, presumably via the PupI and PupR regulatory
proteins, which are homologous to FecI and FecR [15]. The sequenced genome
of Pseudomonas aeruginosa reveals several sets of genes that are homologous
to fecI fecR, the heme transport system of Serratia marcescens seems to be regu-
lated by fecI fecR homologs (C. Wandersmann, personal communication), and
fecI fecR homologs exist in Bordetella pertussis. Fec regulation may become the
paradigm of other similarly regulated gene transcription devices.
Acknowledgments
The important contributions of Annemarie Angerer are gratefully acknowl-
edged.
209
Acknowledgments
Figure 11.2: Model of transmembrane signaling for transcriptional control of the fec sys-
tem. The model indicates activation of FecR by FecA loaded with ferric citrate and activa-
tion of FecI by active FecR. The iron-loaded Fur repressor binds to the promoters, termed
P
fur
, upstream of the fecIR genes and the fecABCDE genes. It is not known whether FecI
and Fe
2+
Fur compete physically at the fecABCDE promoter.
210
References
1. Spiro, T. G., Bates, G., and Saltmann, P. (1967) The hydrolytic polymerization of ferric
citrate. II. The influence of excess citrate. J. Am. Chem. Soc. 89, 55595562.
2. Hussein, S., Hantke, K., and Braun, V. (1981) Citrate dependent iron transport system
in Escherichia coli K-12. Eur. J. Biochem. 117, 4314337.
3. Schultz-Hauser, G., Van Hove, B., and Braun, V. (1992) 8-Azido-ATP labeling of the
FecE protein of the Escherichia coli iron citrate transport system. FEMS Microbiol.
Lett. 95, 231234.
4. Zimmermann, L., Hantke, K., and Braun, V. (1984) Exogenous induction of the ferric
dicitrate transport system of Escherichia coli K-12. J. Bacteriol. 159, 271277.
5. Kim, I., Stiefel, A., Plantr, S., Angerer, A., and Braun, V. (1997) Transcription induc-
tion of the ferric citrate transport genes via the N-terminus of the FecA outer mem-
brane protein, the Ton system and the electrochemical potential of the cytoplasmic
membrane. Mol. Microbiol. 23, 333344.
6. Hrle, C., Kim, I., Angerer, A., and Braun, V. (1995) Signal transfer through three
compartments: transcription initiation of the Escherichia coli ferric citrate transport
system from the cell surface. EMBO J. 7, 14301438.
7. Ochs, M., Veitinger, S., Kim, I., Welz, D., Angerer, A., and Braun, V. (1995) Regulation
of citrate-dependent iron transport of Escherichia coli: FecR is required for transcrip-
tion activation by FecI. Mol. Microbiol. 15, 119132.
8. Welz, D. and Braun, V. (1998) Ferric citrate transport of Escherichia coli: functional re-
gions of the FecR transmembrane regulatory protein. J. Bacteriol. 180, 23872394
9. Lonetto, M. A., Brown, K. L., Rudd, K. E., and Buttner, M. J. (1994) Analysis of the
Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial
RNA polymerase s factors involved in the regulation of extracytoplasmic functions.
Proc. Natl. Acad. Sci. USA 91, 75737577.
10. Angerer, A., Enz, S., Ochs, M., and Braun, V. (1995) Transcriptional regulation of ferric
citrate transport in Escherichia coli K-12. FecI belongs to a new subfamily of s
70
-type
factors that respond to extracytoplasmic stimuli. Mol. Microbiol. 18, 163174.
11. Enz, S., Mahren, S., Stroeher, U. H., and Braun, V. (1999) Surface signaling in ferric
citrate transport gene induction: interaction of the FecA, FecR and FecI regulatory
proteins. J. Bacteriol. 181, 637646.
12. Enz, S., Braun, V., and Crosa, J. (1995) Transcription of the region encoding the ferric
dicitrate transport system in Escherichia coli: similarity between promoters for fecA
and for extracytoplasmic function sigma factors. Gene 163, 1318.
13. Angerer, A. and Braun, V. (1998) Iron regulates transcription of the Escherichia coli
ferric citrate transport genes directly and through the transcription initiation proteins.
Arch. Microbiol. 169, 483490.
14. Ochs, M., Angerer, A., Enz, S., and Braun, V. (1996) Surface signaling in transcrip-
tional regulation of the ferric citrate transport system of Escherichia coli: mutational
analysis of the alternative sigma factor FecI supports its essential role in fec transport
gene transcription. Mol. Gen. Genet. 250, 455465.
15. Koster, M., Van Klompenburg, W., Bitter, W., Leong, J., and Weisbeek, P. (1994) Role
for the outer membrane ferric-siderophore receptor PubB in signal transduction across
the bacterial cell envelope. EMBO J. 13, 28052813.
211
References
12 Structure, Function, Import, and Immunity
of Colicins
Volkmar Braun* and Helmut Pilsl
12.1 Introduction
Colicins are of particular interest regarding their entry into sensitive cells since
due to the impermeability of cell membranes for proteins, colicin import is a
highly specific and active cellular process. Our studies were mainly concerned
with the import of colicins across the outer membrane, and we studied primarily
colicins with target sites within the cytoplasmic membrane into which they ap-
parently integrate spontaneously once they have reached the periplasm. The co-
licins were used as tools to get insights into the receptor-mediated and energy-
dependent protein import in which either the Ton or the Tol system is involved.
Colicins are proteins with molecular weights between 27000 and 75000
that are synthesized by approximately 50% of E. coli strains isolated from nat-
ural sources. They kill other E. coli strains by forming pores in the cytoplasmic
membrane or by degrading DNA or ribosomal 16S RNA [1]. We have sequenced
the activity, immunity and lysis genes of colicins B, D, M, K, S4, U, 5, 10, and of
pesticin and determined their mode of action, their import into sensitive cells
and the immunity of the producer strains. These results contributed to the sub-
division of pore-forming colicins into two groups, colicins A, B, N, and U and co-
licins E1, Ia, Ib, K, 5, and 10. The immunity proteins of the A-group form four
transmembrane segments and the immunity proteins of the E1 group form three
transmembrane segments in the cytoplasmic membrane.
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12.2 Colicin M inhibits murein biosynthesis and thus
displays a unique activity among the colicins
We have studied colicin M because it uses FhuA as receptor and the Ton system
for uptake. It also does not belong to the pore-forming colicins and the nuclease
colicins. Light microscopy and electron microscopy revealed early cell lysis [2]
which is caused by inhibition of murein biosynthesis [3] through inhibition of
the regeneration of the bactoprenylphosphate from bactoprenylpyrophosphate
[4]. Since the same carrier lipid that transfers the murein precursors across the
cytoplasmic membrane also transfers lipopolysaccharide O-antigen precursors
across the cytoplasmic membrane, colicin M inhibits also O-antigen biosynthesis
[5]. These studies have revealed a unique mode of action of colicin M which dif-
fers from the action of any other colicin studied.
Cells that synthesize colicin M are not lysed by colicin M from inside or
when added from outside the cells. Immunity is conferred by an immunity pro-
tein (Cmi) which is contained in the periplasm and anchored by the N-proximal
end to the cytoplasmic membrane. The hydrophobic sequence of Cmi from resi-
dues 323 serves to translocate residues 24117 of Cmi into the periplasm and
anchors Cmi to the cytoplasmic membrane. Replacement of residues 123 of
Cmi by the hydrophobic amino acid sequence 142, that anchors the penicillin
binding protein to the cytoplasmic membrane, results in active Cmi that protects
cells against colicin M [6]. This result demonstrates that translocation and an-
choring of Cmi are not sequence-specific. Substitution of residues 123 by the
cleavable signal sequence of the BlaM b-lactamase results in an active Cmi
which is located in the periplasm. Removal of residues 123 without replace-
ment by a hydrophobic amino acid sequence results in an inactive Cmi which is
located in the cytoplasm. Apparently, the immunity protein inactivates colicin M
in the periplasm before it integrates into the cytoplasmic membrane where it in-
hibits lipid carrier regeneration.
Colicin M is usually encoded together with colicin B on large self-transmis-
sible plasmids [7]. Sequencing of both colicins reveals the gene order cba cbi
cma cmi (colicin B activity gene, colicin B immunity gene, colicin M activity
gene, colicin M immunity gene). The SOS transcription control region, typical
for colicin genes, is located upstream of the cba gene, and the cma gene is tran-
scribed from the same promoter. cba and cma have the same, cbi and cmi have
the opposite transcription polarity. The colicin BM operon encodes no lysis gene
[8] which is usually found downstream of the colicin immunity genes and which
determines a small lysis protein that facilitates release of colicins from producing
cells. Accordingly, 90% of colicins B and M remain cell-associated.
Colicin B was purified to electrophoretic homogeneity and represents a
single polypeptide chain in contrast to a previously published paper. It belongs
to the pore-forming colicins and forms the most stable single channels of all coli-
cins in artificial lipid bilayer membranes [9].
213
12.2 Colicin M inhibits murein biosynthesis
The uptake of colicins B and M requires the Ton system. Both colicins con-
tain a TonB box sequence close to the N-terminal end [10, 11]. Amino acid re-
placements in the TonB box sequences inactivate the colicins because they are
not taken up [12, 13]. The mutations Q160L and Q160K in TonB partially restore
the uptake of the mutated colicins. Suppression of a mutation in one protein by
a mutation in another protein is taken as evidence for an interaction of both pro-
teins through the mutated sites. The cellular receptor proteins FhuA and FepA
also contain TonB box sequences close to the N-terminus. For FhuA it has been
shown that mutations in the TonB box inactivate FhuA with regard to its TonB-
dependent activities but not with regard to the TonB-independent receptor ac-
tivity for phage T5 [14]. The same TonB mutations Q160L and Q160K partially
restore the TonB-dependent FhuA activities. These results demonstrate a dual
participation of the Ton system for colicin M transport which is the first case
where this has been shown. Since we could also demonstrate suppression of
TonB box mutations in colicin B by TonB mutations it is likely that all colicins ta-
ken up by TonB-dependent receptors use the TonB activity in two transport
steps.
12.3 Colicins 5 and 10 are taken up by a novel mechanism
In 1992 Bradley and Howard published a paper in which they provided genetic
evidence that colicins 5 and 10 use the TonB-independent outer membrane re-
ceptor protein Tsx but require TonB for uptake [15]. This finding stood in con-
trast to what we had found with colicins B and M. Therefore, we sequenced the
activity, immunity and lysis genes of colicin 5 [16] and the closely related colicin
10 [17]. In fact, both colicins contain a TonB box close to the N-terminus which
indicates a TonB-dependent uptake. Replacement of the N-terminal sequence
167 of colicin 10 by the N-terminal sequence 1 to 58 of colicin E1 confers to the
hybrid protein a Tol-dependent uptake mechanism typical for colicin E1. Re-
placement of the N-terminal colicin E1 sequence by the N-terminal colicin 10
sequence conferred to the hybrid protein a Ton-dependent uptake. These ex-
periments demonstrate that the N-terminal regions are solely responsible for the
Ton and Tol-dependent uptake routes, respectively, and that receptors need not
to function in a TonB-dependent way to take up colicins by the Ton system. For
these colicins it is sufficient that only they interact with the TonB protein. In ad-
dition, these data support the structure of colicins composed of independent
functional domains.
214
12 Structure, Function, Import, and Immunity of Colicins
12.4 Colicins evolved by the exchange of DNA fragments
which precisely defined functional domains
The modular structure of colicins was first deduced from the comparison of the
amino acid sequences of colicin D and colicin B [18]. The colicin D activity and
immunity genes were sequenced because colicin D shares the outer membrane
receptor and the TonB dependence of uptake with colicin B but it does not form
pores as colicin B. Its mode of action is still unclear. The N-terminal sequences
of both proteins determine TonB-dependent uptake and receptor specificity and
are almost identical (96%) but the C-terminal regions that determine the killing
activities are completely different. The conclusion that during evolution colicins
were assembled from DNA fragments that encode functional regions was
strongly supported by comparison of the pore-forming colicins K and S4 with co-
licins 5 and 10 [19, 20]. The high degree of sequence identity of functionally
equivalent regions can be seen in Fig. 12.1A.
Analysis of the nucleotide sequence of the E. coli colicin S4 determinant
revealed 76% identity to the pore-forming domain of the colicin A protein, 77%
to the colicin A immunity protein, and 82% identity to the colicin A lysis protein.
The N-terminal region, which is responsible for the Tol-dependent uptake of co-
licin S4, has 93.8% identity to the N-terminal region of colicin K. By contrast,
the predicted receptor binding domain shows no sequence similarities to other
colicins (Fig. 12.1B) which reflects its unique binding to OmpW, a minor protein
of unknown function in the outer membrane of E. coli. Colicin S4 is another ex-
ample for the assembly of colicins from DNA fragments that encode functional
domains.
A novel colicin, designated colicin U, was found in two Shigella boydii
strains of serovars 1 and 8 [21]. Colicin U is active against bacterial strains of
the genera Escherichia and Shigella. Colicin U displays sequence similarities to
various colicins (Fig. 12.2). The N-terminal sequence of 130 amino acids has
54% identity to the N-terminal sequence of bacteriocin 28b produced by Serra-
tia marcescens. Furthermore, the N-terminal 36 amino acids share striking se-
quence identity (83%) to colicin A. Although the C-terminal pore-forming se-
quence of colicin U shows the highest degree of identity (73%) to the pore-
forming C-terminal sequence of colicin B, the immunity protein, which interacts
with the same region, displays a higher degree of sequence similarity to the im-
munity protein of colicin A (45%) than to the immunity protein of colicin B
(30.5%). Immunity specificity is probably conferred by a short sequence that
ranges from residue 571 to residue 599 of colicin U; this sequence is not similar
to that of colicin B. Uptake of colicin U by sensitive cells is mediated by the
OmpA protein, the OmpF porin, and core lipopolysaccharide, and is dependent
on the TolA, B, Q, and R proteins [21].
215
12.4 Colicins evolved by the exchange of DNA fragments
Figure 12.1: (A) Differences in the nucleotide sequences, indicated by vertical bars and
as percent of identity, of the genes encoding the activity (termed a), immunity (termed i)
and lysis genes (termed l) of colicins 10 and K, as compared to those of colicin 5 [19]. The
functional domains required for TonB- and TolA-dependent uptake of the colicins via the
Tsx/TolC and the OmpA/OmpF outer membrane receptor proteins, and the pore-forming
domains are indicated. (B) Percent of identity of the nucleotide sequences (within the
bars) and the encoded proteins (above the bars) of colicin A as compared to colicin S4
[20]. The bars indicate the length of the genes and the arrows the transcription polarity.
Note the identical gene arrangements of the colicin determinants.
216
12.5 Pore-forming colicins are inactivated by the cognate
immunity proteins shortly before the formation of the
transmembrane pores
The immunity proteins of the pore-forming colicins reside in the cytoplasmic
membrane, the target site of the colicins. The question is how they inactivate
the colicins. We have studied this question by identifying possible binding sites
on the immunity proteins and the colicins, and by subcellular localization of the
immunity protein binding sites. Mutational analysis of colicin 5 and exchange of
DNA fragments between the most closely related colicins 5 and 10, and be-
tween their immunity proteins localize the regions that determine the reaction
specificity between colicin 5 and its immunity protein to residues 405 to 428 of
colicin 5. This region corresponds to the amphipatic a-helix of the pore-forming
colicins E1 and Ia. The specificity-conferring residues 5558 and 6875 of the
immunity protein are localized in the cytoplasmic loop and the inner leaflet of
the cytoplasmic membrane. The localization of the reactive regions of the immu-
nity protein and the colicin at the inner side of the cytoplasmic membrane sug-
gests that the immunity protein inactivates colicin 5 shortly before the lethal co-
licin pores in the cytoplasmic membrane are opened. This finding is probably
representative for all pore-forming colicins that belong to the colicin 5 sub-
group.
Figure 12.2: Dendrogram of the sequence alignment of the pore-forming colicin proteins
encoded by the activity genes [21]. Note that the sequence similarities are not uniform
along the polypeptide chains but vary strongly for different functional domains, as shown
in Fig. 12.1.
217
12.5 Pore-forming colicins are inactivated by the cognate immunity proteins
The site of interaction of colicin U with the immunity protein was localized
in the tip of the hydrophobic hairpin of the C-terminus which inserts into the cy-
toplasmic membrane [22]. Deletion of the tip resulted in a fully active colicin
that was no longer recognized by the cognate immunity protein. Replacement of
eight residues at the tip of colicin U by the corresponding sequence of colicin B
resulted in a colicin hybrid that was inactivated by the colicin U immunity and
the colicin B immunity protein to the same degree that corresponded to 10% of
the inactivation of wild type colicin U. Since the hairpin forms a loop between
helices 8 and 9 of colicin U and inserts deeply into the cytoplasmic membrane
interaction of the colicin with its immunity protein probably occurs after the coli-
cin has inserted in the cytoplasmic membrane.
The transmembrane topology of the immunity proteins of colicins U and 5
was determined and represents two types with four and three transmembrane
helices, respectively (Fig. 12.3).
An astonishing finding was made when the sequence DGTGW, which we
have proposed to be involved in Tol-dependent uptake of colicin U, was re-
placed by the TonB box DTMVV of colicin B. Uptake of the mutated colicin U
now depended on the Ton system but still required the Tol system (TolQRA) but
no longer TolB. Obviously, the Ton system functionally replaced the TolB activity
[23]. This hybrid protein is the first example of a colicin whose uptake depends
on the Ton and the Tol system. This finding strengthens our results which de-
monstrate the evolutionary relationship between the Ton and the Tol system
[2426].
12.6 Pesticin is a muramidase which is inactivated by the
immunity protein in the periplasm
The sequence of the pesticin gene reveals a bacterial toxin that has no sequence
homology to any of the colicins except the TonB box at the N-terminus which is
identical to the TonB box of colicin B [27]. However, the nucleotide sequences
which flank the pesticin activity and immunity genes are highly homologous to
the sequences flanking the colicin 10 activity, immunity, and lysis genes. This
result is another indication for our notion that during evolution complete bacter-
iocin determinants and DNA fragments encoding functional domains were ex-
changed between plasmids by recombination. In the case of the pesticin deter-
minant containing only a rudimentary fragment of a lysis gene, recombination
may have destroyed the lysis gene. Pesticin is a muramidase with a specificity
as the lysozymes cleave the glycan strands of murein between the C1 position
of muramic acid and the C4 position of N-acetylglucosamine [28]. The immunity
protein is contained in the periplasm and may inactivate pesticin before it
reaches its target.
218
F
i
g
u
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e
1
2
.
3
:
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s
[
1
6
,
2
2
]
.
219
12.6 Pesticin is a muramidase which is inactivated by the immunity protein
Acknowledgments
The authors acknowledge the important contributions of Robin Harkness and
Tobias lschlger.
References
1. V. Braun, H. Pilsl, and P. Gross (1994) Colicins: structures, modes of action, transfer
through membranes, and evolution. Arch. Microbiol. 161, 199206.
2. V. Braun, K. Schaller, and M. R. Wabl (1974) Isolation, characterization, and action of
colicin M. Antimicrob. Agents Chemother. 5, 520533.
3. K. Schaller, J. V. Hltje, and V. Braun (1982) Colicin M is an inhibitor of murein bio-
synthesis. J. Bacteriol. 152, 9941000.
4. R. E. Harkness and V. Braun (1989) Colicin M inhibits peptidoglycan biosynthesis by
interfering with lipid carrier recycling. J. Biol. Chem. 264, 61776182.
5. R. E. Harkness and V. Braun (1989) Inhibition of lipopolysaccharide O-antigen synth-
esis by colicin M. J. Biol. Chem. 264, 1471614722.
6. P. Gross and V. Braun (1996) Colicin M is inactivated during import by its immunity
protein. Mol. Gen. Genet. 251, 388396.
7. T. lschlger, E. Schramm, and V. Braun (1984) Cloning and expression of the activ-
ity and immunity genes of colicins B and M on ColBM plasmids. Mol. Gen. Genet.
196, 482487.
8. G. Thumm, T. lschlger, and V. Braun (1988) Plasmid pColBM-Cl139 does not en-
code a colicin lysis protein but contains sequences highly homologous to the D pro-
tein (resolvase) and the oriV region of the miniF plasmid. Plasmid 20, 7582.
9. U. Pressler, V. Braun, B. Wittmann-Liebold, and R. Benz (1986) Structural and func-
tional properties of colicin B. J. Biol. Chem. 261, 26542659.
10. E. Schramm, J. Mende, V. Braun, and R. M. Kamp (1987) Nucleotide sequence of the
colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins
and receptors. J. Bacteriol. 169, 33503357.
11. J. Kck, T. lschlger, R. M. Kamp, and V. Braun (1987) Primary structure of colicin
M, an inhibitor of murein biosynthesis. J. Bacteriol. 169, 33583361.
12. J. Mende and V. Braun (1990) Import-defective colicin B derivatives mutated in the
TonB box. Mol. Microbiol. 4, 15231533.
13. H. Pilsl, C. Glaser, P. Gross, H. Killmann, T. lschlger, and V. Braun (1993) Domains
of colicin M involved in uptake and activity. Mol. Gen. Genet. 240,103112.
14. H. Schffler and V. Braun (1989) Transport across the outer membrane of Escherichia
coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic
membrane. Mol. Gen. Genet. 217, 378383.
15. D. E. Bradley and S. P. Howard (1992) A new colicin that adsorbs to outer-membrane
protein Tsx but is dependent on the tonB instead of the tolQ membrane transport sys-
tem. J. Gen. Microbiol. 138, 27212724.
16. H. Pilsl and V. Braun (1995) Evidence that the immunity protein inactivates colicin 5
220
immediately prior to the formation of the transmembrane channel. J. Bacteriol. 177,
69666972.
17. H. Pilsl and V. Braun (1995) Novel colicin 10: assignment of four domains to TonB-
and TolC-dependent uptake via the Tsx receptor and to pore formation. Mol. Micro-
biol. 16, 5767.
18. U. Roos, R. E. Harkness, and V. Braun (1989) Assembly of colicin genes from a few
DNA fragments. Nucleotide sequence of colicin D. Mol. Microbiol. 3, 891902.
19. H. Pilsl and Braun V. (1995) Strong function-related homology between the pore-
forming colicins K and 5. J. Bacteriol. 177, 69736977.
20. H. Pilsl, D. Smajs, and V. Braun (1999) Characterization of colicin S4 and its receptor,
OmpW, a minor protein of the Escherichia coli outer membrane. J. Bacteriol. 181,
35783581.
21. D. Smajs, H. Pilsl, and V. Braun (1997) Colicin U, a novel colicin produced by Shigella
boydii. J. Bacteriol. 179, 49194928.
22. H. Pilsl, D. Smajs, and V. Braun (1998) The tip of the hydrophobic hairpin of colicin U
is dispensable for colicin U activity but is important for interaction with the immunity
protein. J. Bacteriol. 180, 41114115.
23. H. Pilsl and V. Braun (1998) The Ton system can functionally replace the TolB protein
in the uptake of a mutated colicin U. FEMS Microbiol. Lett. 164, 363367.
24. K. Eick-Helmerich and V. Braun (1989) Import of biopolymers into Escherichia coli:
nucleotide sequences of the exbB and exbD genes are homologous to those of the
tolQ and tolR genes, respectively. J. Bacteriol. 171, 51175126.
25. V. Braun (1989) The structurally related exbB and tolQ genes are interchangeable in
conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity. J. Bac-
teriol. 171, 63876390.
26. V. Braun and C. Herrmann (1993) Evolutionary relationship of uptake systems for bio-
polymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD
and the TolA-TolQ-TolR proteins. Mol. Microbiol. 8, 261268.
27. H. Pilsl, H. Killmann, K. Hantke, and V. Braun (1996) Periplasmic location of the pes-
ticin immunity protein suggests inactivation of pesticin in the periplasm. J. Bacteriol.
178, 24312435.
28. W. Vollmer, H. Pilsl, K. Hantke, J. V. Hltje, and V. Braun (1997) Pesticin displays
muramidase activity. J. Bacteriol. 179, 15801583.
221
References
13 Structure, Activity, Activation, and Secretion of
the Serratia marcescens Hemolysin/Cytolysin
Volkmar Braun* and Ralf Hertle
13.1 Introduction
When the characterization of the cytolysin (hemolysin) of Serratia marcescens
started [1], it was known that S. marcescens secretes a number of exoenzymes,
proteases, and chitinases, a lipase and a nuclease, in contrast to the Escherichia
coli K-12 laboratory strain, which does not secrete any proteins. Both organisms
belong to the Enterobacteriaceae, and we have been interested in determining
the secretory activities of S. marcescens that are lacking in E. coli. We biochemi-
cally characterized an exoprotease of S. marcescens [2], and during these stu-
dies, we examined whether the unspecific exoprotease lyses erythrocytes to ob-
tain a convenient and sensitive assay for the protease activity. All S. marcescens
strains tested lysed erythrocytes not by the exoprotease, but by an unknown
activity. A hemolytic activity on blood agar plates had occasionally been men-
tioned in the literature, but no data on a hemolysin were available. In a book on
the genus Serratia for clinical microbiologists, infectious disease physicians, and
epidemiologists, the terms hemolysin and cytolysin are not mentioned, which
shows that hemolysis was not considered as a trait of Serratia [3].
The hemolysin was almost neglected because of the very small lysis zones
around colonies on standard blood agar plates, which, as we now know, are
caused by 1) a low diffusion due to the high molecular weight of the hemolysin
[4], 2) its immediate aggregation upon release from the cells [5], 3) degradation
by exoproteases, and 4) synthesis only under iron-limiting growth conditions [6].
On blood agar plates S. marcescens can easily meet its iron requirement by ac-
tive transport through ferric siderophores, heme [7], and hemoglobin [8], result-
ing in repression of hemolysin synthesis. Eleven S. marcescens strains and two
S. liquefaciens strains tested were found to be hemolytic [9], and a subsequent
D-72076 Tbingen
222
ISBN: 3-527-30615-3
survey of nosocomial S. marcescens strains identified 58 hemolytic strains
among 60 strains tested [10]. DNA probes of the S. marcescens hemolysin genes
do not hybridize to DNA of E. coli, Salmonella typhimurium, Proteus mirabilis,
Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella aero-
genes, Klebsiella pneumoniae, Shigella dysenteriae, Yersinia enterocolitica, Yer-
sinia pseudotuberculosis, Listeria sp., Aeromonas sp., Legionella sp., and Me-
ningococcus sp. [9], and polyclonal antibodies against the S. marcescens hemo-
lysin do not react with the rather similar hemolysin of P. mirabilis. Lack of DNA
cross-hybridization and antibody cross-reaction makes the hemolysin a suitable
marker for the diagnosis of S. marcescens and S. liquefaciens, which are the
most frequent Serratia strains among clinical isolates.
The S. marcescens hemolysin (ShlA) represents a new type of hemolysin
and has been studied at the molecular level in great detail with regard to struc-
ture, activation, and secretion. It has nothing in common with the hemolysins
(RTX toxins) of the E. coli type [11, 12]. After we had characterized the S. mar-
cescens hemolysin on the molecular level, hemolysins homologous to the S. mar-
cescens hemolysin were found in P. mirabilis and P. vulgaris [13], Haemophilus
ducreyi [14], and Edwardsiella tarda [15]. Furthermore, sequence motifs shown
to be important for activity and secretion of the S. marcescens hemolysin [16]
are also contained in the filamentous hemagglutinin FHA of Bordetella pertussis
[17], the hemopexin HxuA/HxuB of Haemophilus influenzae [18], and the adhe-
sins HMW2A/HMW2B of Haemophilus influenzae [19] (Table 13.1). Like the he-
molysins of S. marcescens and the related hemolysins, the latter proteins also re-
quire a protein for secretion across the outer membrane; these secretory proteins
display sequence similarity to the S. marcescens secretory protein. Thus, the S.
marcescens hemolysin forms the prototype of a new class of hemolysins and of a
new secretory mechanism.
Table 13.1: Common sequence motifs related to functionally essential regions of the Ser-
ratia marcescens hemolysin.
Serratia marcescens hemolysin ShlA 68-ANPNL
Edwardsiella tarda hemolysin EthA 68-ANPNL
Proteus mirabilis hemolysin HpmA 68-ANSNL
Haemophilus ducreyi hemolysin HhdA 66-ANPHL
Bordetella pertussis filamentous hemagglutinin FHA 65-KNPNL
Serratia marcescens hemolysin ShlA 109-NPNGIS
Edwardsiella tarda hemolysin EthA 108-NPNGIT
Proteus mirabilis hemolysin HpmA 109-NPNGIT
Haemophilus ducreyi hemolysin HhdA 107-NPNGMS
Bordetella pertussis filamentous hemagglutinin FHA 94-NPNGIS
Haemophilus influenzae Type b heme:hemopexin HxuA 86-NPNGVI
Haemophilus influenzae surface protein HMW 150-NPNGIT
Numbering corresponds to the proteins after cleavage of the signal peptide, which is unu-
sually long for FHA (71 residues) and not known for HMW.
223
13.1 Introduction
13.2 Characterization of the S. marcescens hemolysin
(ShlA)
13.2.1 ShlA forms pores in eukaryotic plasma membranes, but not in
prokaryotic plasma membranes
ShlA causes hemolysis by forming pores in erythrocyte membranes [4]. The on-
set of hemolysis is progressively retarded and the hemolysis rate diminishes
when oligosaccharides of increasing molecular weight are added to the assay at
30 mM concentration, which corresponds to the internal osmotic pressure of ery-
throcytes. Maltoheptaose (M
r
1,152) is highly protective, and dextran 4 (M
r
4,000) prevents hemolysis. Removal of dextran 4 and the surplus of hemolysin
immediately results in hemolysis by ShlA, which had been inserted into the ery-
throcyte membrane in the presence of dextran 4. Prevention of hemolysis by oli-
gosaccharides demonstrates formation of ShlA pores of a limited size range
through which water and smaller oligosaccharides flow into the erythrocytes
and cause osmolysis. The larger oligosaccharides prevent osmolysis because
they are too large to enter the erythrocytes through the ShlA pores and thus
counterbalance the internal osmotic pressure of the erythrocytes. Comparison of
the ShlA pores with the structurally defined pores (heptamers) of the Staphylo-
coccus aureus a-toxin has revealed that the ShlA pores are smaller and vary in
size and are larger at 28 than at 08C [20].
Protease digestion experiments have revealed how ShlA is inserted into
the erythrocyte membrane. The hemolysin integrates into the erythrocytes such
that it is not cleaved by trypsin added to erythrocytes and sealed right-side-out
erythrocyte ghosts [21]. In unsealed ghosts and inside-out vesicles, ShlA is
cleaved by trypsin, demonstrating accessibility of ShlA to trypsin from the inside
of the erythrocytes. The most sensitive cleavage site results in a 143-kDa and a
19.5-kDa fragment, both of which remain in the erythrocyte membrane. This
site is close to the C-terminus of ShlA. Upon longer incubations, trypsin yields
fragments of 138, 89, and 58 kDa. Only a few ShlA sites in the C-terminal half
of ShlA, all of which are exposed to the inside of the erythrocytes, are amenable
by trypsin. A genetically engineered N-terminal 72-kDa ShlA fragment adsorbs
to but does not integrate into the erythrocyte membrane, as shown by its com-
plete degradation by trypsin. For insertion of ShlA into erythrocyte membranes,
ShlA must be modified by the outer membrane protein ShlB, and adsorption of
the 72-kDa ShlA depends on co-synthesis with ShlB (Fig. 13.1).
ShlA forms pores in erythrocytes from various animal sources. This raises
the question why ShlA does not kill bacterial producer cells. Activation during
secretion across the outer membrane by ShlB could be a means to protect the
bacterial producer cells. This theory has been tested by transforming stable pro-
toplast-type L-forms of Proteus mirabilis lacking an outer membrane with a
224
13 Structure, Activity, Activation, and Secretion of the Serratia marcescens
plasmid carrying the shlA shlB genes [22]. Inactive ShlA* is secreted by the L-
form cells, and ShlB is associated with the cytoplasmic membrane. Addition of
hemolytic ShlA to the L-form cells has no effect, which suggests that the prokar-
yotic cytoplasmic membrane is resistant to ShlA.
In artificial lipid bilayers, ShlA forms water-filled channels with an inner
diameter of 13 nm, depending on how ShlA is prepared [20]. The data suggest
that pre-formed ShlA monomers and dimers can insert into erythrocyte mem-
branes and that larger oligomers may form in the erythrocyte membranes at
288C. However, oligomerization within the erythrocyte membrane is not re-
quired for pore formation, which occurs more rapidly at 08C than at 288C [21].
At 08C the lateral mobility of integral membrane proteins is greatly reduced so
that oligomerization cannot contribute much to pore formation.
13.2.2 Two proteins determine the S. marcescens hemolytic activity
Bacterial protein toxins are necessarily chimeric proteins they must be hydro-
philic to be soluble when released from the bacteria, but they also have to be
hydrophobic to enter into or through the plasma membrane of eukaryotic cells.
Figure 13.1: Arrangement and transcriptional polarity of the shlA and shlB genes. ShlB
inserts into the outer membrane (OM), and activates and secretes ShlA (a), and the C-
terminally truncated ShlA-255 (c). In the absence of ShlB non-hemolytic ShlA* remains in
the periplasm (b).
225
13.2 Characterization of the S. marcescens hemolysin (ShlA)
These properties frequently result in aggregation of the toxins in aqueous solu-
tion, and the S. marcescens hemolysin is no exception; this makes its biochem-
ical characterization difficult [1]. The breakthrough in the precise description of
many bacterial toxins came with the recombinant DNA techniques, which
yielded accurate data on the number of proteins involved, their molecular
weights, and the transcriptional regulation of protein synthesis, and which
yielded sufficient quantities of the proteins for functional studies. However, in
the case of the S. marcescens hemolysin, biochemical experiments could only
be performed after it was discovered that ShlA can be kept in the soluble form
in 6 M urea without loss of activity and that precipitated ShlA aggregates can
be solubilized in 6 M urea in an active form. ShlA even withstands irreversible
denaturation by 10% trichloroacetic acid, as shown by the hemolytic activity of
TCA-precipitated ShlA solubilized in 6 M urea. In contrast, these treatments de-
nature ShlB; however, ShlB, in contrast to ShlA, can be solubilized in an active
form in mild detergents, such as octylglucoside.
The hemolysin was characterized in transformants of E. coli K-12 that car-
ried both genes encoding ShlA and ShlB (shlA shlB), shlA or shlB, or mutated
shlA and shlB on plasmids. In the cases examined, the properties of the E. coli
transformants agreed with the properties of wild type S. marcescens.
Genetically, the hemolysin trait of S. marcescens is very stable. We have
never observed spontaneous non-hemolytic mutants. A 7.5-kb chromosomal
DNA fragment of S. marcescens renders E. coli K-12 transformants hemolytic.
The nucleotide sequence of the DNA fragment reveals two open reading
frames, named shlA and shlB, which are transcribed from shlB to shlA (Fig.
13.1). shlA encodes the hemolysin, and shlB encodes an outer membrane pro-
tein that is required for the secretion of the hemolytic ShlA protein across the
outer membrane into the culture medium. Mature ShlA is composed of 1578
amino acids, and mature ShlB contains 539 amino acids. Both proteins are
synthesized as precursors that contain signal peptides of 30 and 18 amino acids,
respectively, which are cleaved off during export of the polypeptides across the
cytoplasmic membrane [23].
