JB Accepts, published online ahead of print on 3 November 2014

J. Bacteriol. doi:10.1128/JB.02335-14
Copyright 2014, American Society for Microbiology. All Rights Reserved.

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Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-

Consuming and H2-Producing Reactions in Escherichia coli

Constanze Pinske1, Monique Jaroschinsky2, Sabine Linek2, Ciarn L. Kelly1,3, Frank

Sargent1*, and R. Gary Sawers2*

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Dundee DD1 5EH, Scotland, U.K.

Mothes-Str. 3, 06120 Halle (Saale), Germany

Division of Molecular Microbiology, University of Dundee, College of Life Sciences,

Institute for Biology/ Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-

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Oxford OX1 3QU, U.K.

Present address: University of Oxford, Department of Biochemistry, South Parks Road,

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University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale) Germany; phone +49

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345 5526350; Fax. +49 345 5527010; Email gary.sawers@mikrobiologie.uni-halle.de. or F.

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Sargent, Molecular Microbiology, College of Life Sciences, University of Dundee, Dow

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Street, DD1 5EH Dundee, United Kingdom, phone +44 1382 386463, Email

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f.sargent@dundee.ac.uk.

Address correspondence to: R.G. Sawers, Institute for Biology/Microbiology, Martin-Luther

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Running title: Bi-directional hydrogenase 2 activity

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Key words: fumarate respiration; reverse electron transport; proton reduction; hydrogen

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oxidation; menaquinone; hydrogen-cycling

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Abstract

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Escherichia coli uptake hydrogenase (Hyd) Hyd-2 catalyzes the reversible oxidation of H2 to

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protons and electrons. Hyd-2 synthesis is strongly up-regulated during growth on glycerol or

29

on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric NiFe-

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hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers

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electrons to the quinone pool. Instead Hyd-2 has an additional electron-transfer subunit,

32

termed HybA, with four predicted iron-sulfur clusters. Here we examined the physiological

33

role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used

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menaquinone/ demethylmenaquinone (MQ/ DMQ) to couple hydrogen oxidation to fumarate

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reduction. HybA was essential for electron transfer from Hyd-2 to MQ/ DMQ. H2 evolution

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catalysed by Hyd-2 during fermentation of glycerol in the presence of casamino acids or in a

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fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be

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dependent on both HybA and MQ/ DMQ. The uncoupler carbonylcyanide-m-chlorophenyl

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hydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol indicating the

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requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate

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reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2

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oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes

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still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited

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in extracts derived from cells grown in H2-evolution mode. Our findings suggest that Hyd-2

45

switches between H2-consuming and H2producing modes in response to the redox status of

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the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron

47

transport.

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Introduction

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In 1937 Krebs found that Escherichia coli cells are able to use hydrogen or glycerol to reduce

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fumarate, yielding succinate (1). Today it is known that the enzymes [NiFe]-hydrogenase

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(Hyd), glycerol dehydrogenase and fumarate reductase (FRD) are involved in this process.

53

Under anaerobic conditions E. coli is able to synthesize two uptake Hyd complexes, termed

54

Hyd-1 and Hyd-2, that catalyze the oxidation of H2 to protons and electrons (2, 3). Both

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enzymes face the periplasmic side of the cytoplasmic membrane and are translocated as a

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large- and small-subunit complex by the Tat (twin arginine transport) protein translocation

57

machinery. The Tat signal-peptide is located on the N-terminus of the respective small

58

subunit (4). Both complexes differ in their expression patterns, oxygen tolerance and subunit

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composition when associated with the cytoplasmic membrane (5-7). The Hyd-2 complex has

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an unusual architecture because, in addition to the large- and small-subunit heterodimer of

61

HybC-HybO, a further two subunits, HybA and HybB, are required to complete a

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heterotetrameric complex on the periplasmic side of the membrane (7, 8). The HybA protein

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is a Tat-dependent protein with four predicted iron-sulfur cluster-binding sites, while HybB is

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an integral membrane protein with no known cofactors (7). It is still unknown how the

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assembly of the heterotetramer is co-ordinated subsequent to transport and membrane

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integration of the component parts.

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A third hydrogenase, Hyd-3, forms part of the cytoplasmically oriented hydrogen-evolving

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formate hydrogenlyase complex (FHL), which oxidizes internally produced formate to CO2

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with the aid of formate dehydrogenase H (FDH-H) and uses the electrons to reduce protons to

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H2 (2, 3, 9).

71

All Hyd large subunits contain a bimetallic [NiFe]-cofactor at the active site, which is inserted

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through the concerted action of general Hyp accessory proteins and a further set of

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hydrogenase-specific maturases (2, 3). Maturation of the large subunit is completed with the

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proteolytic cleavage of a peptide at its C-terminus and its subsequent association with the

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small subunit. This occurs prior to membrane association (10).

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With the identification of distinct Hyd enzymes, it became possible to analyze their respective

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protein content and activities after growth under different conditions (11, 12). Thus, growth in

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glycerol-fumarate medium (GF medium) resulted in an increased content of Hyd-2 enzyme

79

compared to growth in glucose medium (Glc medium) (12, 13). In contrast, Hyd-1 is more

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prevalent after growth in Glc medium compared to GF medium. Both uptake Hyd enzymes

81

link H2 oxidation to the reduction of quinones in the respiratory chain (14). As a result,

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hydrogen gas can serve as electron donor for fumarate reductase (FRD) (15), which reduces

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fumarate to succinate. The heterotetrameric enzyme consists of the FrdABCD proteins, with

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FrdA being the catalytic subunit that contains an FAD cofactor. Although the FRD crystal

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structure revealed two quinone binding sites on opposite sides of the membrane, quinone-

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mediated proton translocation is not assumed due to lack of connecting heme groups (16).

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Glycerol can also serve as electron donor during fumarate respiration (1). Glycerol can be

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metabolized in the presence of electron acceptors by an ATP-dependent glycerol kinase

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(encoded by glpK) and subsequently by two quinone-dependent glycerol-3-phosphate

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dehydrogenases (encoded by glpD and glpABC) yielding dihydroxyacetone phosphate, an

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intermediate of glycolysis (summarized in (17)). In addition, anaerobic glycerol utilization in

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the absence of external electron acceptors was shown to be possible if certain requirements

93

were met (18). The key enzyme for glycerol activation to dihydroxyacetone is the NAD+-

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linked glycerol dehydrogenase encoded by gldA (19, 20). At the same time FRD is not

95

required for glycerol fermentation (21). Recent studies have also revealed that during glycerol

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fermentation in the presence of casamino acids, Hyd-2 can evolve hydrogen (22), suggesting

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that under certain conditions the enzyme can function bi-directionally. This is in agreement

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with the electrochemical analysis of purified Hyd-2 (23). Therefore, in this study we wished

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to determine the requirements of Hyd-2 to function in H2 evolution during fermentative

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growth with glycerol and in H2 oxidation during respiratory growth on glycerol and fumarate.

