Transcriptional regulation of the uptake [NiFe]hydrogenase genes in Rhodobacter capsulatus.

Transcription of the hupSL genes, which encode the uptake [NiFe]hydrogenase of Rhodobacter capsulatus, is specifically activated by H(2). Three proteins are involved, namely the H(2)-sensor HupUV, the histidine kinase HupT and the transcriptional activator HupR. hupT and hupUV mutants have the same phenotype, i.e. an increased level of hupSL expression (assayed by phupS::lacZ fusion) in the absence of H(2); they negatively control hupSL gene expression. HupT can autophosphorylate its conserved His(217), and in vitro phosphotransfer to Asp(54) of its cognate response regulator, HupR, was demonstrated. The non-phosphorylated form of HupR binds to an enhancer site (5'-TTG-N(5)-CAA) of phupS localized at -162/-152 nt and requires integration host factor to activate fully hupSL transcription. HupUV is an O(2)-insensitive [NiFe]hydrogenase, which interacts with HupT to regulate the phosphorylation state of HupT in response to H(2) availability. The N-terminal domain of HupT, encompassing the PAS domain, is required for interaction with HupUV. This interaction with HupT, leading to the formation of a (HupT)(2)-(HupUV)(2) complex, is weakened in the presence of H(2), but incubation of HupUV with H(2) has no effect on the stability of the heterodimer/tetramer, HupUV-(HupUV)(2), equilibrium. HupSL biosynthesis is also under the control of the global two-component regulatory system RegB/RegA, which controls gene expression in response to redox. RegA binds to a site close to the -35 promoter recognition site and to a site overlapping the integration host factor DNA-binding site (5'-TCACACACCATTG, centred at -87 nt) and acts as a repressor.

Molecular biology of microbial hydrogenases.

Hydrogenases (H2ases) are metalloproteins. The great majority of them contain iron-sulfur clusters and two metal atoms at their active center, either a Ni and an Fe atom, the [NiFe]-H2ases, or two Fe atoms, the [FeFe]-H2ases. Enzymes of these two classes catalyze the reversible oxidation of hydrogen gas (H2 <--> 2 H+ + 2 e-) and play a central role in microbial energy metabolism; in addition to their role in fermentation and H2 respiration, H2ases may interact with membrane-bound electron transport systems in order to maintain redox poise, particularly in some photosynthetic microorganisms such as cyanobacteria. Recent work has revealed that some H2ases, by acting as H2-sensors, participate in the regulation of gene expression and that H2-evolving H2ases, thought to be involved in purely fermentative processes, play a role in membrane-linked energy conservation through the generation of a protonmotive force. The Hmd hydrogenases of some methanogenic archaea constitute a third class of H2ases, characterized by the absence of Fe-S cluster and the presence of an iron-containing cofactor with catalytic properties different from those of [NiFe]- and [FeFe]-H2ases. In this review, we emphasise recent advances that have greatly increased our knowledge of microbial H2ases, their diversity, the structure of their active site, how the metallocenters are synthesized and assembled, how they function, how the synthesis of these enzymes is controlled by external signals, and their potential use in biological H2 production.

Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H(2)-signaling HupUV hydrogenase in Rhodobacter capsulatus.

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.

Expression of uptake hydrogenase and molybdenum nitrogenase in Rhodobacter capsulatus is coregulated by the RegB-RegA two-component regulatory system.

Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H(2) oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but not nifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required for nifA2 expression, RegA acts as a coactivator of nifA2. We also demonstrated that in reg mutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H(2) oxidation, as well as the energy-demanding processes of N(2) fixation and CO(2) assimilation.

The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two-component HupT/HupR system.

