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In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Shewanella oneidensis MR-1 possesses a periplasmic [NiFe]-hydrogenase (MR-1 [NiFe]-H(2)ase) that has been implicated in H(2) production and oxidation as well as technetium [Tc(VII)] reduction. To characterize the roles of MR-1 [NiFe]-H(2)ase in these proposed reactions, the genes encoding both subunits of MR-1 [NiFe]-H(2)ase were cloned and then expressed in an MR-1 mutant without hyaB and hydA genes. Expression of recombinant MR-1 [NiFe]-H(2)ase in trans restored the mutant's ability to produce H(2) at 37% of that for the wild type. Following purification, MR-1 [NiFe]-H(2)ase coupled H(2) oxidation to reduction of Tc(VII)O(4)(-) and methyl viologen. Change of the buffers used affected MR-1 [NiFe]-H(2)ase-mediated reduction of Tc(VII)O(4)(-) but not methyl viologen. Under the conditions tested, all Tc(VII)O(4)(-) used was reduced in Tris buffer, while in HEPES buffer, only 20% of Tc(VII)O(4)(-) was reduced. The reduced products were soluble in Tris buffer but insoluble in HEPES buffer. Transmission electron microscopy analysis revealed that Tc precipitates reduced in HEPES buffer were aggregates of crystallites with diameters of ∼5 nm. Measurements with X-ray absorption near-edge spectroscopy revealed that the reduction products were a mixture of Tc(IV) and Tc(V) in Tris buffer but only Tc(IV) in HEPES buffer. Measurements with extended X-ray adsorption fine structure showed that while the Tc bonding environment in Tris buffer could not be determined, the Tc(IV) product in HEPES buffer was very similar to Tc(IV)O(2)·nH(2)O, which was also the product of Tc(VII)O(4)(-) reduction by MR-1 cells. These results shows for the first time that MR-1 [NiFe]-H(2)ase catalyzes Tc(VII)O(4)(-) reduction directly by coupling to H(2) oxidation.

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

Background: Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen., Methods and Findings: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon., Conclusions: Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such as the A. caulinodans Hyq endo-hydrogenase, offer promise as biocatalytic agents for H(2) production and/or consumption.

To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H(2)/mol glucose (maximum, 2 mol H(2)/mol glucose).

Sec-independent translocation systems have been characterised in Escherichia coli and other bacteria and differ from the Sec-dependent system by transporting fully folded proteins using the transmembrane proton electrochemical gradient. Proteins transported by this system bear a twin-arginine motif (tat) in the N-terminal signal peptide and include several cofactor-containing proteins. Azotobacter chroococcum strain (MCD124) has a soluble hydrogenase, which exhibited low O(2)-dependent H(2) uptake, and a shift in the pH of the culture to a more alkaline range during growth. We show that the DNA region capable of complementing this strain contains the tatABC genes and that mutations in the tatA gene reproduced the soluble hydrogenase and the culture pH shift phenotypes. We also show that insertional mutation in the tatC gene at a position corresponding to its C-terminal region had no effect on hydrogenase activity, but induced the pH shift of the culture. Sequence and mutagenesis analyses of this genomic region suggest that these genes form an operon that does not contain a tatD-like gene. A mutation in hupZ of the main hup gene region, coding for a possible b-type cytochrome also yielded a soluble hydrogenase, but not the pH-shift phenotype.

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.

In this work, we report the cloning and sequencing of the Azorhizobium caulinodans ORS571 hydrogenase gene cluster. Sequence analysis revealed the presence of 20 open reading frames hupTUVhypFhupSLCDFGHJK hypABhupRhypCDEhupE. The physical and genetic organization of A. caulinodans ORS571 hydrogenase system suggests a close relatedness to that of Rhodobacter capsulatus. In contrast to the latter species, a gene homologous to Rhizobium leguminosarum hupE was identified downstream of the hyp operon. A hupSL mutation drastically reduced the high levels of hydrogenase activity induced by the A. caulinodans ORS571 wild-type strain in symbiosis with Sesbania rostrata plants. However, no significant effects on dry weight and nitrogen content of S. rostrata plants inoculated with the hupSL mutant were observed in plant growth experiments.

