Hydrogenase Gene Research Articles

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Freshly isolated soil bacteria were screened for different characteristics of the H2 metabolism without prior selection for growth on H2. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H2, (2) for chemolithoautotrophic growth on H2 as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum, and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H2. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H2 indicating that the TTC test was not a specific characteristic for H2 oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca, but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H2 oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H2 oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.

Programmed DNA rearrangement of a cyanobacterial hupL gene in heterocysts.

Programmed DNA rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome. Six accessory genes of the cluster, hupABYCDE, begin 4·8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional [NiFe] hydrogenase. The sequencing of the intervening 4·8 kb of hup-specific DNA has now been completed. This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT, and hupV. These genes potentially encode polypeptides with predicted masses of 27·7, 22·3, 11·4, 16·2, 31·38, 8·1, 16·2 and 36·7 kDa, respectively. All eight genes are transcribed from the same strand as hupSL and hupABYCDE. A. chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE. The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome. Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O 2-dependent H 2 oxidation. The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins. HupZ and HupR may both be involved in transferring electrons from hydrogenease to the electron transport chain. A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase. It is now clear that the genes controlling [NiFe] hydrogenase activity in may bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.

From the molecular biology of Desulfovibrio to a novel method for defining bacterial communities in oil field environments

Sulfate-reducing bacteria (SRB) of the genus Desulfovibrio have up to three different hydrogenases all catalyzing the reaction H 2 ⇌ 2H + + 2e. These enzymes allow Desulfovibrio to derive energy for maintenance and growth from the lithotrophic reaction: 4H 2 + SO 4 2− → S 2− + 4H 2O. One of the hydrogenases, the [NiFe] hydrogenase, is present in all of 22 Desulfovibrio species that were investigated. Use of the gene for this hydrogenase as a Desulfovibrio-specific probe allowed the presence of different Desulfovibrio species in oil field production waters to be detected. These results provided only a partial description of the SRB population, because Desulfovibrio is only one of several SRB genera present. These other genera were not detectable with the hydrogenase gene probe. In searching for a more generally applicable DNA hybridization method it was found that the entire chromosomal DNA of a given species, can serve as a specific probe for its detection. Total community DNA isolated from oil field samples can be economically analyzed by a reverse genome probe approach, which has indicated that the sessile population present at metal surfaces is often dominated by selected Desulfovibrio species. This finding is a first step in a more detailed analysis of the roles of these bacteria in metal corrosion and oil field souring, which have a negative impact on the processing of oil.

Organization of the hydrogenase gene cluster from Bradyrhizobium japonicum: sequences and analysis of five more hydrogenase-related genes.

Previously, the deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the Bradyrhizobium japonicum hydrogenase structural genes caused lack of normal-sized hydrogenase (Hup) subunits and complete loss of Hup activity. It was suggested that this region encodes one or more genes required for Hup processing. Sequencing of a 3322-bp XcmI fragment of DNA covering this 2.9-kb EcoRI fragment within the hup gene cluster revealed the presence of five open reading frames (ORFs) designated hupG, hupH, hupI, hupJ and hupK, encoding polypeptides with calculated molecular masses of 15.8, 30.7, 7.6, 18.1 and 38 kDa, respectively. Based on deduced amino acid (aa) sequences, all five products of the hupGHIJK genes showed significant homology with other genes' products in several H2-utilizing bacteria. Of particular interest are HupG and HupI. HupG showed 70% similarity (28% identity) to the HyaE of the Escherichia coli hydrogenase-1 operon which was demonstrated to be involved in the processing of hydrogenase-1. HupI showed strong identity to rubredoxin and rubredoxin-like proteins from many other bacteria. The latter proteins contain two 'C-X-X-C' motifs, which may serve as iron ligands for non-heme iron proteins involved as intermediate electron carriers or in the assembly process for Fe-S (or NiFe-S) clusters.

Sequence and characterization of three genes within the hy drogenase gene cluster of Bradyrhizobium japonicum

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 hypE Gene Completes the Gene Cluster for H 2-oxidation in Azotobacter vinelandii

The nucleotide sequences 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 O 2-dependent H 2-oxidation and affect the amount, processing and localization of the (NiFe) hydrogenase α-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 unidirectionally. 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.

Cloning and sequences 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.

