A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant K m for nitrite ‫؍‬ 0.03 mM), and in which NrfHA (K m for nitrite ‫؍‬ 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.Sulfate-reducing bacteria are frequently exposed to nitrite by interaction with nitrate-reducing, sulfide-oxidizing bacteria in anoxic environments. Because sulfate is often the predominant electron acceptor in anoxic environments (e.g., marine sediments), sulfate-reducing bacteria are primarily responsible for organic carbon oxidation. The sulfide produced is then targeted by nitrate-reducing, sulfide-oxidizing bacteria, which generally use CO 2 as their sole carbon source (4). Thus, sulfate-reducing bacteria and nitrate-reducing, sulfide-oxidizing bacteria symbiotically catalyze the oxidation of organic matter with nitrate through a sulfide intermediate. This symbiosis can potentially be stalled by production of nitrite by the nitratereducing, sulfide-oxidizing bacteria, which is a powerful inhibitor of sulfate-reducing bacteria. Some sulfate-reducing bacteria have a periplasmic nitrite reductase to prevent and/or overcome this inhibition. Cocultures of a Desulfovibrio sp. and the nitrate-reducing, sulfide-oxidizing bacterium Thiomicrospira sp. strain CVO were strongly or transiently inhibited, depending on the absence or presence of nitrite reductase activity in the Desulfovibrio sp. (6). Desulfovibrio vulgaris Hildenborough was very resistant to inhibition by either nitrite or strain CVO and nitrate and had high nitrite reductase activity through the presence of a periplasmic cytochrome c nitrite reductase (NrfHA) that reduces nitrite to ammonium. This reaction is purely detoxifying; no cell growth is associated with t...

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of Desulfovibrio vulgaris Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the hyd mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified D. vulgaris genes encoding proteins active in energy metabolism indicated that the adh gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the adh gene was strongly downregulated in the hyd mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An adh mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The hyd mutant also grew poorly on ethanol, in agreement with its low level of adh gene expression. The adh mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of adh in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of D. vulgaris under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H 2 -heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.The role of the cytoplasmic membrane in the conservation of chemiosmotic energy is fairly well understood for aerobic bacteria, because of similarities to the mitochondrial inner membrane paradigm. However, this role is much less clear for anaerobes. For instance, chemiosmotic energy conservation is crucial for sulfate-reducing bacteria (SRB), when growing with lactate as the electron donor, because the ATP yield of substrate-level phosphorylation exactly balances what is required for sulfate activation (19,21,33). Odom and Peck proposed that SRB of the genus Desulfovibrio conserve chemiosmotic energy by hydrogen cycling (17). In their model, hydrogen generated from cytoplasmic lactate oxidation was proposed to diffuse across the cytoplasmic membrane, where it is oxidized by periplasmic hydrogenase to form a proton gradient with the electrons being conducted back to the cytoplasmic sulfate reduction pathway. Analysis of the genome sequence for Desulfovibrio vulgaris Hildenborough (http://www.tigr.org) has indicated the presence of two cytoplasm-facing, membrane-bound hydrogenases, four periplasmic hydrogenases, and a variety of transmembrane electron-transporting complexes. Hence, all components required for a hydrogen cycling mechanism of energy conservation are ...

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen > 50% hydrogen > lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.Hydrogen is intimately involved in the metabolism of sulfate-reducing bacteria (SRB) of the genus Desulfovibrio (17). In addition to utilizing molecular hydrogen directly as an electron donor for sulfate reduction, hydrogen may play a central role as an intermediate in the generation of a chemiosmotic gradient from the oxidation of organic molecules. This process has become known as hydrogen cycling (18). The current emphasis on developing more efficient energy production strategies has made understanding SRB metabolism a critical endeavor, especially with respect to the SRB connection with hydrogen and the impact of SRB on the petroleum industry through oil reservoir souring and pipeline corrosion.Hydrogenases catalyze the heterolytic cleavage of molecular hydrogen into protons and electrons (H 2 7 2H ϩ ϩ 2e) (9). These enzymes are present ubiquitously in both Archaea and Bacteria, including many SRB. The fully sequenced Desulfovibrio vulgaris Hildenborough has a total of six hydrogenases (14). Four of them are periplasmic and therefore presumably are involved in hydrogen oxidation, including a soluble iron-only [Fe] hydrogenase (Hyd) (16), two membrane-associated nickel-iron [NiFe] hydrogenase isozymes (Hyn1 and Hyn2) (14, 23), and a membrane-associated nickel-iron-selenium [NiFeSe] hydrogenase (Hys) (31). The [NiFe] hydrogenases are widely distributed in SRB (35), but many Desulfovibri...

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 lactateand 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. Sulfate-reducing bacteria of the genus Desulfovibrio contain the genes for several hydrogenases, including the hynBA and hysBA genes for the NiFe-and NiFeSe-hydrogenases, the hydAB genes for Fe-only hydrogenase, and the hndABCD genes for NADP-reducing hydrogenase. The first three enzymes are translocated by the twin-arginine translocation (tat) system and are thus either periplasmic or membrane bound with the active site facing the periplasm. Only the NADPreducing hydrogenase has been shown elsewhere to be cytoplasmic (11). Desulfovibrio vulgaris strain Hildenborough has the Fe-only hydrogenase as well as the NiFe-and NiFeSehydrogenases. The former is a soluble periplasmic enzyme, whereas the latter two are membrane bound. Searching of the database for the D. vulgaris genome at http://www.tigr.org indicated that D. vulgaris has an HndD homolog but lacks the hndA, hndB, and hndC genes. D. vulgaris is thus unlikely to have a cytoplasmic, NADP ϩ -reducing hydrogenase. The hydAB genes encode the 46-kDa ␣ and the 10-kDa ␤ subunit of Fe-only hydrogenase from D. vulgaris (25). The structures of Fe-only hydrogenase have been determined recently both for CpI, the cytoplasmic enzyme from Clostridium pasteurianum (16), and for the periplasmic enzyme from Desulfovibrio desulfuricans (14). The sequences of the ␣ and ␤ subunits of the periplasmic enzyme in D. desulfuricans and D. vulgaris form a contiguous, single polypeptide of 60 kDa in CpI. The splitting of the sequence into two polypeptides in Desulfovibrio spp. is for export: the ␤ subunit has a long twinarginine-type signal sequence for this purpose (24). The function of CpI in the fermentative metabolism of C. pasteurianum is to reoxidize reduced ferredoxin, using protons as the electron acceptor to produce H 2 (2). A similar function has been proposed elsewhere for Fe-only hydrogenase in lactate metabolism by D. vulgaris (23). Reduction of the Fe-only hydrogenase content by expression of hydAB antisense RNA reduced the growth rate and growth yield of D. vulgaris in lactate-and sulfate-containing medium. Observation of a reduced H 2 burst in the initial stages of growth on this medium also pointed to a decreased H 2 production act...

Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H 2 O 2 ) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H 2 O 2 resistance may also be conferred by two other rbr genes in the D. vulgaris genome.