Nature Reviews Microbiology | AOP, published online 10 March 2005; doi:10.

1038/nrmicro1128

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REGULATION AND BIOSYNTHESIS OF CARBAPENEM ANTIBIOTICS IN BACTERIA

Sarah J. Coulthurst, Anne M. L. Barnard and George P. C. Salmond
Abstract | Carbapenem antibiotics are members of the -lactam family of antibiotics, the most important class of antibiotics currently in clinical use. They are active against many important Gram-positive and Gram-negative pathogens. One important feature of carbapenem antibiotics is their resistance to several -lactamases. Thienamycin, isolated from Streptomyces cattleya, was the first carbapenem described. Other well-studied carbapenems were isolated from the Gram-negative bacteria Erwinia carotovora subsp. carotovora, Serratia sp. strain ATCC39006 and Photorhabdus luminescens strain TT01. Here, we review the genetics and biochemistry of carbapenem production in these bacteria. Research into carbapenems could uncover a new repertoire of bioactive molecules and biosynthetic enzymes, and exploiting these novel enzymes could lead to development of new classes of antibiotics with useful chemotherapeutic activities.
Carbapenems belong to the -lactam family of antibiotics, which is the biggest and most important class of clinical antibiotics. Their sales represent an estimated US$15 billion, and as a family they constitute more than 65% of the global antibiotic market1. Interest in the use of -lactam antibiotics to treat bacterial infections was sparked by the discovery of penicillin in 1929 (REF. 2) (TIMELINE). Since then, both naturally occurring and chemically synthesized -lactams have been developed for clinical use and are still widely used today. -Lactams function by inhibiting bacterial cell wall peptidoglycan biosynthesis. Cell wall peptidoglycan is strengthened by the formation of crosslinks, a process that is catalysed by transpeptidase enzymes. -Lactams are thought to function as structural analogues of the transpeptidase substrate, forming relatively stable complexes with the enzyme3. This, in turn, inhibits crosslink formation by the enzyme, and the resulting loss in cell wall integrity can lead to lysis of growing bacterial cells. Since the discovery of penicillin, many naturally occurring and synthetic -lactam antibiotics have been identified. They have a four-membered -lactam ring in common, with different structures attached (TABLE 1). Carbapenem antibiotics (TABLE 2) are characterized by an unsaturated five-membered carbon ring that is fused to the nitrogen and one carbon on the -lactam ring, and the carbapenem biosynthetic pathway has important differences when compared with the biosynthetic pathways of other -lactam antibiotics. Carbapenems are active against a range of Grampositive and Gram-negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Neisseria spp., Haemophilus spp., Pseudomonas aeruginosa, Proteus spp., Enterobacter spp., Bacteroides spp. and Clostridium spp.4. Thienamycin was the first carbapenem antibiotic to be discovered, and was isolated from Streptomyces cattleya5. Recently, the thienamycin biosynthetic genes have been identified. In common with other bacterial antibiotic biosynthetic genes, these genes are clustered in the chromosome with putative regulatory genes6. Other bacteria also produce carbapenem antibiotics, including the Gram-negative plant pathogen, Erwinia carotovora subsp. carotovora, Serratia sp. strain ATCC39006 and Photorhabdus luminescens strain TT01. All of these bacteria produce 1-carbapen-2em-3-carboxylic acid (Car)7,8 (TABLES 1,2).

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK Correspondence to G.P.C.S. e-mail: gpcs@mole.bio.cam.ac.uk
doi:10.1038/nrmicro1128 Published online 10 March 2005.

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Timeline | Key events in the discovery and utilization of -lactam antibiotics

Discovery of cephalosporin C reported54 Cloning and sequencing of the Car biosynthetic gene cluster from E. c. carotovora reported21

Discovery of penicillin reported2

First clinical trials of penicillin reported54

Discovery of nocardicin A reported55

Discovery of thienamycin reported5

1929

1940

1941

1945

1953

1965

1976

1977

1979

1982

1996

2003

First penicillinase activity reported53

Chemical structure of penicillin confirmed54

Mode of action of -lactams elucidated3

Discovery of clavulanic acid (-lactamase inhibitor) reported56

Report of Car production by Erwinia carotovora subsp. carotovora and Serratia sp. strain ATCC39006 (REF. 7)

Cloning and sequencing of the thienamycin biosynthetic gene cluster isolated from Streptomyces cattleya reported6

Car, 1-carbapen-2-em-3-carboxylic acid.

Widespread resistance among clinically important organisms is a problem that is associated with the clinical use of -lactams. Benzylpenicillin was first used therapeutically in 1944, and was active against 95% of S. aureus isolates. Just 5 years later, selection pressure and the spread of resistance reduced this to activity against only 50% of isolates9. Drug resistance might be an inevitable consequence of the clinical application of antibiotics and therefore there is a constant need for development of new antimicrobials. Resistance to -lactam antibiotics often involves -lactamase enzymes, which cleave the -lactam ring and inactivate the antibiotic. Resistance can also involve altered penicillin-binding proteins (a group of proteins that includes the peptidoglycan transpeptidase) or altered cell wall structures (reviewed in REFS 9,10). Many resistance mechanisms, particularly those involving -lactamases, are plasmid-encoded and are easily transmissible among bacteria, ensuring that resistance is readily acquired by susceptible co-infecting organisms. An advantage of the carbapenems is that they tend to be resistant to clinically-encountered -lactamases, including the extended spectrum -lactamases (ESBLs),
Table 1 | The five main classes of -lactam antibiotics
Structure
S N O S N O N O O N O

which are responsible for most of the clinical resistance to penicillin and celphalosporin-related compounds4. Carbapenem-hydrolysing -lactamases, including class B zinc -lactamases, do not occur frequently in clinically important bacteria9,1113. The carbapenem class of -lactams is therefore a promising source of new clinically useful antibiotics. Initially, there was considerable interest in thienamycin production owing to its broad spectrum of activity against Gram-positive and Gram-negative bacteria. However, thienamycin is chemically unstable and is metabolized by a human renal dihydropeptidase enzyme. Instead, a more stable N-formimidoyl derivative of thienamycin (imipenem) (TABLE 2) is used clinically, in combination with cilastatin, which is an inhibitor of the dihydropeptidase enzyme (reviewed in REF. 14). Other carbapenems that have been developed for clinical use, including meropenem and ertapenem, are not good substrates for the renal dihydropeptidase (reviewed in REFS 15,16) (TABLE 2). Penicillins, cephalosporins, cephamycins and their precursors are industrially produced by FERMENTATION and SEMI-SYNTHESIS (reviewed in REF. 1). However, owing

Class Penicillins

Example Penicillin G

Source organism Penicillium chrysogenum

Key biosynthetic enzymes (genes) ACV synthetase (pcbAB), isopenicillin N synthase (pcbC), isopenicillin N acyltransferase (penDE) ACV synthetase (pcbAB), isopenicillin N synthase (pcbC), isopenicillin N epimerase (cefD)

Precursors Cysteine, valine, -aminoadipic acid

Reference 58

Cephalosporins/ Cephalosporin C cephamycins

Acremonium chrysogenum

Cysteine, valine, -aminoadipic acid

58

Monobactams Clavams

Nocardicin A Clavulanic acid

Nocardia uniformis Non-ribosomal peptide subsp. tsuyamanensis synthetases (nocA, nocB) Streptomyces clavuligerus Erwinia carotovora subsp. carotovora Clavaminate synthase, -lactam synthetase Carbapenam synthetase (carA), carboxymethylproline synthase (carB), carbapenem synthase (carC)

Serine, methionine, p-hydroxyphenylglycine Arginine, glyceraldehyde-3phosphate Acetate, glutamate

59 46

Carbapenems
N O

Car

Reviewed herein

ACV, -(L--aminoadipyl)-L-cysteinyl-D-valine; Car, 1-carbapen-2-em-3-carboxylic acid.