The synthesis of the membrane-bound [NiFe]hydrogenase of Rhodobacter capsulatus (HupSL) is regulated negatively by the protein histidine kinase, HupT, and positively by the response regulator, HupR. It is demonstrated in this work that HupT and HupR are partners in a two-component signal transduction system. The binding of HupR protein to the hupS promoter regulatory region (phupS ) was studied using gel retardation and footprinting assays. HupR protected a 50 bp region localized upstream from the binding site of the histone-like integration host factor (IHF) regulator. HupR, which belongs to the NtrC subfamily, binds to an enhancer site (TTG-N5-CAA) localized at -162/-152 nt. However, the enhancer-binding HupR protein does not require the RpoN sigma factor for transcriptional activation, as is the case for NtrC from enteric bacteria, but functions with sigma70-RNA polymerase, as is the case for R. capsulatus NtrC. Besides, unlike NtrC from Escherichia coli, HupR activates transcription in the unphosphorylated form and becomes inactive by phosphorylation. This was demonstrated by replacing the putative phosphorylation site (D54) of the HupR protein with various amino acids or by deleting it using site-directed mutagenesis. Strains expressing mutated hupR genes showed high hydrogenase activities even in the absence of H2, indicating that hupSL transcription is activated by the binding of unphosphorylated HupR protein. Strains producing mutated HupRD54 proteins were derepressed for hupSL expression as were HupT- mutants. It is shown that the phosphorylated form of HupT was able to transfer phosphate to wild-type HupR protein but not to mutated D54 HupR proteins. Thus, it is concluded that HupT and HupR are the partners of a two-component regulatory system that regulates hupSL gene transcription.

Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins.

The photosynthetic bacterium Rhodobacter capsulatus contains a membrane-bound [NiFe]hydrogenase encoded by the hupSL genes. We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein. We also observed that, in contrast to the wild-type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19. Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild-type levels and inducibility by H2. The R. capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively. Active HupUV proteins can catalyze the hydrogen-deuterium exchange reaction. In whole cells, this H-D exchange is distinguishable from the H-D exchange catalyzed by the membrane-bound HupSL proteins by its insensitivity to O2 and to acetylene. By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins. H-D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation. These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2.

Unusual organization of the genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina BBS.

The characterization of a hyd gene cluster encoding the stable, bidirectional [NiFe]hydrogenase 1 enzyme in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium belonging to the family Chromatiaceae, is presented. The heterodimeric hydrogenase 1 had been purified to homogeneity and thoroughly characterized (K. L. Kovacs et al., J. Biol. Chem. 266:947-951, 1991; C. Bagyinka et al., J. Am. Chem. Soc. 115:3567-3585, 1993). As an unusual feature, a 1,979-bp intergenic sequence (IS) separates the structural genes hydS and hydL, which encode the small and the large subunits, respectively. This IS harbors two sequential open reading frames (ORFs) which may code for electron transfer proteins ISP1 and ISP2. ISP1 and ISP2 are homologous to ORF5 and ORF6 in the hmc operon, coding for a transmembrane electron transfer complex in Desulfovibrio vulgaris. Other accessory proteins are not found immediately downstream or upstream of hydSL. A hup gene cluster coding for a typical hydrogen uptake [NiFe]hydrogenase in T. roseopersicina was reported earlier (A. Colbeau et al. Gene 140:25-31, 1994). The deduced amino acid sequences of the two small (hupS and hydS) and large subunit (hupL and hydL) sequences share 46 and 58% identity, respectively. The hup and hyd genes differ in the arrangement of accessory genes, and the genes encoding the two enzymes are located at least 15 kb apart on the chromosome. Both hydrogenases are associated with the photosynthetic membrane. A stable and an unstable hydrogenase activity can be detected in cells grown under nitrogen-fixing conditions; the latter activity is missing in cells supplied with ammonia as the nitrogen source. The apparently constitutive and stable activity corresponds to hydrogenase 1, coded by hydSL, and the inducible and unstable second hydrogenase may be the product of the hup gene cluster.

Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus.

The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons. The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of [gamma-32P]ATP. An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity. This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue.

HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction.

The H-D exchange reaction has been measured with the D2-H2O system, for Rhodobacter capsulatus JP91, which lacks the hupSL-encoded hydrogenase, and R. capsulatus BSE16, which lacks the HupUV proteins. The hupUV gene products, expressed from plasmid pAC206, are shown to catalyze an H-D exchange reaction distinguishable from the H-D exchange due to the membrane-bound, hupSL-encoded hydrogenase. In the presence of O2, the uptake hydrogenase of BSE16 cells catalyzed a rapid uptake and oxidation of H2, D2, and HD present in the system, and its activity (H-D exchange, H2 evolution in presence of reduced methyl viologen [MV+]) depended on the external pH, while the H-D exchange due to HupUV remained insensitive to external pH and O2. These data suggest that the HupSL dimer is periplasmically oriented, while the HupUV proteins are in the cytoplasmic compartment.