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Our studies revealed unforeseen control of Hyd-2 enzyme activity in response to the redox

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status of the menaquinone pool, highlighted the importance of the HybA subunit in both H2

103

oxidation and proton reduction and identified a role for the proton gradient in coupling

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glycerol fermentation with H2 evolution.

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Materials and Methods

107

Strains and growth conditions. All strains used in this study are listed in Table 1. E. coli

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strains were routinely grown at 37 C on LB-agar plates or with shaking in LB-broth (24).

109

Anaerobic growths were performed at 37 C as standing liquid cultures. For phenotypic

110

characterisation the cells were grown for at least 16 h in M9-minimal medium containing

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1x M9 salts, 2 mM MgSO4, 0.1 mM CaCl2, 0.2% (w/v) casamino acids, 3 M thiamine

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hydrochloride, trace element solution SL-A (25), 0.4% glycerol and 25 mM sodium fumarate

113

(26). When required, the

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spectinomycin were added to a final concentration of 100 g ml-1, 50 g ml-1, 12 g ml-1 or

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100 g ml-1, respectively.

116

Genetic manipulations and plasmid construction. Cloning of the 3430 bp frdABCD operon

117

including its native 118 bp promoter region was done using MC4100 genomic DNA as

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template for Herculase II (Agilent, USA) and the oligonucleotides frdA_fw_BamHI 5-

119

GCGGGATCCATCAGACTATACTGTTG-3

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GCGAAGCTTAGATTGTAACGACACCAATC-3. The PCR product was first ligated blunt

121

into pJET1.2 (Thermo Fisher Scientific) and then it was excised and cloned into

122

pACYCDuet-1 using BamHI and HindIII restriction sites. Similarly, the menA gene was

123

cloned into pACYC-Duet-1 including its native promoter using the oligonucleotides

124

menA_fw_BamHI

125

menA_rw_HindIII 5-GCGAAGCTTATGCTGCCCACTGGCTTAG-3. The hybA gene was

antibiotics ampicillin, kanamycin,

and

chloramphenicol

frdD_rw_HindIII

5-GCGGGATCCGACTCCGGTATTAAACGC-3

or

5-

and

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cloned into pJET1.2 including an artificial ribosome binding site using the oligonucleotides

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hybA_fw_BamHI 5-GCGGGATCCAGGAGGATAACCGTGAACAGACGTAATT-3 and

128

hybA_rw_HindIII 5-CGCAAGCTTTCATGACTCATGATCGTCTCC-3. The authenticity

129

of the cloned DNA sequences was verified.

130

Construction of strain CP1A3 was done by transducing the Keio hybA alleles into IC010 by

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P1kc-mediated transduction (24, 27). Similarly, strain CP1034 was constructed by introducing

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the hyfB-R::spec allele into IC010 (28) and IC012 was constructed by introducing the Keio

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menA allele into IC010.

134

Polyacrylamide gel electrophoresis and immunoblotting. Aliquots of 25 g of protein from

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crude extracts were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 10%

136

(w/v) gels (29) and transferred to nitrocellulose membranes as described (30). Antibodies

137

raised against the Hyd-2 subunits (1:20,000), HycG (1:5,000) (31), TatC (1:3,000) and FdhE

138

(1:3,000) were used. Secondary goat-anti-rabbit antibody conjugated with HRP enzyme (Bio-

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Rad, USA) was used for visualization of signals with the Immobilon Western

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chemiluminescent HRP substrate (Millipore, USA). Membrane preparations of crude extracts

141

were prepared as described (11).

142

Enzyme activity assays. Hydrogenase in-gel activity staining using benzyl viologen and 2,3,5-

143

triphenyltetrazolium chloride was done as previously described (32). Determination of total

144

hydrogenase enzyme activity as H2-dependent reduction of benzyl viologen (BV) was

145

performed according to (11) except that the buffer used was 50 mM MOPS, pH 7.0. The

146

wavelength used was 600 nm and an EM value of 7,400 M-1 cm-1 was assumed for reduced

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BV. Measurement of FRD activity as fumarate-dependent oxidation of reduced BV was

148

performed according to (33) in 50 mM MOPS, pH 7.0 at a wavelength of 600 nm. One unit of

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activity corresponded to the reduction of 1 mol of substrate per min. Protein concentration

150

was determined by the method of Bradford (Bio-Rad, USA) (34).

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Cultures for measuring hydrogen production on the electrode were grown anaerobically for 16

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hours in LB + 0.5% (v/v) glycerol and 34 mM fumarate. Cells were harvested and washed

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twice before resuspension in 1 ml per 1 g cells in 0.1 M sodium phosphate buffer, pH 6.8.

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Assays were carried out using a modified Clark-type electrode (Hansatech Oxygraph)

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calibrated with known amounts of H2. The chamber was filled with 2 ml of buffer and cells

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and substrates were added as indicated. In experiments in which methyl viologen (MV)-

157

driven H2 evolution was analysed, cells were prepared as described and MV was added to the

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chamber to a final concentration of 1.2 mM. Aliquots of a freshly prepared 10 mM sodium

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dithionite solution were added until the solution acquired a dark blue colour, after which the

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evolution of hydrogen was monitored. As a negative control the same procedure was

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performed except cells were omitted from the chamber. In experiments where

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carbonylcyanide-m-chlorophenyl hydrazone (CCCP) was used, a 100 mM suspension in

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buffer was prepared to avoid using solvents that cells can use as electron acceptors and it was

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added as a 1:1000 dilution to the electrode chamber where indicated.

165

Gas chromatographic determination of hydrogen content in cultures was carried out in

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Hungate tubes filled with 5 ml of the respective medium and the head-space was flushed with

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nitrogen. An aliquot of 200 l gas phase from the head-space was analyzed on a Shimadzu

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GC-2010 Plus gas chromatograph. Pure nitrogen was used as the carrier gas and the amount

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of produced hydrogen was calculated based on a standard curve.