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Table 2 | Examples of carbapenem antibiotics

Structure
H N O CO2H NH S CO2H OH HH N O CO2H OH O HH N N O CO2H OH O HH N O S CO2H N H NH CO2H S NH CH3 S HN NH

Carbapenem Source Car

Used clinically Reference Reviewed herein

Erwinia carotovora subsp. No carotovora, Serratia sp. strain ATCC39006, Photorhabdus luminescens strain TT01 Streptomyces cattleya No

OH

HH N

Thienamycin

Imipenem

Organic synthesis

Yes

17

Meropenem
CH3

Organic synthesis

Yes

16

Ertapenem

Organic synthesis

Yes

15

FERMENTATION

An industrial process that is used to generate a microbially derived product in a fermenter, in which the growth conditions of the microorganism are tightly controlled and optimized to produce the maximum yield of the required product.
SEMI-SYNTHESIS

Car, 1-carbapen-2-em-3-carboxylic acid.

This describes a process by which molecules of interest are produced by synthetic chemical modification of starting-point compounds that have been produced by microbial fermentation rather than by total chemical synthesis in vitro.
CRYPTIC GENES

to the low titres of thienamycin production and its chemical instability1, commercial production by fermentation is problematic. Carbapenems are currently produced by total organic synthesis 1,17. Recent advances in our understanding of the genetics and biochemistry of carbapenem production in bacteria might lead to production of new carbapenem antibiotics through the exploitation of bacterial enzymes and systems. Here, we review some of the latest findings in this area.
Regulation of carbapenem antibiotic production

Cryptic genes are present in the genome of the microorganism of interest, but are not generally expressed. Crypticity can be due to mutations in regulatory genes. Alternatively, genes that are expressed might have nonfunctional protein products owing to mutations in the coding sequences.
INTRINSIC RESISTANCE

Some bacteria are intrinsically resistant to specific antibiotics and have not acquired antibiotic resistance through gene mutation or horizontal gene transfer from another organism. Intrinsic resistance is particularly important for antibiotic-producing organisms, which must have a mechanism for avoiding the inhibitory effects of the antibiotic that they produce.

There is little data on the genetic, physiological or environmental factors that modulate the production of thienamycin by Streptomyces sp., largely because genetic analysis of the producer strain S. cattleya is difficult. However, the recent cloning and sequencing of the thienamycin biosynthetic cluster6 (see below) is a significant breakthrough that should enable a detailed molecular biological and physiological analysis of thienamycin regulation. In contrast to the paucity of regulatory information for S. cattleya, there is a growing body of information on the regulation of carbapenem production in some species of Gramnegative bacteria. Screening for carbapenem-producing bacteria indicated that, among the Gram-negative bacteria, production of carbapenems might be limited to a tiny subset of species, including strains of E. c. carotovora, Erwinia herbicola and an uncharacterized strain of Serratia spp. (ATCC39006)7,1820. The antibiotic that is produced by E. c. carotovora is Car. It is the simplest naturally occurring carbapenem, and although it is not

itself clinically useful, Car has the same carbapenem nucleus as thienamycin and its derivatives. A cluster of genes named carAH is necessary for Car antibiotic synthesis in E. c. carotovora strain ATCC39048 (REF. 21) (FIG. 1). This cluster was cloned and then used to screen other bacterial strains using Southern hybridization analysis, which revealed that, in many E. c. carotovora strains, the genes that encode Car are present but 22 CRYPTIC (BOX 1). Rarely, genes that encode Car were also detected in Erwinia carotovora subsp. atroseptica strains, although the cluster is absent from the genome of the recently sequenced E. c. atroseptica strain SCRI1043 (REF. 23). Recently, it was reported that the genome of P. luminescens TT01, a bioluminescent insect pathogen, contains a car-like gene cluster8 (FIG. 1). Although the biological role of carbapenem production in microbial communities is unknown, it has been hypothesized that the antibiotic is produced to defend the local ecological niche of producer organisms20.
The Car gene clusters

The car gene cluster of E. c. carotovora ATCC39048 was the first to be sequenced and both functionally and transcriptionally defined21 (FIG. 1). CarAE synthesize Car from precursors (described in more detail below), whereas CarF and CarG are required for INTRINSIC RESISTANCE to the antibiotic by a novel, as-yetuncharacterized mechanism18 (see below). Mutants defective in carH still synthesize normal levels of Car and are intrinsically resistant, so the function of CarH is unknown. The carAH genes are organized as an operon, and transcription initiates at 52 bp upstream from the translational start of carA24 (P1 in FIG. 1).

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presence of OHHL. E . c . carotovora only generates sufficient OHHL to bind to the CarR protein at high cell densities. The CarROHHL complex binds to the carA promoter in the carRA intergenic region and activates expression of the complete carAH operon18,2426. The carR gene is located immediately upstream of the carAH operon and is a separate transcriptional unit, initiating at 108 bp from the translational start24 (PcarR in FIG. 1). However, the QS locus (carI-expR) in E. c. carotovora is not linked to the car biosynthetic locus (FIG. 2). Mutants that lack either carI or carR fail to synthesize Car, but null mutations in the expR gene have no obvious effects on Car production and so its physiological role is unclear in this strain27. Double mutants (carI, expR) are Car-negative, consistent with the requirement for carI (OHHL) to activate CarR, which in turn activates transcription of the carAH cluster. In addition to the QS-dependent promoter P1, there is also a weak QS-independent internal promoter, P2 (FIGS 1,2), located within carD. This resistance promoter, which drives the expression of carEH, might allow CONSTITUTIVE EXPRESSION of the genes that are required for intrinsic carbapenem resistance24. Why might it be necessary to have control of intrinsic resistance independent of QS? One explanation is that, in a population of cells, there might be different rates of antibiotic synthesis. It might therefore make ecological sense to have a basal level of resistance arising from the expression of P2. This would allow producer cells to survive in the presence of antibiotic produced by neighbouring cells, prior to the up-regulated and fully induced biosynthesis of antibiotic and resistance functions that occur when QS is fully operational. A gene cluster containing carAH was also cloned from Serratia ATCC39006 (REF. 28). The conserved organization of this gene cluster implied a similar mode of regulation to that found in E. c. carotovora. However, in Serratia ATCC39006, although production of the antibiotic is QS-dependent, the main QS molecule is butanoyl-L-homoserine lactone (BHL) rather than OHHL29. The smaIsmaR QS locus in Serratia ATCC39006 encodes the SmaI protein, which is responsible for BHL synthesis, and a second LuxR homologue, SmaR. In fact, QS controls transcription of the carR gene in Serratia sp. by a DEREPRESSION mechanism that is mediated, at least in part, through BHL titration by SmaR. The current model suggests that SmaR functions to repress carR transcription in the absence of BHL30. As cell density increases, BHL concentration increases, which is thought to derepress carR transcription. Genetic evidence indicates that, although CarR in Serratia sp. is a LuxR homologue, it functions as a ligand-independent transcriptional activator of the car biosynthetic genes in this bacterium28,30. This mode of regulation is in contrast with that observed for CarR in E. c. carotovora (TABLE 3). Car production by a bacterial strain from a third enteric genus has also been investigated. The cpmAH gene cluster in P. luminescens is similar to the Serratia sp. and E. c. carotovora carAH cluster (the predicted gene products range from having 32.4% to 80.6% similarity