Synthesis of the Rhizobium leguminosarum [NiFe] hydrogenase requires the participation of 16 accessory genes (hupCDEFGHIJKhypABFCDEX) besides the genes encoding the structural proteins (hupSL). Transcription of hupSL is controlled by a -24/-12-type promoter (P(1)), located upstream of hupS and regulated by NifA. In this work, a second -24/-12-type promoter (P(3)), located upstream of the hupG gene and transcribing hupGHIJ genes in R. leguminosarum pea (Pisum sativum L.) bacteroids, has been identified in the hup gene cluster. Promoter P(3) was also active in R. leguminosarum free-living cells, as evidenced by genetic complementation of hydrogenase mutants. Both NifA and NtrC activated P(3) expression in the heterologous host Klebsiella pneumoniae. Also, P(3) activity was highly stimulated by K. pneumoniae NifA in Escherichia coli. This NifA activation of P(3) expression only required the sigma(54)-binding site, and it was independent of any cis-acting element upstream of the sigma(54) box, which suggests a direct interaction of free NifA with the RNA polymerase holoenzyme. P(3)-dependent hupGHIJ expression in pea nodules started in interzone II/III, spanned through nitrogen-fixing zone III, and was coincident with the NifA-dependent nifH expression pattern. However, P(3) was dispensable for hupGHIJ transcription and hydrogenase activity in pea bacteroids due to transcription initiated at P(1). This fact and the lack of an activator recruitment system suggest that P(3) plays a secondary role in symbiotic hupGHIJ expression.

Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme. Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity. This finding prompted us to disrupt the two remaining unstudied hyp genes of H. pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed. In both mutants hydrogenase activity was abolished but urease activity remained unaffected. Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant. In addition, we also disrupted the genes HP0634 (referred as hydD in the H. pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process. In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.

Wolinella succinogenes grows by anaerobic respiration using hydrogen gas as electron donor. The hydE gene is located on the genome downstream of the structural genes encoding the membrane-bound NiFe-hydrogenase complex (HydABC) and a putative protease (HydD) possibly involved in hydrogenase maturation. Homologs of hydE are found in the vicinity of NiFe-hydrogenase-encoding genes on the genomes of several other proteobacteria. A hydE deletion mutant of W. succinogenes does not catalyze hydrogen oxidation with various electron acceptors. The hydrogenase iron-sulfur subunit HydA is absent in mutant cells whereas the apparently processed NiFe subunit (HydB) is located exclusively in the soluble cell fraction. It is suggested that HydE is involved in the maturation and/or stability of HydA or the HydAB complex in some, but not all bacteria containing NiFe-hydrogenases.

The first gene cluster encoding for a membrane bound [NiFe] hydrogenase from a methanotroph, Methylococcus capsulatus (Bath), was cloned and sequenced. The cluster consisted of the structural genes hupS and hupL and accessory genes hupE, hupC and hupD. A DeltahupSL deletion mutant of Mc. capsulatus was constructed by marker exchange mutagenesis. Membrane associated hydrogenase activity disappeared. The membrane associated hydrogenase appeared to have a hydrogen uptake function in vivo.

A 0.8 kb fragment of mbhS2 gene of Aquifex pyrophilus was obtained by PCR with designed primers basing mbhS2 gene of A. aeolicus. It showed 85% homology with the corresponding region of A. aeolicus. Using it as probe, a 5.0 kb Nco I fragment was fished out from the partial genomic library of A. pyrophilus. Then this fragment was cloned, subcloned and sequenced. The result revealed that the fragment contains the full length gene for the mbhS2, the gene orf1 and the first 366 bp of orf2. Comparison with mbhS2 and orf963 of A. aeolicus shows 81% and 60% homologies in amino acid sequence, respectively.

Maturation of [NiFe]-hydrogenases requires the action of several groups of accessory genes. Homologues of one group of these genes, the so-called hyp genes, putatively encoding proteins participating in the formation of an active uptake hydrogenase in the filamentous, heterocyst-forming cyanobacterium Nostoc PCC 73102, were cloned. The cluster, consisting of hypF, hypC, hypD, hypE, hypA, and hypB, is located 3.8 kb upstream from the uptake hydrogenase-encoding hupSL. Gene expression analyses show that these hyp genes are, like hupL, transcribed under N(2)-fixing but not under non-N(2)-fixing growth conditions. Furthermore, the six hyp genes are transcribed together with an open reading frame upstream of hypF, as a single mRNA. Analysis of the DNA region upstream of the experimentally determined transcriptional start site revealed putative -10 and -35 sequence elements and putative binding sites for the global nitrogen regulator NtcA.

