Gene expression by the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough grown on an iron electrode under cathodic protection conditions

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of -1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion.     

Distribution of Hydrogenase Genes in Desulfovibrio spp. and Their Use in Identification of Species from the Oil Field Environment

The distribution of genes for [Fe], [NiFe], and [NiFeSe] hydrogenases was determined for 22 Desulfovibrio species. The genes for [NiFe] hydrogenase were present in all species, whereas those for the [Fe] and [NiFeSe] hydrogenases had a more limited distribution. Sulfate-reducing bacteria from 16S rRNA groups other than the genus Desulfovibrio (R. Devereux, M. Delaney, F. Widdel, and D. A. Stahl, J. Bacteriol. 171:6689-6695, 1989) did not react with the [NiFe] hydrogenase gene probe, which could be used to identify different Desulfovibrio species in oil field samples following growth on lactate-sulfate medium.     

Selenium is involved in regulation of periplasmic hydrogenase gene expression in Desulfovibrio vulgaris Hildenborough

Desulfovibrio vulgaris Hildenborough is a good model organism to study hydrogen metabolism in sulfate-reducing bacteria. Hydrogen is a key compound for these organisms, since it is one of their major energy sources in natural habitats and also an intermediate in the energy metabolism. The D. vulgaris Hildenborough genome codes for six different hydrogenases, but only three of them, the periplasmic-facing [FeFe], [FeNi]1, and [FeNiSe] hydrogenases, are usually detected. In this work, we studied the synthesis of each of these enzymes in response to different electron donors and acceptors for growth as well as in response to the availability of Ni and Se. The formation of the three hydrogenases was not very strongly affected by the electron donors or acceptors used, but the highest levels were observed after growth with hydrogen as electron donor and lowest with thiosulfate as electron acceptor. The major effect observed was with inclusion of Se in the growth medium, which led to a strong repression of the [FeFe] and [NiFe]1 hydrogenases and a strong increase in the [NiFeSe] hydrogenase that is not detected in the absence of Se. Ni also led to increased formation of the [NiFe]1 hydrogenase, except for growth with H2, where its synthesis is very high even without Ni added to the medium. Growth with H2 results in a strong increase in the soluble forms of the [NiFe]1 and [NiFeSe] hydrogenases. This study is an important contribution to understanding why D. vulgaris Hildenborough has three periplasmic hydrogenases. It supports their similar physiological role in H2 oxidation and reveals that element availability has a strong influence in their relative expression.     

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H₂-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate. Received: 17 September 1996 / Accepted: 5 November 1996     

EPR studies with 77Se-enriched (NiFeSe) hydrogenase of Desulfovibrio baculatus. Evidence for a selenium ligand to the active site nickel

The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.     

The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center.

BACKGROUND: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2<-->2H+ + 2e-. They are generally heterodimeric, contain three iron-sulfur clusters in their small subunit and a nickel-iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand. RESULTS: We report here the X-ray structure at 2.15 A resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 A shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared D. gigas enzyme, is absent from the D. baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel-selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between [NiFeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two [NiFe] hydrogenases. CONCLUSIONS: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive 'unready' [NiFe] hydrogenases.     

Hydrogenases in sulfate-reducing bacteria function as chromium reductase

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H₂S produced by the bacteria, and enzymatically, by polyhemic cytochromes c₃. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K_m towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.     

Iron hydrogenases and the evolution of anaerobic eukaryotes

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.     

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach

Hydrogenases are metalloenzymes that catalyze the reversible reaction \textH₂ \leftrightarrows 2\textH ⁺ + 2\texte ^- {\text{H}}_{2} \leftrightarrows 2{\text{H}}^{ + } + 2{\text{e}}^{ - } , being potentially useful in H₂ production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O₂ inhibition and produce more H₂ than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H₂ diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H₂ and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H₂ diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035–2045, 2006). The comparison showed that H₂ density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H₂ to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010–1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H₂ and proton pathways.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.     

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.     

Localization of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris using immunoelectron microscopic procedures.