P1 PcarR Erwinia carR carotovora subsp. carotovora Serratia sp. strain ATCC39006 Intergenic region carA carB carC P2 carD carE carF carG carH

carR

Intergenic region carA

carB

carC

carD

carE

carF

carG

carH

Photorhabdus luminescens strain TT01 Photorhabdus luminescens strain TT01

cpmA cpmB cpmC

cpmD cpmE cpmF cpmG cpmH

cpmI cpmJ cpmK

Figure 1 | Car antibiotic biosynthesis and resistance gene clusters of three Gram-negative bacteria. The carR gene (green) encodes the LuxR-type regulator; carAE (purple) encode biosynthetic enzymes; carF and carG (orange) encode components of the intrinsic resistance mechanism. The function of carH (turquoise) is unknown. The cpm genes in Photorhabdus luminescens encode the corresponding Car homologues. cpmI, cpmJ and cpmK are duplications of cpmF, cpmG and cpmH, respectively, but are of unknown function. The arrows represent transcriptional units of the Erwinia carotovora subsp. carotovora cluster and the three E. c. carotovora promoters are highlighted (PcarR, P1 and P2). Car, 1-carbapen2-em-3-carboxylic acid.

In E. c. carotovora ATCC39048, antibiotic production is cell density-dependent, being regulated by quorum sensing (QS) (reviewed in REF. 20). QS in E. c. carotovora involves the production of the diffusible signalling molecule N-3-(oxohexanoyl)-L-homoserine lactone (OHHL), which is synthesized by CarI, a LuxI family member. CarR (a LuxR homologue) is a DNA-binding transcriptional activator of carAH that functions in the

Box 1 | Carbapenem genes in Erwinia species: function and crypticity Screening programmes for carbapenem-producing Erwiniae uncovered few bacterial isolates that produced carbapenem, which might indicate that the biosynthetic genes are rare. However, once the first car gene cluster was cloned and a gene probe synthesized, genes responsible for Car (1-carbapen-2-em-3-carboxylic acid) biosynthesis were found to be widespread among Erwinia carotovora isolates. All of these strains synthesized N-3-(oxohexanoyl)-L-homoserine lactone, so the inability to produce Car was not attributable to a lack of the quorum sensing (QS) signal molecule22. One strain, Erwinia carotovora subsp. carotovora SCRI193 (FIG. 6) produced Car when the endogenous carR gene was introduced in multicopy in trans , indicating that the cryptic phenotype might be due to a lack of a functional CarR regulator22. The E. c. carotovora SCRI193 carR promoter was able to drive expression of carR in an Escherichia coli-based in vitro expression system, indicating that the defect in CarR in the SCRI193 strain might be due to amino acid substitutions in the protein itself, rather than an effect on transcription or translation of the carR regulatory gene57. Intriguingly, whereas SCRI193 failed to synthesize Car in vitro, the same strain was resistant to the carbapenem that is produced by E. c. carotovora strain ATTn10 (a restrictionless derivative of strain ATCC39048) (FIG. 6). This indicated that the intrinsic resistance genes (carF and carG) were expressed independently of the QS system, consistent with a model in which the internal promoter is transcriptionally active, resulting in low-level intrinsic resistance even in the absence of QS-mediated induction24. In fact, 16% of the E. c. carotovora strains that contain cryptic car genes could synthesize Car when provided with the E. c. carotovora ATCC39048 carR gene in trans22. Cryptic car genes were therefore intact, but lacked a functional transcriptional activator. These studies on crypticity were conducted on strains from a large culture collection, so it would be interesting to probe the distribution of carbapenem producers in natural populations of E. c. carotovora.

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Carbon source

carI

expR ? Temperature

The Rap and Hor transcriptional regulators

CarI
O H N O O

CarR

+ carR + hor Hor Biosynthesis and resistance Resistance (constitutive) carA carB carC carD carE carF carG carH

Temperature

Figure 2 | Summary of known regulators of Car biosynthesis in Erwinia species. CarI is the N-3-(oxohexanoyl)-L-homoserine lactone (OHHL) synthase. OHHL binds to the CarR dimer and the complex (boxed) binds to the carA promoter to activate transcription of the carAH operon. Additionally, CarR activates its own transcription in the presence of OHHL. The Hor protein directly (or indirectly) positively regulates the carAH genes. Transcription of carI is responsive to carbon source (red arrow). Transcription of hor is thermoregulated. Temperature could also affect CarI production or Hor production (or activity, blue arrow). Dotted blue arrow indicates uncertainty. Antibiotic production is also repressed by anaerobiosis (not shown). Car, 1-carbapen-2-em-3-carboxylic acid.

The rap (regulator of antibiotic and pigment) gene of Serratia ATCC39006 and the hor (homologue of rap) gene of E. c. carotovora encode members of the SlyA family of transcriptional regulators34. Members of this protein family, which includes Hor (E. c. carotovora), Rap (Serratia ATCC39006), SlyA (Salmonella typhimurium) and MarR (Escherichia coli), are present in several bacteria and control many physiological functions, including antibiotic production, multidrug resistance, toxin production, virulence in plant and animal pathogens, exoenzymes, and secondary metabolite synthesis34. rap mutants in Serratia spp. and hor mutants in E. c. carotovora do not produce carbapenem antibiotic20,24,25. Assays of lacZ transcriptional fusions to the Serratia ATCC39006 and E. c. carotovora carA genes, have shown that carA transcription is downregulated in rap and hor mutants. This indicates a role for Rap and Hor in activating transcription of car operons in Serratia ATCC39006 and E. c. carotovora24,30. However, Rap does not regulate transcription of smaI in Serratia ATCC39006, and Hor does not regulate transcription of either carI or carR in E. c. carotovora20,24,30,34. So, the Rap and Hor regulators seem to function through QS-independent pathways to control Car production in these two species.
Physiological regulation of Car

CONSTITUTIVE EXPRESSION

This describes a constant level of gene expression rather than gene expression that is induced or repressed in response to a change in a physiological cue.
DEREPRESSION

The removal of repression in response to a physiological signal, resulting in an upregulation of expression of the corresponding target genes.

with the corresponding E. c. carotovora Car proteins)8. The P. luminescens cpmIK gene cluster is homologous to the Serratia ATCC39006 and E. c. carotovora carFH genes (the predicted CpmI, CpmJ and CpmK proteins share 46.9%, 32.4% and 33% identity with E. c. carotovora CarF, CarG and CarH, respectively)8. In addition, there is no evidence for N-acyl homoserine lactone-mediated QS regulation in P. luminescens 8. It is therefore unremarkable that P. luminescens lacks a CarR homologue (FIG.1; TABLE 3). However, there is evidence that a LuxS-type QS system (also known as the AI-2 or furanosyl borate diester system31,32) represses Car production in stationary phase in this insect pathogen8. By contrast, a luxS mutant of E. c. carotovora has no significant difference in antibiotic production, as determined using crude bioassays (S.J.C., unpublished observations), whereas a luxS mutant of Serratia ATCC39006 has reduced Car production, implying genetic or physiological activation through LuxS/AI-2 (REF. 33) (TABLE 3).