The high degree of similarity that exists between all the [NiFe] hydrogenase operons and the near universality of hydrogen metabolism among microorganisms suggest that the microbial ability to metabolize hydrogen is of great importance and ancient origin. The large number of genes present in these operons, which are mostly involved in the maturation of the structural subunit, is indicative of the complexity of the hydrogenase molecular structure. Two main groups of maturation genes can be differentiated based on the resulting phenotypes when mutated: the 'cis-genes', encoding narrow specificity proteins, are mainly located on the same transcription unit as the structural genes, and the 'trans-genes', encoding broad specificity proteins, are located on a different operon. The maturation of the large subunit starts with the formation of a complex with the chaperone HypC, which remains bound to the amino terminus throughout processing. The ligands CN and CO, which are derived from carbamoylphosphate, are then inserted via HypF and probably other accessory proteins. HypB is responsible for nickel atom delivery in a GTP-hydrolysis-dependent reaction. The last identified step in the large subunit maturation process is proteolytic cleavage at the carboxyl terminus. The possible roles of the other maturation proteins are also discussed.

The gene encoding the [Fe]-hydrogenase from the anaerobic bacterium Megasphaera elsdenii has been cloned and sequenced. The gene is monocistronic, in keeping with the protein being a monomer. The translated protein sequence (484 residues, M(r)=53 kDa) comprises a small 2[4Fe-4S] ferredoxin-like domain and a large domain containing the catalytic site. Comparisons with other [Fe]-hydrogenase sequences, including two of which the crystal structures are known, show that the M. elsdenii protein is among the smallest of these enzymes and provide useful indications regarding the basic structural core common to all [Fe]-hydrogenases. It is, nevertheless, to be noted that the genome of Thermotoga maritima encodes a putative [Fe]-hydrogenase that would consist of only 301 residues.

A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H(2) productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3. 9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the alpha subunit of 'reverse' siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.

Rhizobium leguminosarum bv. viciae strain UPM791 induces in symbiosis with peas the synthesis of a nickel-containing hydrogenase which recycles the hydrogen evolved by nitrogenase. The genes required for synthesis of this hydrogenase, hupSLCDEFGHIJKhypABFCDEX, are clustered in the symbiotic plasmid. Analysis of a hypA-deficient mutant showed that HypA is essential for symbiotic hydrogenase activity and required for correct processing of the hydrogenase large subunit. Unlike other microoxically induced hyp genes, the hypA gene was only expressed in pea bacteroids from its own promoter. The hypA mRNA 5' end was mapped 109 bp upstream of the translational start codon. This distinct pattern of expression suggests a different role for HypA and the remaining Hyp proteins in hydrogenase synthesis.

Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1. Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A. eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies. Mutants with lesions in both copies showed clear alterations in hydrogenase activities. Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H2. Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium. Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor. Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes. HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms. Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site.

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX.

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.

Alcaligenes eutrophus H16 produces a soluble hydrogenase (SH) and a membrane-bound hydrogenase (MBH) which catalyze the oxidation of H2, supplying the organism with energy for autotrophic growth. The promoters of the structural genes for the SH and the MBH, PSH and PMBH, respectively, were identified by means of the primer extension technique. Both promoters were active in vivo under hydrogenase-derepressing conditions but directed only low levels of transcription under condition which repressed hydrogenase synthesis. The cellular pools of SH and MBH transcripts under the different growth conditions correlated with the activities of the respective promoters. Also, an immediate and drastic increase in transcript pool levels occurred upon derepression of the hydrogenase system. Both promoters were dependent on the minor sigma factor sigma 54 and on the hydrogenase regulator HoxA in vivo. PSH was stronger than PMBH under both heterotrophic and autotrophic growth conditions. The two promoters were induced at approximately the same rates upon derepression of the hydrogenase system in diauxic cultures. The response regulator HoxA mediated low-level activation of PSH and PMBH in a heterologous system.