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Results

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Hyd-2 exhibits both proton-reducing and H2-oxidizing activities. In order to demonstrate the

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bi-directionality of Hyd-2 in vivo strain IC010 (hyaB hycE), which lacks Hyd-1 and Hyd-3

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(see Table 1), was used. Harvested, washed intact cells were placed in the electrode chamber

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of an H2-sensing electrode and when exogenous glycerol was added the cells initiated H2

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production (Fig. 1). The H2 produced in the electrode chamber could be immediately re-

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oxidized when external electron acceptors such as fumarate, TMAO or nitrate were added to

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the chamber (Fig. 1). This indicates that Hyd-2 can rapidly switch between H2 evolution and

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H2 oxidation modes in vivo.

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Menaquinone and/ or demethylmenaquinone are required for electron transfer to and from

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Hyd-2 in vivo. As Hyd-2 couples hydrogen oxidation with fumarate reduction (12), we

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wanted to examine whether menaquinone (MQ) and demethylmenaquinone (DMQ) are

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involved in electron transfer to and from Hyd-2, the latter of which has been proposed (35)

184

but never demonstrated (36). Therefore, strain IC012 was constructed by introducing a

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menA mutation into strain IC010. Deletion of menA prevents synthesis of menaquinone and

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demethylmenaquinone but has no effect on ubiquinone biosynthesis (37). This strain grew

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very poorly anaerobically with glycerol and fumarate (GF) but sufficient cell material could

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be obtained to analyse the H2-oxidizing activity of Hyd-2 using the viologen dye-linked in-gel

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hydrogenase activity staining procedure after non-denaturing, native-PAGE (11). Surprisingly,

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the results of this analysis (Fig. 2A) revealed that no Hyd-2 activity could be detected in this

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strain after growth in GF medium; however, Hyd-2 activity was visible after fermentative

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growth with glucose (lane labelled Glc in Fig. 2A). Both the original isogenic parent strain

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IC010 and strain IC012 complemented with a plasmid carrying the menA gene revealed Hyd-

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2 activity after growth in GF medium.

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Previous studies have shown that the H2-oxidizing hydrogenases Hyd-1 and Hyd-2 have near-

196

identical biochemical and physiological properties in E. coli wild type strains MC4100 and

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BW25113 (38). Therefore, in order to test whether the deletion of menA caused the same

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Hyd-2 phenotype in the BW25113 background we examined activity of Hyd-2 in strain

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JW3901 (menA). Indeed, no Hyd-2 activity after growth in GF medium could be observed,

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while Hyd-1 activity was unaffected (Fig. S1).

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Next, we examined Hyd-2 dye-reducing activity in strains lacking the genes encoding UbiC

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and UbiF, which are required for only ubiquinone (UQ) and not MQ/ DMQ biosynthesis.

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After growth in GF medium these strains exhibited both Hyd-1 and Hyd-2 enzyme activities

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like the wild type (Fig. S1). As a further control a mutation in ubiE, which prevents both UQ

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and MQ biosynthesis but allows DMQ synthesis (39), was analyzed. Extracts derived from

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the ubiF mutant retained a Hyd-1 and Hyd-2 enzyme activity pattern like that of the parent

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strain MC4100 (Fig. S1). These results demonstrate that the H2: benzyl viologen

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oxidoreductase activity of Hyd-2 is impaired in extracts of menA mutants but is unaffected in

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mutants lacking UQ.

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It could be shown that the lack of measureable viologen dye-reducing activity of Hyd-2 was

211

not due to a defect in Hyd-2 enzyme synthesis in the menA mutant, because when analyzed by

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Western blot with anti-Hyd-2 antibodies, extracts derived from the strain showed near wild-

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type levels of the Hyd-2 large and small subunit antigens after growth in GF medium (Fig.

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S2).

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The effect of the menA mutation on H2 production by Hyd-2 was examined in cells of strain

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IC012 (hyaB hycE menA) (Fig. 2B). The cells of the isogenic parent strain IC010

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produced H2 at a rate of 1.7 nmol min-1 mg-1 when glycerol was used as the electron donor. In

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contrast, cells of IC012 failed to produce H2 gas. This result indicates that MQ/ DMQ

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mediates electron transfer for proton reduction catalysed by Hyd-2. Examination of whole-

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cell H2-oxidizing activity of strains IC010 and IC012 revealed that the menA mutant was

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unable to oxidize H2 with fumarate as electron acceptor (Fig. 2C). Finally, to demonstrate that

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the Hyd-2 enzyme was catalytically active in the menA mutant, IC012 was shown to be

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capable of catalysing H2 oxidation with nitrate as electron acceptor (Fig. 2D). This finding

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perhaps suggests that ubiquinone can couple Hyd-2-driven H2 oxidation to nitrate reduction.

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HybA is essential for reversible electron transfer between Hyd-2 and the quinone pool. The

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results of earlier studies strongly suggested that HybA is required for electron transfer from

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Hyd-2 to the quinone pool (7). To determine whether H2 evolution by Hyd-2 also requires a

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functional HybA subunit, strain CP1A3 (hyaB hybA hycE) was tested and proved to be

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unable to produce H2 at a significant rate (Fig. 3A). Strain CP1034 (hyaB hycE hyfB-R),

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which lacks Hyd-1 and Hyd-3, as well as the genes encoding Hyd-4 (28), was able to produce

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H2 from glycerol at a rate of 4.14 nmol min-1 mg-1 when assayed on the H2-electrode (Fig.

232

3A), and acted as a positive control for the experiment. This H2-evolving activity of CP1034

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was very similar to that determined for IC010 (hyaB hycE) (see Fig. 2B). H2 production

234

stopped abruptly upon addition of fumarate to the chamber and the H2 was rapidly and

235

quantitatively oxidized. Together, these data show that the HybA subunit is required for Hyd-

236

2 to mediate proton reduction.

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To determine whether HybA is required to link H2-oxidation by Hyd-2 to fumarate reduction,

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cells of CP1A3 were grown with glycerol and fumarate, washed and incubated under 600

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nmol H2 and electron transfer to the quinone pool was examined after addition of fumarate

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(Fig. 3B). While strain CP1034 (hyaB hycE hyfB-R) showed H2-oxidizing activity, strain

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CP1A3 (hyaB hybA hycE) lacking HybA failed to oxidize H2. This demonstrates that HybA

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is also necessary for electron transfer from hydrogen to fumarate via menaquinone, as

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previously suggested (7).