In S. marcescens, synthesis of the hemolysin is repressed by iron. In rich
media, iron limitation by the iron-chelator 2,2'-dipyridyl (0.3 mM) strongly in-
creases hemolysin synthesis [5, 6] despite the numerous iron supply systems
that exist in this organism [7]. In E. coli, transcription of the shlB shlA genes is
co-regulated by the Fur protein [24], which, when loaded with Fe
2+
, functions as
a transcriptional repressor of iron-controlled genes. Fur-Fe
2+
binds to the Fur
box, a DNA consensus sequence composed of 19 nucleotides; a similar se-
quence is located in the 30 region of the promoter upstream of shlB. A fur dele-
tion mutant of E. coli, transformed with the shlA shlB genes, produces tenfold
more hemolysin than a fur wild type strain grown in a medium with sufficient
iron [6].
226
13.2.3 Domains in ShlA responsible for secretion, binding to eukaryotic
membranes, and pore formation
The long polypeptide of 1608 amino acids encoded by the shlA gene can be di-
vided into four functional regions. The N-terminal signal sequence serves for
the Sec-dependent export of the ShlA protein [11]. The functional regions of the
mature form have been identified by progressive C-terminal truncation of the
ShlA protein through genetic means. Reduction by 25% results in a hemolysin
that displays 20% of the wild type hemolysis rate. Shorter ShlA fragments cause
only residual or no hemolysis [23]. A 72-kDa fragment (wild type mature pro-
tein: 160,000 kDa) still binds to erythrocytes, but does not lyse them. The frag-
ment can be degraded by trypsin, whereas wild type hemolysin becomes tryp-
sin-resistant upon integration into the erythrocyte membrane [21].
An N-terminal fragment of 238 amino acids (ShlA-238) is the shortest frag-
ment that can still be secreted. ShlA-238 also converts inactive ShlA (ShlA*),
isolated from the periplasm of an ShlB-negative strain, to an active hemolysin.
This kind of activation, which we term complementation because of its similarity
to the a-complementation of b-galactosidase, was discovered when an E. coli
shlB shlA' transformant that secretes a 269-residue ShlA fragment and an E. coli
shlA transformant were streaked in parallel on a blood agar plate. None of the
individual transformants were hemolytic since the ShlA-269fragment secreted
by the ShlB protein is non-hemolytic and ShlA*, which remains in the periplasm
in the absence of ShlB, is also non-hemolytic. After incubation for several days,
a zone of hemolysis appeared around the ShlA*-producing cells, mainly on the
side where the ShlA-269 cells were located. Apparently, ShlA-269 diffused to
the ShlA* cells and gained access to ShlA*, a fraction of which was unspecifi-
cally released by partial lysis of some of the cells during the extended incuba-
tion period. This experiment was then repeated with an ShlA-269-containing
spent medium; ShlA* in a crude cell extract was rendered hemolytic. Activation
of ShlA* by ShlA-269 is reversible, as shown by the inactivation of ShlA* after
chromatographic separation of ShlA* and ShlA-269 [25]. Complementation can
also be achieved with the smaller ShlA-238 and even with a trypsin degradation
fragment of ShlA-269 that consists of only 149 N-terminal residues of ShlA [14].
These data demonstrate that ShlA-238 contains all the information for secretion
and activation of ShlA*. For complementation, secretion of ShlA-238 by ShlB is
required; periplasmic ShlA-238 synthesized in the absence of ShlB does not ac-
tivate ShlA*. Conversion of ShlA* to ShlA by the trypsin fragment also occurs
only when the trypsin fragment is isolated from a secreted ShlA polypeptide.
Phospholipase A2 inactivates ShlA-255 so that it no longer complements ShlA*
to ShlA; this indicates binding of PE to the N-terminus of ShlA, a requirement
for the activation by ShlB and for the hemolytic activity [26].
ShlA contains the sequence ANPN twice; this sequence rarely occurs in
proteins. One of the first asparagine residues of the two tetrapeptides, N-69 or
N-109, was replaced by isoleucine, and N-69 was also replaced by lysine. All
three mutant proteins are not secreted and are not hemolytic in whole cells and
227
after extraction with 6 M urea. The high specificity of these mutations is demon-
strated by a mutant with a substitution of N-111 to isoleucine in which secretion
and hemolytic activity is fully retained. shlA mutants with deletions covering N-
69 (ShlAD6897) and N-109 (ShlAD99117) are also not secreted and are non-
hemolytic. The ShlA derivatives carrying point mutations and deletions gain ac-
tivity by in vitro complementation with ShlA-269 [14]. These results clearly indi-
cate that the N-terminal region of ShlA carries the information for secretion and
activation by ShlB.
Superhemolytic ShlA mutants have been isolated by treatment of plasmid-
encoded shlA with hydroxylamine [27]. Three mutants with hemolysis rates 7-
to 20-fold higher than that of wild type ShlA were studied in some detail. Two
mutants carry single amino acid replacements, glycine to aspartate at position
326 (G326D) and serine to asparagine at position 386 (S386N), and the third mu-
tant contains two mutations (G326D and N236D). The higher activity of the mu-
tant ShlA proteins is mainly due to a greatly reduced aggregation. The half-life
of wild type ShlA activity in the spent medium is 2.5 min and that of the mutant
ShlA proteins is 10, 30, and 40 min. The superhemolytic mutants differ most
strongly from wild type ShlA by their failure to cause hemolysis at 08C when
dissolved in 6 M urea or 6 M guanidinium chloride to prevent spontaneous pre-
cipitation. At 08C, adsorption of the mutant ShlA proteins to erythrocytes is
greatly reduced. At 208C, the mutant ShlA proteins in 6 M urea or 6 M guanidi-
nium chloride lyse erythrocytes with rates similar to that of wild type ShlA.
Since residues G-326 and S-386, and perhaps N-236, contribute to the aggrega-
tion of ShlA, they define important sites of ShlA activity. The strong effects dis-
played by these mutants are surprising if one considers the conservative nature
of the amino acid replacements, and in addition the large size of ShlA.
13.2.4 The dual function of ShlB in secretion and phosphatidylethanol-
amine-dependent activation of ShlA
In an experiment to localize the hemolysin formed by an E. coli shlA shlB trans-
formant, 480 hemolytic units were determined in the spent medium, 205 units
were associated with the cell surface, and 250 units were within the cells. Using
radiolabeled ShlA, 59% of the label was found in the spent medium and 41%
was associated with the cells. In the absence of ShlB, inactive ShlA* remains
completely within cells. Immunogold electron microscopy with anti-ShlA antibo-
dies identified most of the ShlA* in the periplasm and some in the cytoplasm.
The hemolytic activity found in ShlA*-producing cells is 0.1% of the total activ-
ity of ShlA-producing cells [5]. These experiments demonstrate that ShlB is re-
quired for activation of ShlA* and for secretion of ShlA.
If activation of ShlA* is a catalytic reaction and ShlB forms pores for secre-
tion of ShlA, much less ShlB than ShlA should fulfill these two functions. This is
clearly not the case because ShlA* cannot be activated by ShlB when the crude
228
cell extracts used contain much more ShlA* than ShlB. In addition, the ShlB
clone used synthesizes an N-terminal ShlA fragment that is activated by ShlB,
which presumably further decreases the amount of ShlB available to activate
ShlA*. To demonstrate in vitro activation of ShlA* by ShlB uncoupled from se-
cretion, the genes of both proteins have to be overexpressed and the proteins
have to be synthesized in similar amounts [25]; this reflects the in vivo situation
where shlB is transcribed prior to shlA and at least as much ShlB is formed as
ShlA. However, when both ShlA* and ShlB are highly purified by ion-exchange
column chromatography, no active ShlA is formed. The missing component in
the assay is phosphatidylethanolamine (PE), which is the major phospholipid
(90%) of the E. coli outer membrane. Phosphatidylserine, the biosynthetic pre-
cursor of PE, is similarly active, but does not occur in the outer membrane;
phosphatidylglycerol has 10% of the PE activity; and phosphatidylcholine, car-
diolipin, lyso-PE, phosphatidic acid, lipopolysaccharide, and various detergents
have no effect [26]. Approximately four PE molecules bind so tightly to ShlA*
without the help of ShlB that they remain bound to ShlA* during SDS-PAGE
and are only removed by thin-layer chromatography with organic solvents.
Binding of PE to ShlA* does not convert ShlA* to hemolytic ShlA. ShlB has to
be added for ShlA*-PE activation. Removal of the fatty acid at the C2 position of
PE by phospholipase A2 inactivates ShlA and the resulting lyso-PE dissociates
from ShlA. Evidence for the in vivo relevance of these in vitro results comes
from experiments with an E. coli mutant devoid of PE due to a mutation in the
pss gene, which encodes phosphatidylserine synthase [28]. The hemolytic activ-
ity of the pss mutant transformed with an shlA-shlB-encoding plasmid is 9% of
the wild type activity and probably arises from the highly elevated phosphati-
dylglycerol content (46%), which can partially substitute for PE. This inactive
ShlA, termed ShlA8 to differentiate it from the periplasmic ShlA*, is contained
in the culture medium of the pss mutant, and the activity amounts to 16% (mea-
sured by complementation, see below) of the total hemolytic activity of a PE-
synthesizing strain [26].
13.2.5 ShlB has the potential to form membrane pores through which
ShlA might be secreted
To examine the question whether ShlB forms a pore through which ShlA is se-
creted across the outer membrane, ShlB and deletion derivatives of ShlB were
added to artificial lipid bilayer membranes and the change in the membrane
conductance was measured. This procedure has been successfully applied to
FhuA, an outer membrane receptor for various bacterial viruses, bacterial toxins,
and the iron carrier ferrichrome; the conductance was not increased unless a
surface-exposed loop of FhuA was deleted, which converted the FhuA closed
channel into a permanently open channel [29].
229
Various fragments of ShlB were excised by genetic engineering. The re-
sulting proteins were extracted in octylglucoside from the outer membrane and
then highly purified by two ion-exchange column chromatographies to remove
completely the porins, which have a high propensity to form channels in lipid
bilayer membranes. Only two deletion derivatives could be solubilized and puri-
fied. Wild type ShlB causes an increase of the conductance of 1 nS with an irre-
gular frequency and after a few milliseconds, the conductance decreases to the
zero level, which indicates that ShlB has the potential to form a channel [30].
ShlBD87153 and ShlBD65168 increase the membrane conductance stepwise
by 1.2 nS, which demonstrates formation of rather stable single channels. Fre-
quently, two to three channel opening events can be observed, followed by one
or two closing steps, and the open periods last longer than the closed periods.
The deletion derivatives support the channel-forming properties of ShlB, and
we propose that ShlB forms a closed channel which can be opened when ShlA
is secreted.
A topology model of ShlB predicts 20 transmembrane regions intercon-
nected by loops at the cell surface and short turns in the periplasm [30]. This
model is based on the reaction of an antigenic epitope inserted at one of 22 sites
along the ShlB polypeptide. Intact cells of 16 epitope mutants react with the
monoclonal antibody, which demonstrates accessibility of the epitope at the cell
surface. In six mutants, the antibody reacts only with the isolated outer mem-
brane, which indicates a periplasmic or transmembrane location of the epitope.
A computer-assisted program [31] for the prediction of membrane-spanning
b-strands of outer membrane proteins localizes the six latter epitope sites within
the membrane half oriented toward the periplasm. According to this model, the
deletions in the two ShlB mutants that form rather stable channels in artificial li-
pid bilayer membranes comprise portions of the two largest ShlB loops at the
cell surface, extending from residues 60 to 97 and 120 to 213, two transmem-
brane segments, and one periplasmic turn.
Secretion-competent but activation-incompetent ShlB mutants have been
isolated in which secretion is uncoupled from activation. Two tetrapeptide inser-
tion mutants created with a TAB linker at position 136 or 224 of mature ShlB
and a deletion mutant (ShlBD154252) secrete inactive ShlA. A TAB linker mu-
tant at position 332 has a low secretion activity and contains small amounts of
cell-bound active hemolysin. The secreted non-hemolytic ShlA proteins are
completely degraded by trypsin, in contrast to hemolytic ShlA, which is cleaved
into two fragments of 60 and 100 kDa. Secreted non-hemolytic ShlA is con-
verted in vitro into hemolytic ShlA by isolated wild type ShlB and by comple-
mentation with an N-terminal ShlA fragment of 255 residues (ShlA-255). Other
mutants secrete different amounts of active hemolysin. The secretion-competent
but activation-negative mutants define sites in ShlB which are important for ac-
tivation. That active hemolysin remains with the cells suggests that the region
around residue 332 is involved in secretion [32].
230
13.3 Pathogenicity of S. marcescens hemolysin/cytolysin
The hemolysin of S. marcescens is not only responsible for the pathogenicity of
this bacterial strain. S. marcescens is an important opportunistic pathogen that
causes respiratory and urinary tract infections, bacteremia, endocarditis, kerati-
tis, arthritis, and meningitis [33, 34]. In Section 13.2, the biochemical characteri-
zation of the hemolytic activity was presented and the activity was designated
as a hemolysin. However, this hemolysin also is a cytolysin, damages tissues
and causes the release of the inflammatory mediators leucotrienes (LTB4 and
LTC4) from leukocytes and the release of histamine from rat mast cells [35, 36].
ShlA also contributes to the uropathogenicity of the pathogenic E. coli 536/21
after transformation with the S. marcescens shlA shlB genes [37]. The major fac-
tor of this observed pathogenicity is the hemolysin/cytolysin ShlA; however, the
pathogenicity of Serratia strains is a multi-factorial process and also includes ur-
ease, fimbriae, proteases, lipase, and undefined determinants that facilitate in-
vasion. These factors act in concert, and the resulting effects are adherence,
host cell invasion, cytotoxic effects, and final cytolysis. Because the hemolytic
activity is mainly cell associated, its effect arises predominantly after adherence
of the bacteria to the host cell tissue.
13.3.1 Adherence of S. marcescens
Colonization of epithelial tissue requires adherence of bacteria to the target
cells. S. marcescens strains produce type 1 fimbriae [38, 39] or US5 pili [40] that
are involved in adherence to epithelial cells. Non-fimbriated mutants are mark-
edly decreased in their ability to adhere. Fimbriae of S. marcescens also contri-
bute to superoxide production in neutrophils [35, 36] and phagocytosis [41].
Radiolabeled bacteria adhere to purified granulocytes and are subsequently
phagocytized. The superoxide production, determined by chemiluminescence,
is dependent on the fimbriae and also on hemolysin production [35]. However,
S. marcescens also secretes exoproteases and lipases, which also induce chemi-
luminescence. The mutant strain W1436 lacks exoprotease and lipase activity,
but is still active in chemiluminescence although less than the wild type strain
5817 and is more active in causing histamine release [35]. It is not clear
whether these activities are only related to hemolysin. With purified ShlA, no
oxidative burst can be detected; instead, granulocytes continuously increase
chemiluminescence [42]. These data indicate that the oxidative burst detected
with viable hemolytic S. marcescens strains is the sum of cytotoxic effects of var-
ious products secreted by the bacteria and not only of the cytolysin.
The S. marcescens hemolysin contributes to colonization of the urinary
tract epithelium in an experimental rat model [37]. An E. coli 536/21 transfor-
231
mant that produces ShlA is five times more efficient in colonization than the
ShlA-negative recipient strain, but it is not clear whether ShlA acts as a cell-
bound adhesin or facilitates the colonization by its cytolytic activity. On epithe-
lial cell cultures (HEp-2, HeLa), ShlA-producing S. marcescens strains W1128 or
CDC04:H4, for example, are adherent [43]. A non-adherent strain, E. coli BL21,
transformed with the shlAB genes, which lead to the production of 30-fold more
hemolysin than the Serratia wild type, is not adherent. Therefore, it is clear that
adherence is not primarily mediated by the hemolysin.
The adherence of various S. marcescens strains tested in our laboratory is
mainly determined by mannose-sensitive type I fimbriae. We were able to dis-
tinguish between specific adhesion on epithelial cells and unspecific adherence
to polystyrene. LPS rough mutants showed enhanced adherence even in the
presence of mannose, indicating that not only type I fimbriae but also additional
factors, such as LPS, are involved in adherence.
13.3.2 Invasion of S. marcescens in epithelial cells
S. marcescens invades tissue cells [42]. Using the gentamycin protection assay,
cells of the intracellular S. marcescens wild type strain W225, and of strains
W1126 and CDC04:H4 have been isolated from HEp-2, HeLa, and RT112 cells.
Inhibition of eukaryotic protein synthesis by cycloheximide or alteration of the
cytoskeleton by cytochalasin D does not reduce the uptake of the bacterial cells.
The cytotoxicity of these strains mediated by ShlA reduces the number of viable
bacteria protected against gentamycin because the bacteria lyse their host cells
and become amenable to gentamycin. Isogenic hemolytic-negative S. marces-
cens W225 and W1128 strains, constructed by site-directed mutagenesis [43],
display a lower invasiveness than the ShlA wild type and are less cytotoxic in
the LDH release assay.
From the data above, it is concluded that adherence mediated by fimbriae
or pili of the bacteria directs the hemolysin to the target membranes and the
pore-forming toxin gains access to the membrane, but the hemolysin does not
mediate adherence by itself. More likely, pore formation may play a role in inva-
sion, but renders invasion studies difficult due to ShlA cytotoxicity.
13.3.3 Cytotoxic effects and cytolysis by the S. marcescens hemolysin
ShlA is inactive on keratinocytes, endothelial cells, and monocytes, all of which
are specific target cells for other bacterial toxins, such as the staphylococcal
a-toxin [4446]. ShlA induces ATP depletion and potassium efflux in epithelial
cells and in fibroblasts [47]. The depletion of cellular ATP is very strong even
232
with sublytic doses of ShlA. Treatment of HEp-2 or HeLa cells with 1 g ShlA/
ml decreases the initial ATP level to 10% within 30 min. In parallel, intracellular
potassium is released into the supernatant through pores in the plasma mem-
brane formed by ShlA. This is the signal for the Na/K-ATPase in the membrane
to transport potassium into the cell, but the leakage through the ShlA pores is
greater than the import. In an attempt to restore the intracellular potassium
pool, ATP is consumed. This effect on the Hep-2 and HeLa cells, as on erythro-
cytes, is also observed with inactive ShlA* complemented with ShlA255. Pores
formed by ShlA in the eukaryotic cytoplasmic membrane are smaller than the 1
to 3 nm estimated for ShlA pores in artificial black lipid membranes and in ery-
throcytes. The pores do not support the influx of propidium iodide (M
r
668) and
trypan blue, as measured by flow cytometry with cells depleted to 10% of the
initial ATP level. ATP depletion is not affected by osmoprotection with oligosac-
charides, which prevent ShlA-mediated cells lysis [47].
The observed ATP depletion caused by pore formation is reversible up to
80% of the initial ATP level. Depletion of the initial ATP level to less than 20%
decreases the recuperation in HEp-2 cells (40% restored) because the cells be-
gin to lyse. Restoration of the ATP level presumably occurs by the repair or clo-
sure of the ShlA-produced pores in the cytoplasmic membrane. The repair re-
quires protein synthesis, as evidenced by the failure in the presence of cyclo-
heximide [47].
13.3.4 ShlA-mediated vacuolation
The osmotic imbalance caused by ShlA, indicated by potassium efflux, ATP de-
pletion, and cell swelling, results in vacuolation and is observed with epithelial
cells, but not with fibroblasts [47]. Small vacuoles appear all over the cytoplasm;
upon prolonged incubation, the small vacuoles fuse to form large vacuoles with
irregular shape that fill the entire cytoplasm (Fig. 13.2). No lysis, as determined
by the LDH assay, can be seen during vacuolation. Isolated ShlA and hemolytic
S. marcescens strains cause vacuolation, which is thought to be the result of an
osmotically driven influx of water [47]. Oligosaccharides with a molecular mass
up to 1152 Da (maltoheptaose) strongly reduce vacuolation and cytotoxicity, and
oligosaccharides larger than 1400 Da (dextrin 15) completely inhibit vacuolation.
The data demonstrate formation of pores by ShlA and a pore size in nucleated
cells smaller than that in erythrocytes. Removal of the oligosaccharides results
in vacuolation and cytolysis of ShlA-pretreated cells. Unexpectedly, bacteria
found in the large vacuoles formed in the late stage of infection are highly mo-
bile. The reason for this is unknown, but the bacteria may undergo a transition
to a highly mobile swarming state during the infection process. In contrast to va-
cuoles formed by the Helicobacter pylori cytotoxin VacA [48] vacuoles induced
by ShlA are not acidified, vacuolation is not inhibited by bafilomycin, and va-
cuolation cannot been reversed [47].
233
Figure 13.2: Photomicrographs of untreated HEp-2 cells (A) and vacuolation of HEp-2
cells after 10 min (B) and 45 min (C) treatment with ShlA. Cells were cultured in a 24-well
plate until they reached subconfluence. Medium was supplemented with 100 l of a bac-
terial culture supernatant containing ShlA (30 HU/ml). After incubation at 378C under an
atmosphere of 5% CO
2
, cells were examined by phase-contrast microscopy at 320 x mag-
nification.
234
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14 Staphylococcal Lipases:
Molecular Characterization and Use
as an Expression and Secretion System
Friedrich Gtz* and Ralf Rosenstein
14.1 Introduction
The first bacterial lipase gene which has been ever cloned and sequenced was
the lipase gene from Staphylococcus hyicus [1]. Our interest in staphylococcal li-
pases was focused in various directions: we planned to use the staphylococcal
lipase as an expression and secretion system for the food grade S. carnosus, to
study its molecular and biochemical properties, and to study the role of these
enzymes in infection. Among the various exo-enzymatic activities of staphylo-
cocci, the acylglycerol-hydrolyzing activity of lipases and of esterases belongs to
the most frequently detected activities. Lipases or glycerol ester hydrolases (EC
3.1.1.3) are defined as enzymes that hydrolyze emulsions of lipids with long-
chain fatty acids and that show interfacial activation, i. e. a sharp increase in ac-
tivity when the solubility limit of the substrate is reached. An uncertainty exists
as to whether the staphylococcal lipolytic enzymes should be classified as li-
pases or as esterases [2]. Despite the lack of clarity, the designation lipase for
the staphylococcal lipolytic enzymes is commonly accepted in the literature and
will also be used here.
The importance of staphylococcal lipases, like other microbial lipases, re-
sults from their significance in bacterial lipid metabolism and their involvement
in pathogenic processes, and also because they are valuable tools in biotechnol-
ogy [3]. Their potential as biocatalysts is based on enzymatic features, e. g. re-
gio- and enantio-specificity, a broad substrate specificity, and the ability to cata-
lyze not only the hydrolysis, but also the synthesis of fatty acid compounds. The
increasing interest in lipases is reflected by the numerous reviews on this topic
published during the past few years; some of the reviews cover a broad spec-
trum of bacterial lipases (see, for example, [3, 4]). We will focus here on the mo-
lecular characterization of staphylococcal lipases and their use as expression
and secretion system.
238
ISBN: 3-527-30615-3
14.2 Molecular organization of staphylococcal lipases
Up to the present, the nucleotide sequences of nine lipase genes from six differ-
ent staphylococcal species have been published. Three are derived from S. epi-
dermidis (two from S. epidermidis 9 and one from S. epidermidis RP62A), two
from S. aureus (from strains NCTC 8530 and PS54), and one each from S. hae-
molyticus L62, S. hyicus DSM 20459, S. warneri 863, and S. xylosus DSM 20266
[1, 511]. For convenience, the following abbreviations will be used for the li-
pases from these strains: S. aureus NCTC 8530, SAL-1; S. aureus PS54, SAL-2;
S. epidermidis 9 (GehC), SEL-1; S. epidermidis 9 (GehD), SEL-2; S. epidermidis
RP62A, SEL-3; S. haemolyticus, SHaL; S. hyicus, SHyL; S. warneri, SWL; and S.
xylosus, SXL. Five of the sequences were determined in our laboratory.
Since the first description of the primary structure of a bacterial lipase [1],
numerous studies on the molecular and biochemical properties of SHyL have
been undertaken, making SHyL the best-studied staphylococcal lipase. From
the nucleotide sequence a protein of 641 amino acids with a predicted molecu-
lar mass of 71,382 Da was deduced. Analysis of the supernatant of the donor
strain S. hyicus by SDS-PAGE revealed a lipase with an apparent size of 46
kDa. When the SHyL gene was cloned in S. carnosus TM300, a cloning host
with low extracellular proteolytic activity, only an 86-kDa form was detected in
the supernatant. The N-terminal amino acid sequences of the 86- and 46-kDa
forms were determined and a comparison with the SHyL sequence revealed
that both forms were derived from the same precursor protein. The sequence of
the 86-kDa form of SHyL secreted by S. carnosus starts with Asn39, immedi-
ately after a predicted cleavage site (-Ala36-Glu37-Ala38-) for signal peptidase
I. The 46-kDa form produced by S. hyicus starts with Val246 [12]. It soon be-
came clear that SHyL is secreted into the medium as a pro-form (pro-SHyL),
which is subsequently processed to the mature lipase (mature SHyL). This pro-
cessing apparently does not occur in the heterologous host S. carnosus. Based
on the N-terminal sequences of the lipase forms found in the supernatants of S.
hyicus and S. carnosus, it was evident that SHyL is translated as a 641-amino
acid precursor protein with a signal peptide of 38 amino acids and a pro-pep-
tide of 207 amino acids, which is processed to the mature lipase of 396 amino
acids (Fig. 14.1).
All staphylococcal lipases are translated as a pre-pro-enzyme with a leader
peptide of 35 to 38 amino acids, followed by a pro-sequence (207 to 321 amino
acids) and the mature form, i. e. the active lipase that normally appears in the
supernatant of the producing Staphylococcus strain (383 to 396 amino acids).
A multiple alignment of the lipase sequences shows a remarkable sequence
conservation in the region covering the signal peptides, with a motif containing
the perfectly conserved residues Ser, Ile, Arg, and Lys, designated as the SIRK-
motif [1, 13]. It is still unclear, whether the strong conservation of this motif in
the signal peptide of the staphylococcal lipases reflects a biological function in
the secretion process.
239
14.2 Molecular organization of staphylococcal lipases
The various pro-peptides have no striking similarities at the sequence le-
vel, but are distinguished by their overall hydrophilic character. This hydrophili-
city of the pro-peptide is most probably the reason for the observed differences
between the calculated molecular weights of the pro-lipases and the masses es-
timated from SDS-PAGE, with the latter being significantly higher [1, 5, 13].
The molecular masses of the mature lipases, however, show a much better
agreement between theoretical and observed values.
All known staphylococcal lipases reveal the highest sequence similarity to
each other in their mature parts, with identities ranging from 50 to 81%. The
serine, aspartic acid, and histidine residues that are presumably involved in the
lipolytic catalytic triad are well conserved in all sequences.
The active site of lipases consists of three amino acid residues, Ser-Asp
(Glu)-His, which form the catalytic triad. These amino acids always appear in
this order in the amino acid sequence of the lipases, but they are distantly lo-
cated from each other. In the tertiary structure, however, they are arranged near
each other and constitute the active site. The candidates for the amino acids in-
volved in lipolysis, as revealed by sequence similarity, are Ser124, Asp314, and
His355 of the SHyL sequence. Site-directed mutagenesis of any of these amino
acids results either in a drastically reduced lipase activity or in a complete loss
of lipolysis. Since secretion or substrate specificity of SHyL are not hampered by
the mutations, these amino acids are directly involved in catalysis [14]. This sup-
ports the hypothesis that SHyL is a serine hydrolase with the catalytic triad com-
prised of Ser124-His355-Asp314. The corresponding amino acids are conserved
in all known staphylococcal lipases.
Figure 14.1: Organization of the S. hyicus lipase, ShyL, as an example for the pre-pro-
structure of staphylococcal lipases. sp, signal peptide; pp, pro-peptide; M, mature enzyme.
The residues forming the catalytic triad (S
124
, D
314
, H
355
) are indicated. The amino acids
responsible for the phospholipase (region around E
295
, K
298
, and S
356
) activity are indi-
cated below. Amino acids that are involved in calcium binding (D
109/112
) are indicated.
pro-ShyL is processed by the extracellular protease ShpII between T-V
246
; thus the mature
ShyL starts with V
1
. Near the processing site single restriction sites have been inserted fa-
cilitating fusion with heterologous genes.
240
14 Staphylococcal Lipases: Molecular Characterization
In addition, a P-loop consensus sequence, -[AG]-x4-G-K-[ST]-, is found in
all staphylococcal lipases except SEL-2 and SHaL. The P-loop motif commonly
occurs in ATP- or GTP-binding proteins [15]. It is not known, whether this do-
main plays a functional role in the lipases.
14.2.1 Processing of the pro-form
The proteolytic processing of pro-SHyL has been studied in more detail [1618].
Since the 86-kDa pro-SHyL purified from S. carnosus is processed by culture
supernatants of S. hyicus, it became apparent that the processing of the pro-form
by an extracellular protease occurs after secretion. In a co-fermentation of S. hyi-
cus with an S. carnosus recombinant strain that produces pro-SHyL, several in-
termediate degradation products of 72, 55, and 46 kDa were produced during the
processing of the pro-form to the mature lipase [12]. The 86-kDa pro-form and all
intermediary products had lipase activity. In the later growth phase, products
even smaller than 46 kDa that showed no lipolytic activity were observed, indi-
cating that a degradation beyond Val246 affects the catalytic function. The pro-
form and the 72- and 55-kDa products were predominant in the earlier growth
phases, whereas the smaller forms dominated in the stationary phase. This indi-
cates a stepwise processing starting at the N-terminus of pro-SHyL.
The proteolytic activities in the supernatant of S. hyicus were analyzed.
Two proteases, ShpI and ShpII, were identified; ShpII proved to be involved in
pro-SHyL processing [19, 20]. ShpII, a 34-kDa protein with the highest activities
at pH 7.4 and 558C, is a neutral metalloprotease that is strongly inhibited by
chelating compounds. In vitro, ShpII cleaves pro-SHyL between Thr245 and
Val246. In accordance with the proposed role and the biochemical properties of
this protease, the in vivo processing of the pro-lipase by S. hyicus could be in-
hibited in the presence of 300 M EDTA [19]. A similar processing scheme
seems to be involved in the maturation of the other staphylococcal lipases since
in most cases 40- to 48-kDa lipase forms have been detected in the supernatants
of the corresponding donor strains.
14.3 Biochemical characterization of staphylococcal lipases
The biochemical characterization of the staphylococcal lipases will be briefly
mentioned. We had a very close cooperation with the late Prof. Dr. Bert Verheij
(Rijksuniversiteit te Utrecht Centre for Biomembran and Lipid Enzymology, Nie-
derlande), and this cooperation is still ongoing with his remaining coworkers.
241
14.3.1 Ca
2+
-dependency
The activity of most of the staphylococcal lipases increases in the presence of
Ca
2+
, with EDTA correspondingly acting as an inhibitor. SAL-1, SEL-3, SHyL,
and SWL require Ca
2+
for full enzymatic activity. Correspondingly, chelating
compounds, e. g. EDTA or EGTA, act as inhibitors of these lipases. For SAL-1,
Ca
2+
can be replaced by strontium or barium without loss of activity [21]. It was
shown that Ca
2+
is most probably necessary for stabilizing the three-dimen-
sional structure of the lipase during catalysis [21] and that two aspartate resi-
dues are responsible for calcium binding in SHyL (Fig. 14.1). Site-directed mu-
tagenesis of these residues results in a loss of calcium binding, rendering the
corresponding mutant lipases still active at room temperature, but inactive at
higher temperatures [22].
14.3.2 pH optimum
SAL-1 and SEL-3 are active over a broad pH range, with an optimum around
pH 6 [11, 21]. Accordingly, both lipases are stable under acidic conditions,
whereas they are inactivated at pH values above 10. This preference of acidic
conditions is quite unusual among bacterial lipases, which in most cases exert
their highest activities at alkaline pH. For SHyL, a pH optimum of 8.5 has been
reported [23].
14.3.3 Substrate preferences
The differences in pH dependency of SAL-1, SEL-3, and SHyL are also reflected
in the substrate preferences of these lipases. SAL-1 and SEL-3 exhibit a strong
preference for glycerides with short-chain fatty acids. Both lipases have a signif-
icant bias towards substrate molecules with butyric acid esterified to glycerol, p-
nitrophenol, or umbelliferone. Corresponding ester compounds with an acyl
chain length of one methyl group above or below this size, e. g. triacetylglycerol
or tripentanoylglycerol, are poorly hydrolyzed by these enzymes [11, 21, 23].
Until now, SHyL is unique among bacterial lipases in having a very broad sub-
strate spectrum ranging from lipids of various chain lengths to phospholipids
and lysophospholipids [11].
242
14.3.4 Molecular basis of the phospholipase activity of SHyL
Several studies have been undertaken to identify elements in the primary struc-
tures of SHyL that are responsible for its exceptional enzymological properties
[13, 2426]. By construction of a hybrid lipase in which the C-terminal 146
amino acids of SHyL are replaced by 145 amino acids from the C-terminus of
SAL-1, it was demonstrated that the structural elements providing the phospho-
lipase activity must reside within the exchanged element [13]. Attempts were
then made to more narrowly define the regions involved in phospholipase activ-
ity and chain-length selectivity of SHyL by van Kampen et al. [2426]. Various
chimeras between SHyL and SAL-1 were generated by in vivo recombination
and were tested for activity on phospholipids and p-nitrophenyl esters of differ-
ent chain lengths. Three elements in the C-terminal region of SHyL necessary
for phospholipase activity were identified. Furthermore, a central element of
about 70 amino acids was shown to be essential for the chain-length selectivity
of this enzyme. In a more recent study, small stretches of amino acids were ex-
changed between SAL-1 and a synthesized part of SHyL comprising the pre-
viously identified elements to localize single amino acid residues involved in
phospholipase activity. A serine immediately following the catalytically active
histidine had already been shown to be involved in hydrolysis of phospholipids;
van Kampen et al. could now identify a stretch of polar amino acids (position
293300) which, when exchanged for the corresponding, more hydrophobic re-
gion of SAL-1, led to a drastic decrease of phospholipase activity. Two essential
residues, E295 and K298, were identified within this stretch by introducing point
mutations; K298 was shown to be the major determinant for phospholipase ac-
tivity. Interestingly, SAL-1 was made 23-fold more active towards phospholipids
by the introduction of the reverse mutations, thus supporting the determined
role for these amino acid residues. The authors concluded from their results that
the polar stretch between amino acids 293 and 300 lies within a substrate bind-
ing pocket and is involved in the interaction with the polar head group of phos-
pholipids [26].
14.3.5 The catalytic mechanism
Several efforts have been made to crystallize staphylococcal lipases, but the re-
sulting crystals have been of poor quality [27]. Experiments with covalent inhi-
bitors that inhibit SHyL only in the presence of micelles and results obtained
with the substrate analogue p-nitrophenyl-N-alkylcarbamate further support
the hypothesis that SHyL has a lid covering its active site [28, 29].
243
14.4 Role of the pro-peptide region
According to the sequence comparisons, all staphylococcal lipases are predicted
to be primarily synthesized as pre-pro-lipases. While the function of the leader
peptide in secretion is obvious, the role of the pro-peptide remained unclear.
One hypothesis proposed a function in masking the enzyme activity until the se-
cretion process is completed in order to protect the producing cell from detri-
mental effects of the lipase activity. However, the pro-form of SHyL is almost as
active as the mature protein and the pro-lipase can be synthesized intracellu-
larly without hampering the vital functions of the producing strain (G. Thumm,
personal communication). Another possible function is the involvement in secre-
tion, probably as an intramolecular chaperon.
In order to evaluate whether the two functions of the pro-peptide are re-
presented by different sections of the pro-peptide, several derivatives of SHyL
with deletions in the pro-peptide region were constructed and tested for lipase
production in S. carnosus [18]. The results obtained with these lipase mutants
indicate that the SHyL pro-peptide may have two functional domains with each
one located in one half of the pro-region. The N-terminal part seems to be im-
portant for lipase activity and the C-terminal part for translocation and stability.
