Hydrogenases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina

International Journal of Hydrogen Energy 27 (2002) 1463 – 1469

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Hydrogenases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina Korn-el L. Kov-acsa; b; ∗ , Barna Fodora; b , Akos T. Kov-acsa; b , Gyula Csan-adia; b , Gergely Mar-otia; b , Judit Balogha; b , Solmaz Arvania; b , G-abor R-akhelya; b a Department

b Institute

of Biotechnology, University of Szeged, Temesvari krt. 62, Szeged 6726, Hungary of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

Abstract The purple (Sulphur) phototrophic bacterium, Thiocapsa roseopersicina BBS contains several [NiFe] hydrogenases, of which two are membrane bound. Mutant T . roseopersicina cells, carrying deletions in both gene clusters showed hydrogenase activity. This activity was located in the cytoplasm. The structural gene cluster hoxEFUYH was identi:ed and sequenced. In addition, genes homologous to hupUV=hoxBC, the hydrogen sensing hydrogenase have been identi:ed and sequenced. Regulation of hydrogenase biosynthesis was studied in detail for HydSL (renamed HynSL). A random mutagenesis system was optimised for T. roseopersicina. One of the mutations was in a gene similar to that coding for the HypF proteins in other organisms. Inactivation of the hypF gene resulted in a 60-fold increase in hydrogen evolution under nitrogen :xing conditions. In addition to hypF, the following accessory genes were identi:ed: hydD, hupK, hypC1, hypC2, hypDE. Characterisation of the corresponding gene products and search for additional accessory genes are in progress. ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved. Keywords: [NiFe] hydrogenase; Accessory genes; Mutagenesis; Gene expression; Biohydrogen production; Photosynthetic bacteria; Thiocapsa roseopersicina

1. Introduction The rate of fossil fuel formation is much slower than the rate of its exploitation. Thus, the reserves that can be recovered in an energetically feasible manner are shrinking while the global energy demand is growing, presenting a principal diBculty in the sustainable development of mankind. The rapidly depleting fossil fuel reserves, coupled with the detectable signs of global warming, which originates from burning fossil fuels, call for an immediate search for alternative energy sources. The only known, realistic possibility,

∗ Corresponding author. Department of Biotechnology, University of Szeged, Temesvari krt. 62, Szeged 6726, Hungary. Tel.: +36-62-454-351; fax: +36-62-544-352. E-mail address: [email protected] (K.L. Kov-acs).

on a global scale, is the nuclear fusion-type reactor, known as the sun. However, solar energy is “diluted” and intermittent, compared to fossil fuels, thus its storage is a challenging task. Among the alternative energy carriers, hydrogen appears to be the most promising one, since it burns to water when utilised and can be easily transported and stored. Hydrogen can be produced from sunlight in biological processes. The latter may be combined to form hydrogen by an enzyme called hydrogenase. This unique enzyme catalyses the formation and decomposition of the simplest molecule occurring in biology: H2 . Understanding the molecular fundamentals of hydrogen production and utilisation in biological systems is a goal of supreme importance for basic and applied research [1]. It should be noted that hydrogenases can help us in two ways: they may catalyse both H2 generation (e.g., photobiological) and H2 consumption (e.g., in fuel cells). This simple-looking task is solved by a sophisticated macromolecular machinery. Hydrogenases are metalloen-

0360-3199/02/$ 22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 6 0 - 3 1 9 9 ( 0 2 ) 0 0 0 9 7 - 6