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HybA is not required for H2: viologen dye oxidoreductase activity of Hyd-2. A strain with a

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deletion in the hybA gene retains active Hyd-2 enzyme in in vitro assays with viologen dyes

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(7, 40). Analysis of an extract derived from strain FTD671 (hybA) by activity-staining after

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native-PAGE following growth in GF medium or in glycerol medium supplemented with

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casamino acids confirmed that Hyd-2 activity was detectable, but reduced, under both growth

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conditions (Fig. 4A). In contrast to extracts derived from MC4100 grown anaerobically with

250

glycerol, Hyd-2 activity showed relief of inhibition of the H2-dependent BV reductase activity

251

in the hybA strain FTD671 after growth under these conditions (compare Fig. 4A and Fig.

252

2A). Moreover, the pattern of the activity bands was different from that in MC4100, showing

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only the faster migrating species of Hyd-2 activity band (Fig. 4A). This suggests that the

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faster migrating band consists of the catalytically active HybOC complex (see also 7, 40),

255

while the slower migrating species consists of different forms of the heteromeric HybOC-AB

256

complex. Re-introduction of plasmid-encoded hybA into FTD671 restored the more slowly

257

migrating activity band in native-PAGE analysis (Fig. 4A).

258

An experiment comparing the ability of washed, whole cells of IC010 and CP1A3 (hyaB

259

hybA hycE) to use reduced methyl viologen dye as electron donor demonstrated that H2

260

production by a Hyd-2 enzyme lacking HybA was similar to native Hyd-2 (Fig. 4B). Together,

261

these results indicate that HybA is not required for electron transfer to and from artificial

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redox-active viologen dyes but it is necessary for electron flow to and from Hyd-2 via the

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quinone pool.

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The proton gradient drives Hyd-2-dependent H2 evolution. Having demonstrated that H2

265

evolution is dependent on MQ/ DMQ it is important to consider how menaquinol (standard

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redox potential E0 of approximately -80 mV) can drive the endergonic reduction of protons to

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molecular H2 (redox potential E0 of -418 mV). To examine whether this activity is coupled to

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the proton gradient, cells of strain IC010 (MC4100 hyaB hycE) were incubated with

269

glycerol to induce Hyd-2-dependent H2 evolution and then after 10 min incubation the

270

uncoupler CCCP was added (Fig. 5A). The results clearly demonstrate that H2 evolution

271

stopped immediately upon addition of CCCP, which indicates that a proton gradient is

272

required to drive this reaction. After a further 5 min fumarate was added to the cells and H2

273

oxidation commenced (Fig. 5A), indicating that this process was not inhibited by the

274

uncoupler. To confirm that Hyd-2-dependent electron transfer to FRD was independent of the

275

uncoupler CCCP, cells of IC010 were incubated first with H2, then fumarate was added to

276

induce H2 oxidation and after 10 min CCCP was added. The data in Fig. 5B show that CCCP

277

had no effect on fumarate-dependent H2 oxidation, which is in agreement with the fact that

278

hydrogen oxidation coupled to fumarate reduction (fumarate/succinate redox potential E0 of

279

+30 mV) is an exergonic reaction and therefore independent of the proton gradient.

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A frdA strain lacks Hyd-2-linked H2-dependent benzyl viologen reductase activity during

281

respiration with fumarate. Based on the findings described above it is possible to switch

282

Hyd-2 between H2-evolving and H2-oxidizing modes by altering the growth conditions from

283

glycerol fermentation to glycerol-fumarate respiration, respectively. Moreover, it appears that

284

when electron flow to FRD is interrupted, e.g. by a menA mutation (see Fig. 2A), the ability

285

of Hyd-2 to reduce BV is somehow impaired, despite the enzyme being immunologically

286

detectable (Fig. S3). In order to investigate this latter phenomenon in more detail, we decided

287

first to generate strain SAL1 (hyaB hycAI frdA), which lacks FRD, and examine the

288

consequences on Hyd-2 activity measured quantitatively in crude extracts (Table 2). We also

289

examined activity in the in-gel Hyd-2 activity assay after growth in glycerol-fumarate

290

medium (Fig. 6A). While the control strain IC010 showed an activity band corresponding to

291

Hyd-2 after growth in GF medium, no activity band could be observed in extracts of SAL1;

292

note that the hycE and hycAI alleles have identical phenotypes with regard to loss of Hyd-3

293

activity (41). Hyd-2 activity was reduced approximately 7-fold in extracts of SAL1 compared

294

with extracts of IC010 (Table 2). The total hydrogenase activity in IC010 was similar to that

295

in the wild type MC4100, while an extract derived from a hypF mutant (DHP-F2), which

296

lacks all hydrogenases (42) and acted as a negative control, lacked measureable hydrogenase

297

activity. Determination of FRD enzyme activity in the same extracts revealed that strain

298

SAL1 completely lacked activity while both MC4100 (wild type) and IC010 had similar FRD

299

activities (Table 2).

300

To ensure that the effects of the frdA mutation were exclusively due to the deletion of the frdA

301

gene, strain SAL1 was complemented with a plasmid carrying the frdA gene and Hyd-2

302

enzyme activity was restored after growth of the strain in GF medium, as was observed in the

303

in-gel assay (Fig. 6A) and by quantitative measurement of enzyme activity (Table 2). The

304

reappearance of Hyd-2 activity under GF growth conditions correlated with the recovery of

305

FRD activity in the complemented SAL1 strain (Table 2). Together, these results indicate that

13
306

by preventing electron flow to FRD, even in the presence of fumarate, no H2: BV

307

oxidoreductase activity of Hyd-2 could be detected in crude extracts separated in non-

308

denaturing gels. Activity could be restored, however, by re-introducing the frdA-D genes on a

309

plasmid.

310

The E. coli Hyd enzymes are active across a wide range of redox potentials and in the

311

presence of different electron acceptors (23, 32). As well as fumarate, electron transport

312

chains coupling H2 oxidation to DMSO and TMAO have also been described (14). Even in

313

the absence of externally added electron acceptors glycerol can function as sole carbon source

314

in minimal medium as long as the culture is supplemented with casamino acids to permit

315

growth (20). An extract derived from the wild type MC4100 grown anaerobically in glycerol

316

and casamino acids medium lacked Hyd-2 enzyme activity after native-PAGE (Fig. 6B, lane

317

4). Activity of Hyd-1, while reduced compared to extracts of MC4100 cells growth with

318

glucose, was clearly visible. Growth in the presence of either fumarate, TMAO or DMSO

319

revealed that active Hyd-2 and Hyd-1 enzymes were detectable (Fig. 6B; (12)). In the

320

presence of nitrate, a different respiratory chain involving a nitrate-dependent formate

321

dehydrogenase (FDH-N) and nitrate reductase is employed and no Hyd activity is detectable

322

(43). This is due to NarL, the nitrate response regulator, preventing the transcription of both

323

respiratory hydrogenases (44). The results of these experiments indicate that during glycerol

324

fermentation in M9 minimal medium the dye-reducing activity of Hyd-2 is undetectable in

325

stationary-phase cultures, which correlates with Hyd-2 acting in proton-reduction mode.