Several physiological cues that regulate Car production have been described for E. c. carotovora. Specifically, antibiotic biosynthesis is thermoregulated, is under carbon-source-mediated control and is regulated by oxygen availability. Thermoregulation. Car is expressed efficiently when cells are grown at temperatures up to and including 33/34C, but is essentially repressed at 3537C (REFS 20,24). This thermosensitivity is not due to the lability of the Car biosynthetic enzymes, biosynthetic intermediates or Car itself, as car genes that are cloned and expressed in E. coli express functional Car at temperatures up to 42C (REFS 18,21,35,36). Using reporter gene assays, thermoregulation in E. c. carotovora was shown to be exerted at the level of carA transcription24. Further, transcription of both carI and carR is not thermosensitive.

Table 3 | Summary of the effects of two quorum sensing (QS) systems on Car production by three enteric genera
Organism Erwinia carotovora subsp.carotovora strain ATCC39048 Serratia sp. strain ATCC39006 AHL-based QS molecule OHHL Mode of action Physical binding of OHHL by CarR protein; positive activation of carAH transcription carR regulator gene repressed by SmaR protein; BHL thought to relieve this repression; CarR protein function is ligand-independent No carR gene in genome LuxS/AI-2 impact No significant impact Mode of action Not relevant

BHL (and HHL)

luxS mutant shows reduced carbapenem production

LuxS/AI-2 system might activate carbapenem synthesis

Photorhabdus luminescens strain TT01

No detectable AHL system

luxS mutant shows enhanced carbapenem production in stationary phase

LuxS/AI-2 system might repress antibiotic production in stationary phase

AHL, N-acyl homoserine lactone; AI-2, autoinducer-2; BHL, butanoyl-L-homoserine lactone; Car, 1-carbapen-2-em-3-carboxylic acid; HHL, hexanoyl-L-homoserine lactone; OHHL, N-3-(oxohexanoyl)-L-homoserine lactone.

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Oxygen availability. Anaerobiosis represses Car biosynthesis, but whether this effect is mediated through direct sensing of oxygen availability, or whether it is an indirect effect owing to the reduced growth rate of E. c. carotovora under anaerobic conditions, is unclear24.
CO2H NH + H2N CO2H CarB CoA H S O O OH Malonyl-CoA O HO Pyrroline 5-carboxylate

Proline NH

CO2H

CarD? O Glutamate semialdehyde H H2O

HN

CO2H (2S,5S)-carboxymethylproline ATP CarA AMP + PPi H

N CO2H (3S,5S)-carbapenam 2OG + O2 CarC Succinate + CO2 + H2O H

6 7 5 1 2 3

H + N ? CO2H (3S,5R)-carbapenam O

Why quorum sensing? Although Car antibiotic production in Erwinia spp. integrates multiple regulatory inputs, many of these inputs seem to function through modulation of the QS system, highlighting the central physiological importance of QS in the biology of E. c. carotovora. Why should E. c. carotovora synthesize a carbapenem antibiotic under QS control? One rationale would be as follows. Together with the production of the antibiotic, QS also controls the production of plant cell wall-degrading enzymes (reviewed in REF. 20). The degradative effect of the enzymes produces a localized nutritional windfall of cell-wall breakdown products at the site of plant cell wall attack. Competitor bacteria in this environmental niche may be able to exploit the nutritional bonus, and so simultaneous QS activation of production of the broad-spectrum carbapenem by E. c. carotovora might have evolved to defend the nutrient-rich ecological microenvironment that is created by the plant pathogen. Serratia ATCC39006 and P. luminescens TT01 synthesize the same carbapenem antibiotic as E. c. carotovora and have car biosynthetic gene homologues. However, in terms of QS, regulation of Car production in these strains is different from that in E. c. carotovora. These observations, together with the widespread presence of isolate-dependent cryptic car genes in E. c. carotovora strains (BOX 1), highlight the strain-dependent variability of antibiotic regulation systems in Gram-negative, enteric carbapenem producers.
Biosynthesis of carbapenem in Erwinia species

N
4

CO2H (5R)-carbapenem

Figure 3 | Proposed biosynthetic pathway of Car. The central carbon atom involved in the CarC-mediated epimerization reaction is shown in red. Car, (5R)-1-carbapen2-em-3-carboxylic acid; CoA, coenzyme A; 2OG, 2-oxoglutarate; PPi, pyrophosphate.

However, stability of the carI transcript might be temperature sensitive, as addition of synthetic OHHL partially overcomes the thermorepression of carA transcription. However, transcription of hor is tightly thermoregulated, as it is completely repressed at 37C (REFS 20,24). Carbon-source repression. Transcription of carA and production of Car are repressed when E. c. carotovora is grown in the presence of glycerol20,24. The transcription of the carI gene is repressed in the presence of glycerol. As CarI functions to synthesize OHHL, the presence of glycerol downregulates the synthesis of this QS molecule, which in turn downregulates transcription of the car biosynthetic genes20,24. However, glycerol does not affect transcription of either the carR or the hor genes20,24.

Precursor identification, cloning of the biosynthetic genes and the determination of the biosynthetic pathway19,21,37 have revealed that the biosynthesis of Car is distinct from that of the penicillins and cephalosporins38. The Car biosynthetic pathway is shown in FIG. 3; the core biosynthetic enzymes CarA (carbapenam synthetase), CarB (carboxymethylproline synthase) and CarC (carbapenem synthase) are necessary and sufficient to synthesize carbapenem from cellular precursors21,37. CarB converts precursor molecules, which are derived from acetate and glutamate (see below), into (2S,5S)-carboxymethylproline (CMP). CarA then catalyses the formation of the -lactam ring, producing (3S,5S)-carbapenam. Finally, CarC catalyses an epimerization and a desaturation to yield the main product, (5R)-carbapenem (the active antibiotic). In addition, lesser amounts of the inactive (3S,5R)-carbapenam are also produced by CarC. Following disagreement in the literature, the stereochemistry of the carbapenam product of CarA, which is a substrate for CarC and is a central intermediate, was recently confirmed as (3S,5S)carbapenam39,40. Inactivation of either carD or carE results in production of significantly reduced levels of Car21, indicating a possible role for their gene products