The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2 in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated. Bacteroids from FixJ and FixK2 mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein. Bacteroids from a FixK1 mutant exhibit wild-type levels of hydrogenase activity. In beta-galactosidase transcriptional assays with NifA and FixK2 expression plasmids, the FixK2 protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression. The NifA protein does not activate transcription at the hydrogenase promoter. Therefore, FixK2 is involved in the transcriptional activation of symbiotic hydrogenase expression. By using beta-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site.

The genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples were determined in order to show in parallel the existing and active members of Desulfovibrio populations. DNA and total RNA were extracted from different anaerobic bioreactor samples; RNA was transcribed into cDNA. Subsequently, PCR was performed to amplify a ca.-440-bp fragment of the [NiFe] hydrogenase large-subunit gene and its mRNA. Denaturing gradient gel electrophoresis analysis was used to separate the PCR products according to their sequence and thereby to visualize the individual community members. Desulfovibrio strains corresponding to amplified [NiFe] hydrogenase transcripts were regarded as metabolically active, because in pure cultures transcripts were detectable in exponentially growing cells but not in cultures in the stationary phase. DNA sequencing and comparative sequence analysis were used to identify the detected organisms on the basis of their [NiFe] hydrogenase sequences. The genes of characterized Desulfovibrio spp. showed a considerable extent of divergence (ca. 30%), whereas sequences obtained from bacterial populations of the bioreactors showed a low level of variation and indicated the coexistence of closely related strains probably belonging to the species Desulfovibrio sulfodismutans. Under methanogenic conditions, all detected populations were active; under denitrifying conditions, no [NiFe] hydrogenase mRNA was visible. Changes in activity and composition of Desulfovibrio populations caused by changes in the environmental conditions could be monitored by using the approach described in this study.

The nucleotide sequence has been determined for a twelve-gene operon of Escherichia coli designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon is located at 55.8-56.0 min and encodes a putative nine-subunit hydrogenase complex (hydrogenase four or Hyf), a potential formate- and sigma 54-dependent transcriptional activator, HyfR (related to FhlA), and a possible formate transporter, FocB (related to FocA). Five of the nine Hyf-complex subunits are related to subunits of both the E. coli hydrogenase-3 complex (Hyc) and the proton-translocating NADH:quinone oxidoreductases (complex I and Nuo), whereas two Hyf subunits are related solely to NADH:quinone oxidoreductase subunits. The Hyf components include a predicted 523 residue [Ni-Fe] hydrogenase (large subunit) with an N-terminus (residues 1-170) homologous to the 30 kDa or NuoC subunit of complex I. It is proposed that Hyf, in conjunction with formate dehydrogenase H (Fdh-H), forms a hitherto unrecognized respiration-linked proton-translocating formate hydrogenlyase (FHL-2). It is likely that HyfR acts as a formate-dependent regulator of the hyf operon and that FocB provides the Hyf complex with external formate as substrate.

The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.

Among cyanobacteria, the heterocystous, N2-fixing Anabaena variabilis and the unicellular Anacystis nidulans have recently been shown to possess an NAD+-dependent, bidirectional hydrogenase. A 5.0 kb DNA segment of the A. nidulans genome is now identified to harbor the structural genes hoxUYH coding for three subunits of the bidirectional hydrogenase. The gene arrangement in A. nidulans and in A. variabilis is remarkably dissimilar. In A. nidulans, but not in A. variabilis, the four accessory genes hoxW, hypA, hypB and hypF could be identified downstream of hoxH. An insertional homozygous mutant in hoxH from A. nidulans was completely inactive in performing Na2S204-dependent H2 evolution but could utilize the gas with almost 50% of the activity of the wild type. These findings with the first defined hydrogenase mutant in any photosynthetic, 02-evolving microorganism indicate that the unicellular cyanobacterium A. nidulans possesses both an uptake and a bidirectional hydrogenase. The physiological role(s) of the two hydrogenases in unicellular non-N2-fixing cyanobacteria is not yet understood.