326

In order to address the question whether the effects of introducing the frdA allele into strain

327

BW25113 (Table 1) would cause the same Hyd-2 activity phenotype, we analysed extracts of

328

strain JW4115 after growth under the same fermentative and respiratory conditions used to

329

test Hyd-2 activity in MC4100 (Fig. 6B). Clearly, neither fermentative growth with glucose

330

nor respiration with DMSO or TMAO affected either Hyd-1 or Hyd-2 enzyme activities.

331

However, after growth in glycerol and casamino acids no Hyd-2 activity could be detected,

14
332

while after growth in GF medium only a very weak Hyd-2 activity band was observed; in

333

contrast, Hyd-1 enzyme activity was unaffected under these conditions. Western blot analysis

334

of cell extract derived from the frdA mutant revealed near wild-type levels of HybC, the

335

catalytic subunit of Hyd-2, after growth in GF medium (Fig. S3). Thus, the phenotype of a

336

frdA strain grown in GF medium with regard to Hyd-2 activity is comparable with that of the

337

wild type after growth under glycerol fermentation. Note that FRD activity was abolished in

338

an extract of strain JW4115 (frdA) but was recovered by re-introducing the frdA gene on a

339

plasmid (Table 2). Because strain JW4115 has the capacity to synthesize all four

340

hydrogenases, the total hydrogenase enzyme activity of the strain was hardly affected by the

341

frdA mutation because lack of Hyd-2 activity was complemented presumably by a

342

corresponding increase in the activities of Hyd-1, Hyd-3 and/or Hyd-4 (45).

343

Hyd-2 activity can be recovered in the membrane fraction of frdA mutants

344

In order to examine further the biochemical cause of the lack of Hyd-2 dye-reducing enzyme

345

activity in frdA mutants, cells of isogenic strains MC4100, IC010 (hyaB hycE) and SAL1

346

(hyaB hycAI frdA) were grown anaerobically in GF medium and the crude extract was

347

subsequently separated into soluble and membrane fractions by ultracentrifugation and these

348

sub-cellular fractions were assayed for hydrogen-oxidizing activity with benzyl viologen after

349

native-PAGE (Fig. 6C). While no Hyd-2 activity in a crude extract of strain SAL1 was

350

detectable, Hyd-2 enzyme activity was recovered in the membrane fraction after sub-cellular

351

fractionation of the crude extract. H2: BV oxidoreductase activity of Hyd-1 in strain MC4100

352

and of FDH-N/O (46) in all three strains acted as controls demonstrating that all three

353

activities were exclusively detected in the membrane fraction. This result indicates that the

354

apparent inhibition of H2:BV oxidoreductase activity observed in crude extracts in the frdA

355

mutant was reversible.

356

Quantitative analysis of H2 production by Hyd-2.

15
357

Due to the fact that FRD is menaquinone-dependent (47), our findings suggest that Hyd-2 can

358

no longer oxidize hydrogen if electron transfer through the menaquinone pool is impeded.

359

Thus, not only an impediment in menaquinone biosynthesis but also deletion of the genes

360

encoding FRD or growth on glycerol without provision of an external electron acceptor all

361

cause a similar phenotype in which dye-reducing activity of Hyd-2 is inhibited in crude

362

extracts.

363

If electrons cannot be delivered to FRD during growth on glycerol, then it is likely that the

364

electrons will be used to reduce protons and generate hydrogen via Hyd-2 (see e.g. Fig. 2). It

365

has been observed that no H2 accumulates during growth in GF medium, where a respiratory

366

electron acceptor is plentiful (7), despite the findings of recent studies (22) clearly showing

367

that during glycerol fermentation Hyd-2 can contribute to H2 production. Moreover, in vitro

368

studies using purified Hyd-2 have shown that the enzyme can catalyse hydrogen production at

369

low redox potentials (23). Therefore, we analysed H2 production in the culture head-space

370

after growth of various mutants in GF medium. Our experiments showed that during growth

371

of MC4100 in GF medium only a low amount H2 is present in the head-space (Table 3). No

372

hydrogen was detectable in a strain where the main uptake and evolving Hyd enzymes are

373

missing (FTD147, hyaB hybC hycE). In the frdA deletion strain (CP887) more than 3 times

374

as much H2 was produced compared to its parental strain MC4100. A strain deficient in Hyd-

375

2 synthesis (HDK200) showed a similar level of H2 accumulation (0.66 units) to that observed

376

for MC4100, indicating that Hyd-2 was not the only enzyme responsible for H2 production

377

under these conditions. Strain SAL1 (hyaB hycAI frdA) exhibited high levels of H2

378

evolution, similar to those produced by thefrdA mutant CP887 (Table 3).

379

The data in Table 3 show that when MC4100 was grown with glycerol but without fumarate,

380

this also yielded high levels of H2 production (3.15 units). In contrast, strains HDK200

381

(hybBC) and SAL1 (hyaB hycAI frdA) both had approximately 50% of this level of H2

382

production. Together, these results indicate that under conditions where no H2 oxidation by

16
383

Hyd-2 is detectable (see Table 2 and Fig. 6) at least 50% of the H2 generated under these

384

conditions derives from Hyd-2. Furthermore, these findings indicate that Hyd-2 switches

385

between hydrogen consuming and hydrogen producing roles in vivo.

386
387

Discussion.

388

In this study we have investigated the physiology and bioenergetics of bi-directional H2

389

activation by Hyd-2 during anaerobic metabolism of glycerol. Our findings reveal that MQ/

390

DMQ is required for electron transfer to and from Hyd-2 under these conditions, that the

391

HybA subunit is essential to mediate electron transfer between the enzyme the quinone pool

392

and, significantly, the proton gradient drives H2 evolution catalysed by Hyd-2. These features

393

of Hyd-2 are summarized in the working model presented in Fig. 7.