The Rhodobacter capsulatus hupSLC promoter: identification of cis-regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription.

The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H2. H2 activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 5' deletions of the 300bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions. Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography. IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site. The HupR protein binds to one site within the -162 to -152 nt region, which contains the palindromic sequence 5'-TTG-R5-CAA. By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2.

The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus.

The hupT, hupU, and hupV genes, which are located upstream from the hupSLC and hypF genes in the chromosome of Rhodobacter capsulatus, form the hupTUV operon expressed from the hupT promoter. The hupU and hupV genes, previously thought to belong to a single open reading frame, encode HupU, of 34.5 kDa (332 amino acids), and HupV, of 50.4 kDa (476 amino acids), which are >/= 50% identical to the homologous Bradyrhizobium japonicum HupU and HupV proteins and Rhodobacter sphaeroides HupU1 and HupU2 proteins, respectively; they also have 20 and 29% similarity with the small subunit (HupS) and the large subunit (HupL), respectively, of R. capsulatus [NiFe]hydrogenase. HupU lacks the signal peptide of HupS and HupV lacks the C-terminal sequence of HupL, which are cleaved during hydrogenase processing. Inactivation of hupV by insertional mutagenesis or of hupUV by in-frame deletion led to HupV- and Hup(UV)- mutants derepressed for hydrogenase synthesis, particularly in the presence of oxygen. These mutants were complemented in trans by plasmid-borne hupTUV but not by hupT or by hupUV, except when expressed from the inducible fru promoter. Complementation of the HupV- and Hup(UV)- mutants brought about a decrease in hydrogenase activity up to 10-fold, to the level of the wild-type strain B10, indicating that HupU and HupV participate in negative regulation of hydrogenase expression in concert with HupT, a sensor histidine kinase involved in the repression process. Plasmid-borne gene fusions used to monitor hupTUV expression indicated that the operon is expressed at a low level (50- to 100-fold lower than hupS).

Identification of the rpmF-plsX-fabH genes of Rhodobacter capsulatus.

The rpmF-plsX-fabH gene cluster of Rhodobacter capsulatus homologous to that of Escherichia coli was identified. rpmF encodes ribosomal protein L32, plsX plays an undefined role in membrane lipid synthesis, and fabH encodes beta-ketoacyl-acyl carrier protein synthase III. The R. capsulatus plsX gene complemented a defect in an E. coli strain with the plsX50 mutation. Overproduction of the fabH gene product of R. capsulatus in E. coli resulted in dramatically increased beta-ketoacyl-acyl carrier protein synthase III activity. These results indicate that plsX and fabH apparently function the same in R. capsulatus as in E. coli.

Cloning and sequence of the structural (hupSLC) and accessory (hupDHI) genes for hydrogenase biosynthesis in Thiocapsa roseopersicina.

The first molecular biology study on the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is reported, namely, the construction of cosmid libraries and isolation of a hydrogenase gene cluster by hybridization with hydrogenase structural genes from the purple non-sulfur bacterium, Rhodobacter capsulatus. The sequenced gene cluster contains six open reading frames, the products of which show significant degrees of identity (from 40 to 78%) with hydrogenase gene products necessary for biosynthesis of the group-I of [NiFe]hydrogenases. The structural hupSLC genes encode the small and large hydrogenase subunits and a hydrophobic protein shown to accept electrons from hydrogenase in R. capsulatus. They are followed downstream by three genes, hupDHI, which are similar to hydrogenase accessory genes found in other bacteria.

Sequence analysis and interposon mutagenesis of the hupT gene, which encodes a sensor protein involved in repression of hydrogenase synthesis in Rhodobacter capsulatus.