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similarity to these enzymes in the well-conserved -sheet core and to predicted CoA-binding residues. Purified CarB is a trimer, like other crotonase family members41. Purified CarB from E. c. carotovora converts malonyl-CoA and GSA to CMP. With the inclusion of purified CarA and CarC, CMP is converted to active carbapenem41. The reaction proceeds by the decarboxylation of malonyl-CoA to form an enzyme-bound enolate, which can then react with GSA/P5C through several possible pathways. This recent study highlights several aspects of the CarB-mediated reaction that are unusual; for example, the use of malonyl-CoA as a two-carbon source. CarA and CarC can accept different stereoisomers of their natural substrate and CarB produces only a single stereoisomer of CMP. Therefore, it seems that CarB, at least in part, determines the subsequent stereochemical course of the pathway to Car41. Next, CarA catalyses formation of the -lactam ring (FIG. 3). Gerratana et al.42 showed that purified CarA from E. c. carotovora catalyses the ATP-dependent formation of (3S,5S)-carbapenam from CMP. Kinetic analysis of the CarA-mediated reaction showed that ATP binds to the enzyme first, followed by CMP, and that pyrophosphate (PPi) is the final product released. The CarA-catalysed reaction proceeds by the formation of an activated acyladenylate intermediate, followed by intramolecular amide-bond formation to close the -lactam ring (FIG. 4a). The crystal structure of CarA from E. c. carotovora was solved to a resolution of 2.4 with substrate bound (CMP, the non-hydrolysable ATP analogue AMP-CPP (, -methyleneadenosine triphosphate) and Mg2+) and 2.3 without substrate bound43. CarA is a homotetramer, and each monomer has two distinct domains (FIG. 4b). The C-terminal domain contains the active site, which is situated in a cleft between four -strands and five -helices, with an ordered loop that covers, and presumably closes, the active site when substrate is bound. CarA is homologous (sharing 27% identity) to the -lactam synthetase (-LS) that functions in clavulanic acid biosynthesis. -LS catalyses -lactam ring formation in an analagous manner to CarA; that is, substrate adenylation followed by intramolecular amide bond formation to close the -lactam ring44. The structure of the CarA monomer is similar to the structure of -LS, including the highly conserved ATP-binding site. Probable key catalytic residues identified in -LS44 are conserved in CarA; for example, a lysine residue (Lys443) that is proposed to stabilize the negatively charged intermediate/transition state43 (FIG. 4a). CarA can accept a range of substrates42. Unfortunately, in the CarA structure, CMP was not in the correct, or productive, orientation in the active site, limiting the available information on substrate binding and catalysis. We are therefore left with the following question: how does CarA correctly position CMP (or other substrates) in the active site? However, substrate promiscuity might indicate that CarA could represent a versatile tool for engineering novel -lactams. The final and most interesting enzyme in the biosynthetic pathway is CarC. Li et al.37 showed that CarC from E. c. carotovora converts the (3S,5S)-carbapenam

Box 2 | Strategies for the generation of novel carbapenems Several strategies for the generation of novel carbapenems can be envisaged: the use of combinatorial chemistry to generate differently substituted precursors; mixing and matching enzymes from different -lactam biosynthetic pathways; and altering enzyme function or specificity through targeted engineering or directed evolution. The Car (1-carbapen-2-em-3-carboxylic acid) enzymes, most notably CarA, can accept non-natural substrates41,42,49, indicating that such approaches might be feasible. It might also be possible to develop a semi-synthetic method, analogous to that for penicillin production, for the large-scale production of clinically useful carbapenems. This might involve the overexpression of CarAC (or related enzymes) in a bacterial host to obtain the basic bicyclic nucleus (by fermentation), ideally as a chemically-stable derivative, followed by synthetic chemical tailoring to yield a clinically useful molecule. Understanding the regulation of antibiotic production by the natural producer might allow increased production of a precursor or final molecule in the natural host, perhaps complementing one of the above strategies.

in precursor/substrate synthesis. CarD has significant sequence homology to proline dehydrogenase/oxidase enzymes (for example, sharing 32% identity with the proline oxidase precursor of Drosophila melanogaster 21) and CarE is predicted to be a 2Fe-2S ferredoxin that mediates an unidentified electron transfer reaction (for example, sharing 34% identity with a ferredoxin from Synechococcus 21). In the 1980s, the precursors for Car biosynthesis were determined to be acetate (-lactam carbons) and glutamate (pyrrolidine ring) by radiolabel incorporation studies in Serratia spp.19 As discussed below, CarB uses malonyl-coenzyme A (CoA), rather than acetylCoA as previously believed, as a two-carbon source for the generation of CMP. However, as malonyl-CoA can be produced from acetyl-CoA (and therefore from acetate), this finding is consistent with original precursor studies41. Incorporation experiments using radiolabelled L-proline have indicated that the immediate precursor of the pathway and substrate of CarB, glutamate semialdehyde (GSA)/pyrroline 5-carboxylate (P5C), is derived from the oxidation of L-proline40 (which is derived from glutamate). This is consistent with the observation that CarD shows sequence similarity with proline dehydrogenases, although the notion that CarD mediates the conversion of proline to GSA/P5C remains experimentally untested.
Core biosynthetic enzymes: CarA, CarB and CarC

Recent work from the Schofield and Townsend groups has advanced our understanding of how the three central enzymes CarA, CarB and CarC synthesize the active Car molecule from precursors4050. This work has revealed novel and unusual enzymatic mechanisms and should pave the way for generation of novel -lactam molecules (BOX 2). CarB catalyses the first, committed step of Car biosynthesis, which is the formation of (2S,5S)-CMP (FIG. 3). CarB is similar to enoyl-CoA hydrolases (sharing 28% identity with enoyl-CoA hydratase from Sinorhizobium meliloti 21) and other members of the crotonase superfamily, which catalyse reactions involving CoA-activated molecules21. In particular, CarB shows

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a
O

O P O
O

Lys443 H +N HH

O O O

O P O P O O Ad

PPi
O
O

H O :N H CO2
O

NH3
+

AMP
H N+ H CO2 O CO2H H N

CO2

P O O Ad

O P O Ad

ATP

(2S,5S)-CMP

Acyladenylate intermediate

Intermediate/ transition state

(3S,5S)-carbapenam

N-terminal domain

C-terminal domain

Figure 4 | CarA and CarC atomic structures. a | Proposed mechanism of action of CarA. Initially, ATP is attacked by the carboxymethyl group of (2S,5S)-carboxymethylproline (CMP) to yield the acyladenylate intermediate. This adenylated intermediate then undergoes intramolecular amide-bond formation (cyclization/-lactam formation), through a proposed tetrahedral oxyanion intermediate or transition state, to yield (3S,5S)-carbapenam4244. B (Base) facilitates deprotonation of the ammonium ion prior to -lactamization, and this role might be fulfilled by a catalytic dyad composed of Thr345 and Glu380. Lys443 is proposed to stabilize the negatively charged oxyanion intermediate/transition state. Ad, adenosine; PPi, pyrophosphate. b | Crystal structure of CarA43. Left, complete tetramer with each subunit in a different colour. Right, one monomer with bound AMP-CPP (,-methyleneadenosine triphosphate) and CMP, both shown in a ball-and-stick representation, and with Mg2+ shown as a pink sphere. c | Crystal structure of CarC45. Left, complete hexamer with each subunit in a different colour. Right, one monomer with bound 2-oxoglutarate shown in a ball-and-stick representation, and Fe (II) shown as a green sphere.

product of CarA into (5R)-carbapenem (FIG. 3), showing that CarC is responsible for both introducing a double bond between C-2 and C-3 (desaturation) and for the epimerization (stereoinversion) at C-5 (highlighted in FIG. 3). CarC is homologous to 2-oxoglutarate (2OG, or -ketoglutarate)-dependent dioxygenases, which are non-haem, Fe (II)-dependent enzymes. Desaturation

reactions are a known function of this class of enzymes, but the epimerization reaction that is catalysed by CarC is unprecedented in this enzyme family40. Purified E. c. carotovora CarC was confirmed to be a 2OG-dependent, non-haem, Fe (II) oxygenase which, in vitro, mediates the conversion of (3S,5S)carbapenam to Car40.