394

Under respiratory conditions where excess electron acceptor is available Hyd-2 functions as

395

an H2-oxidizing enzyme. During glucose fermentation at high substrate concentration H2 only

396

accumulates in strains that are able to form an intact FHL complex but not in strains where

397

hyc genes are deleted (2). This result shows that strains unable to synthesize FRD are not

398

impaired in glucose fermentation, which agrees with previous observations (48). Under

399

conditions of fumarate-dependent respiration of glycerol, Hyd-2 is synthesized, active, and

400

poised to oxidise H2, passing the derived electrons to the quinone pool. This H2-oxidation

401

activity of Hyd-2 is readily assayed using redox dyes either in solution or after separation of

402

the enzyme complexes in native-PAGE (11). The enzyme is able to re-cycle hydrogen

403

generated, for example, by the FHL complex, coupling H2 oxidation to fumarate reduction

404

(Fig. 7). Glycerol is converted to dihydroxyacetone-3-phosphate (DHAP) by the combined

405

actions of glycerol kinase and glycerol-3-phosphate dehydrogenase (GlpK/ G-3P DH; 17).

406

The reducing equivalents derived from glycerol enter the quinone pool and are re-oxidized by

407

reducing fumarate to succinate catalysed by FRD (33, 39).

17
408

On the other hand, if cells are supplied with only glycerol or if FRD is genetically inactivated,

409

Hyd-2 is still synthesized but catalyses H2 evolution, which presumably prevents over-

410

reduction of the quinone pool during glycerol fermentation (Fig. 7). Anaerobic glycerol

411

oxidation can occur either via GlpK/ G-3P DH or via glycerol dehydrogenase (GldA), which

412

generates NADH (19). Coupling of glycerol-3 phosphate oxidation to proton reduction via

413

MQH2 is possible as depicted in Fig. 7 and this could be linked to H2 evolution by Hyd-2.

414

However, recent studies (19) have suggested that GldA and DHAK also have key roles in

415

glycerol fermentation. Theoretically, NADH oxidation can be accomplished by exclusive

416

conversion of acetyl-CoA to ethanol via alcohol dehydrogenase; however, in wild type E. coli

417

this would obviate the production of H2 by Hyd-2 and instead H2 would be produced from

418

formate via FHL, as we have also observed in this study. Nevertheless, a mutant synthesizing

419

only Hyd-2 was also capable of producing H2 from glycerol, suggesting that if the GlpK/ G-

420

3P DH route is used, G-3P DH couples electron transfer to Hyd-2 via MQ or, alternatively, in

421

the absence of a functional FHL in the mutant, the GldA route operates and formate is

422

excreted into the periplasm where it could be oxidized by FDH-O (46) with concomitant

423

electron transfer to Hyd-2 via MQ. A further alternative would involve generating a mixture

424

of acetate and ethanol; however, complete redox balance could then only be achieved if re-

425

oxidation of the NADH generated by GldA could be coupled to H2 evolution by Hyd-2

426

through a MQ-coupled NADH oxidase activity, which has been observed previously (49).

427

All of these possible routes of linking the excess redox equivalents generated by glycerol

428

fermentation to H2 evolution are complicated by the novel finding of this study that proton

429

reduction catalysed by Hyd-2 is dependent on the proton motive force (pmf). Each of the

430

three possible energy sources, i.e. formate, G-3P or NADH, at physiological concentrations

431

could theoretically allow H2 production by Hyd-2, but this does not readily explain why the

432

pmf is linked to H2 evolution. Hyd-2 has an unusual structure for a modular membrane-

433

associated oxidoreductase in that, as well as having a typical electron-transferring small

18
434

subunit, HybO, it also has the additional electron-transferring HybA subunit. HybA has been

435

shown here to be essential for H2 evolution catalysed by the enzyme. Furthermore, the

436

membrane anchor subunit HybB is atypical in modular oxidoreductases because it lacks

437

cofactors (7). It is conceivable therefore that HybB operates as a conformational proton pump

438

when electrons are channelled through the enzyme to the quinone pool. This would infer that

439

when Hyd-2 operates in the H2-evolving mode, it requires a pmf, while in the opposite H2-

440

oxidizing mode it helps generate a proton gradient. This would also explain why the enzyme

441

is inhibited by an uncoupler when it is working in the H2 evolution direction but not when it

442

functions in the H2-oxidizing direction. Indeed, there is an indication that the uncoupler

443

slightly speeds up fumarate reduction (see Fig. 5), which would support this hypothesis.

444

We have shown here that the HybA subunit of Hyd-2 is required to mediate bi-directional

445

electron transfer with MQ and it is necessary to facilitate reverse electron transport to allow

446

H2 evolution. Surprisingly, we observed a correlation between the enzyme working as a

447

proton reductase and a concomitant inability to detect the enzymes H2-oxidizing activity

448

when assayed with redox dyes in vitro. This suggests that, under certain circumstances,

449

electron flow through Hyd-2 might somehow be modified to maintain a bias towards one

450

reaction direction. It is currently unclear what the underlying mechanistic basis of this

451

catalytic bias might be. Remarkably, however, it was possible to restore dye-reducing activity

452

to Hyd-2 in vitro by separating the membrane and cytoplasmic fractions. One possible

453

explanation for this finding is that H2-dependent dye-reducing activity is reversibly inhibited

454

by over-reduction of the iron-sulfur clusters in the HybO and HybA subunits of the enzyme,

455

although treatment of crude extracts with oxidants such as ferricyanide or performing the

456

electrophoresis under aerobic conditions failed to restore this activity to Hyd-2 in crude

457

extracts (data not shown). An alternative explanation is that Hyd-2 enzyme activity is

458

inhibited by a component in the soluble sub-cellular fraction; however, re-mixing of the

459

soluble and membrane fractions after their physical separation failed to restore inhibition of

19
460

Hyd-2 enzyme activity. Clearly, further experiments will be required to elucidate the

461

biochemical mechanism underlying the reversible inhibition of redox dye-reducing activity.

462

In summary, our findings indicate that Hyd-2 is clearly able to switch between H2 oxidation

463

and H2 production modes without altering protein content and without expending energy on

464

de novo protein synthesis. This is important to allow metabolic flexibility of H2 metabolism

465

and it also possibly explains why E. coli has evolved two routes of anaerobic glycerol

466

utilization (19). The two routes likely operate simultaneously to provide a balanced redox

467

status of the quinone pool and an optimal proton gradient (Fig. 7). H2 production by Hyd-2

468

observed in this study places the recent finding that deletion of genes encoding Hyd-2 has a

469

deleterious effect on H2 production during glycerol fermentation in a new context (17) and

470

provides an explanation for the suggested Hyd-2-dependent H2 during glycerol fermentation

471

in another recent study (50). Moreover, our study provides the first demonstration that H2

472

evolution by Hyd-2 is directly linked to the pmf.