A stabilizing effect of the SHyL pro-peptide has also been observed in an ex-
periment where OmpA of E. coli is fused to the pre-pro-portion of SHyL; in con-
trast to the construct without the lipase secretion signals, no proteolytic degra-
dation occurred after secretion by S. carnosus [30]. A number of experiments
designed to address the question whether the pro-peptide could act also in trans
indicated that the pro-region has to be covalently attached to the mature protein
in order to exert its beneficial effects on translocation, stability, and activity (G.
Thumm, personal communication).
14.5 The use of ShyL as expression and secretion system
The role of the pro-peptide region of SHyL was more thoroughly investigated. A
plasmid carrying the cloned gene encoding SHyL was used for the construction
of various secretion vectors [16]. The Escherichia coli b-lactamase gene (bla) lack-
ing its own signal sequence was fused at various sites along the gene regions en-
coding the pro-peptide and mature forms of SHyL. The amount of b-lactamase
secreted into the supernatant of S. carnosus clones harboring the recombinant
plasmids was measured; only those constructs having at least 160 aa of the SHyL
pre-pro-region fused to b-lactamase secreted an amount of fusion protein com-
244
parable to that of native lipase. All hybrid proteins having a smaller portion of the
lipase fused to b-lactamase remained in the membrane fraction, which indicates
a defect in the translocation, and were prone to extensive proteolytic degrada-
tion. These results support a dual role for the SHyL pro-peptide: an involvement
in protein translocation and a role in stabilization against proteolytic degradation.
Because of these encouraging results, we fused the pre-pro-portion of
SHyL to several other heterologous proteins such as pro-insulin [31] and malaria
antigen [32]. In all cases, the heterologous proteins were successfully and in
good quantities secreted by S. carnosus, thereby showing that the results ob-
tained with E. coli b-lactamase are generally applicable.
Based on the SHyL-gene, we also have constructed a series of plasmid vec-
tors for gene expression and cloning in staphylococci. pPS11 is a promoter probe
plasmid containing a promoterless SHyL-gene. Insertion of a promoter-bearing
DNA fragment at the single BamHI site turns on SHyL-gene expression [33].
The lipase activity can be easily determined to estimate the strength of the in-
serted promoter. pPS11 served also as a basis for the construction of vectors
which allow xylose-inducible gene expression in S. carnosus. In plasmid
pCX15, the SHyL-gene is under transcriptional control of the repressor, XylR,
with the XylR target sequence the xylA promoter/operator. Again, the single
BamHI site in front of the SHyL-gene RBS also makes it possible to put other
promoterless genes under transcriptional control of XylR [33].
The expression vector pCX15 was the basis for improvement and for
further heterologous protein secretion.
In Gram-positive bacteria, a number of surface proteins are covalently an-
chored to the cell wall by an ubiquitous mechanism involving a specific, C-term-
inal sorting signal [34]. This reaction is catalyzed by the sortase.
Normally, non-enzymatic proteins such as IgG- or fibronectin-binding pro-
teins are linked in this way to the cell wall. We asked the question whether an
enzyme, such as ShyL, can be covalently anchored to the cell wall in an active
state [35]. To achieve cell wall immobilization ShyL was fused with the C-term-
inal region of S. aureus fibronectin binding protein B (FnBPB). Indeed, expres-
sion of the hybrid protein in S. carnosus resulted in efficient cell wall anchoring
of enzymatically active lipase. In early stationary cells, 95% of total lipase activ-
ity were covalently anchored to the cell wall, 5% were found to be set free into
the culture supernatant. The cell wall-immobilized lipase (approximately
10000 molecules per cell) retained more than 80% of the specific activity com-
pared to the unmodified S. hyicus lipase secreted by S. carnosus cells. After sol-
ubilization of the hybrid protein by lysostaphin treatment [36] of the cell wall, its
specific activity was indistinguishable from that of the unmodified lipase. These
results demonstrated for the first time that it is possible to immobilize soluble
enzymes on the cell wall of S. carnosus without radically altering their catalytic
activity [35]. Even if the pentaglycine anchor structure of the cell wall was modi-
fied anchoring to the cell wall was not affected [37]. In the meantime, several
other groups [38] are using the ShyL expression and secretion system success-
fully for heterologous protein secretion in S. carnosus which has been worked
out during the collaborative research centre 323.
245
14.5 The use of ShyL as expression and secretion system
14.6 Concluding remarks
Although our knowledge of staphylococcal lipases has steadily increased during
the past years, many aspects of these interesting enzymes remain to be investi-
gated. Very little is known about the regulation of lipase synthesis in staphylo-
cocci. There are controversial reports whether lipase is subject of the global reg-
ulatory system agr (accessory gene regulator), which is known to regulate the
expression of the genes of several exoproteins and cell-wall-associated proteins
in staphylococci or of the alternative sigma factor Sig
B
.
Staphylococcal lipases and especially the ShyL expression and secretion
system have a good potential being applied in biotechnology. However, we
have to spend more work on overproducing the enzymes.
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cus carnosus surface display system by substitution or deletion of a Staphylococcus
hyicus lipase propeptide. FEMS Microbiol. Lett. 179, 131139.
248
15 A Multienzyme Complex Involved in
Murein Synthesis of Escherichia coli
Moritz von Rechenberg, Waldemar Vollmer, and Joachim-Volker Hltje*
15.1 The murein sacculus, a growing molecule
Because of the high intracellular turgor pressure impinging on the cytoplasmic
membrane most bacterial cell walls are mechanically reinforced by a unique
cross-linked biopolymer called murein (peptidoglycan) [13]. Murein consists of
poly-(GlcNAc-b-1,4-MurNAc)glycan strands, which are substituted at the lactyl
group of the muramic acid by short peptides. The presence of a diamino-amino
acid (diaminopimelic acid in the case of E. coli) makes it possible that two pep-
tide moieties can be cross-linked. Cross-linkage is achieved by transpeptidation
of the terminal carboxyl group of one peptide side chain to the free e-amino
group of the diamino-amino acid present in the neighboring peptide side chain
(see Fig. 15.1). Thereby the typical murein netting is formed.
Importantly, the murein net is tailored into a bagshaped structure, called
sacculus, which completely encloses the cell and thus establishes a kind of exo-
skeleton. It follows that growth of the bacterial cell depends on the enlargement
and division of the murein sacculus [1, 2, 4, 5]. These processes must be consid-
ered to be one of the greatest technical challenges for the cell because of two
major problems that have to be solved. First, a structure under extreme tension
has to be enlarged and divided without allowing the sacculus to burst. This pro-
cess is further aggravated since the sacculus of E. coli is believed to be extre-
mely thin, in major parts being a monolayer only [6]. Second, the shape of the
sacculus that determines the species-specific morphology of the bacterium
needs to be maintained and transmitted precisely from generation to generation.
Recently, as will be discussed below, experimental evidence has been obtained
suggesting that this is accomplished by a multienzyme complex, a murein
synthesizing machinery that follows a particular growth strategy [4, 7].
* Max-Planck-Institut fr Entwicklungsbiologie, Abt. Biochemie, Spemannstrae 35,
D-72076 Tbingen
249
ISBN: 3-527-30615-3
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15 A Multienzyme Complex Involved in Murein Synthesis of Escherichia coli
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251
15.1 The murein sacculus, a growing molecule
15.2 Murein growth is accompanied by massive turnover
Growth of the murein sacculus of E. coli has been studied by pulse-chase label-
ing in a diaminopimelic acid auxotrophic strain using
3
H-diaminopimelic acid
[810]. It became clear that about 4050% of the incorporated label is released
during one generation. However, only 48% of the turnover products are lost to
the medium [8, 9], the bulk of the muropeptides accumulating in the periplas-
mic space is taken up across the cytoplasmic membrane into the cytoplasm and
reused for the synthesis of new murein precursor molecules [9, 11, 12]. The re-
cycling of the turnover material is very efficient and quite a number of proteins
are involved (Fig. 15.2). Specific uptake systems have been identified for the up-
Figure 15.2: Murein turnover and recycling in E. coli.
252
take of non-degraded muropeptides (AmpG) [13] as well as for free peptides
(OppA and MppA) [11, 12]. The released turnover products are degraded by
amidases, glucosaminidases and L,D-carboxypeptidases, both in the periplasm
(AmiA) [14] and cytoplasm (AmpD, NagZ, LdcA) [1517]. The free peptides are
then hooked by a specific ligase (Mpl) to UDP-MurNAc [18]. Recently, we have
shown that it is essential that only tripeptides are added to UDP-MurNAc and
no tetrapeptides [17]. In the latter case the resulting UDP-MurNAc-tetrapeptide
intermediate cannot be further processed to the pentapeptide precursor since
the final step is the addition of a D-alanyl-D-alanine dipeptide to the tripeptide
intermediate. Increased incorporation of tetrapeptide precursors into the murein
sacculus results in a decrease of the cross-linkage and causes autolysis of the
cells. A deletion mutant in the L,D-carboxypeptidase LdcA was found to start
lysing when entering the stationary phase of growth. Therefore, the activity of
LdcA is essential for growth of E. coli [17].
15.3 Enlargement and division of a stress bearing structure
The fate of the labeled murein before its release was analyzed by completely
hydrolyzing the murein with lysozyme which was followed by separation of the
degradation products (muropeptides) by reversed phase high-pressure liquid
chromatography [3, 10]. It was found that the structure, i. e. the muropeptide
composition, of the murein changes dramatically during growth. In particular
one structure attracted our interest: cross-linked muropeptides of the type
tris(GlcNAc-b-1,4-MurNAc-peptide), called trimers, which represent the con-
necting piece of three glycan strands. For structural reasons the three glycan
strands at this site have to be placed in two levels, that is one strand either
above or below of the plane defined by the two other strands (Harald La-
bischinski, personal communication). Therefore, one wonders what the meaning
of such a structure could be in a monolayered murein net. It is a minor compo-
nent (about 5%), but interestingly it is completely cleaved in one generation;
thus it represents an intermediate structure [10].
In order to explain murein turnover, the function of the trimers and to ex-
plain how the netting of the monolayered stress bearing murein sacculus might
be enlarged by a mechanism that avoids any risk of lysis, we proposed a strategy
quite analogous to the established growth mechanism of Gram-positive rods [7].
The basic concept of the strategy has been characterized by the motto make be-
fore break [19]. Accordingly, new material is firstly hooked underneath the
stress-bearing layer(s) before it is inserted as a result of the cleavage of some cri-
tical bonds in the stressed murein to which it has been attached. The outcome of
such a mechanism is that the murein sacculus grows from the inside to the out-
side. Whereas complete new layers are added to the stress bearing layers of the
253
15.3 Enlargement and division of a stress bearing structure
thick murein sacculus of Gram-positive bacteria, we propose that only small
patches of new material are added to the thin, monolayered murein sacculus of
Gram-negative bacteria such as Escherichia coli. The newly added patch could
be a murein triplet, that is three murein strands cross-linked to one another (Fig.
15.1). The triplet is hooked to the peptidyl moieties belonging to the neighboring
murein strands to the right and left of a single strand, called the docking strand,
in the growing murein sacculus. Specific hydrolysis of the docking strand results
in the triplet being pulled into the stress bearing layer of the sacculus. Hence,
three new strands replace one old strand. Such a three-for-one growth mechan-
ism is in accordance with the degree of murein turnover. The model also explains
the function of the trimeric muropeptide structure: it represents the attachment
site of the incoming new murein triplets. In addition, it becomes clear why the tri-
mers are all cleaved during growth: this is needed to release the docking strand
from the murein netting (see Fig. 15.1). In analogy to the growth mechanism of
the multi-layered murein of Gram-positive bacteria, the new triplets are added
from the site of the cytoplasmic membrane to which the murein polymerases are
anchored, whereas the old docking strand is degraded from the opposite side by
enzymes bound to the outer membrane. It has recently been detected that most
of the major murein hydrolases of E. coli, the membrane-bound lytic transglyco-
sylases MltA and MltB, are lipoproteins residing in the outer membrane [20, 21].
To find out how the action of the murein polymerases and hydrolases is coordi-
nated is of utmost importance for our understanding of the controlled growth of
the bacterial cell wall. As discussed below, it seems that both classes of enzymes
are combined in a multienzyme complex.
15.4 Interaction of murein hydrolases and synthases
as indicated by affinity chromatography
In order to obtain some experimental data whether the murein hydrolyzing en-
zymes directly interact with the murein polymerases, specific affinity chromato-
graphy was employed to detect protein-protein interactions. This was done by
using various enzymes expected to be involved in murein growth, as specific li-
gands for affinity chromatography. The enzymes were coupled to activated Se-
pharose [2227]. A summary of the obtained results is given in Table 15.1. It was
found that lytic transglycosylases, the soluble Slt70 and the membrane-bound
MltA and MltB and probably also D,D-endopeptidases show specific affinity to
some murein polymerases. This included penicillin-sensitive enzymes that be-
long to the family of penicillin-binding proteins (PBPs). Each of Slt70, MltA and
MltB coupled to Sepharose specifically retained the bifunctional transpeptidase/
transglycosylases, PBP1B and PBP1C, as well as the monofunctional transpepti-
254
dase PBP3. MltA showed additional affinity to the bifunctional PBP1A and the
monofunctional transpeptidase PBP2.
At least one structural protein seems to interact with this group of enzymes
[25, 26]. A protein called MipA (MltA interacting protein) could be detected,
that not only specifically interacted with MltA but in addition with the murein
polymerase PBP1B. No enzymatic activity could be demonstrated for MipA. The
specific protein-protein interactions of murein polymerases and hydrolases
found in vitro may indicate that a multienzyme complex is formed in vivo. This
possibility was therefore studied in more detail employing additional methods.
15.5 Dimerization of the bifunctional transpeptidase/
transglycosylase PBP1B
A major disadvantage of the affinity chromatography method is that binding
constants cannot be determined. Therefore, surface plasmon resonance (SPR)
experiments were performed using a BIAcore 2000 equipment. In the case of
the affinity chromatography experiments the proteins were coupled to activated
Sepharose through primary amino groups present in the proteins. As a result,
the immobilized ligands were oriented in a random fashion on the surface of the
Table 15.1: Summary of the results obtained by affinity chromatography.
Retained protein (specificity
a
) Immobilized proteins
Slt70 MltA MltB PBP1C PBP7
PBP1A (TG/TP) +
b

PBP1B (TG/TP) + + + + +
PBP1C (TG/TP?) + + + / +
PBP2 (TP) + +
b
+
PBP3 (TP) + + + + +
PBP4 (EP) + +
PBP5 (CP)
PBP6 (CP)
PBP6B (CP)
PBP7/8 (EP) +
Slt70 (LT) /
MltA (LT) / + +
MltB (LT) /
a
TG/TP, bifunctional transglycosylase/transpeptidase; TP, transpeptidase; EP, endopepti-
dase; CP, carboxypeptidase; LT, lytic transglycosylase;
b
in the presence of periplasmic proteins.
255
15.5 Dimerization of the bifunctional transpeptidase/transglycosylase PBP1B
matrix material. By contrast, a more controlled method was established for the
BIAcore measurements. We took advantage of the covalent binding of the PBPs
to penicillin. Ampicillin that carries a free amino group was first linked to the
surface of a biosensor chip by a standard amino coupling method. The immobi-
lized ampicillin could then be used to capture and covalently bind PBPs, even
from unfractionated protein preparations [25, 27]. The method not only results
in uniformly oriented ligands but in addition stabilizes the protein by the pre-
sence of a substrate analogue in the active site of the enzyme protein. An inter-
esting finding was that PBP1B binds to immobilized PBP1B (Fig. 15.3a), indicat-
ing dimerization, as has previously been shown by other methods [28].
15.6 Reconstitution of the core particle of a murein
synthesizing machinery
When the interaction of PBP1B with MltA as observed by MltA-Sepharose affi-
nity chromatography [26] was investigated by the SPR method, no specific sig-
nal could be obtained. It was concluded that the interaction depended on addi-
tional specific factors besides PBP1B and MltA. Indeed, addition of MipA re-
sulted in a signal that indicated the formation of a complex between the murein
polymerase PBP1B, MipA, and the murein hydrolase MltA. MipA probably
functions as a scaffolding protein in the assembly of the protein complex. The
kinetics of the formation of the trimeric complex indicates a binding constant of
about 0.850 0.057 M [25, 27] (Figs. 15.3b and c). This value is comparable
with those for cell adhesion molecules.
15.7 Proposed structure of a hypothetical holoenzyme of
murein synthesis
On the basis of the results obtained by affinity chromatography and SPR studies
a hypothetical multienzyme complex can be compiled that could enlarge the
murein sacculus by the safe three-for-one growth strategy [4, 7, 29]. It is as-
sumed that murein polymerizing and murein hydrolyzing enzymes are com-
bined in a kind of yin yang complex. As mentioned above, protein-protein in-
teractions between the bifunctional transpeptidase/transglycosylase PBP1B, the
transpeptidases PBP3 and PBP2 as well as the D,D-endopeptidases PBP4 and
256
Figure 15.3: Kinetic measurements of the binding of (a) PBP1B and (b) of the simulta-
neous binding of MipA together with MltA to immobilized PBP1B. Panel a: At a flow rate
of 10 ml/min 100 ml PBP1B (3 mg/ml) were injected to differently modified flow cells. Curve
1: PBP1B immobilized to ampicillin coated CM5 sensorchips, prepared as described pre-
viously [25]; curve 2: PBP1A immobilized to ampicillin coated CM5 sensorchips; curve 3:
as a control the ampicillin coated sensorchip was digested with b-lactamase; curve 4: the
sensorchip was blocked with ethanolamine. Panel b: Different amounts of an equimolar
mixture of MipA and MltA (ranging from 30 nM to 2.46 mM) were injected at a flow rate
of 10ml/min to a PBP1B loaded sensor chip (see panel a). Panel c: A Scatchard analysis of
the experiment depicted in b is shown.
257
15.7 Proposed structure of a hypothetical holoenzyme of murein synthesis
PBP7 and the lytic transglycosylases Slt70, MltA, and MltB have been demon-
strated partly by affinity chromatography, partly by BIAcore measurements, and
to some extent by both methods. In addition, a complex consisting of PBP1B and
MltA has been reconstituted on the surface of a biosensor chip in the presence
of MipA. Dimerization of PBP1B, PBP3, and PBP4 has been obtained by various
methods in different laboratories [28, 30]. Therefore, the following speculative
complex is supported by quite a number of experimental results (Fig. 15.4). It is
assumed that the synthesizing part of the complex consists of a dimer of a bi-
functional transpeptidase/transglycosylase (TP/TG) that synthesizes two murein
strands and crosslinks these strands by transpeptidation from two sides with a
single strand independently synthesized by a transglycosylase (TG). The pro-
duct would be three murein strands cross-linked with one another, a murein tri-
plet. Also part of the synthesizing sub-complex would be a dimer of a mono-
functional transpeptidase such as PBP3 or PBP2 (TP). The transpeptidases
would be responsible for hooking the murein triplet by transpeptidation under-
Figure 15.4: Hypothetical multienzyme complex supposed to be involved in growth of
the murein sacculus of E. coli. The symbols are the same as in Fig. 15.1. The circles repre-
sent the enzyme molecules: LT, lytic transglycosylase; EP, D,D-endopeptidase; TP, D,D-
transpeptidase; TP/TG, D,D-transpeptidase/transglycosylase; TG, transglycosylase. The
triangle indicates the presence of structural proteins in the complex, such as the recently
identified MipA [25].
Ala
Glu
A
2
pm
Ala
stress-bearing layer
Ala-Glu-A 2pm-Ala A2pm-Glu-Ala
Ala-Glu-A 2pm Ala-A2pm-Glu-Ala
docking strand
NH
2
2
NH
murein triplet
A2pm-Glu-Ala Ala
Ala
A2pm
Glu
Ala
Ala
HN
2
-
Ala
Ala
A2pm
Glu
Ala
HN
2
-
Ala
Glu
A
2
pm
Ala
Ala Ala-Glu-A 2pm
TP
TP/TG
TG
EP
LT
258
neath a murein strand in the murein sacculus. The murein degrading part of the
complex would be responsible for the specific hydrolysis of the docking strand.
A most efficient degradation would be accomplished by a concerted action of ly-
tic transglycosylases (LT) and endopeptidases (EP). Since PBP2 is known to be
responsible for cell elongation and PBP3for septum formation, it is assumed that
two similar complexes exist that differ by the presence of PBP2 in the cell elon-
gation machine and PBP3 in the cell division machine, respectively [4].
15.8 Recent insights in the mechanism of growth of the
murein sacculus reveal novel targets for antibiotics
Being an essential bacterial structure and being unique to prokaryotes, the mur-
ein sacculus and murein metabolism in general remain among the most attrac-
tive targets for antibiotics. Despite of this advantage a surprisingly low number
of new targets have been established in the last years that are involved in mur-
ein metabolism. Even more surprising is that no simple method has been set up
that would allow a high throughput screen for inhibitors of the transglycosyla-
tion reaction. This step is responsible for the polymerization of the precursors to
murein strands, a process that is as important for the formation of the murein
sacculus as the transpeptidation reaction, which is the well known target of the
still most important b-lactam antibiotics. Studying different possibilities to im-
mobilize murein-metabolizing enzymes, we found that PBPs 1A, B, and C bind
to moenomycin coupled to affigel [27]. This finding prompted us to establish an
assay that is based on a competition of test compounds for the binding of moe-
nomycin to the transglycosylation site of the essential PBPs 1A and 1B [31]. The
assay, which can be adapted to high throughput screens, might help to identify
novel structures that could be the basis for new murein synthesis inhibitors of si-
milar therapeutic importance as penicillins.
It is obvious that the assembly pathway of the multienzyme complex, a
multistep process with a number of sites that can be inhibited, could be a target
for powerful antibacterial agents. The observed dimerization of PBP1B is likely
to be a crucial step in the formation of the multienzyme complex. We therefore
considered this reaction to be well suited for a screening of inhibitors by em-
ploying the established BIAcore assay [27, 32]. In collaboration with G. Jung
and K.-H. Wiesmller (Institute for Chemistry, University of Tbingen) first ex-
periments were done with chemically synthesized peptides from a library that
consisted of random peptides from five up to 15 amino acids. Binding of PBP1B
to immobilized PBP1B was followed in the presence of various peptide size
classes at a concentration of 400 mg/ml. A significant decrease of the control sig-
nal was found with peptides larger than eight amino acids. The nonapeptides
259
15.8 Recent insights in the mechanism of growth of the murein sacculus
caused an inhibition by more than 60%. To get an idea of the specific structure
(sequence) that interferes with the dimerization, a library of nonapeptides was
tested. It turned out that a specific position of leucine but not lysine or gluta-
mate in the nonapeptide was most effective in interfering with the dimerization
of PBP1B. As shown in Table 15.2, the nonapeptide X
4
LeuX
4
was also inhibiting
the formation of the trimeric complex consisting of PBP1B, MltA, and MipA. Un-
fortunately, when tested in ether-permeabilized cells, the peptides did not affect
the rate of murein synthesis. This might be explained by the fact that the ether-
treated cells do not grow and therefore synthesize murein only with the help of
the existing, already assembled complexes. It is possible that the peptides do
block the formation of the complex but cannot dissociate preexisting complexes.
Additional in vivo experiments are needed to characterize the antibacterial po-
tency of these peptides. The relatively simple BIAcore-based assay system re-
presents a promising approach to screen for inhibitors of the formation of the
murein synthesizing multienzyme complex and thus for general inhibitors of
murein synthesis and bacterial growth.
Table 15.2: Complex formation in the presence of peptides.
Peptide (400 mg/ml) Formation of protein complex (%)
PBP1B/MipA/MltA MipA/MltA
none 100 100
X
4
LX
4
18 100
X
4
KX
4
108 95
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262
16 The Changing Path of Hopanoid Research:
From Condensing Lipids to New Membrane
Enzymes
Karl Poralla*
16.1 Introduction
A group of important, nearly ubiquitous components in eukaryotic membranes
are the sterols, e. g. cholesterol 1, ergosterol, stigmasterol, or sitosterol. In addi-
tion to their role as precursors for hormones etc., they condense the lipid part of
cell membranes to physiologically appropriate values of viscosity. This contrasts
with the prokaryotes, where with very few exceptions sterols are absent. In pro-
karyotes the condensing function in the membrane is very often adopted by ho-
panoids [31, 32].
Hopanoids 2, 3, 5 show structural similarity to sterols. Both classes of com-
pounds are polycyclic triterpenoids with one or more hydrophilic groups. Their
rigid, polycyclic structure and hydrophobicity is a prerequisite for their conden-
sing function. Glycerolipids, in contrast, have mobile acyl chains and they there-
fore comprise the fluid (less viscous) fraction of the membrane lipids.
Tbingen
263
ISBN: 3-527-30615-3
At the beginning of our work hopanoids had been detected in a few bac-
terial species. Since then they have been found in about 50% of the bacteria ex-
amined, bringing the total to about 100 bacterial strains and species [31]. Poten-
tial hopanoid synthesizing bacteria were brought to light in two genome se-
quencing projects, for Rhizobium NGR234 [33] and Streptomyces coelicolor [34].
In these bacteria the hopanoids are not synthesized under conventional labora-
tory conditions (unpublished results).
This review of our work starting from about 1990 will focus mainly on the
cyclization of squalene to hopene, describing studies from the purification of
squalene-hopene cyclase to its X-ray structure [19, 42] and to directed mutagen-
esis at the active site leading to new enzymes. It will also describe how this
work has contributed to the understanding of cyclic triterpene formation, a clas-
sical field in natural product chemistry. It will also touch on the question of the
biosynthetic precursor of hopene and what is known about the elongation reac-
tions in hopanoid biosynthesis, leading for example to tetrahydroxybacterioho-
pane 5.
264
16 The Changing Path of Hopanoid Research
16.2 The cyclization reaction
In biosynthesis of sterols and hopanoids the key reaction is the cyclization of the
linear polyene compounds squalene and (3S)-2,3-oxidosqualene to hopene and
sterols (Fig. 16.1). An acidic group at the active sites of the corresponding cy-
clases will initiate the reactions by protonation. A carbocationic cyclization cas-
cade will follow and finally the positive charge will be eliminated by proton ab-
straction, or hydroxyl ion from water will be added. In the case of sterol cycliza-
tion ring B of the protosteryl cation is in the strained boat conformation. After
the formation of the protosteryl cation, a rearrangement of hydride and methyl
groups takes place and finally a proton is eliminated.
From mere chemophysical considerations it is evident that the cyclization
has to occur in or at the membrane because the substrates are very hydrophobic
and are therefore dissolved in the hydrophobic inner part of the membrane. It is
also evident that the unordered squalene and oxidosqualene molecules must
be very specifically folded (chaperoned) just prior to cyclization. From chemical
considerations it is obvious that a carbocationic cyclization cascade has to occur
in a shielded milieu where side reactions, especially with water molecules, are
excluded.
In the enzymatic assay it can easily be observed that the cyclization reac-
tion runs to completion without any addition of an energy-rich compound, be-
cause the reaction is highly exergonic. The cyclization reaction ranks among the
Figure 16.1: Reactions catalyzed by squalene-hopene cyclase (SHC) and 3S-(2,3)-oxido-
squalene-lanosterol cyclase (OLaC).
265
16.2 The cyclization reaction
most complex one-step reactions in chemistry and biochemistry. In hopene for-
mation twelve covalent bonds are altered, five cycles formed and nine stereo-
centers established. Despite the high number of stereocenters the specific for-
mation of one main product out of more than 128 possible stereoisomers is ac-
complished.
The squalene-hopene cyclase (= SHC) is the best studied example of a tri-
terpene cyclase and opens the door to understand the reactions of more than
hundred triterpene cyclases which contribute to the high diversity of natural
products in higher plants and ferns.
16.3 Purification of squalene cyclases
When we started our biochemical work on hopene cyclization no triterpene cy-
clase had been successfully purified to homogeneity and no gene of a triterpene
cyclase had been cloned.
In 1987 we started to purify several SHCs in order to clone and sequence
the respective genes and to determine conserved amino acids for site directed
mutagenesis. The ultimate aim was to alter product specificity of the SHC and
thereby to understand the catalysis of hopane skeleton formation. The Alicyclo-
bacillus acidocaldarius SHC was the first triterpene cyclase for which purifica-
tion successfully has been achieved [1]. The decision to investigate the SHC
from this bacterial species (growth optimum at 60
o
C) turned out to be a lucky
choice; the purified enzyme was easy to handle and stable at room temperature.
Because the triterpene cyclases are membrane bound enzymes a critical
step in all purification procedures was the solubilization of the enzyme by var-
ious detergents from the membrane fraction and to find the appropriate deter-
gent for each cyclase. Triton-X-100, CHAPS, or octyl-thioglucoside proved to be
very efficient detergents [13]. Worthwhile to mention is the purification of Rho-
dopseudomonas palustris SHC on a Blue Sepharose column in the last step [8];
Blue Sepharose is normally used for purification of enzymes with a NAD
+
/
NADP
+
binding site. A summary of the different purification procedures is given
in Table 16.1. We observed that each cyclase purification is an individual proce-
dure reflecting the different membrane environments and specific requirements
of the enzymes. In a later stage of the project we purified cloned A. acidocaldar-
ius SHC after insertion of a His-tag at the N-terminus by Ni
2+
-agarose affinity
chromatography [16].
Critical for the purification was the development of a rapid enzyme assay
procedure. We started with a radio thin-layer chromatography method using tri-
tiated squalene. The reaction products were scraped off the thin-layer plates
and the radioactivity was counted [35]. Later, we switched to a thin-layer chro-
matography scanning procedure which proved to be very inefficient because of
266
the low radiation energy of the tritium label [6]. Finally, we developed a gas-
chromatographic assay with unlabelled squalene. This is now the established
method for the SHC assay [1].
We purified the SHC to homogeneity from A. acidocaldarius and R. palus-
tris [1, 8]. Numerous attempts to purify Zymomonas mobilis SHC failed [6]. The
squalene-tetrahymanol cyclase from the protozoon Tetrahymena thermophila
has been efficiently purified in a cooperation project with Guy Ourisson [3]. Tet-
rahymanol 4 is an isomer of diplopterol 3, and therefore, squalene-tetrahymanol
cyclase is a member of the triterpene cyclases.
16.4 Properties of purified cyclases
The molecular weight of all the cyclases purified by our group was about 70
kDa [1, 3, 6, 8]. This was in accordance with the size of the genes. This value
contrasted very much to the values for oxidosqualene-sterol cyclases (= OSCs)
published by other groups which were in the range of 25 to 50 kDa. The correct
value for OSCs is in fact in the range of 86 kDa [36, 37].
SHCs possess a certain degree of unspecificity in that they yield several
products in vitro and also in vivo. Besides hopene 2 (= hop-21,29-ene) it pro-
duces 510% diploterol 3 (= hopane-21-ol). This percentage is independent of
the pH in the assay mixture. R. palustris SHC produces even 50% of diplopterol
and no tetrahymanol 4, although diplopterol and tetrahymanol have been iso-
lated from cells [2], and therefore a production of tetrahymanol in vitro has been
expected. The unspecificity of the SHC is supposed to be biologically meaning-
ful, because diplopterol and presumably also tetrahymanol are able to condense
membranes similar to elongated hopanoids. In contrast, the hydrocarbon
Table 16.1: Solubilization and applied adsorbents for purification of bacterial SHCs and
squalene-tetrahymanol cyclase from Tetrahymena thermophila.
A. acidocaldarius Rh. palustris Z. mobilis T. thermophila
Solubilization in:
Triton X-100 CHAPS Triton X-100 Octylthioglucoside
Column chromatographic method:
DEAE cellulose DEAE Cellulose Octyl Sepharose DEAE Trisacryl
Phenyl Sepharose Octyl Sepharose Isoelectric foc. Hydroxyapatite
Sephacryl S-500 Blue Sepharose Sephacryl S-300 Mono Q
DEAE Cellulose
Preparative PAGE
267
hopene (unable to condense membranes) is probably used in biosynthetic reac-
tions leading to the elongated hopanoids.
With highly purified A. acidocaldarius SHC preparations containing no li-
pid contaminations it was possible to observe traces of additional side products
[22]. In a GC/MS measurement about twelve minor products were detected.
Their relative amounts are mostly 0.5% that of hopene. Since they have molecu-
lar masses of 410, they are supposed to be isomers of squalene and hopene.
These side products turned out not to be artifacts, resulting from the removal of
the SHC from the natural membrane environment or attachment of the His-tag.
They were also detected in the hydrocarbon fraction of A. acidocaldarius.
The structures of five minor hydrocarbons 7, 8, 9, 10, 11 were elucidated
[22]. The 17-isodammaradienes 8 and 10 have yet not been found in nature and
the rest are natural products, mainly from ferns. Four of these products are tetra-
cyclic with a five-membered ring D. Therefore, probably during ring D forma-
tion, the six-membered ring intermediate competes with a five-membered inter-
mediate (Markovnikov cyclization) and this intermediate leads to dead-end pro-
ducts. Furthermore, compounds 7, 8, and 9 show hydride shifts and eupha-7,24-
diene 11 shows two additional methyl shifts before termination of the reaction.
268
Such hydride and methyl shifts do also occur in the formation of sterols. The
double bond introduced by the final deprotonation occurs at different C-atoms.
This result points to a certain unspecificity of the deprotonation reaction and ad-
ventitious deprotonation sites may be postulated. The unspecificity of the depro-
tonation is conceivable if one takes into account the high acidity of C-atoms ad-
jacent to the carbocation.
The side products demonstrate the potential of SHC to produce other and
even new cyclization products. Such catalytic promiscuity may pave the way for
the evolution of new enzymes [38]. By intricate mutagenesis it should be possi-
ble to shift the SHC catalysis to new main products. Thereby, new enzymes will
be obtained. Furthermore, the unspecifity of SHC draws attention to the possibi-
lity that the high diversity of triterpenes in plants is not necessarily correlated
with a high diversity of cyclases.
With the purified A. acidocaldarius SHC we measured a few kinetic prop-
erties. All kinetic constants described are apparent because all reactions occur
in a micellar system; the enzyme, substrate, and inhibitors do not freely diffuse
in a homogeneous phase. The apparent K
m
for squalene is 9 mM [1]. Slightly
higher are the values for the cyclases of T. thermophila and Z. mobilis [3, 6].
The turnover number is in the range of 0.3 sec
1
. This is a very low value,
but it is conceivable by the X-ray structure of SHC and the complex reaction
mechanism [16].
We found that two similar groups of common detergents, namely n-alkyl-
trimethylammonium halides and n-alkyldimethylamine-N-oxides, e. g. 6, are ef-
ficient mechanism-based inhibitors of SHC [1]. By the N-methyl groups and the
positive charge these compounds have a distant relationship to the protonated
squalene during catalysis. The K
i
for the inhibitors with a C
12
-side chain turned
out to be 0.32 and 0.14 mM, respectively. This values are 30 to 60-fold lower
than the K
m
for squalene. The kinetic data show that these are competitive inhi-
bitors. Later, lauryldimethylamine-N-oxide 6 (= LDAO) has been used as a sub-
strate model in the X-ray structure elucidation of SHC [19].
The circular dichroism measurements demonstrated that the A. acidocal-
darius SHC is predominantly composed of a-helices and loops and only a very
low b-sheet proportion [16]. This measurements were confirmed by the X-ray
structure [19]. The circular dichroism measurements further showed that the
point mutants (concerning Asp374, Asp376, and Asp377) of SHC retain their
secondary structure.
269
16.5 Cloning of squalene-hopene cyclases
We cloned the shc gene from A. acidocaldarius [4], A. acidoterrestris [14], Z. mo-
bilis [12], Bradyrhizobium japonicum [15], R. palustris [14, accession no. EMBL
Y09979], and Methylococcus capsulatus [21]. To clone several SHCs was impor-
tant for identifying conserved amino acid residues and was the fundament for
directed mutagenesis. Additional aspects were important for the choice of the
organisms: there was a chance to clone in addition to shc genes also genes for
squalene-tetrahymanol (in R. palustris) and oxidosqualene-lanosterol (in M. cap-
sulatus) cyclase.