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zymes harbouring Ni and Fe, or only Fe atoms, arranged in an exceptional structure. Like most redox metalloenzymes, hydrogenases are usually extremely sensitive to inactivation by oxygen, high temperature, CO, CN and various environmental factors. These properties are not favourable for most biotechnological applications, including biohydrogen production and H2 consumption in fuel cells. Hydrogenases are found in Archaea, Eubacteria and simple Eukaryota. Their physiological function vary: they can serve as redox safety valves to dispose of excess reducing power, or generators of chemical energy by taking up and oxidising H2 , or maintaining a reducing environment for reactions of crucial importance, such as the :xation of atmospheric nitrogen. In some organisms, the numerous functions are performed by the same enzyme, but more frequently, a separate, specialised hydrogenase carries out each in vivo biochemical function. 2. Research on Thiocapsa roseopersicina hydrogenases In metal-containing biological catalysts, it is the protein matrix, surrounding the metal centres, which provides the unique environment for the Fe and Ni atoms and allows hydrogenases to function properly, selectively, and eKectively. Hydrogenases are ancient enzymes, hence their protein matrix is rather conserved. The NiFe hydrogenases are composed of at least two distinct (heterodimer) polypeptides, containing highly conserved metal binding domains. The large subunit harbours the active centre, fastened to the protein by four cysteine ligands. The Fe atom ligates 2 CN and 1 CO diatomic molecules and it is :xed to the Ni atom via sulphur bridges [1]. Similar heterobinuclear NiFe centres are not known in any other metalloenzyme. The presence, the incorporation mechanism, and the function of the CN and CO groups are mysterious as both cyanide and carbon monoxide are poisonous for the micro-organisms and irreversibly inactivate the NiFe hydrogenases themselves when administered externally. The small subunit contains 2–3 Fe4 S4 clusters, L apart, and thus, which are precisely and equally spaced, 15A form a conducting wire inside the protein to facilitate the transport of electrons between the active centre and the protein surface. A major goal for hydrogenase basic research is to understand the intimate protein–metal interaction in this complex structure [2]. In order to develop suitable biocatalysts for future biotechnological applications, the structure–function relationship, biosynthesis and assembly of hydrogenases must be understood. Determination of the protein primary sequences from the structural genes is clearly a necessary, but not suBcient, requirement. A number of other gene-products govern the metal uptake, their attachment into the right place at the right time, formation and ligation of the CN and CO groups, and the incorporation and :xation of this labile inorganic structure into the protein matrix. Our present understanding suggests that the concerted action of, at least, 15 –20 such

accessory proteins is necessary for the formation of an active NiFe hydrogenase [1]. This well organised “assembly-line” works in the molecular dimension. Some of the participating proteins are hydrogenase pleiotrop, called Hyp. They take part in the synthesis of all hydrogenases. Others specifically work on one type of NiFe enzyme and therefore several variants of the similar accessory proteins may exist in the same micro-organism. An almost uniform organisational scheme is observable for the [NiFe] hydrogenase structural genes: the gene coding for the small subunit precedes the one coding for the large subunit and the two genes form one transcriptional unit. Sometimes, the accessory genes are neatly arranged around the structural genes, but most often, they are scattered in the genome. The best sources of hydrogenases, both for basic research and for forthcoming large-scale utilisation, should be micro-organisms that are cheap to cultivate and use sunlight to get energy for their growth. The likely candidates are phototrophic bacteria [3]. Many phototrophs use sulphide (or other reduced sulphur compounds) as electron source. One such phototrophic bacterium is our favourite organism, Thiocapsa roseopersicina. T. roseopersicina is a phototrophic purple sulphur bacterium; the strain marked BBS has been isolated from the cold waters of the North Sea. The bacterium contains a nitrogenase enzyme complex, thus it is capable of :xing atmospheric N2 , a process accompanied by H2 production [4]. Previous studies in our laboratory have revealed that T. roseopersicina contains at least two membrane-associated NiFe hydrogenases with remarkable similarities and diKerences. One of them (HydSL=HynSL) shows extraordinary ◦ stability: it is much more active at 80 C, than around 25 – ◦ 28 C. It is to be noted that T. roseopersicina cannot grow ◦ above 30 C. HydSL=HynSL of T. roseopersicina is also reasonably resistant to oxygen inactivation and stays active after removal from the membrane. The other NiFe hydrogenase, HupSL, is very sensitive to all these environmental factors and thus it resembles the NiFe hydrogenases known from other micro-organisms [4]. The structural genes coding for these enzymes have been cloned and sequenced [5,6]. The translated protein sequences indicate a signi:cant sequence homology between the two NiFe hydrogenases. Despite the pronounced diKerences in stability, the two small subunits are identical in 46% of their amino acids and the two large subunits show 58% sequence identity. In order to understand the physiological roles of these hydrogenases, mutants lacking either or both of them have been generated in our laboratory by marker exchange mutagenesis. Remarkably, the hydrogenase-deleted mutants grew just as well as the wild-type strain. This seemingly odd observation was understood when two additional NiFe hydrogenases were discovered in the cytoplasm of the bacterium, recently. According to our current understanding, there are four distinct NiFe hydrogenase molecular species in T. roseopersicina, representing all hydrogenase forms thus far described in various