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In 2003, the crystal structure of CarC from E. c. carotovora was solved to a resolution of 2.4 in a complex with Fe II and 2OG, and to 2.3 with Fe (II), 2OG and a substrate analogue (N-acetyl-L-proline)45. CarC is a hexamer that is itself composed of two trimers (FIG. 4c), with the active site of each monomer oriented towards the exterior of the hexamer. Each monomer forms a distorted double-stranded -helix (or jellyroll) motif that is characteristic of the 2OG-dependent oxygenase superfamily. Binding of Fe and 2OG in the active site was visualized (FIG. 4c). CarC shares 23% identity with clavaminate synthase (CAS) from Streptomyces clavuligerus, a 2OG-dependent, non-haem, Fe (II) oxygenase that catalyses three separate oxidative reactions (hydroxylation, cyclization and desaturation) during clavulanic acid biosynthesis46. The CarC structure is similar to that of CAS; for example, several important residues binding Fe and 2OG are conserved between CAS and CarC. Unfortunately, the orientation of the substrate analogue in the active site of CarC could not be unambiguously determined45, and we still await the solution of a structure with the natural carbapenam substrate bound. It is also likely that conformational changes occur on substrate binding. Nevertheless, modelling studies combined with observed features of the active site allowed probable key catalytic residues to be identified, and several enzymatic mechanisms consistent with the structural data were proposed45. The mechanism by which CarC performs its intriguing dual reaction remains to be fully clarified, although it seems to involve an Fe IV=O intermediate. Direct hydrogen abstraction at C-5 is believed to initiate epimerization. Desaturation is proposed to follow epimerization, probably in a similar manner to CAS desaturation45,47,48. The overall reaction seems to be driven by coupling it to the reduction of dioxygen40. In addition to Car, CarC also produces small but detectable amounts of (3S,5R)-carbapenam (FIG. 3). It has been proposed 45 that (3S,5R)-carbapenam is a shunt (or side) product, but Stapon et al.47 suggest that it is an intermediate in the reaction to generate Car. It has also recently been shown that purified E. c. carotovora CarC can use all four possible stereoisomers of the natural carbapenam substrate, albeit with differing efficiencies49. It has been proposed that a small molecule reducing agent might be required to rescue CarC from unwanted high oxidation states, or might have an essential role in catalysis48,49. A full understanding of how this enzyme functions will not only be of fundamental mechanistic interest, but might also allow manipulation of CarC to facilitate the generation of novel bioactive molecules.
Comparisons with related systems

Clavulanic acid, a clavam -lactam that is produced by S. clavuligerus, is a potent inhibitor of -lactamases. The part of the biosynthetic pathway that leads to formation of the bicyclic nucleus is well characterized and is distinct from that of penicillins and cephalosporins18,46. Perhaps unexpectedly, there is an evolutionary link between synthesis of the clavam -lactamase inhibitor by this
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Gram-positive bacterium and synthesis of the -lactam antibiotic, Car, by Gram-negative bacterial species. Two of the three essential enzymes in Car biosynthesis, CarA and CarC, have homologues in clavulanic acid synthesis, -LS and CAS, respectively. -LS catalyses the conversion of N2-(2-carboxyethyl) arginine (CEA) to the -lactam deoxyguadinoproclavaminic acid, and CAS catalyses subsequent, separate hydroxylation, cyclization and desaturation reactions18,46. This relationship was first suggested on the basis of sequence similarity21 and has now been confirmed by the structural similarity between CarA and -LS and between CarC and CAS43,45. How these two systems evolved, together or apart, by convergent or divergent evolution, currently remains a matter for speculation. Comparisons of CarA and CarC with the betterstudied -LS and CAS, respectively, have proved valuable in understanding the mechanisms of these two Car enzymes. There are differences as well as similarities between the Car and CAS/-LS enzymes and these can also be informative. For example, the substrate binding pocket in -LS is larger than in CarA to accomodate its larger substrate43. CarC and CAS are not similar in all respects. Although both catalyse oxidative desaturation, presumably by a similar mechanism, CarC performs a unique epimerization reaction and CAS performs two additional oxidative reactions. There is also no CarB homologue in clavulanic acid synthesis, reflecting the different nature of the first committed intermediate (CMP in Car biosynthesis, CEA in clavulanic acid biosynthesis)41. CarA and -LS also share sequence and structural similarities with asparagine synthetase B (AS-B) from E. coli, and both have been proposed to have evolved from this primary metabolic enzyme43,46. The C-terminal domains of -LS/CarA and AS-B catalyse amino-acid adenylation. In -LS/CarA this domain catalyses an intramolecular amide bond formation, and in AS-B it catalyses an intermolecular amide bond formation. The N-terminal domain of AS-B catalyses the hydrolysis of glutamine to glutamate and ammonia. -LS and CarA have maintained a common overall structural fold and a similar C-terminal domain to AS-B. However, differences between AS-B and the -lactam synthetases reveal how the latter might have evolved from AS-B43,50. For example, the N-terminal domains of -LS and CarA lack a functional glutaminase site, and in the C-terminal domain, the substrate-binding clefts of CarA and especially -LS are larger, to accommodate increased substrate sizes. Also, at least in -LS, the spatial arrangement of bound substrates seems to facilitate intramolecular -lactam ring formation50. Another example of predicted CarAC-like proteins has been reported in the Gram-positive phythopathogen Rhodococcus fascians strain D188. In this strain, the att locus is responsible for the production of extracellular autoregulatory signalling compound(s), or inducing factors, which are essential for full virulence51. The att locus contains a predicted biosynthetic cluster of eight genes, which includes homologues of CarB (attD), -LS (attE) and CarC (attF), indicating that the att-dependent

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could also proceed through the initial formation of the same (5R)-carbapenem bicyclic nucleus as produced by CarAC in E. c. carotovora, followed by the incorporation of hydroxyethyl and cysteaminyl side chains41. ThnE shares approximately 40% identity with CarB, and the precursors of carbapenem and thienamycin are the same (glutamate and acetate). Therefore, ThnE probably performs a similar initial precursor condensation step to that of CarB41. However, it has been suggested that methylmalonyl-CoA, rather than malonyl-CoA, might be the substrate for ThnE41. ThnM is 51% similar to CarA and 48% similar to -LS (over 258 and 270 amino acids, respectively), suggesting that this enzyme probably functions in -lactam ring formation. It will be interesting to compare the substrate specificities of ThnM, CarA and -LS, to gain insight into how -lactam synthetases might be engineered to catalyse reactions that involve alternative substrates. Although the cluster lacks a convincing CarC homologue, there are two potential 2OGdependent oxygenases (ThnG and ThnQ) that might catalyse the desaturation of the penam that is produced by ThnE and ThnM41. The Car and thienamycin biosynthetic pathways share similar enzymes and products. However, there are differences between the two systems; first, thienamycin is more complex than carbapenem and therefore requires extra biosynthetic reactions following synthesis of the bicyclic nucleus; second, thienamycin lacks a CarC homologue; last, there are no obvious similarities between the self-resistance mechanisms (see below). So how did simple carbapenem biosynthesis (Gram-negative bacteria) and complex carbapenem production (Gram-positive bacteria) evolve? These enzymes probably evolved from primary metabolic enzymes. CarA/ThnM seem to be derived from AS-B (see above), CarB/ThnE are likely to have evolved from a crotonase family member, whereas CarC and the thienamycin desaturation enzyme (perhaps ThnG or ThnQ) probably evolved from another 2OGdependent oxygenase. Two possible evolutionary scenarios can be envisaged; first, both systems evolved independently (convergent evolution) from primary metabolic enzymes; second, the systems have evolved from a common ancestor, through a horizontally transferred group of genes (divergent evolution). Perhaps CarA and CarB, the first enzymes in the pathway and highly conserved in both systems, arose first, with differing downstream reactions shaping the final molecules.
Resistance mechanisms

ThnG?
9 8

OH H H
6 7 5 1

ThnE

ThnT/V? NH2

2 3

S ThnG/Q? (ThnD/O?)