Different approaches were chosen for cloning the shc genes. For A. acido-
caldarius cyclase the path of reverse genetics had to be taken because no triter-
pene cyclase had been sequenced at that time [4]. From the purified cyclase, we
sequenced the N-terminus and synthesized corresponding DNA-probes. Start-
ing with these probes, transformed E. coli clones were isolated and were shown
to form functional SHC.
For cloning Z. mobilis shc, fragments of A. acidocaldarius shc were used
as heterologous probes in hybridisation experiments [12]. Despite the taxonomi-
cal (phylogenetic) distance and different GC percentages of genomic DNA in
both organisms, a shc containing clone was identified. Upstream of shc the first
hopanoid biosynthesis gene cluster was detected [23]. The same PCR strategy
was applied in the case of shc from A. acidoterrestris [14].
In the case of B. japonicum a fragment of shc was produced by PCR and
sequenced. In a second step, a cosmid-library was checked by PCR with homo-
logous primers designed according to the above fragment for a shc positive
clone [15].
From M. capsulatus producing hopanoids and sterols, a fragment of shc
was amplified. This PCR-product was used as a probe to isolate the shc gene
[21]. The expressed gene was able to convert squalene to hopene and diplop-
terol. But no conversion was observed with oxidosqualene as substrate. The
search by hybridisation techniques with shc fragments as probes for a sterol cy-
clase gene was not successful. Therefore, synthesis of sterol in this bacterium re-
mains a mystery. One may speculate that the M. capsulatus SHC will be post-
translationally altered by an unknown mechanism to form lanosterol from oxi-
dosqualene.
270
16.6 Properties of SHC sequences
SHCs are 631658 amino acids long. The degree of similarity of SHCs roughly
parallels the phylogenetic similarity of the corresponding bacteria. Therefore,
horizontal gene transfer is a rare event for the genes of hopanoid biosynthesis.
SHCs are distantly related by 27% identity and 35% similarity to sterol cyclases,
and both form two separate clusters. It will be of great interest to elucidate also
the sequences of plant hopanoid cyclases. This will clarify the question whether
they are directly derived from bacterial SHCs or from plant OSCs. Lupeol cy-
clase producing a pentacyclic triterpene originates from cycloartenol cyclases
and is not a member of the SHC-cluster [25].
With the help of peptide sequences of rat liver OSC [39], a non-tandem re-
peat could be identified in SHCs and OSCs at about the same positions in each
sequence (Fig. 16.2) [9]. The consensus sequence of the repeat is:
R/K A/G X X F/ Y/W L X X X Q X X X G X W
This motif is called QW-repeat because of the regular occurrence of Q
(Gln) and W (Trp) at the C-terminus. This repeat is highly specific for triterpene
cyclases and therefore has an indicative value. In a later section it will be de-
monstrated that the QW-repeat is connected to the stabilization and not to the
catalysis of SHC. Shortly after the discovery of the QW-repeat it was very sug-
gestive to relate it to catalysis, since a substrate with a repeat unit (isoprene)
would be polycyclized by an enzyme repeat element [7].
A second motif was identified in SHCs and OSCs. Starting from peptidedi-
gest of rat liver OSC which reacted covalently with 29-methylidene-2,3-oxido-
squalene 15 it was possible to identify a DCTA-motif in OSC [39] and the similar
Figure 16.2: Schematic representation of motifs in squalene-hopene cyclase (SHC) and
oxidosqualene cyclases (OSC). The black boxes represent the QW-motifs. For historical
reasons numbering is started from the C-terminus. QW5b occurs in Gram-positive bac-
teria only and QW5c is truncated at the N-terminus. The shaded boxes represent the Asp
containing motifs.
271
16.6 Properties of SHC sequences
DDTA-motif in SHC. They are located at corresponding sequence positions (Fig.
16.2). Thus we have two criteria at hand to identify a triterpene cyclase, similar-
ity and two common motifs.
It is interesting to note that two truncated QW-motifs and the DDTA-motif
occur in kaurene synthase A and abietadiene synthase [40, 41]. These diterpene
cyclases catalyze a cyclization reaction related to triterpene cyclases. Substrate
for these synthases is geranylgeranyl diphosphate. The bicyclization reaction is
not started by a dephosphorylation to generate the first carbocation. The gen-
eration of the first carbocation occurs in the same manner as in triterpene cy-
clases. In contrary, mono- and sesquiterpene synthases (cyclases) start the cycli-
zation reaction by cleavage of the diphosphate group for the generation of the
initial carbocation. These synthases have no similarity or motifs in common with
triterpene cyclases.
16.7 The structure of squalene-hopene cyclase
With the cloned shc from A. acidocaldarius, the mutant Asp376Cys, the purifica-
tion procedure, and enzyme assay, Ulrich Wendt in the group of Georg Schulz
in Freiburg achieved crystallization and X-ray structure elucidation at 2.9 and
2.0 resolution [18, 19, 24, 42].
The SHC is a membrane enzyme and had to be co-crystallized with deter-
gent molecules. Therefore, crystallization was not an easy task. The enzyme is
built mainly from a-helices, loops and turns; also few short b-sheet regions occur
(Fig. 16.3A). The a-helices form two distinctive domains. As shown in Fig.
16.3A, domain 1 is formed by six inner and six outer a-helices (a
6
a
6
barrel).
Domain 2 constitutes a less regular barrel of eleven a-helices. The active site is
located in a large central cavity sandwiched by both domains. A channel struc-
ture with a constriction at the end originates at a hydrophobic surface area (pla-
teau) and leads to the cavity (Fig. 16.3B).
The functional SHC is a dimer formed by two identical monomers [19, 42].
How does this dimeric enzyme interact with the cytoplasmic membrane? Prob-
ably the dimeric SHC dips with its hydrophobic areas (1600 A
2
each) around the
channel entrance from the cytoplasmic side into the hydrophobic part of the
membrane (Fig. 16.3C). Integral membrane proteins that submerge from one
side into the non-polar part of the membrane without protruding through it
were defined as monotopic. By X-ray structure analysis two further monotopic
membrane proteins were identified, namely, two prostaglandin-H
2
synthase iso-
enzymes which have no common sequence features to SHC [43]. The basic
amino acids surrounding mainly the non-polar plateau interact with phospho-
and sulfolipids of the A. acidocaldarius membrane and thereby reinforce the hy-
drophobic membrane/cyclase interaction.
272
A B
C
Figure 16.3: (A) Structure of the monomer of Alicyclobacillus acidocaldarius squalene-
hopene cyclase [41]. Yellow, inner helices; red, outer helices; cyan, b-structure; green,
QW-motifs; brown, the competitive inhibitor LDAO 6; blue, a group of amino acids at the
channel constriction and Asp376 at the top of the catalytic cavity near the methyl
groups of LDAO. (B) Cross section of the channel and the catalytic cavity of squalene-
hopene cyclase. LDAO, cyan; acid residues (red), basic residues (blue), neutral residues
(grey), and hydrophobic residues (yellow). The white arrow indicates the channel. (C) The
homodimeric structure of squalene-hopene cyclase and its hypothetical location in the
membrane. The arrows point to the approximate position of the channel entrances; gray
bars, the polar parts of the membrane.
273
16.7 The structure of squalene-hopene cyclase
It may be hypothesized that squalene diffuses in a twisted, unordered con-
formation in the cytoplasmic membrane and is decoiled when it enters into the
narrow constriction of the SHC channel. Then it may be threaded into the cata-
lytic cavity where it will be properly folded. The catalytic cavity is lined predo-
minately by eight aromatic amino acids which shape a steric matrix for the fold-
ing process and stabilize the intermediate carbocations by their p-electrons or
their electron pairs at the OH-groups of Tyr. Very probably, Asp376 is the proto-
nating residue [16, 44]. Asp377 and Asp374 will facilitate this process by stabili-
zation of the first carbocation. The protonation starts the cyclization cascade
which finally ends in a deprotonation. Responsible for deprotonation is a cluster
of polar amino acids (Gln262:Glu45:Glu93:Arg127) together with some water
molecules at the base of the catalytic cavity. One water molecule has a cataly-
tic function for proton abstraction and will sometimes also spend its OH
to the
last hopenyl cation, thereby forming diplopterol.
It is not clear by which way the product finally leaves the cavity. Possibly,
the product will leave at the end of the exergonic reaction the cavity by passing
the channel constriction, which is surrounded by mobile loops. Details of the re-
action mechanism will be clarified when a co-crystal of the SHC with squalene
or hopene is available.
Since the above mentioned QW-motifs are located at the surface of SHC,
they are not directly involved in catalysis. Six of eight QW-motifs occur in domain
1. The C-terminal moiety of the QW-motif is part of a loop connecting an outer
helix to the next inner helix. This part is forming a net of hydrogen bonds with
the preceding outer helix and the following inner and outer helices. Thereby the
outer helices of domain 1 are not only connected via the peptide chain but also
via a net of at least seven hydrogen bonds of the QW-motif. Thus the barrel of he-
lices is highly stabilized. Likewise two additional QW-motifs in the domain 2 ful-
fill the same role. This stabilization seems necessary because the hopene cycliza-
tion is highly exergonic, producing about 200 kJ/mol (calculated value; see [19]).
This value corresponds to the free energy of about seven phosphate bonds.
16.8 Site directed mutagenesis of squalene-hopene cyclase
On the basis of the elucidated X-ray structure of SHC we mutated amino acids
in the catalytic cavity (Fig. 16.4). This cavity can be divided into three subsites
according to Wendt and Schulz: a protonating site, a site for folding (chaperon-
ing) of squalene and, simultaneously, for stabilizing intermediate carbocations,
and a site for deprotonation [19, 42].
A likely region for protonating is
374
DVDDTA
379
, and Asp376 is the most
probable residue for donating the proton because it is in the neighborhood to
C3 of the modeled hopene molecule. Furthermore, the carboxyl group of
274
Asp376 is pointing from the b-site to C3. Indeed, it was demonstrated that
hopene is protonated at the b-H of C3. The experiment was performed in pure
D
2
O and nearly 100% of the b-H at C3 were deuterated. This experiment
proves that the proton from the deprotonation reaction is not used for protona-
tion (W. Eisenreich, A. Bacher, and K. Poralla, unpublished). The process of pro-
tonation is supposed to be assisted by the hydrogen-bonded couple
Asp376:His451 and by bound water between Asp376 and the OH-group of
Tyr495. Furthermore, it can be suggested that the couple Asp374:Asp377 carries
a negative charge that stabilizes the proton at Asp376. The results in Table 16.2
Figure 16.4: Schematic view of the catalytic cavity of squalene-hopene cyclase with a
modeled hopene molecule (in dark) [41]. The amino acids line the cavity and some men-
tioned in the text are shown in gray. The intermediate carbocations in the hopene mole-
cule are shown as black dots. The b-site of hopene is upside down.
Table 16.2: Mutant SHCs and their properties.
Protonation site:
Asp374Gln slightly reduced activity; wild type product pattern
Asp376Cys very low activity; wild type product pattern
Asp376Glu reduced activity; wild type product pattern
Asp377Glu no activity
His451Ala reduced activity; wild type product pattern
Folding site:
Trp169Phe reduced activity; altered product pattern
Trp312His very low activity; new main product
Tyr420Ala wild type activity; altered product pattern
Phe601Ala wild type activity; altered product pattern
Leu607Lys very low activity; bicyclic main product
Tyr609Phe wild type activity; mainly tetracyclic products
Phe365Leu reduced activity; wild type product pattern
275
show that Asp376 and Asp377 play an important functional role. The exchange
Asp377Glu abolishes activity and Asp376Glu lowers the activity to 10%.
Asp374 possibly takes part in the reaction, but the carboxy group is not essen-
tial since Asp374Asn only slightly reduces the activity (Table 16.2). The impor-
tance of His451 has to be doubted because the exchange His451Ala leads to no
inactivation.
From the effects with the mutations we draw the conclusions that an addi-
tive and cooperative effect leads to the protonating activity of Asp376. This ex-
plains why in most cases a residual activity remains after mutagenesis of the
partners of Asp376. Further arguments for the protonating function are its
conservation in OSCs and their inactivation results merely from mutation of this
specific Asp residue [44].
Effects easier to interpret resulted from several mutations at the folding
site of the catalytic cavity. These mutations showed distinct alterations of the
product pattern. In detail we will discuss four mutations.
1) Tyr420Ala: This mutant produces besides hopene also bicyclic and tricyclic
compounds [29]. a-Polypodatetraene 12 (23% of the hopene peak) and g-poly-
podatetraene 13 (10%) are deprotonated at different C-atoms in ring B. Also
traces of the tricyclic malabaricatriene 14 were detected. This result showed
that the protruding OH-group of Tyr420 is stabilizing the incipient carbocations
at C8 and also at C13 (hopene 2 numbering). When this stabilization is abol-
ished a premature termination of the cyclization cascade will frequently occur.
2) Phe601Ala: This mutant produces besides hopene also the isodammarene 8
(40% of the hopene peak) [28]. The p-electrons of Phe601 are directed towards
the C17 carbocation. When they are lacking the cyclization will end to a signifi-
cant percentage with the production of compound 8. This compound has yet not
been found in nature; but it is known as a minor product of SHC [22], and the
same stereochemistry at C17 has been observed in the transformation of 29-
methylidene-2,3-oxidosqualene 15 to 16 [20]. The same compound has also
been found with mutation Trp169Phe [46]. The above results show clearly that
directed mutagenesis will lead to new products. The approach for the formation
of new compounds may be further elaborated by the use of altered substrates.
3) Leu607Lys: This mutation leads to a new, although low enzyme activity.
Leu607 is protruding into the middle part of the cavity pointing to C8. In the
case of mutation Leu607Lys a premature termination occurs by the introduced
basic amino acid. Lys607 will probably accept a proton from C7, thereby form-
ing g-polypodatetraene 13 by a type of Hofmann elimination reaction (S. Schmitz
and K. Poralla, unpublished). It is very interesting, that in this mutant a single
bicyclic compound is formed, as compared to mutant Tyr420Ala.
4) Tyr609Phe: This mutant produces significantly more tetracyclic compounds
8, 9, 10 than hopene ,and it is therefore justified to designate it as a new enzy-
me. Presumably, Tyr609 stabilizes the cation at C13 by the single electron pairs
at the OH-group together with other aromatic residues. This is a critical point in
276
the cyclization cascade because this stabilization is important for the unfavor-
able anti-Markovnikov cyclization of ring D. Because optimal stabilization is ab-
sent in mutant Tyr609Phe a favorable Markovnikov cyclization will take place
for ring D and therefore a high percentage of tetracycles with a five-membered
ring D will be formed. On the other hand, it may be sterically argued. Phe is
slightly smaller as compared to Tyr. Therefore, the folded squalene alters its po-
sition relative to Phe601 and Trp169 which now insufficiently stabilize the cation
at C13 with consequently premature termination of the cyclization cascade [30].
Other mutants are listed in Table 16.2 for which the side products have not
been identified.
277
16.9 Hopanoid biosynthesis gene clusters
In B. japonicum, Z. mobilis, and M. capsulatus to shc additional ORFs corre-
sponding to genes for hopanoid biosynthesis (hpn genes) were sequenced (see
Fig. 16.5) [21, 23]. Very similar clusters do also occur in Rhizobium NGR234 and
Streptomyces coelicolor [33, 34].
In general, the ORFs overlap with their putative stop and start codons. Pu-
tative termination sites were found downstream of shc. The hpnC gene has
been characterized as a squalene synthase gene [23]. E. coli transformed with
hpnC was able to produce squalene; in E. coli the precursor farnesyl diphos-
phate is produced but not squalene. The reducing cofactor for this first bacterial
squalene-synthase has still to be characterized in a cell-free system.
Yet unknown is the function of hpnD which shows similarity to genes for
squalene, dehydrosqualene, and phythoene synthases; the latter ones are in-
volved in carotenoid biosynthesis. Neither B. japonicum nor Z. mobilis contain
carotenoids. Presumably, hpnD codes for a dehydrosqualene synthase which
may be reduced by HpnE (similar to phythoene dehydrogenase) to squalene.
These two pathways for squalene formation possibly use two different reducing
agents. This is perhaps of value for bacteria which have a high requirement of
reducing power for N
2
-fixation. The upstream adjacent gene to shc in M. capsu-
latus was also characterized as a squalene synthase. This squalene synthase
and the squalene synthase of B. japonicum have a very low identity of 32%.
The four synthases of B. japonicum and Z. mobilis belong in the phylogenetic
tree to a cluster of prokaryotic and eukaryotic phythoene synthases. We suppose
therefore that some bacterial squalene synthases originate from phytoene
synthases which have changed their function to squalene synthases.
Figure 16.5: The DNA regions from Zymomonas mobilis and Bradyrhizobium japonicum
coding for hopanoid biosynthesis (hpn) genes. Features of the genes: hpnA, putative oxi-
doreductase of sugars; hpnB, putative glycosyl transferase; hpnC, gene for squalene
synthase; hpnD, putative squalene or dehydrosqualene synthase; hpnE, similar to phy-
toene desaturase; hpnF, squalene-hopene cyclase.
278
The ORFs hpnA and hpnB seem to be connected with the elongation reac-
tions in hopanoid biosynthesis. Elongated hopanoids contain at least one pen-
tose and some additionally also a derivative of glucose. HpnA has similarity to
UDP-glucose-4-epimerase and HpnB to glycosyl transferases [23].
The hopanoid biosynthesic gene cluster in Rhizobium NGR234 has the
same structure as compared to B. japonicum. Both clusters are differently regu-
lated. Under laboratory conditions the cluster of B. japonicum is expressed [13],
whereas the cluster of Rhizobium NGR234 is not expressed (E. Kannenberg, un-
published results).
16.10 Miscellaneous results
Since the detection of hopanoids in Frankia [45] there existed a greater interest for
hopanoid formation in N
2
-fixing bacteria. In cooperation we detected hopanoids
in two Beijerinckia species and Azotobacter vinelandii and we also found that
free-living B. japonicum and some relatives produce hopanoids [11, 13, 17]. It is
tempting to speculate that the condensing function of hopanoids lowers the O
2
-
diffusion across the cell membrane and thereby prevents nitrogenase inactivation.
Normally, tetrahymanol 4, an isomer of diplopterol 3 is found in lower eu-
karyotes. To our surprise it was also detected in the bacterium R. palustris [2]. In
B. japonicum, besides tetrahymanol, methylated tetrahymanol species do exist
(M. Perzl, J.-M. Bravo, E. Kannenberg, M. Rohmer, and K. Poralla, unpublished
results). Organic geochemists are highly interested in this result, because now
tetrahymanol and gammacerans (possessing no OH-group) in sediments are no
longer indicative for the former occurrence of lower eukaryotes.
In Staphylococcus aureus a dehydrosqualene synthase gene has been
characterized as a member of the C
30
-carotenoid biosynthesis cluster [10]. De-
hydrosqualene is a homologue to phythoene, and therefore the corresponding
synthases are similar to squalene synthase which is an essential enzyme in ho-
panoid biosynthesis.
As was mentioned above, wild type and mutant SHCs may be useful for
the synthesis of new and unusual cyclic isoprenoids. An illustration for this is
given by the transformation of a 29-methylidene-2,3-oxidosqualene 15 by SHC to
a tetracyclic isodammarenoid 16 containing an additional cylohexane system [20].
In the genome project for Streptomyces coelicolor A3(2) an isoprenoid bio-
synthesis cluster of genes was described [34]. In this organism also two ORFs
are occurring with significant similarity to phythoene and squalene synthases.
The cluster also includes a gene for SHC. In liquid culture S. coelicolor does not
produce hopanoids, but hopanoids are present when it sporulates on agar med-
ium. All tested white mutants (B, G, and J) which are able to produce aerial my-
celium but no spores synthesize hopanoids. Mutants with the property to pro-
279
16.10 Miscellaneous results
duce no aerial mycelium (bald) can be split into two groups. Only bald mutants
(A, C, D, G, and H) which are defective in the production of an extracellular sig-
nal cascade are also deficient in the production of hopanoids, whereas bald B
produces at least minute amounts of hopanoids (K. Poralla, G. Muth, and T.
Hrtner, unpublished results).
16.11 Outlook
Besides the described major achievements several important questions still re-
main to be solved. For the solution of these questions bacteria will be helpful for
which genetic methods are established, as it is the case with B. japonicum and
S. coelicolor. With the symbiotic B. japonicum the following questions should be
answered: 1) is hopanoid synthesis essential during growth outside the host
plant, and 2) what is the role for hopanoids in symbiosis? With S. coelicolor the
question will be answered if hopanoid biosynthesis is essential for sporulation
by insertion mutagenesis of the shc gene.
Nothing is known about the regulation of the hopanoid biosynthesis clus-
ter in B. japonicum and in Rhizobium NGR234. Also unclear is the observation
that some organisms have two genes similar to squalene synthase. In our opin-
ion only a single gene seems sufficient for hopanoid biosynthesis.
Anewstraight forward strategy for mutagenesis is an evolutionaryapproach.
Hopefully, this will lead to the isolation of altered SHCs with new substrate and
product specificity. Plasmids may be constructed for Saccharomyces cerevisiae
mutants deficient in oxidosqualene-lanosterol cyclase (erg7). By this way mutant
SHCs may be isolated forming lanosterol and thus repeating in a short time the
evolution from hopene to sterol cyclases. In this type of experiments it will be fig-
ured out which alterations are necessary unbiased by mutations not related to cat-
alysis. This goal was one motivationfor the ongoing research.
Acknowledgments
Since 1990 the following students forwarded the described project: Peter Grt-
ner, Dietmar Ochs, Cord Tappe, Gisela Kleemann, Jrg Saar, Corinna Feil, Mi-
chael Perzl, Annette Tippelt, Ok-Byung Choi, Angelika Rahn, Thorsten Merko-
fer, Susanne Schmitz, Christine Fll, and Michael Knigge. Most important for
280
many tasks was Thomas Hrtner. I am thankful to them, who contributed so
much. Also my French colleagues helped a lot: Guy Ourisson, Michel Rohmer,
and Catherine Pale-Grosdemange. Corinna Kaletta, Glenn D. Prestwich, Ikuro
Abe, Hermann Sahm, and Georg Sprenger made essential contributions to the
project. I am also thankful to Ulrich Wendt and Georg Schulz who enormously
strengthened the project by their X-ray analysis. Gnther Jung (collaborative
research centre 323) was always very helpful. Without the financial help of the
DFG this project would have never been started and continued. I like to remem-
ber the comments of the DFG-referees. I wish to thank Elmar L. Kannenberg for
his input of ideas and critical reading of the manuscript.
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References
17 Genetic and Biochemical Analysis of the
Biosynthesis of the Orange Carotenoid
Staphyloxanthin of Staphylococcus aureus
Friedrich Gtz*
17.1 Introduction
The yellow to orange colony color of Staphylococcus aureus is one of the classi-
cal species criteria. As early as 1882, Ogston connected the yellow-orange ap-
pearance of pus with the color of the infecting microorganisms [1]. Later it was
shown that the pigment should not be the only basis for classification, since it is
not a very stable character. Pigmentation is usually apparent after 18 to 24 h of
growth at 378C, but is more pronounced when cultures are held at room tem-
perature for further 24 to 48 h. In particular those S. aureus strains isolated from
bovine or which are multiply antibiotic resistant, are yellow-pigmented. Non-
pigmented (white) derivatives of S. aureus are often found in subcultures of
stored organisms, and non-pigmented clones can arise in pigmented colonies,
giving a sectored appearance [2]. Non-pigmented variants are more susceptible
to desiccation and to linolenic acid than the corresponding wild type strains [3].
Treatment with nitrosoguanidine can result in irreversible loss of the capacity to
synthesize pigment [4]. Although pigment production is a rather unstable char-
acter, it has been ruled out that the respective genes are encoded on typical
plasmids [3].
Marshall and Wilmoth [5] isolated the pigments of S. aureus S41 and deter-
mined their chemical structure, identifying 17 compounds which are all triterpe-
noid carotenoids possessing a C
30
- instead of the C
40
-carotenoid structure found
in most other organisms. The main pigment is staphyloxanthin, an alpha-D-
glucopyranosyl-1-O-(4,4'-diaponeurosporene-4-oate)6-O-(12-methyltetradecano-
ate), in which glucose is esterified with both, triterpenoid carotenoid carboxylic
acid and a C
15
fatty acid.
The postulated biosynthetic pathway of staphyloxanthin starts with a
head-head condensation of two molecules of farnesyl diphosphate to form dehy-
284
ISBN: 3-527-30615-3
drosqualene (= 4,4'-diapophytoene) or squalene. 4,4'-Diaponeurosporene is
formed by three or four successive dehydrogenation steps and is the first yellow
carotenoid intermediate. The oxidation of the terminal methyl group of 4,4'-di-
aponeurosporene to form the carboxylic acid 4,4'-diaponeurosporene-4-oic acid
proceeds via the aldehyde 4,4'-diaponeurosporene-4-al. The acyl compound
staphyloxanthin, as the final orange pigment in S. aureus, is formed via gluco-
syl-4,4'-diaponeurosporenoate [5]. This pathway has been verified by the analy-
sis of various S. aureus mutants, in which intermediary products either were ab-
sent or accumulated [6].
The aim of our studies was to genetically characterize the steps of staphyl-
oxanthin biosynthesis in staphylococci. Having identified the genes we are able
to create deletion mutants and to study the function and regulation of staphyl-
oxanthin. We chose S. carnosus TM300 as the cloning host because the colonies
of this strain are white and should therefore not produce staphyloxanthin or col-
ored intermediates. Staphyloxanthin-producing clones of S. carnosus harboring
pOC1 were easily detected by their orange-colored colonies. In a first paper we
described the characterization of two genes, crtM and crtN, involved in the bio-
synthesis of the yellow carotenoid 4,4'-diaponeurosporene and proposed a path-
way for its biosynthesis by analysis of intermediary products in the various
clones [7]. In a second paper (submitted) we analyzed the function of three
further genes crtO, crtP, and crtQ. All five genes have the same orientation and
form an operon (crtOPQMN) with a s
B
-dependent promoter upstream of crtO
and a termination region downstream of crtN. The analysis of the sequences of
CrtO, CrtP, and CrtQ and the spectrophotometric analyses of the carotenoids
produced by Staphylococcus carnosus clones carrying crtMNQ, or crtMNOQ, or
crtMNOPQ suggest that CrtP is responsible for the oxidation of 4,4'-diapo-
neurosporene to 4,4'-diaponeurosporen-4-oic acid and that the glucosylation of
the carbon acid is catalyzed by CrtQ. All five genes including crtO were shown
to be required for the formation of staphyloxanthine [8]. We also have investi-
gated the survival of S. aureus strain Newman and its non-pigmented mutant
DcrtM after exposure to UV light and to oleic acid to determine the function of
the carotenoid. However, so far we have not got any clue as to the function of
staphyloxanthin, therefore, these studies will be continued.
17.2 Cloning of the carotenoid biosynthetic genes from
S. aureus Newman in S. carnosus and E. coli
S. aureus Newman chromosomal DNA was partially digested with MboI. DNA
fragments of 5 to 25 kb were ligated to BamHI-digested pCA44. The ligated
DNA was transformed into the non-pigmented S. carnosus TM300; several yel-
285
17.2 Cloning of the carotenoid biosynthetic genes from S. aureus Newman
low- and orange-pigmented S. carnosus colonies were detected. Restriction ana-
lysis of one isolated plasmid, pOC1, revealed that the orange-pigmented S. car-
nosus clone carried a 12-kb DNA insert in that plasmid [7].
Subcloning of fragments of the 12-kb insert in E. coli and S. carnosus led
to yellow and non-pigmented clones in both organisms. The smallest fragment
which caused yellow pigmentation was a 3.5-kb fragment containing the crtM
and crtN genes. Expression of these two genes in E. coli (pUG1) or S. carnosus
(pOC21) led to the formation of a yellow pigment. Analysis of this pigment re-
vealed that it was the deep-yellow carotenoid 4,4'-diaponeurosporene. This is
the major pigment produced by Staphylococcus aureus Newman which is after
prolonged cultivation in part converted to the orange end product, staphyl-
oxanthin.
17.3 Function of CrtM and CrtN
CrtM encodes a 254 aa protein of M
r
30,121 which is rather hydrophobic (39%
hydrophobic aa). The deduced amino acid sequence of CrtM was compared
with other enzymes involved in carotenoid biosynthesis. There are three boxes
of pronounced similarities with the phytoene synthase of Synechococcus [9] and
the squalene synthases of two Erwinia species, Saccharomyces cerevisia and of
human. One domain represents the postulated prenyl (farnesyl) diphosphate
binding motif [10].
CrtN encodes a 448 aa protein of M
r
50,853. This protein is rather hydro-
phobic too (38% hydrophobic aa), however, obvious membrane-spanning do-
mains characteristic of integral membrane proteins are absent. The deduced
amino acid sequence was compared with other carotenoid biosynthetic en-
zymes. CrtN shows a pronounced similarity (3035%) to the phytoene desa-
turases (CrtI) of Erwinia herbicola and Rhodobacter capsulatus. The NH
2
-termi-
nus of CrtN (aa positions 229) is distinguished by a conserved amino acid se-
quence with homology to FAD-, NAD(P)-binding domains of a series of dehy-
drogenases and oxidases. The phytoene desaturases catalyze the dehydrogena-
tion reactions from phytoene to neurosporene (lycopene).
286
17 Genetic and Biochemical Analysis of the Biosynthesis
17.4 Identification of carotenoids in S. carnosus (pOC21),
E. coli (pUG1), and S. aureus Newman
In the yellow-pigmented E. coli (pUG1) 4,4'-diaponeurosporene was identified
by its characteristic absorption spectrum as a main carotenoid. 4,4'-diapo-
7,8,11,12-tetrahydrolycopene and 4,4'-diapolycopene were present in lower
amounts. These results agree with the presence of the two genes, crtM and
crtN, on plasmid pUG1. In radio-TLC, with farnesyl diphosphate as a substrate
and E. coli (pUG1) extracts, dehydrosqualene and 4,4'-diaponeurosporene are
produced; indicating that the proposed dehydrosqualene synthase (CrtM) is cat-
alyzing the condensation of two farnesyl diphosphates to dehydrosqualene, and
the proposed dehydrosqualene desaturase (CrtN) is catalyzing all oxidation
steps between dehydrosqualene and 4,4'-diaponeurosporene.
In the yellow-colored S. carnosus (pOC21) we found essentially the same
carotenoid spectrum as in E. coli (pUG1), identifying 4,4'-diaponeurosporene as
the main product at 22.3 g/g cell dry weight.
In S. aureus Newman extracts we found that the concentration of staphyl-
oxanthin was only 50% of that of 4,4'-diaponeurosporene. This can be ex-
plained by the observation that 4,4'-diaponeurosporene is already formed after
12 h cultivation, while staphyloxanthin is produced later after 24 h incubation.
The amount of the main carotenoids was estimated spectrophotometrically using
their specific extinction coefficients.
17.5 Identification of dehydrosqualene in E. coli (pUG1)
and E. coli (UG9)
In E. coli JM83 extracts no squalene was detectable. On the other hand, we
identified squalene in S. carnosus TM300 extracts, which is in agreement with
earlier observations [11]. This indicates that a squalene synthase must be chro-
mosomally encoded in S. carnosus. The finding of squalene in S. carnosus ex-
tracts raised the question as to the enzymatic activity of CrtM which exhibits si-
milarities with phytoene and squalene synthases.
In order to answer the question whether the C
30
-carotenoid biosynthesis
starts with squalene or, in analogy to the C
40
-carotenoid biosynthesis, with dehy-
drosqualene (= 4,4'-diapophytoene) we constructed plasmid pUG9 containing
only the intact crtMgene. In enzyme assays with cell free extracts of E. coli (pUG9)
we never obtained squalene, irrespectively of the presence of NAD(P)H. However,
we could identify a compound with a slightly lower R
f
-value compared to squalene
287
17.5 Identification of dehydrosqualene in E. coli (pUG1) and E. coli (UG9)
which was found in a very hydrophobic fraction of the lipid extraction of E. coli
(pUG9). The samples were separated by HPLC and analyzed by diode array spec-
troscopy. Only one peak occurred, showing an absorption maximum at 287 nm
and shoulders at 275 and 297 nm. Based on the characteristic absorption spectrum
[6] we identified this compound as dehydrosqualene. No dehydrosqualene was
detected in control experiments with E. coli (pUC19). The isolated dehydrosqua-
lene revealed in GLC/MS-analysis two peaks of similar retention times with mole-
cular ions at 408 m/z, corresponding to C
30
H
48
. The fragmentation patterns are
nearly identical, suggesting the two compounds to be the cis- and trans-isomers of
dehydrosqualene. Since there is no squalene found in the E. coli (pUG9) extracts
these results showthat CrtMrepresents a dehydrosqualene synthase.
17.6 Squalene is very likely no substrate for CrtN,
the proposed dehydrosqualene desaturase
With squalene as a substrate and with cell extracts of either E. coli (pUG1) or S.
carnosus (pOC21) we never observed in an in vitro assay the formation of 4,4'-
diaponeurosporene, irrespectively of the presence of cofactors (such as FAD,
NADP, NAD, FMN) or various divalent metal ions. We therefore think that the
proposed dehydrosqualene desaturase is specific for dehydrosqualene.
17.7 The crt operon
The caroteinoid biosynthesis genes on plasmid pOC1 were further sequenced,
and an operon containing five genes (crtOPQMN) was identified; in addition to
the known genes crtM and crtN three further genes upstream of crtM were
found. By analysis of deletion mutants it turned out that all five genes are neces-
sary for the formation of the orange staphyloxanthin. The organization of the op-
eron is shown in Fig. 17.1.
Figure 17.1: Organization of the staphyloxanthin biosynthesis genes of Staphylococcus
aureus strain Newman.
288
17.8 Homology of CrtO, CrtP, and CrtQ
CrtO (M
r
20.3 kDa) shows no similarities to any sequences of other carotenoid
biosynthesis proteins known. However, the deduced amino acid sequence of
CrtP (M
r
50.8 kDa) shows similarities to sequences of diapophytoene dehydro-
genase of Heliobacillus mobilis (identity: 29%; similarity: 93%) [Takaichi 1997
#34], phytoene dehydrogenases from Myxococcus xanthus (identity: 30%; simi-
larity: 63%) [12], Erwinia herbicola (identity: 28%; similarity: 62%) [13], Phyco-
myces blakesleeanus (identity: 28%; similarity: 61%) [14], and of CrtN of S. aur-
eus strain Newman [7].
CrtQ (M
r
42.5 kDa) shows similarities to the sequences of galactosyl and
glycosyl transferases of Bradyrhizobium japonicum (identity: 30%; similarity:
62%) [15], and to the processive glycosyl transferases of S. aureus (identity:
23%; similarity: 41%) [16] and S. epidermidis (identity: 22%; similarity: 41%)
[17]. Five strictly conserved amino acids are involved in the catalytic function of
processive glycosyltransferases. A model for beta-glycosyl transferase shows
that the catalytic amino acids are organized in two domains: domain D1 con-
tains one amino acid and domain D2 contains four amino acids [18]. Non-pro-
cessive glycosyl transferases (transferases that transfer only one monosacchar-
ide) have only the one conserved amino acid in domain D1. The alignment of
the CrtQ sequence with that of the processive glycosyltransferase IcaA from S.
aureus [16] and the corresponding gene from S. epidermidis [17] shows that
CrtQ has only the conserved amino acid in domain D1. No sequence similarities
to the strictly conserved amino acids of domain D2 were detected. Therefore,
CrtQ appears to be a member of the non-processive glycosyl transferase family.