473
474

Acknowledgements

475

This work was supported by the BBSRC grants BB/I02008X/1 and BB/L008521/1 to FS and

476

by the Deutsche Forschungsgemeinschaft (No. SA 494/3-2) to RGS.

477
478

Authors contributions

479

SL carried out experiments in Fig. 6 and Table 2. SL and CP carried out the strain

480

constructions for this study. MJ and CK carried out the H2 head-space measurements. CK

481

performed the H2 electrode experiments leading to Fig. 1. CP carried out all other experiments.

482

CP, FS and RGS drafted the manuscript and conceived the study. All authors read and

483

approved the final manuscript.

484

20
485

Competing interests

486

The authors declare that they have no competing interest.

487
488

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633

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634

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635
636

26
637

Tables

638

Table 1: Strains and plasmids.

Strain

Genotype

Reference

BW25113

F- (araD-araB)567 lacZ4787( : : rrnB-3) - rph-1 (27)

(rhaD-rhaB)568 hsdR514

MC4100

F-, araD139, (argF-lac)U169, -, rpsL150, relA1, (51)

deoC1, flhD5301, (fruK-yeiR)725(fruA25), rbsR22,
(fimB-fimE)632(::IS1)

DHP-F2

MC4100 hypF

(42)

FTD147

MC4100 hyaB hybC hycE

(52)

FTD671

MC4100 hybA

(7)

HDK200

MC4100 hybBC

(53)

IC010

MC4100 hyaB hycE

(52)

IC012

MC4100 hyaB hycE menA::kan

This study

JW4115

BW25113 frdA::kan

(27)

JW3901

BW25113 menA::kan

(27)

JW5713

BW25113 ubiC::kan

(27)

JW5581

BW25113 ubiE::kan

(27)

JW0659

BW25113 ubiF::kan

(27)

CP887

MC4100 frdA::FRT

This study

CP1034

MC4100 hyaB hycE hyfB-R::spec

This study

CP1037

MC4100 hyaB::kan hycAI::cat

This study

SAL1

MC4100 hyaB::FRT hycAI::FRT frdA::FRT

This study

CP1A3

MC4100 hyaB hycE hybA::kan

This study

27
Plasmids
pACYCDuet- pACYCDuet-1, frdA promoter, frdABCD+, Cmr

This study

frdAD
pmenA

pACYCDuet-1, menA promoter, menA+, Cmr

This study

pJET-hybA

pJET1.2, RBS-hybA+, Apr

This study

639
640
641

Table 2 Analysis of hydrogenase and fumarate reductase activity complementation.

Strain/ plasmida

Specific

hydrogenase Specific

fumarate

reductase

activity in U mg protein-1 activity in U mg protein-1

standard deviationb

standard deviationb

MC4100

0.19 0.03

0.20 0.03

DHP-F2 (hypF)

< 0.01

n.d.

CP1037 (hyaB hycAl)

0.15 0.05

n.d.

IC010 (hyaB hycE)

0.15 0.06

0.20 0.10

SAL1 (hyaB hycAl frdA)

0.02 0.01

< 0.01

SAL1/pACYCDuet-frdAD

0.11 0.02

0.42 0.04

JW4115 frdA)

0.23 0.16

< 0.01

JW4115

0.25 0.02

0.40 0.20

frdA)/pACYCDuet-frdAD
642

n.d. not determined

643

644

fumarate.

645

646
647

Strains were grown for 16 hours in M9-minimal media containing 0.4% glycerol and 25 mM

The mean and standard deviation of at least three independent measurements are shown.

28
648

Table 3: Hydrogen production during glucose, glycerol and glycerol/fumarate growth.

Strain*

0.4% glycerol

0.4% glycerol

0.8% glucose

25 mM fumarate
MC4100

3.15 0.87

0.67 0.09

1.93 0.52

CP887 (frdA)

2.99 0.95

2.27 0.30

1.56 0.25

HDK200 (hybBC)

1.64 0.43

0.66 0.35

2.36 0.58

SAL1 (hyaB hycAI

1.84 0.33

2.17 0.91

0.04 0

< 0.01

< 0.01

< 0.01

frdA)
FTD147hyaB hybC
hycE)
649

* samples were drawn from the head space after 26 hours of growth and calculated

650

according to the optical density as U ml-1 OD600 nm-1

651
652

29
653

Figures

654

Figure 1: Hydrogenase 2 generates H2 from glycerol and oxidizes H2 using different

655

electron acceptors. Cells for hydrogen production analysis on the hydrogen electrode were

656

grown and prepared as described in Material and Methods. Aliquots of 25 mg of cells from

657

strain IC010 (hyaB hycE), lacking Hyd-1 and Hyd-3 but synthesizing Hyd-2, was added at

658

point A. After equilibration, glycerol was added to a final concentration of 170 mM at point B.

659

After approximately 20 min, TMAO (filled squares, 67.5 mM final) was added at point C or

660

fumarate (open triangles, 25 mM final) or nitrate (filled circles, 60 mM final) were added at

661

point D.

662
663

Figure 2: MQ/ DMQ acts as physiological electron donor and acceptor for Hyd-2. A:

664

The strains IC010 (hyaB hycE) and IC012 (hyaB hycE menA), as well as IC012

665

complemented with pmenA, were grown in GF medium (0.4% w/v glycerol and 25 mM

666

fumarate) or Glc medium (0.8% w/v glucose) and extracts equivalent to 25 g of protein were

667

loaded on native-PAGE and subsequently stained for hydrogenase activity as described in

668

Material and Methods. The migration position of Hyd-2 is labelled on the right hand side of

669

the gel. B: The strains IC010 (black line) and IC012 (gray line) were grown in GF medium

670

and cells corresponding to 5 mg of protein were analyzed for hydrogen production on the

671

electrode. Glycerol was added to a final concentration of 170 mM and fumarate to 25 mM as

672

indicated. C: The same cells as in B (IC010 black; IC012 grey) were applied to the electrode

673

and H2-saturated buffer was added as indicated prior the addition of 25 mM fumarate. D: H2-

674

saturated buffer was added to strain IC012 on the electrode and DMSO (175 mM final) and

675

nitrate (60 mM final) were subsequently added as indicated.