ThnK/L/P

ThnM

CO2H thnE

b
thnA thnB thnC thnD thnF thnG thnH thnI thnJ thnK

thnL

thnM

thnN

thnO

thnP

thnQ thnR

thnS

thnT

thnU thnV

Figure 5 | Thienamycin and the thn gene cluster. a | The structure of thienamycin and putative roles for thn gene products in its assembly6,41. ThnE could generate the pyrrolidine ring and ThnM the -lactam ring. ThnK, ThnL and/or ThnP might add methyl groups to generate the hydroxyethyl side chain, ThnT and/or ThnV might be involved in addition of the cysteaminyl side chain, ThnG might hydroxylate the hydroxyethyl side chain, and ThnG/Q (or ThnD/O) might insert the double bond to form a carbapenem. b | The thn gene cluster6. Genes whose products are predicted to be involved in assembly of the thienamycin molecule (biosynthetic genes) are shown in blue, those predicted to be involved in regulation of thienamycin production are in purple, those predicted to be involved in export and/or resistance are in green, and those of unknown function are in white. Note that thnK and thnL are contiguous.

molecule might be a -lactam compound that is related to Car or clavulanic acid. However, no -lactam antibiotic or -lactamase inhibitory activity has been detected so far. Such a -lactam-like autoinducer molecule would form a novel class of signalling (including QS) molecules.
Thienamycin biosynthesis

It has been reported that thienamycin is the most potent, most broad-spectrum, natural antibacterial known38. The genetic intractability of its producer, S. cattleya, hampered efforts to elucidate the biosynthesis of this antibiotic. However, the gene cluster for thienamycin biosynthesis was recently identified and sequenced6. Using bioinformatics, twenty-two genes (thnAV) were identified in the thienamycin cluster and were assigned putative roles in thienamycin synthesis. Insertional inactivation of three of the thn cluster genes in S. cattleya (thnL, thnN or thnO) resulted in loss of thienamycin production. Homologues of CarA (ThnM) and CarB (ThnE) probably catalyse -lactam ring formation and initial precursor condensation, respectively, and three predicted methyltransferases might be involved in forming the S-adenosyl methionine (SAM)-derived hydroxyethyl side chain on C6 (FIG. 5). Two potential cysteine transferases might be involved in the generation of the cysteaminyl side chain on C2, and there are also candidates for the several oxidoreduction steps and hydroxylation that are required for thienamycin synthesis. The cluster also contains several genes that are potentially involved in export of, or resistance to, thienamycin, such as a putative membrane transporter/efflux pump and a putative class A -lactamase, respectively. Based on the thn cluster sequence and a pathway proposed earlier, a speculative biosynthetic pathway for thienamycin synthesis has been suggested6,52. In this pathway, the -lactam ring is formed early on, followed by addition of the cysteaminyl side chain, methylation and hydroxylation. A late oxidation step then converts the penam to the penem. However, thienamycin synthesis

The last genes in the E. c. carotovora car operon, carFH, are not involved in Car biosynthesis. Instead, carF and carG confer self-resistance to Car (FIG. 6), whereas the function of carH is unknown. In E. c. carotovora, the periplasmic proteins CarF and CarG are required for full resistance, and their stoichiometry seems to be important36. The mechanism by which these proteins confer resistance is not known but does not seem to involve any previously characterized mechanism of -lactam resistance. CarF and CarG do not produce any detectable -lactamase activity and are not homologous to known -lactamases, penicillin-binding proteins or
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a
i ii

b
i ii

c
i ii

iii

iii

iii

Figure 6 | Car production, resistance and quorum sensing in Erwinia carotovora subsp. carotovora and Serratia sp. strain ATCC39006. a | Car (1-carbapen-2-em-3-carboxylic acid) production is shown by zones of clearance (cell death) on a lawn of supersensitive Escherichia coli strain ESS that are observed around colonies of E. c. carotovora strain ATTn10 (i) (a restrictionless derivative of strain ATCC39048) and Serratia ATCC39006 (ii). By contrast, E. c. carotovora strain SCRI193 does not produce Car (iii). b | Production of the quorum sensing (QS) molecule N-3-(oxohexanoyl)-L-homoserine lactone (OHHL) by E. c. carotovora ATTn10 (i) and E. c. carotovora SCRI193 (iii), and production of butanoyl-L-homoserine lactone (BHL) by Serratia ATCC39006 (ii). OHHL is detected using a Chromobacterium violaceum sensor strain CV026 and BHL is detected by Serratia ATCC39006 sensor strain (a smaI mutant derivative of Serratia ATCC39006). E. c. carotovora or Serratia ATCC39006 cultures are spotted onto a lawn of the appropriate bacterial sensor strain. QS molecule production is indicated by coloured halos, as the diffusible signal molecule induces pigment production by the sensor strains (which do not produce QS molecules themselves). c | Resistance to Car is conferred by the car gene cluster. The Car+ E. c. carotovora ATTn10 can kill Car-sensitive bacteria when spotted onto a bacterial lawn of E. coli ESS (i) or E. c. carotovora SM10 (ii), a derivative of E. c. carotovora ATTn10 that lacks the car operon. However, E. c. carotovora ATTn10 does not kill E. c. carotovora SCRI193 (iii), as this strain expresses the carF and carG resistance genes from a cryptic car cluster (BOX 1).

outer membrane porins36. Database searches reveal that CarF contains a conserved domain of unknown function (DUF323) and shares similarity with various putative proteins36. They also show that CarG has no homologues (apart from CpmG/J). CarF and CarG do not confer resistance to other -lactams or other carbapenems such as meropenem36. How such a specific resistance mechanism functions is still unclear but is probably intimately linked to the mechanism by which Car is exported from the producer cell without causing lethality. Production of extracellular Car by E. coli cells expressing the car cluster21 indicates that machinery sufficient to export Car is either present in E. coli or in the car operon. Owing to the high specificity of the resistance mechanism, transfer of Car resistance through horizontal gene transfer to other bacterial community pathogens is unlikely to cause clinical -lactam resistance problems, especially as Car itself is not clinically useful. However, the possibility remains that this resistance mechanism could evolve to accommodate different substrates. CarH has no homologues (apart from CpmH/K) but although conserved in all three car operons present in E. c. carotovora, Serratia ATCC39006 and P. luminescens, it seems to be unnecessary for Car production or resistance, at least in E. c. carotovora36.
Conclusion