17.9 Construction of crtM mutants of S. aureus
strain Newman
By using plasmid pRB573SXDcrtM, the crtM gene in the chromosome of S. aur-
eus strain Newman was exchanged with the gene encoding chloramphenicol
transferase (cat) through a double-crossover event. The resulting mutant, S. aur-
eus strain Newman DcrtM did not produce C
30
-carotenoids and formed colorless
colonies on TSB agar plates. This loss of pigmentation also showed that no alter-
native pigment biosynthesis pathway exists in this strain. No differences between
the growth of the wild type strain and of the DcrtM mutant were observed.
The inserted cat gene, which lacks a promoter, was also used as a reporter
gene to study the regulation of the pigment biosynthesis genes in S. aureus
289
17.9 Construction of crtM mutants of S. aureus strain Newman
strain Newman. In the DcrtM mutant, the cat gene is under the control of the
promoter of the crt operon. Therefore, the transcriptional regulation of the crt
operon was studied by following Cat activity during the growth of the culture.
The Cat activity increased significantly at the beginning and during the station-
ary phase, which is in agreement with earlier observations of a marked increase
in the pigmentation of the wild type strain only after 24 to 36 h of growth [7].
17.10 s
B
-regulated promoter of the crt operon from
S. aureus strain Newman
The loss of the sigB gene and the regulatory genes rsbV and rsbW leads to a
loss of pigmentation in S. aureus strain Newman. In addition, the colorless
S. aureus strain RN4220 carrying plasmid pIK57 (a pTX15 derivative with an in-
ducible sigB gene [19]) is pigmented after sigB induction. Based on these re-
sults, a s
B
-regulated crt promoter has been hypothesized [19, 20]. Further char-
acterization of the crt operon promoter region indicated that indeed the crt op-
eron promoter is regulated by s
B
. The transcription start point of the crt operon
was identified using primer-extension analysis.
17.11 The carotenoid biosynthesis genes
All of the carotenoid biosynthesis genes were cloned together into vector pTX15
under the control of the xylA promoter, forming plasmid pTXSX. With this sys-
tem, it was possible to induce a strong pigment formation in S. carnosus. To in-
vestigate the function of carotenoid synthesis genes and the metabolic products,
subsets of genes were cloned into pTX15 behind the xylA promoter, forming
plasmids pTXMNPQ (carrying crtMNPQ), pTXMNQ (carrying crtMNQ), and
pTXMN (carrying crtMN).
After cultivation of the recombinant strains, the carotenoids were ex-
tracted and separated by preparative TLC, and the absorption spectrum of
each individual pigment band was recorded. From the data obtained, it was
possible to assign the gene products of crtP, crtQ, and crtO to the steps of the
hypothetical biosynthesis pathway (Fig. 17.2). As shown previously, the gene
products of crtM and crtN are responsible for the formation of the first yellow-
colored C
30
-carotenoid, 4,4'-diaponeurosporene. The pigments produced by
290
S. carnosus (pTXMNQ) and S. carnosus (pTXMN) were similar, and differences
only in the non-pigmented compounds were observed after exposing the de-
veloped TLC plates to UV light. This change can be explained by an unspeci-
fic glycosylation by CrtQ. 4,4'-Diaponeurosporene was only modified when the
crtP gene product was also present in S. carnosus (pTMNPQ). The spectral
data indicated that CrtP catalyzes the oxidation of a terminal methyl side
group, forming 4,4'-diaponeurosporen-4-oic acid, and the carboxyl group is
then glycosylated by CrtQ; the last step in the pathway is the esterification
with a fatty acid by CrtO.
Figure 17.2: Proposed enzymatic pathway of the S. aureus staphyloxanthin biosynthesis:
CrtM, dehydrosqualene synthase; CrtN dehydrosqualene desaturase (major intermediary
product is 4,4'-diaponeurosporene); CrtP, diapophytoene dehydrogenase; CrtQ, non-pro-
cessive glycosyl transferase; CrtO, esterification with a fatty acid (last step in staphyl-
oxanthin biosynthesis).
291
17.11 The carotenoid biosynthesis genes
17.12 Function of the pigments in S. aureus strain Newman
The survival of S. aureus strain Newman and its non-pigmented mutant DcrtM
after exposure to UV light and to oleic acid was tested to determine the function
of the carotenoid. After treatment with UV light, the survival of S. aureus strain
Newman DcrtM and DsigB mutants was only slightly lower than that of the wild
type strain. This shows that pigmentation does not protect against UV light. The
oleic-acid-killing assay was previously used by Chamberlain et al. [21] in their
studies of the pigmented S. aureus strain 18Z and an undefined colorless mutant
strain 18Z-76. Mutant 18Z-76 has a higher sensitivity to oleic acid than the wild
type strain. The authors concluded that there is a relationship between pigment
production and the resistance of S. aureus to the cell-damaging influence of
oleic acid. In our tests, there was no difference in the sensitivity of S. aureus
strain Newman wild type and the DcrtM mutant to treatment with oleic acid.
The pigmentation is therefore probably not responsible for the higher resistance
of the colorless mutant 18Z-76 to oleic acid. The difference seen was probably
due to a mutation in a gene that controls pigment formation such as sigB, rsbU,
or rsbW as well as other function(s) that rendered the cells more sensitive to
oleic acid.
17.13 Distribution of pigment biosynthesis genes among
staphylococcal species
The distribution of the C
30
-carotenoid biosynthesis genes among staphylococci
was examined using the S. aureus strain Newman crtM gene, which encodes
the key enzyme for the synthesis of the C
30
-carotenoid, as a probe in cross-spe-
cies DNA hybridization experiments. Seven species formed pigmented colonies
and gave a positive signal with the crtM probe.
292
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294
18 Second Messenger Systems in Paramecium
Joachim E. Schultz* and Jrgen Linder
18.1 Introduction
The world of microbes has proven to be a large and mainly unopened chest,
which contains a rich panoply of metabolites representing chemically highly di-
verse structures. So far perhaps less than 1% of all bacteria thought to exist has
been investigated in this respect. For those engaged in the development of no-
vel lead compounds for therapeutic purposes the bacterial producers of second-
ary metabolites turned out to be gold mines. Yet, discriminating between real
nuggets and fools gold has become a more and more difficult task. We are fa-
cing the fact that isolation and structural elucidation of novel structures is only
one side of the coin, the other being the daunting task to screen for a biological
activity, which may be of potential therapeutic value. In a somewhat daring and
certainly novel approach we wished to use a well known protozoan unicell,
Paramecium, as a eukaryotic model to assay mixtures of and purified com-
pounds of microbial origin for an activity on neurophysiological membrane
events. The rationale for this approach was based on several lines of thinking.
First, considering the universality of governing principles apparent in modern
biology we felt that there exist universal mechanisms of excitation, adaptation
and sensory transduction in nature, which can be examined in a lower eukar-
yote such as Paramecium. Second, Paramecium has been a favorite neurobiolo-
gical model cell to study behavior because the genetics and the electrophysiol-
ogy are well understood [14]. Like neuronal cells, Paramecium has an excitable
membrane and generates action potentials, which are graded to the strength of
the stimulus. In addition, the stereotypic swimming behavior of this ciliated
creature is a direct correlate of electrophysiological events and can, therefore,
be easily observed under a stereomicroscope without obligatory measurements
* Pharmazeutische Biochemie, Universitt Tbingen, Auf der Morgenstelle 8, D-72076T-
bingen
295
ISBN: 3-527-30615-3
of electrogenesis by impaling the cells with microelectrodes [3]. An avoiding re-
sponse, i. e. backward swimming, is always associated with a depolarizing Ca
2+
-
inward current whereas hyperpolarization is correlated with accelerated forward
swimming. Third, Paramecium responds to chemical, mechanical and thermal
cues, i. e. the machinery for different sensory modalities is present. Finally, Para-
mecium is a fresh-water protozoan, which displays several typical protozoan fea-
tures known from parasitic protozoa such as a variable glycoprotein surface
coat. It was our hope that data generated with this harmless protozoan model
may become applicable to some of the dreaded parasitic protozoans, which are
responsible for a high fraction of diseases afflicting human beings.
As early as 1980, we established as a first step conditions for an axenic
massculture of Paramecium, which allowed the production of a cell mass suffi-
cient for protein biochemistry to be carried out [5, 6]. This allowed the identifi-
cation and characterization of biochemical targets, which may be applicable for
screening purposes. This should permit then to use a triple strategy for testing
novel compounds of bacterial origin with respect to unknown biological activ-
ities: 1) in vivo studies such as observation of an intrinsic activity to elicit stereo-
typically characterized changes in swimming patterns, which would indicate an
interpretable action on electrogenesis in Paramecium or an activity (or toxicity)
to impair behavioral responses to canonical stimuli such as sustained depolari-
zation caused by addition of Ba
2+
and high K
+
or hyperpolarization caused by
dilution of the external K
+
or an increase of the external Ca
2+
(calcium paradox)
[7] concentrations; 2) ex vivo studies by measuring the effect of compounds on
the intracellular generation of the second messengers cAMP and cGMP, which
are biochemical correlates of electrical events in this ciliate [812], and 3) in vi-
tro studies, in which the effect of novel compounds or compound mixtures on in-
dividual, isolated or recombinant components of the second messenger signal
transduction system, such as adenylyl and guanylyl cyclases, phosphoprotein
phosphatases, phosphodiesterases, and protein kinases, is examined [1315].
18.2 Identification and characterization of cGMP and
cAMP second messenger signaling systems in
Paramecium
18.2.1 Regulation of cGMP levels
Cyclic nucleotides are used universally as second messengers. In metazoan cells
regulation of cyclic nucleotide biosynthesis routinely involves a first messenger,
that interacts with an extracellularly oriented membrane receptor, which in turn
296
couples to adenylyl cyclase via heterotrimeric G-proteins or directly activates a
guanylyl cyclase catalyst by a receptor intrinsic to the membrane-bound enzy-
me. Soluble guanylyl cyclases are activated by the intracellular first messenger
nitrogen oxide radical [16, 17]. In a free-living freshwater protozoan signal sen-
sing and signal responses can be expected to concern overwhelmingly environ-
mental changes such as in the nutrient situation, the ion composition of the pool
or a predator/prey relationship. We wished to determine, which stimuli affect
and regulate intracellular cAMP and cGMP formation in the ciliate Paramecium.
Basically, we found that sudden changes in the external ion composition are the
most reliable effectors of cyclic nucleotide biosynthesis in the ciliate.
cGMP levels are regulated by a depolarizing Ca
2+
-inward current elicited,
e. g. by the addition of Ba
2+
-ions or an increase in the concentration of external
K
+
, i. e. the Ca
2+
-inward current serves a dual role as a simple charge carrier and,
conceptually, as a first messenger, which activates a membrane bound guanylyl
cyclase [10]. The Ba
2+
-activated Ca
2+
-inward current is responsible for a short-
lived 6-fold increase in intracellular cGMP in conjunction with a behavioral
avoiding response, i. e. repeated bouts of backward swimming (Fig. 18.1). The
importance of this current for cGMP biosynthesis was identified by using Parame-
cium mutants with identified defects in Ca
2+
-channel regulation. The pawn mu-
tants are unable to respond to a depolarizing stimulus with backward swimming
because the voltage-operated Ca
2+
-channel cannot be opened and remains shut
[1, 1820]. The biochemical readout of the missing Ca
2+
-inward current in these
mutant cells is a lack of cGMP generation although the guanylyl cyclase enzyme
activity in the mutants seems to be perfectly normal (Fig. 18.1) [10]. Paramecium
Figure 18.1: Depolarization as a hormone surrogate stimulates cGMP formation in Para-
mecium. Time-dependent increase in intracellular cGMP concentrations in Paramecium
tetraurelia as stimulated by a BaCl
2
-elicited depolarization (wild type 51s (*), double mu-
tant pawn A/pawn B (t) and mutant dancer (&)). For stimulation 250 l 6 mM BaCl
2
in
equilibration buffer (5 mM KCl, 50 M CaCl
2
, 10 mM MOPS, pH 7.2) were added to 250
l of cells. Note the transient response in wild type cells, the extended response in dancer
mutant cells which have a defect in Ca
2+
-channel closure, and the absence of a response
in pawn mutant cells which have a defect in opening the voltage-gated Ca
2+
-channel
(adapted from [10]).
297
18.2 Identification and characterization of cGMP and cAMP second messenger
dancer mutants have a genetic defect opposite to the pawn mutation, i. e. they
lack the ability to quickly close the activated Ca
2+
-channel and consequently
show extended bouts of ciliary reversal even upon minor depolarizing stimuli [21,
22]. The biochemical correlate of this genetic defect is an exaggerated and sus-
tained intracellular cGMP formation upon depolarization (Fig. 18.1) [10]. We
have characterized intensively the enzymatic properties of a particulate guanylyl
cyclase of Paramecium, which is localized to a large extent in the excitable ciliary
membrane [23, 24]. The enzyme is stimulated by micromolar concentrations of
Ca
2+
. This effect most likely is mediated by an accessory protein initially assumed
to be calmodulin [24]. Several experimental observations put this conclusion into
doubt, e. g. M concentrations of exclusively protozoan calmodulins are required
and mammalian calmodulins were inactive despite extensive sequence identities.
The protein, which actually mediates the Ca
2+
-effect on the guanylyl cyclase may
be a minor, heat-stable, as yet unidentified protein contaminant, which is present
in calmodulin preparations specifically from Paramecium and Tetrahymena. Cer-
tainly, further work is required to elucidate the details of the Ca
2+
-regulation of
the protozoan guanylyl cyclases.
18.2.2 Regulation of cAMP levels
The regulation of intracellular cAMP formation in Paramecium was even more
astounding. Control of cAMP levels in Paramecium is not governed by hor-
mones, but by changes in the concentration of extracellular ions that cause hy-
perpolarization [9, 11, 25]. As a freshwater ciliate, Paramecium must actively
control its K
+
-resting conductance to maintain the resting membrane potential
within narrow limits in shifting environments. Therefore, the extent of a hyper-
polarization that is caused by dilution of external cations, is dependent on the
composition of the equilibration buffer, which determines the existing resting
conductance, mainly a K
+
-conductance. We investigated whether the K
+
-resting
conductance itself may be coupled to activation of a membrane-bound adenylyl
cyclase. When Paramecia are adapted for 4 hours to a solution containing 16
mM external K
+
, in which the resting conductance is high, an instantaneous di-
lution of K
+
to 2 mM causes a rapid, about four-fold rise of cAMP levels starting
within milliseconds [9]. It reaches its maximum within 5 to 10 s and declines to a
new steady state level after about 240 s. (Fig. 18.2A). This biochemical response
is accompanied by a fast swimming response, which persists for longer periods
of time than the actual cAMP response indicating that both must not be
coupled. Interestingly, in cells adapted to 1 mM K
+
, in which the resting conduc-
tance is low, dilution of K
+
to one eight of its original concentration does not en-
hance cAMP formation. Similarly, the behavioral response is almost absent. Ap-
parently the stimulation of cAMP formation is correlated with hyperpolarizing
changes of the resting membrane potential and suggests that both are coupled.
In fact, hyperpolarization and cAMP production were quantitatively related to
298
Figure 18.2: Hyperpolarization as a hormone surrogate stimulates cAMP formation in
Paramecium. (A) Time-dependent formation of intracellular cAMP elicited by hyperpolari-
zation. Cells were equilibrated in a buffer containing 16 mM KCl, 0.5 mM CaCl
2
, 10 mM
MOPS, pH 7.2. For stimulation, KCl was diluted to one eighth of its original concentration
by transfer of 50 l of cells into 350 l of potassium-free buffer. (B) Hyperpolarization-dose
response curve for cAMP formation. Cells equilibrated in 16 mM KCl were stimulated by
dilution of external KCl proportionally as indicated on the abscissa and cAMP levels were
determined 5 s later. (C) Hyperpolarization-stimulated cAMP formation in Paramecium
wild type and K
+
-channel mutant cells (restless). Cells were equilibrated in 4 mM KCl and
stimulated by an eight-fold dilution of KCl to 0.5 mM. cAMP was determined after 5 s
(open bars: control cells; black bars: dilution-stimulated cells). Addition of the K
+
-channel
blocker tetraethylammonium (8 mM; patterned bar) with the dilution buffer abolished the
exaggerated response in restless mutant cells (data are adapted from [11]).
299
18.2 Identification and characterization of cGMP and cAMP second messenger
each other. When graded stimuli were applied to Paramecia by dilution of exter-
nal K
+
to different extents, hyperpolarization response curves for cAMP forma-
tion were obtained, which were essentially equivalent to hormone dose re-
sponse curves observed in mammalian cells and tissue preparations (Fig.
18.2B). This means that conceptually hyperpolarization of Paramecium is a sti-
mulus, which corresponds to a hormonal adenylyl cyclase stimulation in a me-
tazoan cell.
To investigate the directness of coupling between the K
+
-resting conduc-
tance and cAMP generation we used classical K
+
-channel blockers. Tetraethyl-
ammonium, Cs
+
-ions or quinine, which block K
+
-channels in many cells includ-
ing Paramecium, inhibited cAMP production upon dilution of external K
+
dose-
dependently (Fig. 18.2C). Further, a mutant of Paramecium, restless, is over-
reactive to low K
+
. The mutant cannot control its K
+
-resting conductance and
behaves like a K
+
-electrode [20, 26]. E.g. at 4 mM K
+
-restless has a membrane
resistance of 30 MO compared to 60 MO in wild type cells. When incubated in
low K
+
medium it will die because of the incessant loss of cellular K
+
. Conse-
quently, restless adapted to 4 mM K
+
responds to an eight-fold dilution of K
+
with a large accumulation of cAMP whereas wild type cells do not (Fig. 18.2C).
The shift in the K
+
-equilibrium potential as brought about by dilution is
sufficient to transiently hyperpolarize the cell. In principle, a net ion current is
not required. Nevertheless, a sudden hyperpolarization affects those inwardly
and outwardly directed ion currents, which are at equilibrium at rest, i. e. are
electrically balanced. Application of the Nernst equation indicates that only a
K
+
-efflux is possible that may flow through K
+
-channels, which carry the major
resting conductance, and is afflicted in restless mutant cells. This resting current
then appears to serve a non-electrical function in that it is involved in regulating
cAMP formation.
18.3 Biochemical properties of an adenylyl cyclase
The above data were of particular interest considering the proposed membrane
topology of mammalian adenylyl cyclases. The currently accepted topology com-
prises two hydrophobic membrane cassettes of six a-helical transmembrane
spans, designated M1 and M2, and a catalytic center formed by two conserved
cytoplasmic domains of approximately 250 amino acids, C1a and C2a, that are
downstream of M1 and M2, respectively. C1a and C2a precede poorly conserved
cytosolic segments of variable length, designated C1b and C2b, the latter is
missing in some isoforms. The overall topology of mammalian adenylyl cyclases
can thus be abbreviated as M1C1abM2C2a(b) [17] (see Fig. 18.3A, right, for a
model depiction). Immediately after this membrane topology was recognized it
was speculated that the bulky membrane anchors of the adenylyl cyclase may
300
Figure 18.3: (A) Putative membrane topology of the cloned guanylyl cyclase from Para-
mecium (Tetrahymena and Plasmodium). The transmembrane spans are depicted as bar-
rels and the C-terminal cytosolic portions of the guanylyl cyclase are shown doughnut
shaped for the C1a/C2 heterodimer [31]. Like in several mammalian adenylyl cyclase iso-
forms, a distinct C2b region is absent in the Paramecium guanylyl cyclase. The short-
hand nomenclature used for mammalian adenylyl cyclases has been adopted. M1 and M2
designate the two membrane anchors with six transmembrane spans, the C1a- and C2-
positioned domains form the catalytic center. (B) A local alignment of the catalytic C1a
and C2a-positioned domains of the Paramecium guanylyl cyclase with the corresponding
mammalian adenylyl (bovine type VII) and guanylyl cyclase (rat soluble) sequences shows
that the C1a and C2-positioned cytosolic domains of the Paramecium guanylyl cyclase are
inverted. The signature motifs GDCY and TYMA are shaded, substrate defining amino
acid residues are enlarged. Data are adapted from [48].
301
18.3 Biochemical properties of an adenylyl cyclase
possess an intrinsic transport activity [27]. Similar topologies were known to be
present in the multi-drug resistance gene, various membrane transporters such
as ABC-transporters and voltage-operated ion channels. Quite obviously, our
lines of thinking were influenced by these finding. We hypothesized that the
membrane-bound adenylyl cyclase of Paramecium may have retained an ances-
tral ion conductance. Therefore, we axenically cultivated Paramecia in bioreac-
tors of up to 200 liters [6]. Because the cilia constitute only 1% of cellular protein,
yet contain 50% of adenylyl cyclase activity, they were used as starting material
for adenylyl cyclase purification. About 20 ng of purified adenylyl cyclase were
obtained from 170 g of Paramecium with a specific activity of 25 mol/mg/min
[11]. Analysis by SDS-polyacrylamide gel electrophoresis showed a single band
at 96 kDa, i. e. in a range compatible with an adenylyl cyclase of mammalian
membrane topology. Next, we tested the purified enzyme in artificial lipid-bilayer
membranes for pore-forming and ion-conductance capacity. When purified en-
zyme was used for reconstitution, an ion conductance of 320 60 pS was meas-
ured [11]. The relative permeability ratios for K
+
: Cs
+
: Na
+
: Ca
2+
: Li
+
: Mg
2+
were
determined to be 1: 1: 0.5: 0.3: 0.25: 0.2, respectively. Tetraethylammonium did
not pass the pore. We were unable to reliably test whether K
+
-channel blockers
affected the reconstituted ion conductance. The observed conductance was im-
permeable for anions such as chloride or acetate. The enzymatic activity and
pore-forming capacity were strictly interdependent. All purification steps that in-
terfered with adenylyl cyclase activity also impaired pore-forming activity, which
co-purified through six distinct separation steps. This data strongly suggested
that the protozan adenylyl cyclase is not only regulated by the resting membrane
potential, but is the transmembrane ion pore, which is responsible for setting the
resting potential of the cell. Thus, it may possess an intrinsic secondary, regula-
tory function compatible with the metazoan adenylyl cyclase membrane topol-
ogy. Obviously, cloning of the gene corresponding to the protozoan adenylyl cy-
clase would clarify the exact relationship of this cyclase to its metazoan conge-
ners and eventually yield clues what a presumably primordial ion conductance of
the adenylyl cyclase membrane anchor may look like.
18.4 A guanylyl cyclase disguised as an adenylyl cyclase
Initially, we were determined to purify enough protein to obtain amino acid se-
quence information, which could be used to develop degenerate primers and
clone the gene using PCR and a Paramecium cDNA library as a template. All ef-
forts toward that goal were, however, unsuccessful. This was highly discoura-
ging because despite the universality of cAMP as a second messenger, at least
three phylogenetically separate classes of adenylyl cyclases exist (a forth and
possibly a fifth class are currently discussed) and notwithstanding the above ar-
302
guments there was no certainty that the presumed adenylyl cyclase of Parame-
cium would belong to the class III isoform family present in all eukaryotes. The
three classes of adenylyl cyclases, which are defined on the basis of sequence
identities and similarities in their putative catalytic domains, share no sequence
similarity and the current view is that they evolved independently [17, 28].
The class I adenylyl cyclases evolved in bacteria, e. g. in E. coli or Yersinia
and are represented by cytosolic or membrane-attached forms. Class II isoforms
comprise the so-called bacterial toxins from Bordetella pertussis and Bacillus an-
thracis. The extracellularly secreted enzymes of up to 200 kDa are bifunctional
[29, 30]. Notably, these toxins are linked with a faintly hemolysin-like domain,
which inserts itself as a membrane pore into the eukaryotic cell membrane and
uses this gate to deliver the catalytic domain to the cytosol. The class III cy-
clases, comprising adenylyl as well as guanylyl cyclases, are present in essen-
tially all eukaryotes looked at so far. However, members of this cyclase family
also occur in bacteria such as Brevibacterium, Mycobacterium, Stigmatella and
Anabaena [28, 31]. Most recently, it was reported that a soluble adenylyl cyclase
related to class III enzymes is present in rat testis in a very large protein back-
ground of unknown function [32]. This indicates that class III adenylyl cyclase
catalysts may occur in different protein backgrounds connected to additional
functional and potentially regulatory or regulated domains.
For a homology cloning approach we used a 328 bp genomic DNA se-
quence from Paramecium, which displayed 29% amino acid identity to the cata-
lytic C2a region of a rat type III adenylyl cyclase isoform (kindly provided by C.
Russel and R. D. Hinrichsen) [33], i. e. we set out to clone an adenylyl cyclase,
which would conform to the mammalian membrane topology M1C1abM2-
C2a(b). Using cDNA library screening we obtained a huge 7.2 kb DNA frag-
ment with a 3'-TGA stop, a poly-A tail and a continuous open reading frame ex-
tending all the way to the 5'-end. A genomic DNA library was used to obtain
the ATG start, which was just 12 bp upstream. The completed clone coded for a
protein of 2412 amino acids with a calculated molecular mass of 282.6 kDa (Fig.
18.3A; GenBank accession number AJ238859). Hydrophobicity analysis indi-
cated five domains with a total of 22 putative transmembrane spans. Similarity
searches demonstrated that the protein consisted of two large units: an N-term-
inal half of 1319 amino acids (155 kDa) with topological and amino acid se-
quence similarities to P-type ATPases and a C-terminal half of 982 amino acids
(115 kDa) with a membrane topology identical to the prototypical mammalian
adenylyl cyclase and unequivocal sequence similarity in the catalytic loops
(Fig.18.3A). Both units were linked by a polypeptide of about 100 amino acids.
To exclude the possibility of an artifact cDNA accidentally joined during library
preparation, we verified the genomic structure by cloning a 2.5 kb EcoRI frag-
ment from gDNA, which was intronless and fully covered the intradomain re-
gion. Thus we unequivocally established the presence of this gene at the gDNA
level.
The Paramecium cyclase unit contained hydrophobic M1 and M2 regions
with six transmembrane spans each, which were followed by C1a- and C2a-po-
sitioned domains with sequence similarity to the catalytic loops of metazoan
303
adenylyl cyclases (Fig. 18.3A). The C1a-positioned domain was followed by a
hydrophilic stretch of 221 amino acids reminiscent of a C1b region, a distinct
C2b region was absent. All mammalian adenylyl cyclases have GDCY as a sig-
nature sequence in the C1a domain. In the protozoan cyclase GDCY is present
in the C2a-positioned domain (22762279), i. e. close to the C-terminal. Simi-
larly, the motif TYMA is invariant in all C2a regions of mammalian adenylyl cy-
clases whereas in the ciliate cyclase it was located in the C1a-positioned do-
main (16361639), i. e. toward the 5'-end (Fig. 18.3B). Further, mammalian ade-
nylyl cyclases contain a VKGKG motif in their C2a catalytic region. The first ly-
sine presumably binds the g-phosphate of ATP [34, 35] and is essential for cata-
lysis. In the Paramecium gene a similar motif,
1793
AKGKG
1797
, was found in the
C1a-positioned domain. Therefore, we concluded that the C1a- and C2a-posi-
tioned loops of the Paramecium cyclase were inverted compared to mammalian
adenylyl cyclases resulting in a (mammalian nomenclature) M1C2aC1bM2C1a
architecture.
In 1997/1998, the amino acids in the heterodimeric catalytic pocket of me-
tazoan adenylyl and guanylyl cyclases, which are important for substrate speci-
ficity, K, D, and Q in adenylyl cyclases, E, C, and R in guanylyl cyclases, have
been determined by X-ray crystallography and site-directed mutagenesis [34
37]. To our surprise, in the Paramecium cyclase these crucial amino acids are
guanylyl cyclase-like with a substitution of C by S1698 (Fig. 18.3B). On the
other hand, several amino acids, which form a second, non-catalytic pocket in
mammalian adenylyl cyclases, are conserved in the protozoan cyclase, i. e. were
like those in adenylyl cyclases. These results imply that the cloned gene repre-
sents a novel guanylyl cyclase, which by topology and primary structure is most
closely related to metazoan adenylyl cyclases.
P-type ion transport ATPases were the only proteins with significant simi-
larity to the N-terminal half of this Paramecium nucleotide triphosphate cyclase.
The predicted membrane topology was identical to that of P-type ATPases, i. e.
two cassettes of two putative transmembrane spans in the N-terminal region
(amino acids 63110 and 349407) and one set of six transmembrane spans close
to the C-terminus (amino acids 11211319; Fig. 18.3A) [38, 39]. In the cytosolic
domains of the P-type ATPase family, which has more than 150 members, sev-
eral sequence blocks are conserved [40]. Four of these align to corresponding
cytosolic regions of the Paramecium guanylyl cyclase. DKTGT(L/I)T, which is lo-
cated within the large cytoplasmic loop, is an invariant signature sequence in
all P-type ATPases [41]. For ion transport to occur the aspartate must be phos-
phorylated [42]. In the Paramecium protein DKTGTLT was conserved at the
equivalent position (amino acids 461467). A conserved GDGXND motif present
in the hinge region of P-type ATPases was
1025
GDSFSD
1030
in the Paramecium
gene and the (TSND)GE(SNT) block in the transduction domain was retained
with a significant E to N change as
190
SGNT
193
virtually excluding ATPase func-
tion [41, 43]. Finally, the ATP-binding domain in nearly all eukaryotic P-type
ATPases involves an indispensable aspartate in a block of moderately conserved
amino acids [44]. The corresponding amino acid in the Paramecium P-type
ATPase-like domain was Glu848. These decisive deviations of the Paramecium
304
sequence from the ATPase consensus imply that this guanylyl cyclase domain
has no ATPase activity and probably has adopted another function. Indeed,
ATPases have been cloned from Paramecium, which conform in all respects to
the canonical structure [45, 46]. So far, transfection of Paramecium is not possi-
ble. Yet, heterologous expression of Paramecium genes is impossible because it
uses the universal stops TAA and TAG for glutamine [47].
We changed all 99 TAA and TAG codons to CAA/CAG by site-directed
mutagenesis. The repaired cDNA was inserted into the bicistronic expression
vector pIRES1neo. However, upon transfection of HEK293 cells we obtained no
successful transcription, i. e. no functional mRNA. We assumed that the problem
may be caused by the high AT-content of the Paramecium guanylyl cyclase
gene (66%). Therefore, we resynthesized the ciliate cDNA coding for M1 and
M2 using the standard mammalian codon usage [48]. The partially synthetic
gene in pIRESneo was transfected into HEK293 cells and yielded a G418-select-
able cell population. Cell homogenates had membrane-bound guanylyl cyclase
activities of up to 150 pmol 7mg
1
7min
1
. The K
M
for MgGTP was 50 M. Ade-
nylyl cyclase activity was not observed with MgATP as a substrate, yet was de-
tectable using Mn
2+
as a metal cofactor. Forskolin did not enhance enzyme ac-
tivity. These results provided the final proof that the cyclase domain of the
cloned gene constituted a guanylyl cyclase in the disguise of what hitherto had
been considered to be a prototypical mammalian adenylyl cyclase topology.
Up to now, we were unable to express the full-length Paramecium guany-
lyl cyclase gene. Accordingly, we are unable to speak to the role of the P-type
ATPase domain and a potential functional interplay between both domains. We
may get a hint by examining the pore and cation-binding helices TM4, 5, and 6.
TM4 and 5 differ substantially from the corresponding helices of ion transporters
and the ciliate TM6 lacks amino acids, which supposedly are involved in cation-
binding [39]. We believe that the protozoan pore region does not bind inor-
ganic ions. Perhaps it associates with organic molecules as does another family
of P-type ATPases, the putative aminophospholipid transporters. The protozoan
P-type ATPase-like domains would then constitute a receptor-like entity remi-
niscent of the atrial-natriuretic receptor-guanylyl cyclase couple in mammals.
However, presently this must be marked as a speculation. Using PCR we de-
tected several additional genes coding for guanylyl cyclases of the same struc-
ture (see below).
Is the occurrence of this disguised guanylyl cyclase unique to Paramecium
or do similar enzymes exist in other organisms? We were not surprised to be able
to clone similar genes from the ciliate Tetrahymena because regulation of second
messenger cyclic nucleotides has been shown to be essentially identical to Para-
mecium [25; GenBank accession #AJ238858]. In addition, we searched in data-
bases for the presence of guanylyl cyclases with this architecture in other organ-
isms. In the GenBank we detected a 2.797 bp fragment designated as an adenylyl
cyclase from chromosome 11 of Plasmodium falciparum (accession No. U33118).
The open reading frame codes for a cluster of six transmembrane spans (M2) and
a C2a-positioned domain. At nucleotide 1966 a substrate-specifying arginine is
encoded, which strongly indicates that this gene, contrary to the given definition,
305
codes for a guanylyl cyclase with mammalian adenylyl cyclase topology. This de-
position did not specify a C1a-positioned domain. Further, the data, which have
been released, yet not edited, from the Malaria Genome Project, show the pre-
sence of a guanylyl cyclase sequence on chromosome 13 of Plasmodium falci-
parum, which exactly matched the Paramecium guanylyl cyclase topology, i. e. it
contained a P-type ATPase-like domain fused with a guanylyl cyclase of mamma-
lian adenylyl cyclase topology with the C1a and C2a positions inverted [48]. The
alignment of the C1a-positioned region with the Paramecium and Tetrahymena
guanylyl cyclases clearly identified GTP as the substrate.
The data allow us to discuss evolutionary steps during the development of
nucleotide triphosphate cyclases. The fact that cyclases exist with catalytic C1a
and C2a domains arranged in both ways, supports the hypothesis that initially
membrane-anchored monomers formed a homodimeric adenylyl cyclase. This
structure is still in existence in several bacteria and may actually represent a
common ancestor of mammalian and ciliate nucleotide triphosphate cyclases
[31, 49]. Upon gene duplication separate evolution led to the formation of a het-
erodimeric adenylyl cyclase in eukaryotes before the monomers were fused in a
single peptide chain. Evidently, the order of subsequent monomer linkage oc-
curred either way. Only after this event did the protozoan guanylyl cyclase
evolve by a few point mutations in the purine binding pocket from an ancestor
adenylyl cyclase module. All guanylyl cyclase isoforms detected in Paramecium,
Tetrahymena and Plasmodium likely evolved from a common ancestor. On the
other hand, the soluble heterodimeric guanylyl cyclases in mammals must have
diverged from an ancestral cyclase homodimer before the bulky membrane an-
chors were added and before heterodimers evolved because the guanylyl cy-
clases have retained identical amino acids at positions, which determine sub-
strate specificity and at equivalent positions of a non-catalytic pocket (see Fig.
18.2) [35]. In summary then, the protozoan guanylyl cyclases evolved from a
membrane-anchored, heterodimeric adenylyl cyclase with inverted catalytic
centers whereas mammalian guanylyl cyclases most likely descended from a
primordial cytosolic cyclase homodimer. The acquisition by the protozoan gua-
nylyl cyclases of an ATPase-like domain may have occurred prior to or after the
change in substrate specificity [48].
18.5 On the way to an adenylyl cyclase with an intrinsic ion
conductance
Irrespective of the novelty of the above findings we realized that we missed the
original target of our intended studies, cloning of a ciliate adenylyl cyclase with
an ion transport capacity [10, 50]. Therefore, we began a renewed search for the
306
protozan enzyme, which converts ATP to cAMP. In keeping with the initial idea
of the existence of a mammalian class III adenylyl cyclase with an ancestral ion
conductance we started a homology cloning search to detect a cyclase, in which
the decisive, substrate defining amino acids located in the C1a-positioned do-
main would define ATP as a substrate. Sequencing about 100 PCR clones
yielded only another dozen of guanylyl cyclase isozymes, which had a serine,
not an aspartate, as one of the three crucial amino acids that specify GTP as a
substrate, i. e. these genes coded for guanylyl cyclases. The extremely con-
served regions around the GTP-specifying regions (20 to +2) were nearly iden-
tical in all protozoans variants and highly conserved with respect to the corre-
sponding region in mammalian adenylyl cyclases. The anterior 40 amino acids
were diverged (2193% identity) and had approximately the same degree of di-
versity as that observed in the nine mammalian adenylyl cyclase isoforms [48].