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hupR hupS hupL hupC hupD hupH hup I

hydS

isp1

isp2

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hydL

Thiocapsa roseopersicina

hupT hupU

hupV

hoxF

huxU

hoxY

hoxH

Fig. 1. Hydrogenases in T. roseopersicina and the organisation of the gene clusters coding for the corresponding proteins. The location of the enzymes is indicated schematically within the cells of this purple sulphur bacterium. The structural genes are marked in black, genes coding for proteins that form a stable complex with the hydrogenase subunits are coloured dark grey, genes coding for accessory proteins are light grey, regulatory genes are faint grey and genes coding for proteins of unknown function are white.

micro-organisms, in a single cell. This makes T. roseopersicina one of the best candidates for studies of NiFe hydrogenase structure–function relationships and assembly. The outstanding situation allows us to address speci:c questions concerning the assembly of each of these enzymes and the regulation of their biosynthesis. Answers to these questions will be of direct relevance in designing an optimal catalyst for biological hydrogen production and=or utilisation and to protein engineering of scienti:cally intriguing and biotechnologically important redox enzymes in general. The characteristics of the four NiFe hydrogenases are summarised in Fig. 1. Stable hydrogenase (HydSL=HynSL): Commonly called Hyd, a recent nomenclature revision proposes a new abbreviation of Hyn for these enzymes [7]. In addition to its outstanding stability, this enzyme is noted also for the unusual organisation of the structural genes coding for the Hyd-hydrogenase. Unlike most hydrogenase structural gene clusters, the gene coding for the small subunit, hydS=hynS, is separated from hydL=hynL with an approximately 2 kb long DNA segment, containing genes for two putative proteins. The role of this arrangement and the properties of the putative proteins are subjects of ongoing research in our lab. HydSL=HynSL has been puri:ed as an active hydrogenase heterodimer to homogeneity [8]. The puri:ed enzyme is stable, similarly to the membrane associated in vivo state. In vitro HydSL=HynSL catalyses both H2 -evolution and H2 -uptake, but it functions primarily in H2 consumption in the living bacterium. Unstable hydrogenase (HupSL): HupSL also functions in the H2 -uptake direction in vivo. Its sequence shows high homology to HydSL, but it is so unstable that we have not yet been able to isolate the protein from the membrane in