In recent years there have been significant advances in our understanding of how carbapenem antibiotics are synthesized and how their production is regulated in

response to environmental cues. These studies have revealed new and interesting enzymatic reactions and have provided insights into how control of carbapenem production is linked to global gene regulation systems. Studies on the biosynthetic enzymes from Erwinia spp. have shed light on how the basic carbapenem nucleus can be constructed, and studies on thienamycin have shown how this nucleus can be elaborated to give a highly bioactive molecule. However, much remains to be revealed, from a full atomic-level understanding of novel catalytic conversions, through to physiological and ecological questions about the fitness advantages conferred by the possession of the corresponding biosynthetic, resistance or regulatory genes. Why is there such strain-dependent variation among the enterics? What are the evolutionary links between emergence of carbapenem genes in such taxonomically-distant hosts as the enteric plant pathogen E. c. carotovora and the Gram-positive soil bacterium, S. cattleya? How widely distributed in nature are producers of carbapenems? Given the evolution of penicillin and cephalosporin gene clusters in both prokaryotes and some eukaryotic fungi, it might not be surprising to find new fungal sources of carbapenem genes. We anticipate that the characterization of other carbapenem gene clusters could reveal a rich repertoire of bioactive molecules and novel enzymatic building blocks for the biotechnological development of new antibiotics.

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Elander, R. P. Industrial production of -lactam antibiotics. Appl. Microbiol. Biotechnol. 61, 385392 (2003). Fleming, A. On the antibacterial action of cultures of a Penicillium, with special reference to their use in the isolation of B. influenzae. Br. J. Exp. Pathol. 10, 226236 (1929).

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Tipper, D. J. & Strominger, J. L. Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-D-alanyl-D-alanine. Proc. Natl Acad. Sci. USA 54, 11331141 (1965). Bradley, J. S. et al. Carbapenems in clinical practice: a guide to their use in serious infection. Int. J. Antimicrob. Agents 11, 93100 (1999).

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Kahan, J. S. et al. Thienamycin, a new -lactam antibiotic. I. Discovery, taxonomy, isolation and physical properties. J. Antibiot. (Tokyo) 32, 112 (1979). Nunez, L. E., Mendez, C., Brana, A. F., Blanco, G. & Salas, J. A. The biosynthetic gene cluster for the -lactam carbapenem thienamycin in Streptomyces cattleya. Chem. Biol. 10, 301311 (2003).

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This long-awaited paper describes the cloning and sequencing of the thienamycin biosynthetic gene cluster from S. cattleya. This discovery paves the way for a comprehensive analysis of thienamycin biosynthesis and regulation. Parker, W. L. et al. SQ 27,860, a simple carbapenem produced by species of Serratia and Erwinia. J. Antibiot. (Tokyo) 35, 653660 (1982). Derzelle, S., Duchaud, E., Kunst, F., Danchin, A. & Bertin, P. Identification, characterization, and regulation of a cluster of genes involved in carbapenem biosynthesis in Photorhabdus luminescens. Appl. Environ. Microbiol. 68, 37803789 (2002). Livermore, D. M. -Lactamase-mediated resistance and opportunities for its control. J. Antimicrob. Chemother. 41 (Suppl. D), 2541 (1998). Hawkey, P. M. The origins and molecular basis of antibiotic resistance. BMJ 317, 657660 (1998). Livermore, D. M. Acquired carbapenemases. J. Antimicrob. Chemother. 39, 673676 (1997). Nordmann, P. & Poirel, L. Emerging carbapenemases in Gram-negative aerobes. Clin. Microbiol. Infect. 8, 321331 (2002). Rasmussen, B. A. & Bush, K. Carbapenem-hydrolyzing -lactamases. Antimicrob. Agents Chemother. 41, 223232 (1997). Edwards, J. R. & Betts, M. J. Carbapenems: the pinnacle of the -lactam antibiotics or room for improvement? J. Antimicrob. Chemother. 45, 14 (2000). Livermore, D. M., Sefton, A. M. & Scott, G. M. Properties and potential of ertapenem. J. Antimicrob. Chemother. 52, 331344 (2003). Shah, P. M. & Isaacs, R. D. Ertapenem, the first of a new group of carbapenems. J. Antimicrob. Chemother. 52, 538542 (2003). Miller, T. W. Crystalline N-formimidoylthienamycin. US Patent 4260543 (1981). McGowan, S. J., Bycroft, B. W. & Salmond, G. P. Bacterial production of carbapenems and clavams: evolution of -lactam antibiotic pathways. Trends Microbiol. 6, 203208 (1998). Bycroft, B. W., Maslen, C., Box, S. J., Brown, A. & Tyler, J. W. The biosynthetic implications of acetate and glutamate incorporation into (3R,5R)-carbapenam-3carboxylic acid and (5R)-carbapen-2-em-3-carboxylic acid by Serratia sp. J. Antibiot. (Tokyo) 41, 12311242 (1988). Whitehead, N. A., Barnard, A. M., Slater, H., Simpson, N. J. & Salmond, G. P. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25, 365404 (2001). McGowan, S. J. et al. Analysis of bacterial carbapenem antibiotic production genes reveals a novel -lactam biosynthesis pathway. Mol. Microbiol. 22, 415426 (1996). This is the first paper to describe the cloning and sequencing of a carbapenem biosynthetic gene cluster. Holden, M. T. et al. Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator. Microbiology 144, 14951508 (1998). Bell, K. S. et al. Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. 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A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens. Microbiology 144, 201209 (1998). 29. Thomson, N. R., Crow, M. A., McGowan, S. J., Cox, A. & Salmond, G. P. Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Mol. Microbiol. 36, 539556 (2000). 30. Slater, H., Crow, M., Everson, L. & Salmond, G. P. Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum-sensing-dependent and-independent pathways. Mol. Microbiol. 47, 303320 (2003). This report describes the regulatory control of antibiotic production in Serratia species. The study showed that the QS-mediated control of carbapenem biosynthesis operates in a different way from that in Erwinia species. 31. Coulthurst, S. J., Whitehead, N. A., Welch, M. & Salmond, G. P. Can boron get bacteria talking? Trends Biochem. 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Acknowledgements
The authors would like to thank the BBSRC, Sygen International Plc and NERC for financial support, and would also like to thank Martin Welch and members of the Salmond group for valuable discussions. We dedicate this paper to Barrie Bycroft, on his retirement, for stimulating our collaborative interest in carbapenems.

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Competing interests statement

The authors declare no competing financial interests.

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Online links
DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/Entrez Erwinia carotovora subsp. atroseptica | Photorhabdus luminescens strain TT01 SwissProt: http://www.ca.expasy.org/sprot CarA | CarB | CarC | CarD | CarE | CarF | CarG | CarH FURTHER INFORMATION George P. C. Salmonds laboratory: http://www.bio.cam.ac.uk/~salmond Access to this interactive links box is free online.

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REGULATION AND BIOSYNTHESIS OF CARBAPENEM ANTIBIOTICS IN BACTERIA

Sarah J. Coulthurst, Anne M. L. Barnard and George P. C. Salmond
Nature Rev. Microbiol. 3, 295306 (2005)

The correct chemical structure of (3S,5S )-carbapenam is shown below.

N O CO2H