None of the PCR products identified a potential adenylyl cyclase.
Another candidate adenylyl cyclase prototype with a potential ion conduc-
tance may be the bacterial class II toxins as exemplified by the secreted en-
zymes from Bordetella pertussis and Bacillus anthracis [28, 30]. Both enzymes
share little sequence identity. As mentioned above these cyclase toxins form a
membrane pore in the eukaryotic target cell, which is used to deliver the cataly-
tic domain into the cytosol. We developed degenerate oligonucleotide primer
pairs using the Paramecium codon usage, which covered the two most con-
served sequence stretches. A Paramecium cDNA library was used as a template.
Analysis of about 100 PCR products subcloned into pBluescript did not indicate
the presence of a class II adenylyl cyclase in Paramecium.
We reasoned that class I adenylyl cyclases, which so far have been exclu-
sively detected as soluble bacterial enzymes, were unlikely candidates for a cili-
ate isoform with an ion conductance and did not warrant a homology cloning
approach. Next we centered our attention on bacterial class III adenylyl cy-
clases, which share to some extent identities in their catalytic domains with
mammalian adenylyl cyclases. Some bacterial isoforms are linked or unlinked
homodimers and have different membrane anchors. Using this approach we ob-
tained a cDNA clone from Paramecium, which may constitute an adenylyl cy-
clase monomer. The cDNA codes for a protein, which is partitioned into several
domains. Structural predictions indicate the presence of an ion channel, poten-
tially a K
+
-conductance, an adenylyl cyclase catalytic domain, and a C-terminal
tetratricopeptide segment, which could pin the monomer unit to structural pro-
teins underneath the cell membrane. The skewed codon usage of Paramecium
precludes heterologous expression, and we will be faced with the same pro-
blems alluded to above to remanufacture this gene for expression in heterolo-
gous systems, i. e. in HEK293 and Sf9 insect cells and as a cRNA in Xenopus
oocytes. The functional proof of the existence of a Paramecium adenylyl cyclase
containing an intrinsic, regulatory ion conductance has, therefore, not yet been
established.
307
18.5 On the way to an adenylyl cyclase with an intrinsic ion conductance
18.6 Downstream of second messengers
After the generation of cyclic nucleotides the predominant intracellular chain of
events involves reversible phosphorylation of specific target proteins by respec-
tive cylic AMP- and cGMP-activated protein kinases and dephosphorylation by
protein phosphatases. Several investigations have shown that regulation of ion
channels can proceed by reversible phosphorylation. Thus, Paramecium with a
number of behaviorally, electrophysiologically and genetically characterized ion
channels may in fact represent a good model system to study. We and others
have detected several protein kinases in Paramecium, among those also cAMP
and cGMP-dependent varieties [5155]. We have then focussed our work on pro-
tein phosphatases as these enzymes have received much less attention than pro-
tein kinases. A large fraction of cytosolic protein phosphatases in eukaryotes has
been categorized in four major groups. This classification, which was originally
based on enzymological criteria using specific peptide inhibitors, activators and
substrate proteins, defined protein phosphatases type 1, 2A, 2B (calcineurin) and
2C. Molecular cloning revealed that protein phosphatases types 1, 2A, and 2B
belong into one gene family, whereas the type 2C enzymes are defined as a sepa-
rate gene family. Initially, our work was spurned by the surprising identification
of the dinoflagellate polyketal okadaic acid as a highly potent and specific inhibi-
tor of protein phosphatase type 1 and 2A [56]. We speculated that phosphoryla-
tion/dephosphorylation cycles may be decisive in opening and closing the vol-
tage-operated Ca
2+
-channel. First, we demonstrated by protein biochemistry the
presence of a protein phosphatase type 1 in Paramecium and the specific inhibi-
tion of the isolated enzyme by okadaic acid [14, 57]. At 1 M of the inhibitor the
protozoan type protein phosphatase was essentially blocked whereas other pro-
tein phosphatases present in Paramecium, such as protein phosphatase 2A and
2C were not affected. The failure of the toxin to inhibit protein phosphatase 2A
came as a considerable surprise. Protein phosphatases type 1 and 2A are mem-
bers of the same gene family and the mammalian enzymes share about 50% se-
quence identity. Whatever the explanation for this may be, it was fortuitous be-
cause it meant that okadaic acid is a specific inhibitor of protein phosphatase
type 1 in Paramecium. This unique property allowed us to prove as an exemplary
case the validity of our initial approach to use Parameciumas an in vivo model for
screening of novel compounds. We demonstrated that okadaic acid did not affect
the normal forward swimming pattern of Paramecium [14]. An increase in the ex-
ternal K
+
-concentration from 1 to 20 mM triggered backward swimming as ex-
pected from a depolarization-activated Ca
2+
-influx. In the absence of okadaic
acid the backward swimming response was transient and >90% of stimulated
cells had returned to the forward swimming mode within 20 s (Fig. 18.4). In the
presence of 34 M okadaic acid, however, >95% of cells were still swimming
backward 2 min after the increase of K
+
. This dramatic effect required a 15 min
preincubation with okadaic acid and, as in metazoans, higher concentrations of
okadaic acid compared to those needed to inhibit protein phosphatase type 1 in
308
vitro. This may be due to higher local phosphatase concentrations in vivo com-
pared to those present in in vitro assays, as well as to slow and insufficient diffu-
sion of okadaic acid across the thickly protein-coated surface of Paramecium.
The effect of okadaic acid most likely was directed at the voltage-operated Ca
2+
-
channel because chelation of external Ca
2+
by addition of EGTA 2 min after K
+
-
depolarization in the presence of okadaic acid caused an immediate termination
of backward swimming and resumption of forward movement, i. e. okadaic acid
treatment enhanced the influx of Ca
2+
and did not affect the motile apparatus of
the cilia itself [14]. The effects of okadaic acid in Paramecium are compatible
with a model for regulation of the voltage-operated Ca
2+
-channel in the ciliary
membrane, which accounts for all the behavioral, electrophysiological and bio-
chemical observations. With the membrane potential at rest the Ca
2+
-channel is
in a closed state, which is susceptible to activation by depolarization. Depolariza-
tion triggers a conformational change of the channel converting it to an open
state within 12 ms, which leads to a depolarizing Ca
2+
-influx and drives the re-
versal of the ciliary beat, hence backward swimming, by an okadaic acid-insensi-
tive mechanism. We propose that the Ca
2+
-channel is phosphorylated in the
closed groundstate, which is inaccessible to dephosphorylation. By contrast, the
open state of the channel is dephosphorylated rapidly converting it to an inacti-
vated, dephosphorylated state, which does not permit further Ca
2+
-influx. This
Figure 18.4: Okadaic acid, a specific protein phosphatase type 1 inhibitor, impairs Ca
2+
-
channel closure in Paramecium in vivo. Cells were equilibrated in buffer containing 1 mM
KCl, 50 M CaCl
2
, 10 mM MOPS, pH 7.2. Okadaic acid (final concentration 34 M in 1%
DMSO) was added 15 min prior to stimulation. Swimming behavior was monitored micro-
scopically. Cells were depolarized by addition of KCl (20 mM). Okadaic acid sustained the
Ca
2+
-influx as measured by the prolonged backward swimming response. Addition of
75 M EGTA abruptly ended the avoiding reaction due to chelation of external calcium
(from [14]).
309
18.6 Downstream of second messengers
reaction sequence is responsible for termination of the backward swimming re-
sponse after about 10 s in the absence of okadaic acid. In the presence of okadaic
acid protein phosphatase 1 is inhibited, dephosphorylation attenuated and a
Ca
2+
-inward current is sustained, which is sufficient to support ciliary reversal
unless Ca
2+
-influx is prevented by chelation of extracellular Ca
2+
. The reconver-
sion of the inactivated dephosphorylated state to the closed phosphorylated state,
which resensitizes the channel to depolarizing stimuli, is a slow process requiring
510 min at ambient temperature. These data, though encouraging, also demon-
strate that characterization of a molecular target prior to in vivo screening can be
a most useful way to define in vivo reaction cascades and support the tendency to
approach drug development by reverse pharmacology.
The extent of overall conservation among protein phosphatase 2C en-
zymes from different phyla, for which sequence data are available, is limited.
The considerable sequence disparities may indicate a functional diversity in dif-
ferent phyla. In Paramecium, the protein phosphatase 2C is membrane-asso-
ciated, a considerable fraction of the enzyme is localized to the cilia, the highly
specialized motile organelle of the ciliate [58]. Microsequencing of six tryptic
peptides of the purified enzyme revealed a relationship to other PP2C isoforms.
The Paramecium PP2C gene was obtained using degenerate oligonucleotide
primers. The gene coded for a 33 kDa-protein with 300 amino acids, i. e. it is
one of the smallest PP2C isoforms [58]. A C-terminal truncation by about 80
amino acids is responsible for the small size of the Paramecium PP2C compared
to isoforms from other organisms. We defined three core regions of high conser-
vation to be present in all PP2C enzymes. These account for about 25% of the
protozoan primary sequence. After mutation of nine ciliate Q codons (TAA) to
CAA the Paramecium gene was expressed as an active protein in E. coli. The
catalytic core region was defined by N- and C-terminal deletions to represent
284 amino acids, i. e. the ciliate PP2C mainly comprises the catalytic core [59].
The data support the notion that the three-dimensional structure of the Parame-
cium PP2C is identical to that determined with the recombinant human PP2C
despite an overall meager amino acid conservation (31%), which is more or less
restricted to the three conserved regions mentioned above. In the X-ray struc-
ture an unordered loop region is in front of the b9-strand [60]. We introduced a
factor Xa protease cutting site (IEGRA) in this domain to define the catalytically
active center by generation of proteolytically truncated versions of the PP2C.
Surprisingly, this construct, in which the sequence QLII (212215) was changed
to IEGR, thus generating in conjunction with Ala
216
a factor Xa cutting site, was
inactive. Individual amino acid exchanges at each of the changed positions
showed that exclusively the I214G conversion was responsible for the loss of
function (Table 18.1). In an I214A variant 62% of wild type activity was retained,
and an I214L product was as active as the wild type enzyme. Strikingly, the ad-
jacent isoleucine 215 was not crucial for enzymatic activity. Neither an I215R
nor an I215G conversion abolished phosphatase activity. Similar mutation ex-
periments were then carried out with the bovine type 2Ca protein phosphatase,
which contains a hydrophobic valine at an equivalent position. AV215G conver-
sion also inactivated the mammalian enzyme (Table 18.1). In this respect our
310
data clearly go beyond the picture, which has emerged from the crystal struc-
ture of PP2C. We have no clue why a mutation that replaces a hydrophobic
amino acid at position 214 (Paramecium PP2C numbering) with glycine results
in inactivation. The loss of a hydrophobic side chain may alter the stability or a
directed movability of this part of the protein, which has no defined tertiary
structure in the crystal, such that the active site centers cannot sufficiently stabi-
lize an enzyme/substrate transition state. In contrast to PP1/PP2A, there are cur-
rently no specific inhibitors available for protein phosphatase 2C. So far, we
have tested a number of synthetic and biological compounds, yet could not un-
equivocally identify a specific inhibitor of PP2C, which would aid to pinpoint a
specific physiological function for this protein phosphatase like okadaic acid did
for PP1. We also examined the subcellular localization of PP2C [59]. The major
fraction is cytosolic, partly it is associated with the macronucleus, and a minor
part is structurally bound to the cilia where it was associated with microtubulus
and dynein, i. e. it was clearly targeted to the ciliary motor, implicating a motor
component as one of the PP2C substrates. It seems reasonable to consider that
the cytoplasmic and nuclear localization of PP2C may be related to processes in-
volving cellular cargo transport [59].
18.7 In vivo screening of bacterial secondary metabolites
We tested more than 100 bacterial secondary metabolites for their toxicity and
registered qualitatively and quantitatively by digitized motion analysis changes
in the swimming behavior of Paramecium. In part, the assays were marred by
Table 18.1: A single amino acid replacement (I214G) in a region without defined second-
ary structure [60] abolishes enzymatic activity of Paramecium protein phosphatase 2C. A
corresponding replacement in the bovine type 2Ca isoform (V215G) also leads to an inac-
tive enzyme (adapted from [59]).
Enzyme variant Specific activity
(mU/mg)
PtPP2C wild type 15.7
PtPP2C L213E 12.3
PtPP2C I214G 0
PtPP2C I214A 9.7
PtPP2C I214L 19.2
PtPP2C I215R 11.2
bov. PP2Ca wild type 2.5
bov. PP2Ca V215G 0
311
18.7 In vivo screening of bacterial secondary metabolites
the poor solubility of the compounds and the need to use organic solvents,
mainly dimethylsulfoxide, for bath application. Although we have identified
several compounds, e. g. depsichlorin, T 3580 and T 3586, to distinctly affect
evoked swimming patterns of the ciliate, clear-cut actions were not obtained,
which would have permitted to deduce a mechanism of action on a single com-
ponent of the second messenger signal transduction pathway.
Acknowledgments
Supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Che-
mischen Industrie. Sequence data for P. falciparum chromosome 13 was ob-
tained from The Sanger Centre website at http://www.sanger.ac.uk/Projects/
P_falciparum/. Sequencing of P. falciparum chromosome 13 was accomplished
as part of the Malaria Genome Project with support by The Wellcome Trust.
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References
Chemical Synthesis
and Structure Elucidation
ISBN: 3-527-30615-3
19 Structure Elucidation and Chemical Synthesis
of Microbial Metabolites
Roderich D. Smuth, Jrg Metzger, and Gnther Jung*
19.1 Introduction
The introduction of the collaborative research centre 323 opened a fruitful and
interesting cooperation between biologists and chemists, wherefrom both sides
have greatly benefited. This successful cooperation profited from the expertise
of the co-working chemists in the fields of biochemical analytics and chemical
synthesis.
From the chemists view three achievements extraordinarily contributed to
the work of the collaborative research centre 323. The first was the introduction
of automated parallel peptide synthesis. The development of robotic systems
made the synthesis of hundreds of different peptides within several days feasi-
ble and enormously accelerated the production of peptide libraries and peptido-
mimetics.
The second important innovation was the electrospray mass spectrometer
(ESI-MS) installed in 1989, and the successive development and performance of
so called hyphenated analytical techniques, such as HPLC-ESI-MS. The intro-
duction of the electrospray mass spectrometer at that time completely revolutio-
nized the possibilities of analyzing biological samples and extracts.
Finally, the installation of the 600 MHz NMR spectrometer enabled the de-
termination of peptide and small protein 3D-structures.
However, the impetus by these developments was given not only to the
groups directly cooperating within the collaborative research centre 323, but
also to new cooperations in related fields e. g. immunology (collaborative re-
search centre 510) and finally the introduction of combinatorial chemistry in T-
bingen (BMBF project 03 D 0037). The resonance towards the introduction of
these new techniques reached far beyond the cooperating research groups of
* Institut fr Organische Chemie, Universitt Tbingen, Auf der Morgenstelle 18,
D-72076 Tbingen.
319
ISBN: 3-527-30615-3
microbiology in and outside of Tbingen. Due to the early introduction of these
new methods in Tbingen, some key reviews [14], scientific contributions and
books were published, which had impact on the research of related fields.
The following gives an overview only of the most significant experimental
scientific contributions within the collaborative research centre 323.
19.2 Development of methods
19.2.1 Peptide synthesis
Shortly after the invention of the solid phase synthesis by Nobel laureate R. B.
Merrifield in 1963, the Institute of Organic Chemistry introduced this new tech-
nique at the University of Tbingen (G. Jung, E. Bayer in 1965). Automated
peptide synthesizers were introduced in 1989. As one of the first academic
groups in Europe G. Jungs group used these synthesizers for parallel synthesis
of hundreds of peptides in mg-quantities. As a consequence of this development
and after the introduction of the library concept, partly or fully randomized pep-
tide libraries were synthesized. From the rapidly established key technologies
in Tbingen, cooperating research groups in biochemistry, immunology, and
sensorics gained considerable profit from protein and substrate mapping ex-
periments, the finding of sequence motifs of MHC ligands and T cell epitopes,
and chemosensor developments. Peptides or peptide libraries synthesized in
parallel were extensively used in the collaborative research centre 323, e. g.
with the groups of V. Braun [5] and F. Gtz [6], but also in cooperation with the
immunology group of H.-G. Rammensee and many external research groups
within Europe.
A further important branch was the expertise on the synthesis of unusual
peptides, e. g. alamethicin in 1984 and lipopeptides vaccines in 1986. This con-
tinuous knowledge was decisive for the success of several projects, e. g. synth-
esis of heterocyclic backbone modifying amino acids as a part of the microcin
B17 structure [7] and the total synthesis of microcin B17 in 1996 [8].
320
19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites
19.2.2 Characterization of peptide libraries with electrospray mass
spectrometry
The collaborative research centre 323 obtained the first ESI-MS instrument in
Europe. This opened new ways for the mass spectrometric characterization of
natural products, especially of peptides (Fig. 19.1) and proteins, but also of oligo-
nucleotides, oligosaccharides, and polymers. In the projects of J. Metzger (project
C3) and G. Jung (project C2) several hyphenated techniques, e. g. HPLC-MS
(Fig. 19.2) and autosampler-ESI-MS were established. The first mass spectro-
metric investigation of randomized peptide libraries has been performed [9] and
further extensive investigations of these libraries confirmed ESI-MS as a method
of choice for synthesis control of mixtures containing few to several thousands of
components [10]. Moreover, the first LC-ESI-MS characterization of a 9-mer pep-
tide library was performed, revealing side reactions, incomplete side chain de-
protection, etc. resulting from peptide synthesis and cleavage from the resin [10,
11]. It was shown that mass spectrometric tandem experiments such as daughter
ion scan, parent ion scan and neutral loss scan are suitable methods for the detec-
tion of undesired side reactions and byproducts. In further studies mass spectra
of the synthetic peptide libraries were compared with theoretically expected
Figure 19.1: Identification of tert-butylated peptides with electrospray-tandem mass
spectrometry in the octapeptide library SNYTFX1X2X3(X1 = T,I,E,S)(X2 = N,K,Q)(X3 =
L,M,I,V). Top: Q1-spectrum of the mixture displaying the [M+H]
+
-signals of the theoreti-
cally expected 48 peptides and the tert-butylated byproducts. Bottom: Selective detection
of the tert-butylated byproducts using neutral loss scan (loss of isobutene 56).
0
100
75
50
25
0
R
e
l
.
I
n
t
.
(
%
)
987
1001
1015
1030
1043
1058
1075
1089
100
75
50
25 R
e
l
.
I
n
t
.
(
%
)
931
945
959
973
987
1001
1015
1019
1030
1044
1058
[M+H]
[M+H+56]
+
+
[M+H+56]
+
*
(= tBu)
(= tBu)
321
mass distributions, using computer-assisted programs, which have been devel-
oped especially for this purpose [12]. With the help of these programs, which im-
plement a basis set of proteinogenic amino acids, protecting groups, as well as
user-defined amino acid residues, the purity of a peptide library can be qualita-
tively estimated. These fundamental investigations on peptide library analysis
formed the basis for subsequent contributions of other groups, e. g. on combinato-
rially synthesized small molecule libraries reported by Rebek et al. [1315].
19.2.3 Automated sequential Edman degradation combined with
ESI-MS detection
The major focus of the group of G. Jung and his coworkers has been the struc-
ture determination of complex peptides. One of the most advanced instrumental
sequencing methods is Edman degradation, which is still of essential importance
in determining the primary structure of lantibiotics. However, post-translational
modifications, e. g. N-terminal blocking and intra-chain bridging via lanthio-
nines as they occur in lantibiotics made the application of standard protocols im-
possible. In the case of lanthionines, e. g. nisin, bearing a,b-didehydroamino
acids sequence abortion occurs due to desamination, thus resisting routine Ed-
man degradation. As a consequence important information about sequences
could not be obtained. A second problem arose for lanthionine amino acids, be-
cause for these amino acids no PTH-derivative could be detected. To circumvent
Figure 19.2: Total ion chromatogram of an HPLC-MS run containing a 48 peptide mix-
ture. The peptides were separated on a nucleosil C-18-column (26100 mm, 5 m; linear
gradient 520% B in 40 min; A 0,1% TFA aq., B 0.1% TFA in acetonitrile). Top: The total
ion chromatogram displays the elution of 48 peptides in the mass range of m/z 8001200.
Bottom: The ion chromatogram of m/z 1041 displays the elution of eight peptides with
identical nominal mass (isobaric peptides).
total ion chromatogram
m/z 800-1200
100
75
50
25
0
ion chromatogram
m/z 1041
0.0 5.0 10.0 15.0 retention time [min]
100
75
50
25
0
16.1
15.2
14.5
16.4
R
e
l
.
i
n
t
e
n
s
i
t
y
[
%
]
R
e
l
.
i
n
t
e
n
s
i
t
y
[
%
]
322
both problems, a chemical derivatization procedure prior to Edman sequencing
was developed [16], which enabled the complete determination of the amino
acid sequences of the lantibiotics gallidermin and Pep5.
However, analysis of other peptides with unusual amino acids with stan-
dard Edman degradation faced problems since the obtained information was
not sufficient for compound characterization because no PTH-standards are
available for modified amino acids, and their Edman degradation products are
often overseen or misinterpreted. The routine identification by HPLC retention
times is only standardized for the 19 essential amino acids. As a consequence,
the sequencer-ESI-MS-coupling was developed for more sophisticated sequence
analysis [17] (Fig. 19.3). The continuous effluent of the HPLC of the Edman se-
Figure 19.3: Sequencer-ESI-MS coupling. UV-absorbance chromatogram and total ion
chromatogram (TIC) of an optimized gradient of PTH-standard amino acids (despite Cys).
5,0 10,0 15,0 20,0 25,0
238 475 713 950 1187
Time (min)/Scan
100
75
50
25
0
R
e
l
.
I
n
t
.
(
%
)
100
75
50
25
0
R
e
l
.
I
n
t
.
(
%
)
Asp
Leu
Thr
Asn
Ser
Gln
Glu, Gly
Ala
Tyr
Lys
Val
Phe
Ile
Met
Trp, His
Arg
Pro
Asp
Leu
Asn
Ser
Gln, Thr
Ala
Tyr
Lys
Val
Phe
Ile
Met
Trp, His
Arg
Pro
Glu, Gly
UV
TIC
Edman-sequenator ESI - mass spectrometer
323
6
,
0
8
,
0
1
0
,
0
1
2
,
0
1
4
,
0
1
6
,
0
1
8
,
0
2
0
,
0
2
2
,
0
2
4
,
0
t
/
m
i
n
1
0
0
7
5
5
0
2
50
R e l . I n t . ( % )
9
,
2
1
0
,
5
1
7
,
1
2
0
,
1
2
1
,
4
2
2
,
1
2
2
,
5
2
3
,
0
d
m
t
u
d
m
p
t
u
2
0
0
2
5
0
3
0
0
3
5
0
4
0
0
4
5
0
m
/
z
1
0
0
7
5
5
0
2
50
R e l a t i v e I n t e n s i t y ( % )
3
3
1
,
03
4
9
,
0
[
M
+
H
]
+
[
M
+
N
H
4
]
+
3
3
3
,
0
1
9
2
,
6
2
0
0
2
5
0
3
0
0
3
5
0
4
0
0
4
5
0
m
/
z
1
0
0
7
5
5
0
2
50
R e l a t i v e I n t e n s i t y ( % )
2
0
9
,
4
3
8
5
,
2
4
0
2
,
0
[
M
+
N
H
4
]
+
[
M
+
H
]
+
[
2
M
+
H
]
+
[
2
M
+
N
H
4
]
+
P
T
H
-
G
l
y
P
T
H
-
D
i
d
e
h
y
d
r
o
C
h
t
F
i
g
u
r
e
1
9
.
4
:
S
e
q
u
e
n
c
e
r
-
E
S
I
-
M
S
c
o
u
p
l
i
n
g
:
U
V
c
h
r
o
m
a
t
o
g
r
a
m
o
f
a
n
E
d
m
a
n
d
e
g
r
a
d
a
t
i
o
n
c
y
c
l
e
o
f
3
-
c
h
l
o
r
o
-
b
-
h
y
-
d
r
o
x
y
-
t
y
r
o
s
i
n
e
(
C
h
t
)
a
n
d
c
o
r
r
e
s
p
o
n
d
i
n
g
m
a
s
s
s
p
e
c
t
r
a
o
f
i
d
e
n
t
i
f
i
e
d
P
T
H
-
d
e
r
i
v
a
t
i
v
e
s
.
324
quencer was directly introduced into the ESI source. The on-line coupling guar-
antees a complete overview over all products after the degradation cycle. As an
additional and most important information, the total ion chromatogram (TIC)
contains the molecular mass data of the PTH-amino acids. With this method,
peptides bearing unusual amino acids, e. g. 4-hydroxyphenylglycine (Hpg), 3,5-
dihydroxy-phenylalanine, ornithine, lanthionine, [2,3]-didehydro-asparagine,
etc. have been analyzed. This method turned out to be practicable for a great
variety of linear and cyclic peptides, e. g. lantibiotics or even microheterogenous
peptides. Furthermore, because of the extremely low amounts of sample re-
quired this method is suitable for the analysis of natural metabolic peptides of
microbial and mammalian origin.
The sequencing method based on mass spectrometric detection was of im-
portance in the structure elucidation of CDA, a calcium dependent antibiotic
[18], confirming its primary structure, which was not clearly solved from the se-
quence information obtained by two-dimensional HMBC- and NOESY-NMR
spectra. Moreover, this method is generally applied for the structure elucidation
of new peptides bearing post-translational modifications.
The application of the sequencer-ESI-MS coupling sometimes leads to sur-
prising results which are, however, clearly explicable through the mass spectro-
metric data. For example, Fig. 19.4 displays the degradation cycle of 3-chloro-b-
hydroxy-tyrosine (Cht), an amino acid which was found during the structure elu-
cidation of intermediates of the balhimycin biosynthesis [19]. Almost no PTH-de-
rivative of 3-chloro-b-hydroxy-tyrosine was detected. Instead, as a major product
PTH-glycine and minor amounts of the 2,3-didehydro-3-chloro-tyrosine PTH-de-
rivative were unequivocally identified. We could explain these results by assum-
ing a retro-aldol reaction under basic conditions (formation of the PTC-peptide)
and a dehydration under acid conditions (formation of the ATZ-amino acid and
conversion to the PTH-amino acid) during Edman degradation (Fig. 19.5).
19.3 Structure elucidation
Over the past years several dozen structures of a variety of different metabolites
from microbial sources have been determined. They comprise molecules of a
wide range of compound classes, e. g. antibiotics, siderophores, and signaling
molecules. They were either isolated in the course of a targeted screening by
the cooperating groups, or represented biosynthetic intermediates produced by
mutants genetically generated with the methods of molecular biology.
As tools for structure elucidation a variety of analytical methods have been
used: electrospray mass spectrometry (ESI-MS) and respective hyphenation tech-
niques, 2-dimensional NMR spectroscopy, GC- and LC-chromatograpy, chiral
GC-MS, amino acid analysis, Edman degradation and CD spectroscopy.
325
19.3.1 Structure elucidation of metabolites of microbial origin
The following chapter comprises a selection of some of the structures belonging
to different classes of metabolites. Compounds presented had no greater impact
on the main research projects which mainly concerned siderophores and lanti-
biotics. However, they represent interesting structures and some of these com-
pounds developed into research fields of other groups.
The following peptidic compounds have been characterized (Fig. 19.6): the
rhizocticins, small phosphono-oligopeptides [20], the fengycins lipopeptide anti-
biotics [21] and the chlorotetains, which all were isolated from Bacillus subtilis
strains [22], the lipoglycopeptidic herbicolins from Erwinia herbicola [23], echi-
noserin from Streptomyces tendae [24], and aborycin, a tricyclic 21-peptide anti-
Figure 19.5: Retro-aldol and dehydration reaction for the Edman degradation of 3-chloro-
b-hydroxy-tyrosine.
326
biotic from Streptomyces griseoflavus [25]. For the latter antibiotic the complete
solution 3D-structure was also determined by NMR spectroscopy.
In addition, the structures of non-peptidic compounds (Fig. 19.7), e. g. of
polyol macrolide antibiotic kanchanamycin from Streptomyces olivaceus [26],
the antifungal lactam antibiotic maltophilin produced by Stenotrophomonas
maltophilia [27], spirofungin from Streptomyces violaceusniger [28], and boophi-
line from Boophilus microplus [29] were determined. The early structure deter-
minations were done with the help of FAB-MS, and
1
H- and
13
C-NMR spectro-
scopy. However, with the availability of ESI-MS and straightforward 2-dimen-
sional NMR-techniques, the structure elucidation was facilitated and speeded
up significantly.
Figure 19.6: Peptidic metabolites isolated from different microorganisms.
327
Figure 19.7: Non-peptidic metabolites isolated from different microorganisms.
328
19.3.1.1 Siderophores
Rhizoferrin from Rhizopus microsporus [30], ferrirhodin from Botrytis cinerea
[31], yersiniabactin from Yersinia enterocolitica [32], and staphyloferrin A from
Staphylococcus hyicus [33] were isolated and their structure determined.
Staphyloferrin A consists of two citric acid moieties and D-ornithin (Fig. 19.8).
From dicitrylputrescin contained in rhizoferrin, a number of analogs has
been obtained by directed fermentation [34]. The chirality of these analogs and
their iron complexation properties have been characterized spectroscopically.
The complexation modes of citryl-containing carboxylate siderophores for a
number of different transition metal ions have been studied with rhizoferrin as a
model compound [35]. From Ralstonia pickettii DSM 6297, the enantiomer of
fungal rhizoferrin has been isolated [36, 37]. This opened up a new field of re-
search on the chirality in citric acid-containing carboxylate siderophores and
comparative studies on the biosynthetic pathways in fungal and bacterial side-
rophore producers. New developments in the structure elucidation, synthesis,
and function of peptide siderophores have been summarized in a recent review
by H. Drechsel and G. Jung [38].
Figure 19.8: Isolated and characterized siderophores.
329
19.3.1.2 Lantibiotics
The lantibiotics represent a class of antimicrobials extensively studied at the
University of Tbingen through the cooperation of the groups of Prof. Jung,
Prof. Entian, Prof. Gtz, and Prof. Zhner. They are polypeptide antibiotics, pro-
duced by Gram-positive bacteria which contain intra-chain thioether bridges
formed by lanthionine or methyllanthionine. These covalent thioether links im-
pose conformational constraints and stability against protease degradation.
Within the collaborative research centre 323 the lantibiotics Pep5 [39], epider-
min [40], actagardine [41], gallidermin [42], and SA-FF22 [43], were elucidated
as shown in Fig. 19.9.
The structures of epidermin and Pep5 had to be determined by enzymatic
degradation and derivatization to smaller, sequencable subfragments. With 2-di-
mensional NMR-experiments, using a 600 MHz NMR-spectrometer, in addition
to ESI-MS and sequencer-MS, 3-dimensional solution structures of duramycin
B, C [44], actagardin [45], and gallidermin [46] were determined by distance
geometry and restrained-molecular-mechanics calculations (Fig. 19.10).
Figure 19.9: Sequences and thioether bridging patterns of lantibiotics elucidated by che-
mical and enzymatic degradation, mass spectrometry, and 2-dimensional NMR spectro-
scopy.
Epidermin
Ala Gly Pro Ala Ile Arg Ala Ala Val Lys Gln Ala Gln Lys Dhb Leu Lys Ala Dhb Arg Leu Phe Abu Val Ala Ala Lys Gly Lys Asn Lys Ala Lys
O
O
S S
S
Gallidermin
Pep5
Actagardin
S S
Ala Ser Gly Trp Val Ala Abu Leu Abu Leu Glu Ala Gly Abu Val Ile Ala Ala Ala
S S
O
Ile Ala Ala Lys Phe Ile Ala Abu Pro Gly Ala Ala Lys Dhb Gly Ala Phe Asn Ala Tyr Ala
S S S
N
H
S
Ile Ala Ala Lys Phe Leu Ala Abu Pro Gly Ala Ala Lys Dhb Gly Ala Phe Asn Ala Tyr Ala
S S S
N
H
S
330
Knowledge of the lantibiotics structures prompted microbiologists to inves-
tigate the biosynthesis of the lantibiotics which is described in Chapter 3.
19.3.1.3 Microcin B17
The structure determination of the 43-peptide antibiotic microcin B17
(Fig. 19.11) from E. coli revealed the structure of the first known gyrase inhibitor
(topoisomerase II inhibitor) of peptidic nature. The structure elucidation turned
out to be extremely sophisticated because of eight backbone modifications and
a nonaglycine sequence. The post-translational backbone modifications [47, 48]
of bicyclic oxazole/thiazole-rings protected the major part of peptide from Ed-
man degradation. Fully
13
C,
15
N-labeled microcin B17 had to be prepared for
crucial heteronuclear 2D-NMR experiments on the backbone of the heterocyclic
polypeptide.
In subsequent work, synthesis of the novel oxazole and thiazole amino
acids had to be achieved [7], which ended up in the total synthesis of microcin
B17 in 1996 [8]. The total synthesis of microcin B17 was performed on solid sup-
port using fragment condensations in order to form the nonaglycine sequence,
which was not accessible by stepwise successive standard solution phase chem-
istry. The purity of the crude 43-peptide was about 50%. Chemical data and
antibiotic activity were identical with those of natural microcin B17 [8]. In addi-
tion to the natural gyrase inhibitor some analogs have been synthesized for
structure-activity studies. The structure of microcin B17 formed the basis to pos-
tulate a biosynthesis mechanism for the post-translational modification leading
to the oxazole/thiazole rings [47, 48]. The investigation of the microcin biosynth-
esis is still pursued [4951].
Figure 19.10: Solution structures of the lantibiotics gallidermin and actagardine.
Figure 19.11: Structure of microcin B17 [48].
N
S N
O
GGQGG
VGIGGGGGGGGG
S
N
S
N
N
O N
S
G
SN
GGNG
O
N
G
O
N
GSHI
331
19.3.1.4 CDA (Calcium Dependent Antibiotic)
Further interesting secondary metabolites are the peptides of the CDA (Calcium
Dependent Antibiotic) group, isolated from Streptomyces coelicolor. These cyc-
lic peptides inhibit the growth of Gram-positive bacteria in the presence of
Ca
2+
-ions. The cyclic 11-mer peptide antibiotic (Fig. 19.12) consists of seven pro-
teinogenic and four non-proteinogenic amino acids: D-tryptophan, 4-D-hydroxy-
phenylglycine, phospho-asparagine and D-methylglutamic acid [18]. Initial at-
tempts to perform Edman degradation failed due to N-terminal blocking with
an epoxyhexanoyl group which was identified in a later period of structure elu-
cidation by NMR. Therefore, the peptide was treated with BNPS-skatol, which
cleaves the peptide at the N-terminal bond of tryptophan residues. The peptide
fragments obtained were further investigated by Edman degradation using the
above mentioned sequencer-ESI-MS coupling. The structure elucidation of
CDA required the combination of a sophisticated set of analytical methods
(HPLC-MS, MS/MS, 2D-NMR) and chemical modification reactions. At present
the biosynthesis of CDA is investigated by the groups of Hopwood and Mara-
hiel [52].