an active form. A comparative study with HydSL, at both molecular biology and protein biochemistry level [5], will hopefully shed light on the structural basis of the stability diKerences. NiFe hydrogenases related to HupSL have been found and studied in a number of micro-organisms [1,3,4]. In other systems, biosynthesis of HupSL is linked to the nitrogen :xation process and the generally assumed physiological role of HupSL is to recycle the excess H2 produced by the nitrogenase enzyme complex [2,5]. In T. roseopersicina, however, the biosynthesis of HupSL is apparently unrelated to nitrogen :xation and H2 does not regulate how - T. many copies of this enzyme are present in the cell (A. Kov-acs et al., unpublished results). Soluble hydrogenase (HoxYH): This is one of the cytoplasmic hydrogenases discovered recently in T. roseopersicina. In fact, the structural gene cluster predicts that it is a four or :ve subunit enzyme, coded by the hoxEFUYH gene cluster. The hox gene products are related to HoxFUYH, described in detail in Ralstonia eutropha, a chemolithotrophic bacterium [9]. The corresponding structural genes have been sequenced, the puri:cation and characterisation of this hydrogenase from T.roseopersicina is in progress. The soluble hydrogenase functions primarily in the direction of H2 production, therefore its study should give us information, which will be useful in designing biocatalysts for biohydrogen production. Sensor hydrogenase (HupUV): A set of genes homologous to hupTUV=hoxJBC in Rhodobacter capsulatus and R. eutropha, respectively, has been identi:ed and sequenced from T. roseopersicina. HupUV senses H2 in the environment of R. capsulatus and triggers the biosynthesis of the only hydrogen-uptake enzyme, HupSL in this organism [10]. A similar function has been assigned to the homologous

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HoxBC protein in R. eutropha [9]. Interestingly, external H2 signals do not appear to regulate the biosynthesis of either of the NiFe hydrogenases in T. roseopersicina. Therefore, a H2 sensing hydrogenase has no apparent function in this bacterium. From the fragmented information available, there is no clear answer as to why T. roseopersicina needs so many distinct hydrogenases. Our working hypothesis links this abundance of various NiFe hydrogenases to the fact that this bacterium should be able to perform various metabolic activities (photoautotrophic, photoheterotrophic, heterotrophic metabolism) in order to survive in its natural habitat [11]). Having numerous hydrogenases at hand increases the chances of survival for the bacterium and increases our chances to understand basic phenomena of hydrogenase catalysis. The advantages of having at least four types of NiFe hydrogenases in T. roseopersicina are counter-balanced by the fact that most of the accessory genes needed for their assembly and biosynthesis are scattered in the genome. The downstream region of the hupSL structural gene cluster contains some of these genes: hupCDHI and hupR have been identi:ed on the basis of their sequence homology to the corresponding accessory genes in other micro-organisms [5]. No accessory genes have been found around the other three structural gene clusters. This situation is not unique among the micro-organisms, but due to the numerous NiFe enzymes present in T. roseopersicina, a large number of accessory genes must be found and characterised in order to locate the elements of the hydrogenase “assembly-line”. Since some of these genes are pleiotropic, the number of missing genes is estimated to be between 20 and 30. The majority of these genes and their protein products are needed for the macromolecular structure–function studies; therefore a systematic search has been launched in our research team using two approaches. We have begun sequencing the genome of T. roseopersicina and applying random mutagenesis and screening for altered hydrogenase phenotypes. This combination of approaches will allow us to identify those genes that play a signi:cant role in the formation of the functionally intact enzymes. Random mutagenesis is a straightforward approach, as long as there is a good method available to screen the mutants, and the mutation causes phenotypic change(s). It should be noted that the two approaches provide complementary information and their simultaneous application is therefore justi:ed. The mutagenesis approach will be discussed in the following section. First , genetic tools appropriate for use in T. roseopersicina had to be developed, since no molecular genetic work had been done on this micro-organism. An eBcient conjugative gene transfer system was employed [12]. Second, a random transposon mutagenesis system was adopted using a wide host range plasmid and Tn5 transposon derivative. Third, an eBcient screening method was necessary to detect the mutants with altered hydrogenase activity within a

Heat treatment (optional) 75oC, 1-3hr, air

H2

Fig. 2. Screening for hydrogenase de:cient phenotype. The light grey colonies of T. roseopersicina are lifted on a :lter paper, transferred onto a stack of :lter papers soaked with oxidised redox dye (benzyl viologen) under air. Following heat treatment, the cells containing heat stable, active enzyme turn blue (indicated as black dots) under hydrogen atmosphere, those containing defected hydrogenase remains purple (shown as light grey) [12].