The hupT gene, which represses hydrogenase gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus, has been identified and sequenced. The nucleotide sequence of hupT and of the contiguous downstream open reading frame, hupU, is reported. The HupT protein of 456 amino acids (48,414 Da) has sequence similarity with the FixL, DctB, NtrB, and ArcB proteins and is predicted to be a soluble sensor kinase. Insertional inactivation of the hupT gene led to deregulation of transcriptional control, so that the hydrogenase structural operon hupSLC became overexpressed in cells grown anaerobically or aerobically. The HupT- mutants were complemented in trans by a plasmid containing an intact copy of the hupT gene. The hupU open reading frame, capable of encoding a protein of 84,879 Da, shared identity with [NiFe]hydrogenase subunits; the strongest similarity was observed with the periplasmic hydrogenase of Desulfovibrio baculatus.

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants.

A 25 kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium leguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF-hupS-hupL-hupM-hu pD-hupF-hupG-hupH-hupJ-hupK-hypA- hypB-hupR1- hypC-hypD-hypE-ORF19-ORF20, all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup- mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD gene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant prevented the expression of a hupS::lacZ translational fusion from being stimulated by H2 as it is observed in the wild-type strain B10. It is inferred that the HypF protein is a factor involved in H2 stimulation of hydrogenase expression.

Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2.

The Escherichia coli beta-galactosidase enzyme was used as a reporter molecule for genetic fusions in Rhodobacter capsulatus. DNA fragments that were from the upstream region of the hydrogenase structural operon hupSLM and contained 5' hupS sequences were fused in frame to a promoterless lacZ gene, yielding fusion proteins comprising the putative signal sequence and the first 22 amino acids of the HupS protein joined to the eight amino acid of beta-galactosidase. We demonstrate the usefulness of the hupS::lacZ fusion in monitoring regulation of hydrogenase gene expression. The activities of plasmid-determined beta-galactosidase and chromosome-encoded hydrogenase changed in parallel in response to various growth conditions (light or dark, aerobiosis or anaerobiosis, and presence or absence of ammonia or of H2), showing that changes in hydrogenase activity were due to changes in enzyme synthesis. Molecular hydrogen stimulated hydrogenase synthesis in dark, aerobic cultures and in illuminated, anaerobic cultures. Analysis of hupS::lacZ expression in various mutants indicated that neither the hydrogenase structural genes nor NifR4 (sigma 54) was essential for hydrogen regulation of hydrogenase synthesis.

A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression.

A gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2.

The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase.

The hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin-resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzylviologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.

Identification and sequence analysis of the hupR1 gene, which encodes a response regulator of the NtrC family required for hydrogenase expression in Rhodobacter capsulatus.

The hupR1 gene from Rhodobacter capsulatus was cloned and sequenced. It can encode a protein of 53,843 Da which shares significant similarity with several transcriptional regulators and activates transcription of the structural hupSL genes of [NiFe]hydrogenase, as shown by the use of a translational fusion of lacZ with the hupSL promoter. A Hup- mutant having a point mutation in the hupR1 gene is described.

Molecular biology studies of the uptake hydrogenase of Rhodobacter capsulatus and Rhodocyclus gelatinosus.

In the photosynthetic bacteria, as in other N2-fixing bacteria, two main enzymes are involved in H2 metabolism: nitrogenase, which catalyses the photoproduction of H2, and a membrane-bound (NiFe) hydrogenase, which functions as an H2-uptake enzyme. The structural genes for Rhodobacter capsulatus and Rhodocyclus gelatinosus uptake hydrogenases were isolated and sequenced. They present the same organization, with the gene encoding the small subunit (hupS) (molecular masses 34.2 and 34.6 kDa, respectively) preceding the gene encoding the large one (hupL) (molecular masses 65.8 and 68.5 kDa, respectively). The two hupSL genes apparently belong to the same operon. The deduced protein sequences of the small and of the large subunits share nearly 80% and maximally 70% identity, respectively, with their counterparts in uptake hydrogenases found in N2-fixing bacteria. However, unlike in Bradyrhizobium japonicum, R. gelatinosus or Azotobacter chroococcum, another open reading frame (ORFX) was found downstream and contiguous to the R. capsulatus hupSL whose transcription seemed to depend on the same hup promoter as hupSL. ORFX contained 786 nucleotides capable of encoding a hydrophobic polypeptide of 262 amino acids (30.2 kDa).