676

30
677

Figure 3: The role the HybA subunit of Hyd-2 in electron transport to and from the

678

quinone pool. A: Strains CP1034 (hyaB hycE hyfB-R; black curve) and CP1A3 (hyaB

679

hybA hycE; gray curve) were grown in M9-minimal medium with 0.4% (w/v) glycerol and 25

680

mM fumarate, the cells were harvested and washed before an amount equivalent to 5 mg

681

protein was added to the chamber of a hydrogen electrode. After a period of equilibration,

682

glycerol was added to a final concentration of 170 mM as indicated. After a further 20 to 30

683

min equilibration, fumarate was added to a final concentration of 25 mM as indicated by the

684

arrows. B: Strains CP1034 (hyaB hycE hyfB-R; black curve) and CP1A3 (hyaB hybA hycE;

685

gray curve) were grown in M9-minimal medium with 0.4% (w/v) glycerol and 25 mM

686

fumarate, the cells were harvested and washed before an amounts equivalent to 5 mg protein

687

was added to the chamber of a hydrogen electrode. An aliquot of hydrogen-saturated buffer

688

corresponding to 600 nmol was added after one minute of equilibration. After a further

689

equilibration of the signal of between 5 and 10 minutes, fumarate was added to a final

690

concentration of 25 mM as indicated by the arrow.

691
692

Figure 4: Hyd-2 lacking the HybA subunit retains H2: viologen dye oxidoreductase

693

activity. A: Strains MC4100, FTD671 (hybA) and the complemented FTD671 with pJET-

694

hybA were grown in GF medium or in glucose medium (Glc) and 25 g of protein were

695

subjected to native-PAGE and subsequently stained for hydrogenase activity as described.

696

The migration patterns of Hyd-1 and Hyd-2, as well as the H2-oxidizing activity of FDH-O

697

and FDH-N, are shown on the right side. B: Strains IC010 (hyaB hycE; black curve) and

698

CP1A3 (hyaB hybA hycE; gray curve) were prepared as in A and an amount of cells

699

corresponding to 5 mg protein was added to the electrode chamber. After a short equilibration,

700

MV was added to the chamber to a final concentration of 1.2 mM and subsequently reduced

701

with sodium dithionite to a dark blue (indicated by an arrow and DTH). Buffer without cells

702

served as a negative control (dotted curve).

31
703

Figure 5: Glycerol-dependent H2 evolution by Hyd-2 requires a proton gradient. Strain

704

IC010 (hyaB hycE) was grown with 25 mM fumarate and 0.4% (v/v) glycerol and cells

705

corresponding to 5 mg of protein were applied to the electrode. A: After equilibration,

706

glycerol was added to a final concentration of 170 mM as indicated. When H2 production was

707

linear CCCP was added to a final concentration of 100 M as indicated. After about 5 min,

708

fumarate was added to a final concentration of 25 mM as indicated by an arrow. B: Strain

709

IC010 was added to the electrode and H2-saturated buffer was added as indicated. When the

710

signal attained a constant level, fumarate was added to 25 mM final concentration as indicated.

711

After 10 min of H2 oxidation, CCCP was added to 100 M and the signal was recorded for

712

another 20 min.

713
714

Figure 6: A frdA strain lacks hydrogen: BV-oxidoreductase activity of hydrogenase 2.

715

A: Strains MC4100, IC010 (hyaB hycE) and SAL1 (hyaB hycAI frdA) and SAL1

716

complemented with pACYC-frdAD were grown anaerobically in M9-minimal media

717

containing either 0.8% (w/v) glucose (Glc) or 0.4% (v/v) glycerol and 25 mM fumarate (GF).

718

Extracts with 25 g of protein were subjected to native-PAGE and subsequently the gel was

719

stained for hydrogenase activity as described in Material and Methods. The migration patterns

720

of the hydrogenase-independent formate dehydogenases O and N (FDH-O/N) as well as Hyd-

721

1 through Hyd-3 are labelled on the right hand side of the gel. B: Cells of MC4100, DHP-F2

722

(hypF) and JW4115 (frdA) were grown in M9-minimal medium as described in the

723

Materials and Methods section with 0.8% (w/v) glucose or 0.8% (v/v) glycerol as carbon

724

source. The electron acceptors sodium fumarate, sodium nitrate (NO3-), TMAO or DMSO

725

were added where indicated to a final concentration of 25 mM. Equivalent amounts of protein

726

(25 g) were loaded on native-PAGE and after electrophoresis the gel was stained for

727

hydrogenase activity. The bands are labelled as FDH-O/N for hydrogenase-independent

728

formate dehydrogenase O and N activities, Hyd-2 and Hyd-1 for the hydrogenases 2 and 1,

32
729

respectively. C: Strains DHP-F2 (hypF), MC4100, IC010 and SAL1 were grown 0.4% (v/v)

730

glycerol and 25 mM fumarate (GF) and the crude extracts as well as cytosolic fraction

731

(soluble) and membrane fraction (membrane) were prepared as described in Material and

732

Methods. Aliquots of the sub-cellular fractions (25 g of protein) were loaded on a native-

733

PAGE and subsequently subjected to hydrogenase activity staining. The migration pattern of

734

FDH-O/N, Hyd-1 and Hyd-2 are labelled on the right of the figure.

735
736

Figure 7: Model depicting the function of Hyd-2 during glycerol fermentation and

737

glycerol-fumarate respiration. A schematic representation of the anaerobic metabolism of

738

glycerol is shown in the lower portion of the Figure. The reduced products of anaerobic

739

glycerol metabolism are shown in red letters. Note that the enzyme glycerol dehydrogenase

740

(GldA), glycerol kinase (GlpK) and glycerol-3 phosphate dehydrogenase (G-3P DH) are

741

normally membrane-associated enzymes. The upper portion of the figure depicts how Hyd-2

742

is involved in glycerol fermentation (left side) or glycerol respiration (right side). The red

743

dashed line indicates that a proton motive force (pmf) drives the electron transfer from

744

glycerol via menaquinol to Hyd-2. The filled red line indicates that CCCP blocks H2

745

production by Hyd-2. The horizontal dashed black line signifies that hydrogen generated by

746

the formate hydrogenlyase (FHL) complex can be re-oxidized by Hyd-2. The Hyd-2 enzyme

747

comprises the HybOABC subunits and HybA, which is required for electron transfer to and

748

from the quinone pool (see text), is highlighted in red. Electron transfer within the Hyd-2

749

complex is represented by dashed white lines. The other protein complex represented in

750

purple is fumarate reductase (FRD). Abbreviations: DHAK, dihidroxyacetone kinase; DHAP,

751

dihydroxyacetone phosphate; QH2, reduced quinone; MQ/ MQH2, menaquinone/ menaquinol.

752
753