large mutant library. The H2 -uptake activity of colonies was observed using the selective and indicative colour change of the redox dye, methyl viologen (Fig. 2). 2.1. Mutants identi
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[14] indicating that the hydrogenase “assembly-line” has been conserved through huge evolutionary distances. Based on indirect evidence gathered in E. coli, the HypD protein is assumed to participate in the incorporation of Ni into the active centre [15]. It is worth noting that it starts with GUG start codon instead of the commonly used AUG start codon and contains 11 conserved cysteine residues. 2.1.3. hypE It has been found in several bacteria. The protein encoded by this gene plays an important pleiotropic role in the formation of an active NiFe hydrogenase, but its speci:c function is not known. In T. roseopersicina it is located in the vicinity of hypCD. 2.1.4. hypF This gene codes for a fairly large protein that has a central role in the assembly of NiFe hydrogenases. The homologous protein in E. coli was shown to have an acyl-phosphatase consensus sequence and a domain typical of enzymes performing O-carbamoylation [16]. In addition, it contains a putative chaperone domain. These domains are clearly distinguishable in the translated hypothetical T. roseopersicina HypF protein, as well [12]. Taken the various, conserved properties together, HypF is believed to be the protein that synthesises and incorporates the CO and CN ligands into the active centre. When hypF is deleted, none of the NiFe hydrogenases are synthesised in an active form. The RhypF mutant of T. roseopersicina produces large amounts of H2 under nitrogen :xing conditions, indicating that the majority of the NiFe hydrogenase activity is in the H2 -uptake direction in vivo. The RhypF mutant of T. roseopersicina was the :rst direct evidence showing that this bacterium can be “engineered” to release signi:cant amounts of biohydrogen, although in this case, the evolved H2 originated predominantly from the nitrogenase complex [12]. 2.1.5. hupK Interestingly, this gene has been found in a very limited number of bacteria so far [17]. In those instances where hupK is present, it is indispensable for the formation of the active enzyme. Based on circumstantial evidence, a role in “handling” the Fe atom has been assigned to HupK, although this does not explain how other strains, lacking HupK, assemble the active centre of their NiFe hydrogenases. We have demonstrated unequivocally, that HupK takes part in the targeting of the enzymes into the membrane (G. Mar-oti et al., unpublished results), hence it provides the “:nishing touches” on the membrane-bound hydrogenases, rather than inserting a metal atom at an earlier stage. Indeed, bacteria that do not contain membrane-bound hydrogenase, do not have hupK, and the ones synthesising membrane-bound enzymes do.