Alcaligenes eutrophus H16 produces two [NiFe] hydrogenases which catalyze the oxidation of hydrogen and enable the organism to utilize H2 as the sole energy source. The genes (hoxK and hoxG) for the heterodimeric, membrane-bound hydrogenase (MBH) are located adjacent to a series of eight accessory genes (hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, and hoxV). In the present study, we generated a set of isogenic mutants with in-frame deletions in the two structural genes and in each of the eight accessory genes. The resulting mutants can be grouped into two classes on the basis of the H2-oxidizing activity of the MBH. Class I mutants (hoxKdelta, hoxGdelta, hoxMdelta, hoxOdelta, and hoxQdelta) were totally devoid of MBH-mediated, H2-oxidizing activity. The hoxM deletion strain was the only mutant in our collection which was completely blocked in carboxy-terminal processing of large subunit HoxG, indicating that hoxM encodes a specific protease. Class II mutants (hoxZdelta, hoxLdelta, hoxRdelta, hoxTdelta, and hoxVdelta) contained residual amounts of MBH activity in the membrane fraction of the extracts. Immunochemical analysis and 63Ni incorporation experiments revealed that the mutations affect various steps in MBH maturation. A lesion in hoxZ led to the production of a soluble MBH which was highly active with redox dye.

A 2.8-kbp DNA region of Clostridium acetobutylicum ATCC 824 containing the putative hydrogenase gene (hydA) was cloned and sequenced. The 1,745-bp hydA encodes a 64,415-Da protein and presents strong identity with the [Fe] hydrogenase genes of Desulfovibrio and Clostridium species. The level of the putative hydA mRNA was high in cells from an acidogenic or an alcohologenic phosphate-limited continuous culture, while it was comparatively very low in cells from a solventogenic phosphate-limited continuous culture. These results were in agreement with the hydrogenase protein level, indicating that expression of hydA is regulated at the transcriptional level. Primer extension analysis identified a major transcriptional start site 90 bp upstream of the hydA start codon. The position of a putative rho-independent transcription terminator immediately downstream of the termination codon is in agreement with the size of the hydA transcript (1.9 kb) determined by Northern (RNA) blot experiments and confirms that the gene is transcribed as a monocistronic operon. Two truncated open reading frames (ORFs) were identified downstream and upstream of hydA and in opposite directions. The amino acid sequence deduced from ORF2 presents strong identity with ortho phosphoribosyl transferases involved in pyrimidine synthesis. The amino acid sequence deduced from ORF3 presents no significant similarity to any sequence in various available databases.

The hydA locus of Escherichia coli is known to encode some function necessary for formation of hydrogenase activity. The locus contains two open reading frames, hydN and hypF. In this communication, an analysis of the regulation of these two genes and of the phenotype of respective mutants is presented, Both genes were expressed in a T7 promoter/polymerase system, yielding a 19-kDa (HydN) and a 81-kDa (HypF) protein. In-frame deletions were constructed for each gene and transferred to the chromosome by homologous recombination. The mutation in hydN led to a decrease of the activity of formate dehydrogenase H (FDH-H) in crude extracts, but the activity and maturation of hydrogenases were nearly unaffected. In contrast, a deletion in hypF resulted in the loss of hydrogenase activity and in the synthesis of the large subunits of the hydrogenase isoenzymes 1, 2 and 3 in the inactive precursor form. For hydrogenase 3, it was shown that this is due to a lack of incorporation of nickel into the large subunit. hydN and hypF are organised in an operon that is a member of the formate regulon. Transcription was shown to be dependent on sigma 54 and FhlA, and an FhlA-binding site upstream of hydN was identified. The sigma 54-dependent promoter shows a rare deviation from the consensus at positions -24/-12, namely GG/GA instead of GG/GC. In conclusion, the product of hydN appears to have some role in electron flow from or to FDH-H, and the product of hypF is connected with maturation of all three hydrogenases of E. coli.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.

The membrane-bound hydrogenase (Hdg)-encoding structural genes were isolated from the hydrogen-utilizing bacterium Pseudomonas hydrogenovora (Ph). Nucleotide sequence analysis revealed two genes, hupS and hupL. The hupS gene encoded a 363-amino-acid (aa) polypeptide (40.4 kDa). The deduced aa sequence contained a putative 43-aa leader peptide sequence. The hupL gene started at 55 bp downstream from the stop codon of hupS and encoded a 622-aa polypeptide (69.3 kDa). The disruption of Ph hupS resulted in the loss of Hdg activity.