19.3.1.5 Peptide pheromones of Staphylococcus epidermidis
The agr quorum-sensing system (accessory gene regulator) is responsible for the
regulation of several virulence factors in staphylococci. Novick et al. [53, 54] char-
acterized the agr-system of S. aureus strains and in accordance with the genomic
sequence data, a released peptide factor was presumed to activate the agr-sys-
tem. Indeed, small amounts of peptides have been isolated from S. aureus strains
to perform Edman degradation and mass spectrometry. A conserved cysteine was
presumed to form a thiollactone with the carboxy terminus of the peptide. How-
ever, insufficient amounts were obtained for a full structure and function analysis.
Since we and F. Gtzs group were also unable to isolate sufficient amounts from
S. epidermidis strains, we decided to synthesize the proposed molecule.
We showed that the cyclic thiollactone DSVc[CASYF] (Fig. 19.13) indeed
activated the agr-system in nanomolar concentrations [55]. Non-cyclic peptides
were completely inactive, and shortened or elongated cyclic thiollactones
Figure 19.12: Structure of CDA [18].
332
(GDSVc[CASYF] and SVc[CASYF]) showed a far lower activity as DSVc[CASYF].
Furthermore, we showed that the S. epidermidis thiollactone peptide DSVc
[CASYF] inhibited the activation of the agr-system of S. aureus strains [55]. The
replacement of the bridging cysteine, forming the thiollactone bond, by serine
(DSVc[SASYF]) or 1,3-diamino propionic acid (DSVc[DprASYF]) had only little
influenced the inhibition or activation of the agr-system.
Administration of such thiollactone peptides against S. aureus infected
mice reduced the infection [56]. It remains to be shown whether such quorum-
sensing blockers are attractive lead structures for the development of antibiotic
drugs aimed at treating staphylococcal infections.
19.3.2 Investigation of biosyntheses
19.3.2.1 Nikkomycin biosynthesis
Nikkomycin biosynthesis has been of major interest, because nikkomycins act
as competitive inhibitors of chitin synthetases from fungi and insects. Nikkomy-
cins might be attractive drugs and highly diverse lead structures for the agro-
chemical and pharmaceutical industries. Earlier works on structure elucidation
and synthesis of nikkomycine derivatives have been published by W. A. Knig
and H. Hagenmaier [57, 58] in cooperation with H. Zhners group. More re-
cently, in cooperation with C. Bormann, biosynthesis of nikkomycins, produced
by Streptomyces tendae T 901 was investigated by characterization of novel
biosynthetic intermediates formed by mutants inactivated in specific nikkomy-
cin-biosynthesis genes. Additionally, mutasynthetically generated compounds
from genetically engineered mutants have been characterized.
Within this cooperation the function of the nikF nikkomycin biosynthesis
gene encoding a P450 monooxygenase was elucidated [59]. Nikkomycins L
x
Figure 19.13: Structure of the quorum sensing signal peptide DSVc[CASYF] [55].
333
and L
z
produced by NikF inactivated mutants are analogous structures to nikko-
mycins X and Z formed by the parent strain, but lack the hydroxy group at the
pyridyl residue (Fig. 19.14). The structure of the biosynthetic intermediate
formed by a nikO-inactivated mutant was determined as ribofuranosyl-4-formyl-
4-imidazolone, which represents a novel nucleoside. This finding indicated that
the nikO encoded putative enolpyruvyl transferase catalyzes the initial step in
the biosynthesis of the nucleoside moieties of nikkomycin. Structure elucidation
of two biosynthetic intermediates isolated from the culture filtrate of mutants
blocked in the biosynthesis of the nucleoside moieties is in progress. The struc-
ture of a novel intermediate produced by a nikK-mutant that is unable to intro-
duce the amino group to the nucleoside moiety was determined as 4-formyl-4-
imidazolin-2-one. This is the base which is incorporated to yield nikkomycins
containing this base.
Feeding benzoic acid to a mutant deficient in the nikC gene which en-
codes lysine-2-aminotransferase catalyzing the initial step in the biosynthesis of
the peptidyl moiety led to production of nikkomycin B
x
and B
z
[60]. These nik-
komycins, which are the most potent compounds among known nikkomycins re-
vealed good activity against Candida albicans.
19.3.2.2 Epidermin biosynthesis
The characterization and structure elucidation of lantibiotics had an impact on
the microbiologists within and outside the collaborative research centre 323.
The gene cluster for the biosynthesis of epidermin, epiA-D, epiP, and epiQ was
sequenced. The prepeptide EpiA, consisting of 52 amino acids, is ribosomally
Figure 19.14: Structure of isolated nikkomycins L
x
[59] and B
x
[60].
334
synthesized and transformed into the biologically active 22mer peptide by sev-
eral post-translational modifications, including oxidative decarboxylation, dehy-
dration, formation of thioether bridges, and cleavage of the leader peptide.
In cooperation with T. Kupke and F. Gtz the enzyme EpiD has been iso-
lated and characterized. With EpiA substrates it has been shown that EpiD cata-
lyzes the oxidative decarboxylation (H
2
, CO
2
) of C-terminal cysteine residues
to form S-[(Z)-2-aminovinyl]-D-cysteine [61]. This hitherto unknown enzymatic
oxidative decarboxylation reaction of EpiA substrates with EpiD has been de-
tected by electrospray mass spectrometry. Subsequently, the substrate specificity
of this reaction has been determined with precursor peptides of mutants and
chemically synthesized 7mer peptides and peptide libraries [62]. The substrate
specificity of EpiD was clearly shown by neutral loss scan experiments using
randomized peptide libraries. This was the first application of neutral loss scan
to identify products of an enzymatic reaction. Moreover, kinetic studies of the
conversion reaction have been performed with electrospray mass spectrometry.
The enzymatic reaction of EpiD with
13
C-labeled peptide substrate has been fol-
lowed by HSQC-NMR spectroscopy [63] (Fig. 19.15).
Figure 19.15: Reaction of the
13
C-labeled substrate KKSFNSYTC with the enzyme EpiD
in projections along the F1 (
13
C) axis of the HSQC spectra recorded during the course of
the reaction. The product signal at d(
13
C) = 120.5 ppm increases in intensity with time [63].
C N C
O H
C
13
H S H
H
C N C
O H
C
13
H
SH
H
COOH
H
0 min
75 min
195 min
375 min
675 min
/ ppm 20 40 60 80 100 120
335
19.3.2.3 Biosynthesis of glycopeptides: balhimycin
Paying attention to the dramatically increasing number of antibiotic resistant mi-
croorganisms, vancomycin and the related family of glycopeptides constitute
antibiotics of the last resort, especially in the case of infections caused by methi-
cillin-resistant staphylococci. The cyclic glycopeptides are also of interest as
model receptors [64] for the investigation of ligand-receptor interactions, and
they are widely used as chiral selectors in chromatography [65]. Considering the
stereochemistry of the glycopeptides, the total synthesis of these molecules is one
of the most challenging enterprises for synthetically working chemists [66, 67].
The target molecule of the group of W. Wohlleben and S. Pelzer is balhi-
mycin, a vancomycin-type glycopeptide antibiotic, produced by Amycolatopsis
mediterranei, from which the biosynthesis gene cluster was sequenced [68].
After identification of the biosynthesis gene cluster, in 1998 the first gene-dis-
Figure 19.16: Structures of the linear biosynthesis intermediates of balhimycin, a vanco-
mycin type tricyclic glyco-peptide antibiotic [19].
336
ruption mutants were cloned in the oxygenase genes oxyA and oxyB and in the
glycosyltransferase gene bgtfB. In contrast to the glycosyltransferase mutant,
the oxygenase mutants showed no antibiotic activity. The culture filtrates from
both types of mutants were investigated, and according to the characteristic iso-
topic pattern of the two-fold chlorination of the detected compounds they were
assigned to the expected biosynthesis intermediates. The compounds were iso-
lated and investigated with ESI-MS, Edman degradation, amino acid analysis,
and 2-dimensional NMR experiments [19].
The compounds produced by the oxygenase mutants revealed the first
known linear peptide intermediates of glycopeptides (Fig. 19.16). They give a
first insight into the biosynthesis pathway of the glycopeptide antibiotics [19,
68] (Fig. 19.17). The compounds produced by the bgtfB-mutant showed a com-
plete lack of glycosylation.
In-frame mutations of each single gene, of the oxygenases OxyA/B/C and
the glycosyltransferases BgtfA/B/C are currently generated. The culture filtrates
from the mutant strains are investigated with HPLC-ESI-MS for the presence of
the biosynthesis intermediates, and the structures of these metabolites are deter-
mined after purification. The goal of the ongoing cooperation is to inactivate the
halogenase gene bhaA to unravel the so far unknown function of a haloperoxi-
dase and to demonstrate its substrate specificities.
19.4 Summary
During the past decade of the collaborative research centre 323, efficient analy-
tical and synthetic methods were developed and applied, e. g. ESI-MS and cou-
pling techniques, multiple peptide synthesis, 2-dimensional NMR spectroscopy,
and molecular modeling. With these new methods it was possible to perform a
high level of biological and biochemical research. Frequently, cooperation with
biologically working groups showed that an expertise in both analytical chem-
istry and synthesis was extremely valuable or even decisive for the success of a
project. Either the synthetic and analytical work supported the value and mean-
ing of biological results or paved the way for the biological research.This fruitful
constellation, the close cooperation between microbiology and chemistry re-
sulted in numerous journal publications.
With the installation of the Fourier-transform-ion-cyclotron resonance mass
spectrometer in 1998 it has been once more possible to establish a modern ana-
lytical technique at the Institute of Organic Chemistry [69]. The installation of
these key technologies is extremely important to keep pace with the rapidly
growing requirements of biologists for powerful analytical methods.
337
19.4 Summary
Figure 19.17: Proposed biosynthesis pathway of glycopeptides with the example of bal-
himycin.
NH
2
N
H
O
O H
Cl
O
N
H
O
N H
2
O
N
H
N
H
OH
O
N
H
OH
O
O H
Cl
O
O H
OH OH
NH
2
N
H
O
O H
Cl
O
N
H
O
N H
2
O
N
H
N
H
OH
O
N
H
OH
O
O H
Cl
O
N
H
OH OH
OH O H
O
O H
oxygenases
oxyA/B/C
glycosyltransferases
bgtfA/B/C
NH
2
OH O H
O
O H
SP-969
SP-1134
HD-1112/1126
HD-1128/1142
-6H
polyketidsynthase ?
aminotransferase ?
3,5-dihydroxy-
phenylglycine
Balhimycin
R
1
= -H / -CH
3
R
2
= -H / -OH
peptidesynthetases ?
O H OH
O
N H
HO
2
C
OH
O
O H
Cl
NH
N
H
O
O
N
H
O
N H
2
O
N
H
N
H
O
N
H
OH
O
O
R
R
Cl
2
1
O
O
N H
2
O
C H
3 CH
3
O
O
CH
2
OH
O H
O H
O H
O H OH
O
N H
HO
2
C
O
Cl
NH
N
H
O
O
N
H
O
N H
2
O
N
H
N
H
O
N
H
OH
O
O
CH
3
OH
Cl
338
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342
Documentation
ISBN: 3-527-30615-3
20 Documentation of the Collaborative
Research Centre 323
20.1 List of institutes involved
Lehrstuhl fr Mikrobiologie/Biotechnologie, Universitt Tbingen
Lehrstuhl fr Mikrobiologie/Membranphysiologie, Universitt Tbingen
Lehrstuhl fr Mikrobielle Genetik, Universitt Tbingen
Lehrstuhl fr Physiologische Chemie/Biochemie, Universitt Tbingen
Lehrstuhl fr Pharmazeutische Chemie, Universitt Tbingen
Lehrstuhl fr Pharmazeutische Biologie, Universitt Tbingen
Medizinisch-Naturwissenschaftliches Forschungszentrum, Universitt Tbingen
Max-Planck-Institut fr Entwicklungsbiologie, Abt. Biochemie, Tbingen
Max Planck-Institut fr Biologie, Abt. Infektionsbiologie, Tbingen
Institut fr Organische Chemie, Universitt Tbingen
Lehrstuhl fr Hydrochemie und Hydrobiologie, Universitt Stuttgart
Robert Koch-Institut des Bundesgesundheitsamtes, Wernigerode
20.2 List of supported project areas
Project area A
TP A1 Mikrobieller Sekundrstoffwechsel
Prof. Ing. Hans Zhner, Mikrobiologie/Biotechnologie 19861996
TP A2 Fermentation, Aufarbeitung und Analytik
niedermolekularer Metabolite 19861988
Fermentationstechnik, Naturstoffisolierung,
Naturstoffanalytik 19891990
345
ISBN: 3-527-30615-3
Grundlagen der Produktionsoptimierung und
Analytik mikrobieller Sekundrmetabolite 19911993
Fermentation und Produktionsoptimierung
mikrobieller Naturstoffe (Mikrobiologie/Biotechnologie) 19941999
Prof. Dr. Hans-Peter Fiedler, Mikrobiologie/Biotechnologie
TP A3 Biotechnologische Produkte von Peptiden in Hefe 19861988
PD Dr. Karl-Dieter Entian, Biochemie
TP A4 Genetik und Biochemie der Nikkomycin-Biosynthese
in Streptomyces tendae 19911993
Genetische und biochemische Charakterisierung
eines antifungisch wirksamen Proteins aus
Streptomyces tendae (Mikrobielle Genetik)
Biosynthese des Antibiotikums Nikkomycin und
extrazellulre Proteine bei Streptomyceten 19971999
Dr. Christiane Bormann, Mikrobiologie/Biotechnologie
TP A5 Hopanoid-Cyclasen 19911996
Synthese und Funktion von Hopanoiden 19971999
Prof. Dr. Karl Poralla, Mikrobiologie/Biotechnologie
TP A10 Molekulargenetische und biochemische Analyse
der Sekundrmetabolit-Biosynthese in Aktinomyceten 19971999
Prof. Dr. Wolfgang Wohlleben, Mikrobiologie/
Biotechnologie
TP A11 Molekularbiologische Untersuchungen zur Biosynthese
des Cytostatikums Landomycin A, gebildet von
Streptomyces cyanogenus (DSM 5087)
Untersuchungen zum Aufbau der Zuckerseitenkette
Untersuchungen zur Biosynthese der Desoxyzucker
L-Rhodinose und D-Olivose 19971999
Dr. Andreas Bechthold, Pharmazeutische Biologie
TP A12 Kontrolle der autolytischen Enzyme in Escherichia coli 19971999
Prof. Dr. Joachim-Volker Hltje, MPI fr Entwicklungs-
biologie, Abt. Biochemie
Project area B
TP B1 Metabolische Wechselwirkungen zwischen Pro-
und Eukaryonten 19861996
Eisen als Umweltsignal
Prof. Dr. Volkmar Braun und PD Dr. Klaus Hantke,
Mikrobiologie/Membranphysiologie
Regulierte Transport- und Signaltransfer-Kanle
in der ueren Membran Gram-negativer Bakterien 19971999
Prof. Dr. Volkmar Braun, Mikrobiologie/Membranphysiologie
346
20 Documentation of the Collaborative Research Centre 323
TP B2 Regulation der interzellulren Kommunikation in
Zellen des Nervensystems durch eu- und prokaryon-
tische Wirkstoffe 19861990
Auffindung, Isolierung und Charakterisierung
von second messenger-Systeme regulierenden
bakteriellen Wirkstoffen 19911991
Prof. Dr. Bernd Hamprecht, Biochemie
TP B3 Molekulare Mechanismen der Aktivierung von
Makrophagen zur Phagosytose und Bakterizidie
durch mikrobielle Oberflchenkomponenten und
synthetische Analoga 19861988
Prof. Dr. Wolfgang Bessler, Mikrobiologie/
Membranphysiologie
TP B4 Chemie der Reizverarbeitung in Paramecium 19861990
Prof. Dr. Joachim Schultz, Pharmazie
PD Dr. Susanne Klumpp, Pharmazie
Funktion von second messengern in Paramecium 19971999
Prof. Dr. Joachim Schultz, Pharmazeutische Chemie
TP B5 Molekularbiologische Charakterisierung und
Regulation von Exoproteinen und Exopeptiden
bei Stapylokokken 19881990
von Exopeptiden und Staphylokokken 19911993
Biosynthese und Regulation von Exoproteinen,
Exopeptiden und Membranpigmenten bei
Staphylokokken 19941996
von Exopeptiden/Exoproteinen und Carotinoiden
bei Staphylokokken 19971999
Prof. Dr. Friedrich Gtz, Mikrobielle Genetik
TP B6 Eisentransport bei Gram-positiven und Gram-
negativen Bakterien 19911999
Prof. Dr. Klaus Hantke, Mikrobiologie/
Membranphysiologie
TP B7 Proteinphosphatasen in Paramecium 19911995
PD Dr. Susanne Klumpp, Pharmazeutische Chemie
TP B8 Analyse der IgA Protease b-Domne, ein Vehikel
fr den Proteinexport durch die uere Membran
von Gram-negativen Bakterien 19911993
Mechanismus und Bedeutung der natrlichen
Transformationskompetenz bei Neisserien 19941996
PD Dr. Thomas Meyer, MPI Biologie
347
20.2 List of supported project areas
TP B9 Intrazellulre Freisetzung und Transport von
A-Proteinen pathogener Neisserien in eukary-
ontischen Zellen 19911993
PD Dr. Johannes Pohlner, MPI Biologie
TP B10 Genetische und biochemische Charakterisierung
des Cytotoxins von Helicobacter pylori 19941996
Dr. Rainer Haas, MPI Biologie
Project area C Chemical structure elucidation
TP C2 Chemie von Antibiotika und Immunmodulatoren 19861990
Chemie der Mikroorganismen 19911996
Peptid- und Proteinchemie der Mikroorganismen 19971999
Prof. Dr. Gnther Jung, Organische Chemie
TP C3 Naturstoffaufklrung 19911993
Analytik und Strukturaufklrung von Naturstoffen 19941999
PD Dr. Jrg Metzger, Organische Chemie,
spter Hydrochemie/Hydrobiologie
TP YE1 Dr. Reissbrodt, RKI Wernigerode 19921993
TP YW1 Prof. Braun, Tbingen, Mikrobiologie 19921993
20.3 Promotion of members of the collaborative
research centre
The following project leaders left for other positions outside the collaborative re-
search centre:
Annette Beck-Sickinger, C4, University of Basel.
Wolfgang Bessler, C3, Immunology, University of Freiburg.
Karl-Dieter Entian, C3, Microbiology, University of Frankfurt.
Rainer Haas, C3, Medical Microbiology, University of Munich.
Knut Heller, C2, Microbiology, University of Konstanz.
Susanne Klumpp, C3, Pharmaceutical Chemistry, University of Marburg.
Jrg Metzger, C4, University of Stuttgart.
Thomas F. Meyer, C4, Infection Biology, MPI Infection Biology, Berlin.
Johannes Pohlner, Evotec BioSystems GmbH, Hamburg.
Oliver Potterat, C2, University of Lausanne.
348
20.4 Recruitment of new project leaders
The project leaders who left were replaced by the following new project leaders:
Andreas Bechthold, Pharmaceutical Biology, University of Tbingen.
Christiane Bormann, Microbial Genetics/Microbiology-Biotechnology, Univer-
sity of Tbingen.
Klaus Hantke, Microbiology-Membrane Physiology, University of Tbingen.
Joachim-Volker Hltje, Biochemistry, MPI of Developmental Biology, Tbingen.
Karl Poralla, Microbiology-Biotechnology, University of Tbingen.
Wolfgang Wohlleben, Microbiology-Biotechnology, University of Tbingen.
20.5 Alphabetical list of members and participants
Name Prename Acad. degree Institute Project area Participation
Allgaier*, Hermann Dipl. Chem. Organ. Chemie C2 8690
Alvarado*, Maria Dipl. Biol. Biologie A1 8689
Andres*, Nikolaus Dipl. Biol. Biologie A1 8889
Angerer*, Annemarie Dipl. Biol. Biologie B1 8891
Anton*, Helga Apothekerin Pharmazie B4 9195
Auge, Ulrike Dipl. Biol. Biologie B5 99
Augustin*, Johannes Dipl. Biol. Biologie B5 8890
Bauch, Angela Dipl. Biol. MPI, Infektions. B9 93
Baumgartner*, Angelika Apothekerin Biologie B4 86
Barten*, Roland Dipl. Biol. Biologie B8 9496
Bayer*, Anja Dipl. Biol. Organ. Chemie C2 8894
Bayer, Ernst Dr. rer. nat. Phys. Chem. B2 8687
Bechthold, Andreas PD Dr. rer. nat. Pharmazeut. Bio. A11 9799
Beck-Sickinger, Annette Dipl. Chem. Organ. Chemie C2 88
Beitz*, Eric Apotheker Pharmazie B4 9495
Bessler, Wolfgang Prof. Dr. Biologie B3 8688
Beyer*, Angelika Apothekerin Pharmazie B4 89
Bielecki, Jarek Dr. rer. nat. Biologie A1/GW 86
Blanck*, Wolfgang Dipl. Biol. Biologie A1 8687
Blind*, Brigitte Dipl. kotroph. Phys. Chem. B2 8791
Bs*, Christoph Dipl. Chem. Biologie B1 9697
Bormann, Christiane** Dr. rer. nat. Biologie A1 8690
Bormann, Christiane Dr. rer. nat. Biologie A4 9199
Braun*, Dieter Dipl. Biol. Biologie A1 9093
Braun,Volkmar Prof. Dr. Biologie B1 8699
Breisch*, Monika Dipl. Biol. Phys. Chem. B2 86
349
Britten* Uwe Dipl. Biol. Biologie A1 8991
Brooks*, Mark Dipl. Biol. Organ. Chemie C2 9499
Brckner, Reinhold Dipl. Biol. Biologie B5 8999
Bruntner*, Christina Dipl. Biol. Biologie A4 9597
Brutsche*, Sandra Dipl. Biol. Biologie B1 9797
Burckhard*, Renate Dipl. Biol. Biologie B1 86
Cebulla*, Ingeborg Dipl. Biol. Biologie A1 9295
Chehadeh*, Heidi Dipl. Biol. Biologie B1 86
Choi*, Ok-Byung Dipl. Biol. Biologie A5 9295
Cullmann*, Hans-Jrgen Dipl. Biol. Biologie A1 9294
Decker*, Heinrich Dr. rer. nat. Biologie A1 8695
Demleitner*, Gaby Dipl. Biol. Biologie B5 8892
Deres*, Karl Dipl. Biol. Organ. Chemie C2 8892
Domann*, Silvie Dipl. Biol. Biologie A11 9798
Drautz, Hannelore Dr. rer. nat. Biologie A1 8692
Drechsel*, Hartmut Dipl. Chem. Organ. Chemie C2 8999
Dringen*, Ralf Dipl. Biochem. Phys. Chemie B2 8890
Drr, Hansjrg Dipl. Chem. Organ. Chemie C2 Stip.
Eick-Helmrich*, Katrin Dipl. Biol. Biologie B1 8689
Engel*, Peter Dipl. Biol. Pharmazie B4 95
Entian**, Karl-Dieter Dr. rer. nat. Phys. Chem. A3 8688
Enz*, Sabine Dr. rer. nat. Biologie B1 9599
Fauth*, Ursula Dr. rer. nat. Biologie A1 8690
Faust*, Bettina Dipl. Biol. Pharmazie A11 9798
Feil*, Corinna Dipl. Biol. Biologie A5 9396
Fels*, Johannes Dipl. Biol. Biologie A1 9193
Flechsler*, Insa Dipl. Chem. Organ. Chemie C2 9398
Fleckenstein*, Burkhard Dipl. Biochem. Organ. Chemie C2 9399
Fiedler**, Hans-Peter Prof. Dr. Biologie A2 8699
Fiedler,* Waltraud Dipl. Biol. Biologie B1 8687
Fischer, Eckhard Dr. rer. nat. Biologie B1 86
Franz, Brigitte Dipl. Chem. Organ. Chemie C2 88
Freund*, Stefan Dipl. Chem. Organ. Chemie C2 8893
Freund*, Wolf-Dietrich Dipl. Biochem. Pharmazie B4 8791
Friedl, Anette Dipl. Biochem. Phys. Chem. B2 8687
Freund, Stefan Dipl. Chem. Organ. Chemie C2 88
Freund*, Wolf-Dieter Dipl. Biochem. Pharmazie B4 8691
Fridrich*, Gerald Apotheker Pharmazie B4 89
Frosch, Ingrid Dr. rer. nat. Biologie A1 8687
Frchtel*, Jrg Dipl. Chem Organ. Chemie C2 9497
Fussenegger*, Martin Dipl. Biol. Biologie B9 9395
Gaisser*, Sabine Dipl. Biol. Biologie B6 9193
Gierlich, Doris Apothekerin Pharmazie B4 86
Gtz, Friedrich Prof. Dr. Biologie B5 8699
Groeger*, Wolfram Dipl. Biol. Biologie B1 9898
Gro, Matthias Dipl. Biol. Biologie B5 9899
Grothe*, Kirsten Apothekerin Pharmazie B4 9598
Gombert*, Frank Dipl. Biochem. Organ. Chemie C2 9091
Gro*, Patricia Dipl. Biol. Biologie B6 9395
Gnther*, Karola Dr. rer. nat. Biologie B1 8789
Guo*,Yinglan Dipl. Chem. Pharmazie B4 8699
350
Haag*, Hubert Dipl. Biol. Biologie A1 8992
Haag*, Sabine Dipl. Biol. Biologie A1 9295
Habeck*, Martina Dipl. Biol. Biologie B1 9898
Hsler*, Peter Dipl. Biol. Biologie A1 8688
Hambach, Kristina Apothekerin Pharmazie B4 9898
Hamprecht, Bernd Prof. Dr. Biochemie B2 8691
Handschuh, Dieter Dr. rer. nat. Biochemie B2 8691
Hansen*, Martin Apotheker Pharmazie B4 9899
Hantke**, Klaus Prof. Dr. Biologie B1 8690
Hantke, Klaus Prof. Dr. Biologie B6 9199
Harder, Michael, Dr. rer. nat. Biologie A2 9495
Harkness, Robin Dr. rer. nat. Biologie B1 8688
Hartjen*, Uwe Dipl. Biol. Biologie A2 8990
Hatzelmann, Armin Dipl. Biochem. Pharmazie B4 86
Heckmann*, Dorothee Dipl. Biol. Biologie A10 9598
Heidel, Martina Dipl. Biol. Pharmazie B4 86
Hertle, Ralf Dr. rer. nat. Biologie B1 9699
Heuermann*, Dorothee Dipl. Biol. Biologie B10 9596
Hilger*, Martina Dipl. Biol. Biologie B1 9495
Hille*, Matthias Dipl. Biol. Biologie B5 9699
Hobbie*, Silke Dipl. Biol. Biologie B1 9093
Hltje, Joachim-Volker, Prof. Dr. Biochemie A12 9799
Hltzel*, Alexandra Dipl. Chem. Organ. Chemie C2 9599
Hrner*, Thomas Dr. rer nat. Biologie A2 8690
Hrr*, Ingmar Dipl. Biol. Organ. Chemie C2 9599
Hoff*, Hubert Dipl. Biol. Biologie A1 8890
Hofmann,* Hans-Joachim Apotheker Pharmazie B4 8689
Hoffmann*, Helmut Dipl. Biol. Biologie B1 86
Hoffmann*, Thomas Dipl.Chem. Pharmazie B4 9597
Holz, Brbel Dipl. Biol. Biologie A1 9094
Huhn*, Wolfgang Dipl. Biol. Biologie A1 8687
Hummel*, Rolf-Peter Dipl. Biochem. Organ. Chemie C2 87
Hwang-Kim*, In-Sook Dipl. Biol. Biologie B1 9193
Ihlenfeldt*, Hans-Georg Dipl. Chem. Organ. Chemie C2 9095
Isselhorst-Scharr*,
Caroline Dipl. Biol. Biologie A1 8890
Jack, Ralph-Wilson Dr. rer. nat. Organ. Chemie C2/GW 9199
Jung, Gnther Prof. Dr. Biochemiker C2 8699
Jung*, Oliver Dipl. Biol Biologie A1 9194
Kabatek* Ursula Dipl. Biol. Organ. Chemie C2 8687
Kaiser*, Dietmar Dipl. Chem. Organ. Chemie C2 9598
Kammler*, Meike Dipl. Biol. Biologie B6 9294
Kannenberg, Elmar Dr. rer. nat. Biologie A5 9899
Kapitza, Susanne Dipl. Biol. Organ. Chemie C2 9899
Katzer*, Werner Dipl. Biol. Biologie A1 8791
Kellner*, Roland Dipl. Chem. Organ. Chemie C2 8689
Kempf, Markus Dipl. Biol. Biologie A2 97
Kempter*, Christoph Dipl. Chem. Organ. Chemie C2 9396
Kern, Armin Dr. rer. nat. Organ. Chemie C2 86
Kies, Stefanie Dipl. Biol. Biologie B5 9899
Killmann*, Helmut Dr. rer. nat. Biologie B1 9099
351
Kim*, In-Sook Dipl. Biol. Biologie B1 93
Klauser, Thomas Dr. rer. nat. MPI-Biologie B8 9193
Kleemann*, Gisela Dipl. Biol. Biologie A5 8992
Klumpp**, Susanne Dr. rer. nat. Pharmazie B4 9195
Knigge, Michael Dipl. Biol. Biologie A5 99
Koebnik*, Ralf Dipl. Biol. Biologie B1 9091
Knninger*, Ulrich Dipl. Biol Biologie B1 9498
Kster*, Wolfgang Dipl. Biol. Biologie B1 8687
Krmer, Joachim Dipl. Biol. MPI Infekt. Biol. B8 9193
Kremer*, Stephan Dipl. Biol. Biologie A1 8790
Krismer*, Bernhard Dipl. Biol. Biologie B5 9599
Koch*, Ulrike Dipl. Chem. Organ. Chemie C2 8688
Kster**, Wolfgang Dr. rer. nat. Biologie B1 8690
Konetschny-Rapp*, Silvia Dr. rer. nat. Organ Chemie C2 8690
Koss, Dieter Dipl. Biol. Biologie B6 9497
Krger*, Thomas Dipl. Chem. Pharmazie B4 9397
Kugler*, Martin Dipl. Biol. Biologie A1 8686
Khn*, Sabine Dipl. Biol. Biologie B1 9195
Kupke*, Thomas Dipl. Biochem. Biologie B5 8995
Langenberg, Uwe Dipl. Biochem. MPI Infekt. Biol. B9 91
Langer*, Monika Dipl. Biol. Biologie A1 9296
Lauer*, Bettina Dipl. Biol. Biologie A4 9599
Lechner*, Max Dipl. Biol Biologie B5 89
Leipert, Dietmar Dipl. Chem. Organ. Chemie C2 9498
Linder*, Jrgen Dipl. Chem. Pharmazie B4 9899
Lipps*, Hans-Peter Apotheker Pharmazie B4 89
Lohmann, Susanne Dr. rer. nat. Phys. Chem. B2 86
Maerker, Christian Dipl. Biol. MPI Infekt. Biol. B9 91
Mahnke*, Marion Dipl. Biol. Biologie A1 8687
Marquardt*, Udo Dipl. Chem. Organ. Chem. C2 9899
Mayer*, Irene Dipl. Biol. Biologie B1 8990
Merkofer, Thorsten Dipl. Biol. Biologie A5 9698
Mehrkhler, Christian Dipl. Biol. Phys. Chemie B2 8991
Meiwes*, Johannes Dipl. Biol. Biologie A1 8790
Mende*, Jasmin Dipl. Biol. Biologie B1 8687
Metzler*, Monika Dipl. Biol. Biologie A1 8689
Metzger**, Jrg Dr. rer. nat Organ. Chemie C2 8690
Metzger, Jrg Dr. rer. nat. Organ. Chemie C3 9199
Meyer, Thomas Dr. rer. nat. MPI Infekt. Biol. B8 91
Mhrle*,Volker Dipl. Biol. Biologie A4 9195
Mller, Andreas Dipl. Chem. Phys. Chem. B2 8690
Mller*, Kerstin Dipl. Biol. Biologie B6 9497
Mller, Judith Dipl.Lebensm.Ing. MPI Biochem. A12 99
Mutard, Denise Apothekerin Pharmazie B4 91
Muth, Gnther Dr. rer. nat. Biologie A10 9599
Neubauer, Heike Dipl. Biol. Biologie A4 96
Noda, Shigeru Dr. med. vet. Pharmazie B4 86
Ochs*, Dietmar Dipl. Biol. Biologie A5 90
Ochs*, Martina Dipl. Biol. Biologie B1 9195
Odenbreit, Stefan Dipl. Biol. MPI, Biochem. B10 9495
lschlger*, Thobias Dipl. Biol. Biologie B1 8690
352
tzelberger, Karin Dipl. Biol. MPI Biochem. B9 9193
Ondrazcek, Roland Dipl. Biol. Biologie B1 91
Ottenwlder, Birgit Dipl. Biol. Biologie B5 9596
Otto*, Michael Dipl. Chem. Biologie B5 9397
Otto*, Susanne Dipl. Biol. Pharmazie B7 9093
Pantel, Iris Dipl. Biol. Biologie A4 95
Patzer*, Silke Dipl. Biol. Biologie B6 9499
Peschel, Andreas Dipl. Biol. Biologie B5 9495
Perzl*, Michael Dipl. Biol. Biologie A5 9399
Petersen*, Frank Dipl. Biol. Biologie A1 8891
Pfefferle*, Uwe Dipl. Biol. Biologie A2 8990
Pfeiffer, Brigitte Dr. rer. nat. Phys. Chemie B2 8690
Pilsl*, Holger Dr. rer. nat. Biologie B1 9498
Pfeifer,Volker Dipl. Biochem. Biologie A10 99
Plaga*, Armin Dr. rer. nat. Biologie A2 8690
Plantoer*, Stefan Dipl. Biol. Biologie B1 98
Pohlner**, Johannes Dr. rer. nat. MPI Biochem. B9 9193
Potterat, Oliver Dr. rer. nat. Biologie A1 9194
Poralla, Karl Prof. Dr. Biologie A5 9199
Preler*, Uwe Dipl. Biol. Biologie B1 86
Pultar*, Thomas Dipl. Biol. Biologie A1 8688
Probst, Katrin Dipl. Chem. Organ. Chemie C2 99
Rabenhorst*, Jrgen Dipl. Biol. Biologie A1 8687
Rak, Gabriele Dr. rer. nat. Biologie A1 8790
Rauch*, Beatrix Dipl. Biol. Biologie A1 8991
Rapp* Claudius Dipl. Chem. Organ. Chemie C2 8688
Rechenberg*, Moritz von Dipl. Biochem. MPI Biochem. A12 9799
Reeger, Eva Dipl. Biol. Biologie B6 97
Reissbrodt, Rolf Dr. rer. nat. R. Koch Inst. YE1 9193
Reuschenbach,*, Peter Dipl. Biol. Biologie A2 8688
Reuschenbach*, Petra Dipl. Biol. Biologie A1 86
Reutter*, Felix Dipl. Chem. Organ. Chemie C3 9699
Rexer, Hans-Ulrich Dipl. Biol. Biologie A10 9799
Richter*, Monika Dipl. Biol. Biologie A2 95
Rhl*, Franz Dipl. Biol. Biologie A1 8687
Rsch, Hartmut Dipl. Biol. Pharmazie B4 90
Rohling*, Anette Dipl. Biol. Biologie A10 97
Roos*, Margareta Dipl. Biol. Biologie A1 8992
Roos*, Ulrich Dipl. Biol. Biologie B5 9193
Rose, Andreas Dipl. Biochem. Phys. Chem. A3 86
Rosenstein, Ralph Dr. rer. nat. Biologie B5 8699
Ruan,Yuan Dipl. Biol Biologie B1 92
Russwurm*, Roland Dipl. Biol. Biologie A4 9799
Sauer*, Martin Dipl. Biol. Biologie B1 8687
Sauter, Simon Dipl. Biol. Biologie B1 99
Schade, Uwe Apotheker Pharmazie B4 8690
Schffer*, Sven Dipl. Biol. Biologie B1 8687
Scharr*, Jrgen Dipl. Biol. Biologie A1 8791
Schaude*, Renate Dipl. Biochem. Organ. Chemie C2 8789
Schiller*, Max Dipl. Biol. Phys. Chemie B2 8690
Schiffer*, Guido Dr. rer. nat. Biochemie A12 9798
353
Schmidt, Gnther Dipl. Biochem. Phys. Chemie A3 89
Schmitz, Susanne Dipl. Biochem. Biologie A5 9799
Schneider*, Ursula Apothekerin Biologie A1 86
Schneider, Richard Dipl. Biol. Biologie B1 9495
Schnell, Norbert Dipl. Biochem. Phys. Chemie A3 89
Schffler*, Harald Dipl. Biol. Biologie B1 8689
Schn*, Claudia Dipl. Biol. Biologie B1+B6 8790
Schnborn, Christoph Apotheker Pharmazie B4 9091
Schnefeld*, Ulrich Dipl. Biochem. Pharmazie B4 8688
Schnherr*, Roland Dipl. Biol. Biologie B1 9293
Schuerhoff-Gters*,
Wilhelm Apotheker Pharmazie B4 8690
Schz*, Traugott Dipl. Biol. Biologie A1 86
Schz*, Traugott Dr. rer. nat. Biologie A2 8794
Schultz*, Gabriele Dipl. Biol. Biologie B1 8892
Schultz, Joachim Prof. Dr. Pharmazie B4 8699
Schwarz*, Wolfgang Dipl. Biol. Biologie A4 9799
Schwartz, Dirk Dr. rer. nat. Biologie A10 9599
Seiffert*, Andreas Dipl. Biol. Biologie A1 9092
Selke*, Dagmar Dipl. Biol. Pharmazie B4 9195
Sommer*, Patricia Dipl. Biol. Biologie A4 9495
Sorg, Gerhard Dipl. Chem. Organ. Chemie C3 9899
Sprengler, Siegried Dipl. Chem. Phys. Chemie B2 8688
Stahl, Bernd Dipl. Biochem. Phys. Chemie B2 8687
Stanger*, Andrea Dipl. Biol. Biologie A1 86
Staudenmaier*, Horst Dipl. Biol. Biologie B1 86
Steinlen*, Siegfried Dipl. Biochem. Pharmazie B4 8992
Stephan*, Holger Dipl. Chem. Organ. Chemie C2 9195
Stefanovic*, Stefan Dipl. Biochem. Organ. Chemie C2 8992
Stegmann*, Evi Dipl. Biol. Biologie A10 9899
Stiefel*, Alfred Dipl. Biol. Biologie B1 9799
Stmpfel*, Joachim Dipl. Biol. Biologie A1 8688
Smuth*, Roderich Dipl. Chem. Organ. Chemie C2 9599
Surovoy, Andrey Dr. rer. nat. Organ. Chemie C2/GW 9097
Tappe*, Cord-Henning Dipl. Biochem. Botan. Instit. A5 9193
Tejmar-Kolar, Liana Dr. rer. nat. Biologie A1 86
Templin, Markus Dr. rer. nat. Biochemie A12 9799
Teufel*, Pia Dipl. Biol. Biologie B5 8992
Theobald, Uwe Dr. Ing. Biologie A2 9599
Thumm*, Gnter Dipl. Biol. Biologie B5 8893
Tippelt*, Anette Dipl. Biol. Biologie A5 9697
Traub*, Irene Dipl. Biol. Biologie B1 9092
Trefzer, Axel Dipl. Biol. Pharmazie A11 9899
Troeger*, Wilfried Dipl. Chem. Organ. Chemie C2 8991
Tschen, Shu Yuan Dipl. Biol. Biologie A1 91
Tschierske*, Martin Dipl. Biol. Biologie A1 9294
Ungermann*,Volker Dipl. Biol. Biologie A1 8890
Vlkel*; Helge Dipl. Biochem. Pharmazie B4 9296
Voges, Brigitte Dr. rer. nat. Organ. Chemie C2 8688
Voges, Klaus-Peter Dr. rer. nat Organ. Chemie C2 8688
Vollmer*,Waldemar Dr. rer. nat. Biochemie A12 9799
354
Van hove*, Brundhild Dipl. Biol. Biologie B1 89
von der Mlbe, Florian Dipl. Biochem. Organ. Chemie C2 9799
Veitinger*, Sabine Dipl. Biol. Biologie B1 8992
Vierling*, Silke Dipl. Biol. Biologie A10 9799
Videnov, Georgi Dipl. Biol. Organ. Chemie C2/GW 9195
Walker, Georg Dipl. Biol. Biologie B1 9799
Walz*, Franz Dr. rer. nat. Biologie A2 8688
Wang-Tschen*, Shu-Yuan Dipl. Biol. Biologie B1 9192
Wasiliu*, Michal Dipl. Biol. Biologie A1 8686
Weber, Tillmann Dipl. Biol. Biologie A10 99
Weitnauer, Gabriele Dipl. Biol. Biologie A11 9899
Welz*, Dietrich Dipl. Biol. Biologie B1 9295
Wieland*, Bernd Dipl. Biol. Biologie B5 8993
Wieland*, Karsten-Peter Dipl. Biol. Biologie B5 9699
Wiesinger, Heiner Dr. rer. nat. Phys. Chem. B2 8691
Wiesmller*, Karl-Heinz Dipl. Chemiker Organ. Chemie C2 8692
Witke, Claudia Dipl. Biol. Biologie B5 9194
Woelk*, Uwe Dipl. Biol. MPI Infektions. B8 9596
Wohlleben, Wolfgang Prof. rer. nat. Biologie A10 9599
Zhner, Hans Prof. Dr. Ing. Biologie A1 8696
Ziegelmaier-Kemmling,*
Dagmar Dipl. Biol. Biologie A1 8690
Zimmermann, Luitgard Dr. rer. nat. Biologie B1 86
Zimmermann, Norbert Dipl. Chem. Organ. Chemie C2 91
* Graduation with PhD during the collaborative research centre 323.