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2.1.6. hydD=hynD This was probably the most fortunate :nd among the mutants. The HydD=HynD [15] protein is most likely an endopeptidase. Its function is to clip oK a peptide segment from the C-terminus of the stable hydrogenase large subunit HydL=HynL. When the heterobinuclear metal centre, together with the CN and CO diatomic ligands, is properly inserted into the protein core, the C-terminal processing cuts oK a peptide necessary to keep the complex open. After elimination of the C-terminal peptide, the remaining tail of the HydL=HynL polypeptide Sips over and locks the active centre into the protein matrix. The complex metal centre can only be removed from the assembled hydrogenase after an irreversible inactivation of the enzyme. It is worth noting that in the T. roseopersicina hydD gene, no typical transcriptional start codon or ribosome binding site could be identi:ed yet, suggesting that a thorough functional study is warranted. 2.2. Heterologous complementation studies The genes identi:ed, sequenced and partially characterised using transposon mutagenesis and sequence alignment need also to be analysed for their physiological function. One straightforward method for the functional tests is to transfer the gene in question, cloned on a suitable plasmid vector, into a homologous or heterologous host cell, which lacks this gene and check for the restored hydrogenase activity by complementation. Since T. roseopersicina strains lacking the accessory genes are not available yet, most of the complementation experiments have been done using the appropriate E. coli, R. capsulatus and =or R. eutropha deletion strains, obtained from our collaborating partners within the European basic research network COST Action 841 (Prof. Barbel Friedrich, Humboldt University, Berlin, DE; prof. Paulette M. Vignais CEA=CENG Grenoble, FR; Prof. August BTock, University of MTunich, DE). It is reasonable to assume that the accessory genes, particularly the pleiotropic ones will be functionally active in the heterologous host cell. The experiments have been completed in the case of hypC1, hypD and hypF. HypD from T. roseopersicina could not restore the corresponding function in E.coli, indicating signi:cant diKerences between the “assembly-line” of the two bacteria. In the case of hypC1, a strong background activity is observed in E. coli. HypC only participates in the assembly of the third hydrogenase of E. coli [13], the other two hydrogenases remained active in the RhypC E. coli strain and interfered with the measurements. This observation questions the truly pleiotropic nature of HypC in E. coli and, in agreement with this hypothesis, we have found a second hypC gene in T. roseopersicina. This suggests distinct hypC genes for the assembly of, at least some of, the Ni–Fe hydrogenases both in T. roseopersicina and in E. coli. Complementation with hypF gene from T. roseopersicina in a RhypF strain of R. capsulatus was successful, a clear

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demonstration that a functionally active form of this Thiocapsa gene product is synthesised by the R. capsulatus cells from the “foreign” template. The same experiment using a RhypF E. coli strain resulted in barely detectable complementation. We conclude that there must be strain-dependent variations in the complementation capacity and that the most thoroughly studied bacterium, E. coli, may not be the best choice for such complementation studies of hydrogenase assembly and biosynthesis.

Using the information, gathered on the function of the accessory proteins, reconstruction of the fascinating precise and miniature molecular assembly line becomes a realistic task. It is a long-term aim to discern how the H2 producing=consuming biocatalysts are made by Nature. Understanding the structure and function of this class of macromolecules and the elementary biosynthetic steps forming the functionally active hydrogenase oKers a significant advance towards the development of renewable and clean fuel, and thus, sustainable development supported by biotechnology.

3. Conclusions Properly engineered hydrogenases will be indispensable components of biohydrogen production and utilisation biotechnological devices that will be used in generation and utilisation of renewable energy for sustainable development. The research programme of our team centres on the NiFe hydrogenases: understanding the structure and function, assembly and regulation of biosynthesis of these complex macromolecules. Excellent laboratories in Europe, and worldwide, work on solving the complicated puzzle of biohydrogen catalysis. In collaboration with our EU partners within a highly eBcient scienti:c network (COST Action 841), and partly based on their experience, we have chosen the T. roseopersicina system to study the basic phenomena of assembling the NiFe hydrogenases containing a unique active centre composed of metal ions. T. roseopersicina turned out to be a very promising and suitable model system for several reasons: • The bacterium is cheap to cultivate, utilising sunshine to support its growth. • It contains every known form of NiFe hydrogenases and thus permits the study of their various properties without the disturbing eKects of strain-dependent variations. • It contains an exceptionally stable NiFe hydrogenase, which could be a good starting point in deciphering structure–function relationship of a stable macromolecular biocatalyst. Uncovering the way in which the hydrogen activating biocatalyst is assembled is a scienti:cally attractive challenge for basic and applied research. This knowledge will help us design future macromolecular catalysts for practical applications and it will contribute to our understanding of a speci:c class of macromolecules, the redox metalloenzymes. The molecular assembly line yielding an active and properly manufactured NiFe hydorgenase has many components. The most thrilling aspect of NiFe hydrogenase biosynthesis is the action of the accessory gene products that build the inactive polypeptides into a functional and active biocatalyst. Some of the accessory genes were identi:ed by transposon mutagenesis. The function of the gene products has been studied and is being studied by functional investigations: complementation and protein expression.