The putative products of six Azotobacter vinelandii chromosomal open reading frames (ORFs) were suggested to be involved in dihydrogen (H2) metabolism [Chen and Mortenson (1992) Biochim Biophys Acta 1131, 199-202]. A promoterless lacZ-containing cassette was used to disrupt the ORFs. Qualitative analysis revealed that the lacZ genes were expressed only in those mutants where the directions of the inserted lacZ were identical to those of the ORFs, showing that the six ORFs were transcribed as predicted. Unlike wildtype (w.t.), none of the mutants could perform dioxygen (O2)-dependent H2-oxidation, even though Western immunoanalyses showed that the hydrogenase large subunit was present although in amounts less than in w.t. Only one of the mutants (a hypB mutant), grown in nickel-enriched media, showed meaningful restoration of the H2-oxidizing ability. From the above observations it is concluded that (a) the six-ORF region is transcriptionally active and involved in H2-oxidation, (b) the product of hypB is needed for nickel activation of hydrogenase, and (c) the six ORFs (genes) belong to two or more operons. Possible roles of the gene products for the assembly, modification, and processing of hydrogenase from its apoproteins and metal centers are discussed.

The hydBGDA genes, which encode the four subunits beta, gamma, delta and alpha of the [Ni-Fe] hydrogenase from the archaeon Pyrococcus furiosus, have been isolated and sequenced using a PCR/IPCR-based strategy. From the sequence analysis it appears that the four structural genes are tightly linked and organized in a single transcription unit. The hydD and hydA gene products are related to the small and the large subunits of several archaeal and eubacterial [Ni-Fe] hydrogenases with an overall degree of sequence relatedness ranging from 35% to 50% (identity + similarity). In particular, the amino acid sequence motifs involved in the accommodation of nickel and iron-sulfur clusters are conserved. In addition, the database search revealed that the hydB and hydG gene products are homologous to the asrA- and asrB-encoded subunits of the sulfite reductase enzyme from Salmonella typhimurium. This is particularly interesting in view of the recent finding that the P. furiosus hydrogenase appears to be a bifunctional enzyme endowed with both proton- and sulfur-reducing activities.

Using a transcriptional fusion to the lacZ gene, we have analyzed the anaerobic regulation of the hydrogenase 1 (hya) operon in response to different anaerobic growth conditions and to mutations in regulatory genes. We found that the transcription of the hya operon was induced when the growth condition was changed from aerobic to anaerobic and that this induction was independent of Fnr but dependent on regulators AppY and ArcA. Furthermore, we found that the transcription of the hya operon was not regulated by the cyclic AMP-cyclic AMP receptor protein complex. Investigation of the effects of different anaerobic growth conditions on the expression of the hya operon showed that expression was induced by formate and repressed by nitrate. Formate induction was not mediated by the fhlA gene product, and nitrate repression was not mediated by the narL gene product. We found a high level of anaerobic expression of the hya operon in glucose medium supplemented with formate and in glycerol medium supplemented with fumarate, suggesting that hydrogenase isoenzyme 1 has a function during both fermentative growth and anaerobic respiration.

A 2.0-kb DNA fragment downstream from the hydrogenase-encoding structural genes within the hydrogenase gene cluster of Bradyrhizobium japonicum was sequenced. Analysis of the nucleotide (nt) sequence revealed three open reading frames (ORFs), designated hupC, hupD and hupF, which encode polypeptides of 28, 21 and 10.7 kDa, respectively. Based on analysis of the nt sequence and physiological studies, hupSL (hydrogenase structural genes) and hupCDF are organized as a single transcriptional unit. Plasmid pRY12 carrying hupSL genes did not complement (restore) hydrogenase activity of the hupSL deletion mutant strain (JHCS2), whereas the activity of the mutant was considerably restored by pLD22 harboring the entire hydrogenase operon (hupSLCDF genes). Western blots revealed a very low level of hydrogenase protein in JHCS2 containing pRY12. The results suggest that the products of the hupCDF genes may be involved in either stabilizing the hydrogenase peptides (i.e., from degradation) or in post-translational regulation of hydrogenase production. The products of hupC and hupD were successfully expressed in Escherichia coli by a phage T7 promoter system, although the apparent sizes of the gene products were slightly larger than those calculated from the deduced amino-acid sequences.

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.