The intracellular location of membrane-associated (NiFe) and (NiFeSe) hydrogenases of Desulfovibrio vulgaris was determined using pre-embedding and post-embedding immunoelectron microscopic procedures. Polyclonal antisera directed against the purified (NiFe) and (NiFeSe) hydrogenases were raised in rabbits. One-day-old cultures of D. vulgaris, grown on a lactate/sulfate medium, were used for all experiments in these studies. For post-embedding labeling studies cells were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde, dehydrated with methanol, and embedded in the low-temperature resin Lowicryl K4M. Our post-embedding studies using antibody-gold or protein-A-gold as electron-dense markers revealed the location of the two hydrogenases exclusively at the cell periphery; the precise membrane location was then demonstrated by pre-embedding labeling. Spheroplasts were incubated with the polyclonal antisera against (NiFe) and (NiFeSe) hydrogenase followed by ferritin-linked secondary antibodies prior to embedding and sectioning. The observed labeling pattern unequivocally revealed that the antigenic reactive sites of the (NiFe) hydrogenase are located in the near vicinity of the cytoplasmic membrane facing into the periplasmic space, whereas the (NiFeSe) hydrogenase is associated with the cytoplasmic side of the cytoplasmic membrane.     

Hydrogen metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of an uptake hydrogenase for hydrogen-dependent dissimilatory azoreduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni-Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis.     

Ferric iron reduction by Desulfovibrio vulgaris Hildenborough wild type and energy metabolism mutants

Desulfovibrio vulgaris Hildenborough wild type and its hyn1, hyd and hmc mutants, lacking genes for periplasmic [NiFe] hydrogenase-1, periplasmic [FeFe] hydrogenase or the transmembrane high molecular weight cytochrome (Hmc) complex, respectively, were able to reduce Fe(III) chelated with nitrilotriacetic acid (NTA), but not insoluble ferric oxide, with lactate as the electron donor. The rate and extent of Fe(III)-NTA reduction followed the order hyn = WT > hmc >> hyd, suggesting that reduction of soluble Fe(III) is a periplasmic process that requires the presence of periplasmic [FeFe] hydrogenase. Reduction of Fe(III)-NTA was not coupled to cell growth. In fact cell concentrations declined when D. vulgaris was incubated with Fe(III)-NTA as the only electron acceptor. Wild type and mutant cells reducing a limiting concentration of sulfate (2 mM), reduced Fe(III)-NTA with similar rates. However, these were similarly incapable of catalyzing subsequent lactate-dependent reduction of Fe(III)-NTA to completion. Periplasmic reduction of Fe(III)-NTA appeared to inhibit the productive, sulfate-reducing metabolism of D. vulgaris, possibly because it prevents the cycling of reducing equivalents needed to achieve a net bioenergetic benefit.     

Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria

The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.     

Development of a genetic system for the chemolithoautotrophic bacterium Thiobacillus denitrificans

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.     

Biodiversity of Hydrogenases in Frankia

Eighteen Frankia strains originally isolated from nine different host plants were used to study the biodiversity of hydrogenase in Frankia. In the physiological analysis, the activities of uptake hydrogenase and bidirectional hydrogenase were performed by monitoring the oxidation of hydrogen after supplying the cells with 1% hydrogen and the evolution of hydrogen using methyl viologen as an electron donor, respectively. These analyses were supported with a study of the immunological relationship between Frankia hydrogenase and other different known hydrogenases from other microorganisms. Uptake hydrogenase activity was recorded from all the Frankia strains investigated. A methyl-viologen-mediated hydrogen evolution was recorded from only four Frankia strains irrespective of the source of Frankia. From the immunological and physiological studies, we here report that there are at least three types of hydrogenases in Frankia: Ni-Fe uptake hydrogenase, hydrogen-evolving hydrogenase, and [Fe]-hydrogenase. An immunogold localization study, by cryosection technique, of the effect of nickel on the intercellular distribution of hydrogenase proteins in Frankia indicated that nickel affects the transfer of hydrogenase proteins into the membrane.