Acknowledgements The research team is supported by EU 5th Framework Programme projects (QLK5-1999-01267, QLK3-200001528, QLK3-2001-01676) and by domestic sources (OTKA, FKFP, OMFB, OM KFHAT). International collaboration the EU network COST Action 841 is greatly appreciated. We thank Profs. Barbel Friedrich, Paulette M. Vignais and August BTock for the strains and DNA samples provided. References [1] Cammack R, Robson RL, Frey M. editors. Hydrogen as a fuel: learning from nature. London: Taylor & Francis, 2001. [2] Kov-acs KL, Bagyinka CS. Structural properties and functional states of hydrogenase from Thiocapsa roseopersicina.. FEMS Microbiol Rev 1990;87:407–12. [3] Sasikala K, Ramana ChV, Rao PR, Kov-acs KL. Anoxygenic photosynthetic bacteria: physiology and advances in hydrogen production technology. Adv Appl Microbiol 1993;68:211–95. [4] Vignais PM, Toussaint B, Colbeau A. Regulation of hydrogenase gene expression. In: Blankenship RE, Madigan MT, Bauer CE, editors. Anoxygenic photosynthetic bacteria. Dordrecht: Kluwer Academic Publishers, 1993. p. 1175–90. [5] Colbeau A, Kov-acs KL, Chabert J, Vignais PM. Cloning and sequencing of the structural (hupSLC) and accessory (hupDHI) genes for hydrogenase biosynthesis in Thiocapsa roseopersicina.. Gene 1998;140:25–31. [6] R-akhely G, Colbeau A, Garin J, Vignais PM, Kov-acs KL. Unusual gene organization of HydSL, the stable [NiFe] hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina.. J Bacteriol 1998;180:1460–5. [7] Vignais PM, Billoud B, Mayer J. Classi:cation and phylogeny of hydrogenases. FEMS Microbiol Rev 2001;25:455–501. [8] Kov-acs KL, Tigyi G, Thanh LT, Lakatos S, Kiss Z, Bagyinka CS. Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina. J Biol Chem 1991;266:947–51. [9] Friedrich B, Schwartz E. Molecular biology of hydrogen utilization in aerobic chemolithotrophs. Annu Rev Microbiol 1993;47:351–83. [10] Elsen S, Colbeau A, Chabert J, Vignais PM. The hupTUV operon is involved in the negative control of hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol 1996;178:5174–81.

K.L. Kov0acs et al. / International Journal of Hydrogen Energy 27 (2002) 1463 – 1469 [11] ImhoK JF. True marine and halophilic anoxygenic phototrophic bacteria. Arch Microbiol 2001;176:243–54. - Kov-acs KL. Transposon [12] Fodor B, R-akhely G, Kov-acs AT, mutagenesis in purple sulfur photosynthetic bacteria: identi:cation of hypF, encoding a protein capable to process [NiFe] hydrogenases in ;  and  subdivision of proteobacteria. Appl Environ Microbiol 2001;67:2476–83. [13] Drapal N, BTock A. Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation. Biochemistry 1998;37:2941–8. [14] Tak-acs M, R-akhely G, Kov-acs KL. Molecular characterization and heterologous expression of hypCD, the :rst two [NiFe]

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hydrogenase accessory genes of Thermococcus litoralis.. Arch Microbiol 2001;176:231–5. [15] Theodoratou E, Paschos A, Mintz-Weber S, BTock A. Analysis of the cleavage site speci:city of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia Coli.. Arch Microbiol 2000;173:110–6. [16] Paschos A, Glass RS, BTock A. Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases. FEBS Lett 2001;488:9–12. [17] KortlTuke C, Horstmann K, Schwartz E, Rohde M, Binsack R, Friedrich B. A gene complex coding for the membrane bound hydrogenase of Alcaligenes eutrophus H16. J Bacteriol 1992;174:6277–89.