The nucleotide sequence was obtained for the hypE gene in the cluster of structural and accessory genes required for the assembly and functioning of the membrane-bound, dimeric, (NiFe)hydrogenase in Azotobacter vinelandii. The hypE gene encodes a polypeptide of 341 amino acid residues which is rich in alanine, glycine, valine and proline and appears to be involved in maturation of the enzyme because chromosomal mutations in hypE block O2-dependent H2-oxidation and affect the amount, processing and localization of the (NiFe) hydrogenase alpha-subunit. The complete nucleotide sequence for the hydrogenase gene cluster in A. vinelandii has now been assembled into a contiguous sequence of 13,914 bp containing 16 potential genes which appear to be transcribed undirectionally. They are arranged in the order hoxK, hoxG, hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, hoxV, hypA, hypB, hypF, hypC, hypD and hypE. This cluster closely resembles those described for comparable (NiFe) hydrogenases in other bacteria.

The nucleotide sequence of a 2.2 kb region downstream of the hydrogenase structural genes in Bradyrhizobium japonicum was determined. Four genes encoding predicted polypeptides of 27.8 (HupC), 21.4 (HupD), 10.6 (HupF) and 15.8 (HupG) kDa were identified, of which the first three probably belong to the same operon as the hup structural genes, hupS and hupL. HupC is homologous to the hydrophobic polypeptides with four potential transmembrane regions that are encoded by open reading frames following the hydrogenase structural genes in Rhodobacter capsulatus, Escherichia coli, Azotobacter vinelandii, Wolinella succinogenes, Rhizobium leguminosarum and Alcaligenes eutrophus. Also HupD, HupF and HupG are homologous to genes involved in processing, maturation, functioning and regulation of hydrogenase activity in various hydrogen-oxidizing bacteria.

The gene locus nuo of the proton-translocating NADH: ubiquinone oxidoreductase in Escherichia coli was identified by means of a DNA probe made by the polymerase chain reaction. The primers used for the reaction were derived from consensus sequences of the NAD(H)-binding subunit of mitochondrial NADH: ubiquinone oxidoreductase and the NAD(+)-reducing hydrogenase of Alcaligenes eutrophus. The nuo locus lies between minutes 49.2 and 49.6 on the E. coli chromosome and contains a cluster of 14 genes. They are bordered upstream by a sequence resembling sigma 70-dependent promoters and downstream by a sequence resembling rho-independent terminators. All 14 proteins derived from the nuo-genes are related to subunits of mitochondrial NADH: ubiquinone oxidoreductase, among which all subunits presumed to bind the substrates and to harbour the redox groups are found, as well as all seven mitochondrially encoded subunits. The E. coli enzyme seems to represent the minimal form of a proton-translocating NADH: ubiquinone oxidoreductase. The gene order in the nuo locus is conserved in comparison with other bacterial genomes and the chloroplast genome of higher plants. To some extent, the gene order correlates with the topological arrangement of the encoded subunits. The conception of modular evolution of NADH: ubiquinone oxidoreductase is further supported by the arrangement of the nuo-genes.

The Azotobacter chroococcum chromosome contains a region spanning about 14 kb associated with hydrogen-uptake (Hup) activity. The small and large subunits of the hydrogenase are encoded by the structural genes hupS and hupL. Two other genes, hupD and hupE, are located 8.9 kb downstream from hupL and are required for the formation of a catalytically active hydrogenase. In this study, we determined the nucleotide sequence of a 3.8-kb region immediately upstream from hupD. This revealed four additional closely linked ORFs which we designated hupA, hupB, hupY and hupC; these genes potentially encode polypeptides with predicted masses of 12.6, 33.3, 80.4 and 9.0 kDa, respectively. This cluster of genes was shown to be essential for hydrogenase activity by insertion mutagenesis using antibiotic-resistance gene cassettes and a Tn5 derivative carrying a promoterless lacZ gene. A 10.5-kb fragment of DNA beginning 3.4 kb downstream from hupL, and including the sequenced region, was able to complement hupA and hupY mutants, supporting earlier evidence for a promoter downstream from hupSL. The deduced amino acid sequences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively. Of particular interest is the fact that there is no homologue of the hupY gene product in the E. coli hyp operon. Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fusion, showing that hupY and hupB are not major factors in regulating the transcription of the hydrogenase structural genes.

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX18CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH- and MBH-specific antigen. Growing the cells in the presence of 63Ni2+ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.