** Habilitation during the collaborative research centre 323.
20.6 Support of young scientists
List of promotions resulting from the collaborative research centre
TP A1
Alvarado, Maria (1990): Monomeres und dimeres Cinnachinon aus Streptomy-
ces griseoflavus (T 2482).
Andres, Nikolaus (1989): Hormaomycin, ein Peptidlacton mit morphogener Wir-
kung auf Streptomyceten.
Blank, Wolfgang (1987): Untersuchungen zur biologischen Modifikation der Ba-
filomycine.
Braun, Dieter (1993): Enzymatische Halogenierung von mikrobiellen Metaboli-
ten und Etablierung eines Photokonduktivittsscreening.
355
Cebulla, Ingeborg (1995): Gewinnung komplexbildender Substanzen mittels
Amycolatopsis orientalis.
Cullmann, Hans Jrgen (1994): Depsichlorine und andere Metabolite aus Strep-
tomyces antibioticus T 1661.
Decker, Heinrich (1989): Untersuchung zur Struktur-Wirkungsbeziehung der
Nikkomycine und Isolierung neuer Nikkomycine.
Fauth, Ursula (1987): Galbonolid A und B, neue antifungische Makrolid-Anti-
biotika.
Fels, Johannes (1994): Suche nach Chitinase-Inhibitoren und Charakterisierung
der Hemmstoffe aus Streptomyces tendae T 2774 und Streptomyces sp. T
3566.
Haag, Hubert (1992): Neue Eisenkomplexbildner aus Staphylokokken und Yer-
sinia enterocolitica. Screening, Fermentation, Isolierung und Charakterisie-
rung.
Haag, Sabine (1996): Gentransfer zur Produktion neuer Naturstoffe am Beispiel
der Tetracenomycine und Urdamycine und CDA ein Calcium-abhngiges
Antibiotikum aus Streptomyces coelicolor A3(2).
Hsler, Peter (1988): Streptonolide. Sekundrmetabolite aus Streptomyces oliva-
ceus T 2108.
Hoff, Hubert (1990): Obskurolide: neue Butyrolactone aus Streptomyceten.
Huhn, Wolfgang (1986): Maduraferrin, ein neues Siderophor aus Actinomadura
madurae. Screening, Isolierung und Fermentation.
Isselhorst-Scharr, Caroline (1995): T 3010/1. Ein neues Thiolactonantibiotikum
aus Streptomyces olivaceus ssp. gelaticus T 3010.
Jung, Oliver (1993): Screening nach neuen Siderophoren und die Charakterisie-
rung eines hochaffinen Eisenaufnahmesystems bei Staphylococcus aureus.
Katzer, Werner (1991): Bromo- und Chlorotetain aus Bacillus amyloliquefaciens.
Kremer, Stefan (1990): Untersuchungen zur Biosynthese von ungewhnlichen
Macrolidantibiotika am Beispiel der Bafilomycine.
Kugler, Martin (1986): Rhizocticin. Ein neues Antibiotikum aus Bacillus subtilis
ATCC 6633.
Langer, Monika (1996): Biotinantagonisten und Siderophore aus Streptomyces
griseoflavus ssp. griseoflavus.
Mahnke, Marion (1990): Inhibitoren der Lysin-N
6
-Hydroxylase aus Escherichia
coli MM 128. Screening, System und Charakterisierung.
Meiwes, Johannes (1989): Neue Siderophore von Staphylococcen und Strepto-
myceten.
Metzler, Monika (1989): Untersuchungen an sekundren Metaboliten verschie-
dener Streptomyceten.
Petersen, Frank (1991): Germicidin B ein autoregulatorischer Keimungshemm-
stoff aus Streptomyces viridochromogenes.
Plaga, Armin (1990): Studien zur mikrobiologischen Produktion von Phosphino-
thricin aus Streptomyces viridochromogenes T 494.
Pultar, Thomas (1988): Lysolipin X und I. Fermentation, Isolierung, biologische
Wirkung und Interaktion mit Mg
2+
.
Rabenhorst, Jrgen (1986): Valclavam ein antifungisches b-Lactam.
356
Rauch, Beatrix (1992): Hormaomycin und andere differenzierungsaktive Sub-
stanzen aus Streptomyceten.
Reuschenbach, Peter (1986): Studien zur Fermentation der phosphorsuretri-
esterhaltigen Lactone aus T 1718.
Rhl, Franz (1986): Untersuchungen zur Wirkungsweise von Clavam Antibiotica.
Roos, Margareta (1994): Untersuchungen zur Differenzierung bei Streptomyces
griseus und Streptomyces antibioticus.
Scharr, Jrgen (1993): Hohe Zelldichten bei Bacillus thuringiensis var. israelensis.
Schz, Traugott (1990): Pelletbildung bei Streptomyces tendae T 901/S2566
und verfahrenstechnische Optimierung der Nikkomycin-Fermentation.
Seiffert, Andreas (1992): Untersuchungen zum Eisentransport bei verschiede-
nen Bakterien.
Stmpfel, Joachim (1988): Pyrrolam. Ein gamma-Lactam aus Streptomyces
olivaceus T 3082. Fermentation, Isolierung und biologische Wirkung.
Tschierske, Martin (1994): Studien zur Produktion von Rhizoferrin und analogen
Verbindungen mit Cunninghamella elegans.
Ziegelmaier-Kemmling, Dagmar (1990): Vergleichende Untersuchungen zur
Wirkungsweise von Tetramsure-Verbindungen.
TP A2
Incorporated in TP A1
TP A4
Bruntner, Christina (1997): Molekularbiologische Untersuchungen zur Biosyn-
these von Nikkomycin D in Streptomyces tendae T 901.
Lauer, Bettina (1997): Charakterisierung von Nikkomycin-Biosynthesegenen
und genetische Manipulation des Biosyntheseweges in Streptomyces tendae
T 901.
Mhrle, Volker (1995): Klonierung und Charakterisierung von Nikkomycin-Bio-
synthesegenen aus Streptomyces tendae T 901.
Roos, Ulrich (1993): Histidin-Aminotransferase-Aktivitt in Streptomyces ten-
dae: Korrelation zur Nikkomycin-Produktion, Reinigung und Charakterisie-
rung.
Schwarz, Wolfgang (2000): Untersuchungen zur transkriptionellen Regulation
der Nikkomycin Synthese in Streptomyces tendae T 901.
Sommer, Patricia (1995): Klonierung, Sequenzierung und Charakterisierung
eines Lipasegens aus Streptomyces cinnamomeus T 89.
Russwurm, Roland (2000): Genetische Untersuchungen zur Nikkomycin-Biosyn-
these in Streptomyces tendae T 901.
Tesch, Cornelia (1995): Klonierung, Sequenzierung und Charakterisierung eines
Esterasegens aus Streptomyces diastatochromogenes T 20.
TP A5
Choi, Ok-Byung (1995): Experimente zur Klonierung, Sequenzierung und Ex-
pression der Squalen-Hopen-Cyclase aus Rhodopseudomonas palustris und
Alicyclobacillus acidoterrestris.
357
Feil, Corinna (1996): Ortsspezifische Mutagenese zur Identifizierung katalytisch
aktiver Aminosuren der Squalen-Hopen-Cyclase von Alicyclobacillus acido-
caldarius.
Kleemann, Gisela (1992): Hopanoidgehalt und Fettsuremuster zweier Rhodo-
pseudomonas-Arten und Reinigung der Squalen-Hopen-Cyclase aus Rhodo-
pseudomonas palustris.
Perzl, Michael (1996): Biochemische und molekularbiologische Untersuchungen
zur Hopanoid-Biosynthese in Bradyrhizobium und zur Tetrahymanol-Biosyn-
these in dem Ciliaten Tetrahymena.
Schmitz, Susanne (2000): Charakterisierung des Hopanoidbiosynthese-Operons
aus Bradyrhizobium japonicum und der Squalen-Hopen-Cyclase aus Alicyclo-
bacillus acidocaldarius.
Tappe, Cord Henning (1993): Squalen-Hopen-Cyclasen: Reinigung, Charakteri-
sierung und Inhibitor-Experimente.
TP A10
Burger, Annette (1997): Isolierung und Charakterisierung einer Genregion mit
Zellteilungs- und Differenzierungsgenen aus Streptomyces coelicolor A3(2).
Maas, Ruth Maria (1997): Molekulargenetische Analyse des Plasmids pSG5 aus
Streptomyces ghanaensis DSM 2932.
Schwartz, Dirk (1997): Molekulargenetische Analyse der Phosphinothricin-Tri-
peptid-Biosynthese in Streptomyces viridochromogenes T 494.
Stegmann, Efthimia (1999): Molekulargenetische und biochemische Untersu-
chungen des EDDS-Produzenten Amycolatopsis japonicum MG417-CF17.
Vierling, Silke (2000): Molekulargenetische Analyse des recA/recX-Operons in
Streptomyces lividans TK 64.
TP A11
Gaisser, Sibylle (1998): Molekularbiologische und biochemische Untersuchun-
gen zur Avilamycin-Biosynthese und Resistenz in Streptomyces viridochromo-
genes T57.
Doman, Silvie (1998): Untersuchungen zum Wirkmechanismus der Landomy-
cine und zur Biosynthese der Desoxyzucker D-Olivose und L-Rhodinose in
den Angucyclin-Antibiotika Landomycin A und Urdamycin A.
Faust, Bettina (1998): Untersuchungen zur Biosynthese von Urdamycin A und
Herstellung neuer Naturstoffe mittels molekularbiologischer Methoden.
TP A12
Schiffer, Guido (1998): Identifizierung und funktionale Charakterisierung des
Penicillin-Bindeproteins 1C als Mureinpolymerase in Escherichia coli.
Rechenberg, Moritz von (1998): Untersuchungen der Protein-Protein Wechsel-
wirkungen zwischen Murein Hydrolasen und Penicillin-bindenden Proteinen
in Escherichi coli.
Vollmer, Waldemar (1998): Identifizierung und Charakterisierung eines Struk-
turproteins in Multienzymkomplexen aus Mureinsynthasen und Mureinhy-
drolasen in Escherichia coli.
358
TP B1
Hoffmann, Helmut (1986): Proform und reifes FhuA Rezeptorprotein von E. coli
K-12: Reindarstellung und biologische Eigenschaften.
Kster ,Wolfgang (1986): Eisenhydroxamattransport von E. coli: Nukleotidse-
quenz des fhuB Gens. Identifizierung und Lokalisierung des FhuB Proteins.
lschlger, Tobias (1986): Genetische Analyse des colM Lokus und Nukleotid-
sequenz des Colicin M Immunittsproteins.
Burkhardt, Renate (1987): Molekulare Charakterisierung der Gene fhuA, fhuC
und fhuD des Ferri-Hydroxamat-Transportsystems bei E. coli.
Fiedler, Waltraud (1987): Physiologische Bedeutung periplasmatischer Oligosac-
charide (membrane derived oligosaccharides, MDO) bei Escherichia coli
K-12: Untersuchungen an Mutanten der MDO Biosynthese.
Preler, Uwe (1987): Genetische Charakterisierung des Bindeprotein-abhngi-
gen Eisen-Dicitrattransports.
Eick-Helmerich, Katrin (1989): Untersuchungen zur Struktur und Funktion der
Gene exbB und exbD bei Escherichia coli K-12.
Sauer, Martin (1989): Eisen(III)-Aufnahme von Escherichia coli K12: Nukleotid-
sequenz des fhuE Gens und Untersuchungen zur Funktion konservierter Be-
reiche bei TonB-abhngigen Rezeptoren.
Schffer, Sven (1989): Untersuchung des Fur-Eisenrepressors von Escherichia
coli K12.
Schffler, Harald (1989): Transport durch die uere Membran vonEscherichia coli.
Staudenmaier, Horst (1989): Exogene Induktion des Eisendicitrat-Transportsy-
stems von Escherichia coli.
Gnter, Karola (1990): Zur Intermembran-Kopplungsfunktion des TonB Proteins
von Escherichia coli.
Mende, Jasmin (1990): Colicin B: Untersuchungen zum Exportverhalten Coli-
cin-produzierender Zellen und zur TonB-abhngigen Aufnahme durch die
uere Membran.
Schn, Claudia (1990): Untersuchung des Aufnahmesystems fr zweiwertiges
Eisen bei Escherichia coli.
Van hove, Brunhilde (1990): Bindeprotein-abhngiger Transport und Transmem-
branregulation des Eisen(III)Dicitrat-Transportsystems.
Chehade, Heidi (1990): Untersuchungen zur Wechselwirkung von Escherichia
coli Wildstmmen und Mutanten in umweltregulierten Genen bei bakterizi-
den Komponenten von Humanserum.
Angerer, Annemarie (1991): Ein neues Eisentransportsystem in Serratia marce-
scens.
Gaisser, Sabine (1992): Das TonB Gen aus Serratia marcescens.
Koebnik, Ralf (1992): Ferrichrom-Aufnahme in Bakterien: Membrantopologie
des Ferrichromrezeptors von Escherichia coli und Struktur des Ferrichromre-
zeptors und des TonB Proteins von Yersinia enterocolitica.
Schultz-Hauser, Gabriele (1992): FhuC, die konservierte Komponente des
Eisen(III) Hydroxamat-Transports.
Traub, Irene (1992): Untersuchung funktioneller Domnen des TonB-Proteins
von Escherichia coli.
359
Veitinger, Sabine (1992): Das Eisen(III)-Dicitrat-Transportssystem: Kartierung
auf dem Chromosom von Escherichia coli K-12 und Untersuchungen zur
Transmembran-Regulation.
Hobbie, Silke (1993): Untersuchungen zur Struktur und Funktion des ShlB Pro-
teins, der transportierenden und aktivierenden Komponente des Hmolysins
von Serratia marcescens.
Killmann, Helmut (1993): Eisen(III)Hydroxamat-Transport in Escherichia coli.
Funktionsdomnen des Rezeptor-Proteins FhuA und Untersuchungen zur
dreidimensionalen Grundstruktur des Proteins in der ueren Membran.
Schnherr, Roland (1993): Aktivierung und Sekretion des Hmolysins von Ser-
ratia marcescens.
Ochs, Martina (1994): Untersuchungen zur Regulation des Eisen-Dicitrat-Trans-
portsystems von Escherichia coli K-12.
Kim, In-Sook (1995): Exogene Signaltransduktion des Eisen(III)-Dicitrat-Trans-
portsystems von Escherichia coli K-12.
Khn, Sabine (1995): Rhizoferrin-Aufnahme in Morganella morganii.
Gro, Patricia (1996): Untersuchungen zur Struktur und Funktion des Colicin
M-Immunittsproteins (Cmi).
Pilsl, Holger (1996): Domnenstruktur, Evolution und Immunitt der Colicinde-
terminanten 5, 10 und K von Escherichia coli und der Pesticindeterminanten
von Yersinia pestis.
Bs, Christof (1997): In vivo Charakterisierung des Ferrichrom-Rezeptor-Pro-
teins FhuA aus Escherichia coli mittels thiolspezifischer Reagenzien.
Brutsche, Sandra (1997): SigX ein neuer Sigmafaktor der ECF-s
70
Subfamilie
aus Bacillus subtilis.
Enz, Sabine (1997): Transkriptionsregulation des Eisen(III) Dicitrat-Transportsy-
stems von Escherichia coli K-12.
Welz, Dieter (1997): Funktion und Lokalisierung der Induktionsproteine FecI
und FecR von Escherichia coli K-12.
Groeger, Wolfram (1998): Eisen(III)-Hydroxamat-Transport in Escherichia coli:
Topologie des integralen Transportproteins FhuB.
Habeck, Martina (1998): Energiekopplung durch TonB im Eisen(III)Dicitrat-
Transportsystem von Escherichia coli K-12.
TP B4
Hirschhausen, Heinrich Reginhard von (1986): Die Phosphodiesterasen in Para-
mecium.
Schade, Uwe (1988): Zur physiologischen Rolle von cyclischem 3',5'-Guanosin-
mono-phosphat in Paramecium tetraurelia.
Hofmann, Hans-Joachim (1990): Anreicherung und Charakterisierung einer
membranstndigen, ciliren Guanylatcyclase aus Paramecium tetraurelia.
Schrhoff-Goeters, Wilhelm (1990): Zur Entstehung cyclischen AMPs infolge hy-
perpolarisierender Puffervernderungen bei Paramecium tetraurelia.
Freund, Wolf-Dietrich (1991): Inositol-Phospholipide und Inositol-Phosphate in
Paramecium tetraurelia.
360
Steinlen, Siegfried (1992): Untersuchungen zur Regulation der partikulren Gua-
nylatcyclasen der Ciliaten Paramecium und Tetrahymena durch Calmodulin.
Friderich, Gerald (1992): Reinigung und Charakterisierung der Proteinphospha-
tase Typ 1 aus den Cilien von Paramecium tetraurelia.
Vlkel, Helge (1992): Adenylatcyclasen aus ciliren Geweben und dem retina-
len Pigmentepithel.
Beyer, Angelika (1993): Proteinphosphatase Typ 2C aus Paramecium tetraurelia:
Lokalisation, Isolierung, Teilsequenzierung und Charakterisierung.
Schnborn, Christoph (1993): Untersuchungenzur Regulationvon cyclischemAde-
nosin 3',5'-monophosphat bei Parameciumtetraurelia und Ionenkanalmutanten.
Otto, Susanne (1994): Reinigung und Charakterisierung einer Adenylatcyclase
der Retina.
Vlkel, Helge (1995): Adenylatcyclasen aus ciliren Geweben und dem retina-
len Pigmentepithel.
Selke, Dagmar (1995): Proteinphosphatase 2C aus der Rinderretina Aufreini-
gung, Charakterisierung, Klonierung und Expression.
Hanke, Cordula (1996): Proteinphosphatase Typ 2C aus Paramecium tetraurelia.
Guo, Yinglan (1996): Zur Ca
2+
-abhngigen Regulation der Bildung von cGMP,
des Schwimmverhaltens und der Lokalisation von Ca-Kanlen in Paramecium
tetraurelia.
Anton, Helga (1996): Proteinphosphatase Typ1 aus der Rinderretina Reini-
gung, Charakterisierung und Expression.
Linder, Jrgen (1997): Klonierung einer Adenylatcyclase aus Paramecium.
Beitz, Eric (1997): Zur cAMP-Signaltransduktion des retinalen Pigmentepithels
des Rindes und des Innenohrs der Ratte.
Krger, Thomas (1997): Klonierung von Adenylatcyclasen aus dem Ciliaten Te-
trahymena pyriformis.
Grothe, Kirsten (1998): Mutationsanalyse und Lokalisation der Proteinphospha-
tase Typ 2C aus Paramecium tetraurelia.
Hoffmann, Thomas (1999): Membranstndige Guanylatcyclasen aus Parame-
cium und Tetrahymena: Klonierung und bakterielle Expression der katalyti-
schen Bereiche.
Engel, Peter (1999): Klonierung und Expression einer Guanylatcyclase aus Para-
mecium tetraurelia.
TP B5
Demleitner, Gabi (1992): Die Exolipase von Staphylococcus hyicus: Identifizie-
rung der katalytisch aktiven Aminosuren und Untersuchungen zur Funktion
des Propeptids.
Krismer, Bernhard (1999): Studium der Funktion der sekretierten Proteine SceA
und SceB, Analyse des Galaktoseoperons galRKET und Kontruktion von Se-
kretions- und Expressionsvektoren in Staphylococcus carnosus.
Kupke, Thomas (1992): Posttranslationelle Modifikation bakterieller Peptide
proteinchemische Untersuchungen zur Epiderminbiosynthese.
Lechner, Max (1989): Klonierung und Charakterisierung des Gens fr die Phos-
phatidylinositol-spezifische Phospolipase C von Bacillus thuringiensis.
361
Otto, Michael (1997): Epidermin: Biochemische Untersuchungen zur Biosyn-
these, Regulation und Immunitt.
Teufel, Pia (1993): Isolierung; Sequenzierung und Charakterisierung einer Me-
talloprotease aus Staphylococcus epidermidis und Sekretions- und Regulati-
onsstudien bei Staphylococcus carnosus.
Thumm, Gnther (1996): Molekularbiologische Charakterisierung von Lysosta-
phin und Lysostaphin-Immunitts-Faktor (Lif).
Wieland, Bernd (1993): Der xylA Promotor aus Staphylococcus xylosus als Grund-
lage der transkriptionellen Regulation vonGenen in Staphylococcus carnosus.
Wieland, Karsten-Peter (1999): Organisation und Genexpression der Carotin-
oid-Biosynthesegene aus Staphylococcus aureus Newman und Untersuchun-
gen zur Funktion von Staphyloxanthin.
TP B6
Kammler, Meike (1994): Sequenzierung und Charakterisierung des Aufnahme-
systems fr zweiwertiges Eisen von Escherichia coli.
Mller, Kerstin (1997): FhuF, ein neuartiges, eisenreguliertes Eisen-Schwefel-
Protein von Escherichia coli.
Patzer, Silke (1999): Regulation durch Metallionen in Escherichia coli, insbeson-
dere Identifizierung und Charakterisierung des hochaffinen Zink-Aufnahme-
systems ZnuABC und des zinkabhngigen Regulators Zur.
TP C2
Bayer, Anja (1993): Produktion, Isolierung und Strukturaufklrung des glycin-
reichen Polypeptidantibiotikums Microcin B17.
Brooks, Marc (1999): Neue DNA-Gyrase und Humane Type II DNA-Topisome-
rase-Inhibitoren.
Deres, Karl (1992): MHC-Klasse-1-restringierte Peptide.
Drechsel, Hartmut (1993): Strukturaufklrung neuer Siderophore vom Carboxy-
lat-Typ und Synthese von Siderophor-Antibiotika-Konjugaten.
Flechsler, Insa (1998): Transfektion von Nukleinsuren mit neuen kationischen
Lipiden und Vergleich verschiedener Antisense-Strategien.
Fleckenstein, Burkhard (1997): Kombinatorisch aufgebaute Peptidkollektionen
zum Studium der HLA-Klasse II-Peptid-Interaktion und der Antigenerken-
nung autoreaktiver, humaner T-Zellen.
Hltzel, Alexandra (1999): Structure Elucidation of Secondary Metabolites and
of the Loop Sequence 316333 of the ThuA Receptor.
Hrr, Ingmar (1999): RNA-Vakzine zur Induktion von spezifischen cytotoxischen
T-Lymphozyten und Antikrpern.
Ihlenfeldt, Hans-Georg (1995): Die B-und T-Zell-Epitope des Hepatitis C-Virus.
Kabatek, Ursula (1987): Eine neuartige Teichonsure aus Streptomyces vene-
zuelae.
Kaiser, Dietmar (1998): Strukturaufklrung und Konformationsanalyse biolo-
gisch aktiver Sekundrmetabolite.
Kellner, Roland (1989): Lantibiotika ribosomal synthetisierte Polypeptidanti-
biotika mit Sulfidbrcken und Dehydroaminosuren.
362
Kempter, Christoph (1996): Strukturaufklrung mikrobieller Sekundrmetabo-
lite durch Elektrospray-Massenspektrometrie und mehrdimensionale Kernre-
sonanzspektroskopie.
Koch, Ulricke (1988): Fengycin, Strukturaufklrung eines mikroheterogenen Li-
popeptolidantibiotikums.
Konetschny-Rapp, Sylvia (1990): Neue mikrobielle Eisenkomplexbildner, Screen-
ing, Isolierung, Strukturaufklrung und komplexchemische Untersuchungen.
Metzger, Jrg (1988): Immunstimulierende Lipopeptide als Membrananker fr
Haptene und biologisch aktive Wirkstoffe.
Rapp, Claudius (1988): Neue antifungische Phosphono- und Chloro-Oligopep-
tide aus Bacillus subtilis und ein neues Thiolactonantibiotika aus Streptomy-
ces olivaceus Isolierung und Strukturaufklrung.
Stephan, Holger (1995): Strukturaufklrung mikrobieller Sekundrstoffwechsel-
produkte durch mehrdimensionale Kernresonanzspektroskopie.
Stevanovic, Stefan (1992): Multiple Sequenzanalyse Ein neuer Ansatz in der
Peptidsequenzierung: Isolierung, Synthese und Strukturaufklrung von mi-
krobiellen Metaboliten aus Amycolatopsis mediterranei, Staphylococcus epi-
dermidis und Streptomyces lividans.
List of habilitations resulting from the collaborative research centre
Bormann, Christiane (1997): Biosynthese des Antibiotikums Nikkomycin.
Brckner, Reinhold (1997): Katobolitrepression in Staphylokokken.
Bechthold, Andreas (1998): Streptomycetengenetik, Grundlage fr die Herstel-
lung neuer Antibiotika: Klonierung und Charakterisierung der Biosynthese-
gencluster von Avilamycin, Landomycin, Urdamycin und Granaticin.
Fiedler, Hans-Peter (1988): Isolierung und Analytik niedermolekularer mikro-
bieller Sekundrmetabolite.
Klumpp, Susanne (1989): Charakterisierung von Phosphatasen und Entdeckung
eines Inhibitor-Proteins.
Kupke, Thomas (1998): Mikrobielle Enzyme neuartige Reaktionen und Kataly-
semechanismen.
Metzger, Jrg (1994): Naturstoffanalytik und Strukturaufklrung mit modernen
Methoden der Massenspektrometrie.
Graduiertenkollegs
Mikrobiologie
Analytische Chemie
Zellbiologie in der Medizin
363
20.7 Alphabetical list of guests
Barnet, James Prof. A1-Zhner USA 8788
Bielecki, Jarek Dr., Univ. Warsaw A1-Zhner Poland 86
Focareta, Antonio Dr.,University of Adelaide B1-Braun Australia 8990
Howard, Stephen Peter, University of Regina B1-Braun Canada 99
Klmannczhelyi, Attila, Bukarest A4-Bormann Bulgaria 91
Kim, In-Sook Dr. rer. nat B1-Braun South-Korea 91
Jack, Ralph-Wilson Dr. rer. nat., Univ. of Otago C2-Jung N. Zealand 902000
Milla, Paola, Univ. Turin A5-Poralla Italy 98
Noda, Shigeru Dr. rer. nat., Univ. Gieen B4-Schultz (China) 86
Potterat, Olivier, Dr. rer. nat. A1-Zhner Switzerland 9192
Reinhard, Peter Dr. rer.nat., Univ. Canberra B2-Hamprecht Australia 86
Sarem, Aslani Dr. rer. nat., Univ. Wrzburg B2-Hamprecht Egypt 88
Shi, Liangru Dr. A1-Zhner P. R. China 90
Smaijs, David Dr., Masaryk University B1-Braun Czech Rep. 97
Stojiljkovic, Igor Dr. med., Univ. Zagreb B1-Braun Croatia 9293
Surovoy, Andrey Dr. med., Shemaykin-Inst. Moscow C2-Jung Russia 9097
Tschen, Shu-Yuan Dr. rer. nat., Chengdu A1-Zhner P. R. China 91
Videnov, Georgi Dr. rer. nat., Molecular Biology,
Sofia C2-Jung Bulgaria 9195
20.8 International cooperation
(A5)
Prof. M. Rohmer, University of Strasbourg, France
Prof. L. Cattel, Institute of Applied Pharmacie, University of Turin, Italy
Prof. G. D. Prestwich, Dep. Chemistry, University at Stony Brook, New York
(A11)
Prof. Dr. J. A. Salas, Oviedo, Spain
Prof. Dr. P. Leadlay, Cambridge, UK
Prof. Dr. K. Ichinose, Tokyo, Japan
Prof. Dr. H. G. Floss, Seattle, USA
Eli-Lilly and Company Limited, Hampshire, UK
Combinature-Biopharm AG, Berlin, Germany
Glaxo Wellcome, Stevenage, UK
(A12)
Prof. Dr. Miguel A. de Pedro, Laboratory for Cell Envelopes, Centro de Biologia
Molecular Severo Ochoa, Facultad de Ciencias UAM, Campus de Canto-
blanco, 28049 Madrid, Spain
364
(C2)
Nijmegen SON Research Center for Molecular Structure, Design and Synthesis
University of Nijmegen, The Netherlands
Shemyakin Institute of Bioorganic Chemistry, University of Moscow, Russia
Laboratoire Nationale de Sant, Luxembourg
Laboratory of Microbial Technology, Agricultural University of Norway, As, Nor-
way
Ecole Polytechnique Federale de Lausanne, Department de Chimie, EPFL-
Ecublens, Lausanne, Switzerland
Groupe RdMN, Institut Pasteur de Lille, Lille, France
German-Israel Foundation for Scientific Research & Development, Weizmann In-
stitute Rehovot, Israel
20.9 International conferences
Sekundrmetabolite aus Mikroorganismen. Tbingen 1989.
Microbial Secondary Metabolism. Interlaken 1994.
Vectorial Transport Across Bacterial Membranes. Tbingen 1995.
1. Import, Export and Sorting
2. Protein and Peptide Channels
Biologie der Actinomyceten. Tbingen 1996.
Plasmide und Genregulation. Blaubeuren 1997.
Tbinger/Gttinger Gesprche zur Chemie von Mikroorganismen. Blaubeuren,
each year.
20.10 Funding
The collaborative research centre 323 has been supported by grants of the
Deutsche Forschungsgemeinschaft totalling DM 35 678 000 in the period 1986
1999.
365
20.10 Funding

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) is used to treat iron-overload diseases, phosphinothricin (trade

) is a herbicide, and the nikkomycins are potent inhibitors of

(mesylate salt of desferri-fer-

mutants and development

mutants of S. epidermidis T 3298 by ethyl-methane-

-like phenotype were selected with the aim of

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