ThehupB gene of theAzotobacter chroococcum hydrogenase gene cluster is involved in nickel metabolism

InAzotobacter chroococcum the hydrogenase gene (hup) cluster spans about 14 kb of DNA. The genes coding for the small and large subunits,hupSL, are located at the 5′ end, and a cluster of genes,hupABYCDE, resembling theEscherichia coli hyp operon, is located at the 3′ end. In this study, we determined the effect of adding nickel to the medium used for the growth ofhup mutants. Hydrogenase activity was restored tohupA andhupB mutants, but nothupY, hupD, orhupE mutants, by the addition of nickel to the growth medium, suggesting that the products ofhupA andhupB are somehow involved in nickel metabolism. The restoration of hydrogenase activity to thehupB mutant required protein synthesis.

Hydrogen uptake by Robinia pseudoacacia nodules

Hydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H2 produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae.

Hydrogenase in Bradyrhizobium japonicum: genetics, regulation and effect on plant growth

A region of 15 kbp DNA located on the chromosome of Bradyrhizobium japonicum is essential for a Hup(+) phenotype and contains the hydrogenase structural genes. During the last few years, other genes with various functions related to hydrogenase oxidation, such as Ni(2+) incorporation into the hydrogenase enzyme, electron transport from hydrogenase to O2, and regulation of hydrogenase expression in free-living conditions, have been identified in this region. A region in front of the hydrogenase structural genes is necessary for transcriptional regulation of hydrogenase expression by O2, H2 and Ni(2+). In addition, the N2 fixation system and the tertiary structure of the chromosome seem to be involved in the expression of the hydrogenase genes. The effect of legume inoculation with Hup(+) rhizobia has been evaluated. The possible benefits of a Hup(+) phenotype, such as the regeneration of chemical energy in the form of ATP or reductants, and the removal of O2 and H2 from the active site of the nitrogenase enzyme where they might inhibit the nitrogenase reaction, are discussed. The data indicate that a H2-uptake system is beneficial in soybean nodules, but host plant and environmental factors may interfere with the effects of H2 cycling in the plant.

Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16.

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.

Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae.

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.

The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC

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 Tn 5 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 hyp A, 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 β-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.

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.

Nucleotide sequence and organization of an H 2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames

The nucleotide sequence of a 3·2 kb region following the hydrogenase structural operon ( hupSLCDEF) in the H 2-uptake gene cluster from Rhizobium leguminosarum bv 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 H 2-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 hupGHTJK 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.

Identification of Distinct Communities of Sulfate-Reducing Bacteria in Oil Fields by Reverse Sample Genome Probing

Thirty-five different standards of sulfate-reducing bacteria, identified by reverse sample genome probing and defined as bacteria with genomes showing little or no cross-hybridization, were in part characterized by Southern blotting, using 16S rRNA and hydrogenase gene probes. Samples from 56 sites in seven different western Canadian oil field locations were collected and enriched for sulfate-reducing bacteria by using different liquid media containing one of the following carbon sources: lactate, ethanol, benzoate, decanoate, propionate, or acetate. DNA was isolated from the enrichments and probed by reverse sample genome probing using master filters containing denatured chromosomal DNAs from the 35 sulfate-reducing bacterial standards. Statistical analysis of the microbial compositions at 44 of the 56 sites indicated the presence of two distinct communities of sulfate-reducing bacteria. The discriminating factor between the two communities was the salt concentration of the production waters, which were either fresh water or saline. Of 34 standards detected, 10 were unique to the fresh water and 18 were unique to the saline oil field environment, while only 6 organisms were cultured from both communities.

Hydrogen production by cyanobacteria

Cyanobacteria are capable of biophotolysis, the light-driven splitting of water into hydrogen and oxygen, in reactions which involve the enzymes nitrogenase and hydrogenase. Anabaena cylindrica possesses an integral membrane hydrogenase and a soluble hydrogenase, both of which are nickel-dependent. The release of nitrogenase-catalyzed hydrogen can be controlled chemically and also by manipulating nickel levels in the growth medium, suggesting that selection of uptake hydrogenase-deficient mutants is an important objective for maximizing biophotolysis. We have cloned and sequenced the hydrogenase gene for the soluble hydrogenase of A. cylindrica with a view to genetically manipulating the hydrogen metabolism of this and other cyanobacteria.

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.

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.