Tungsten and molybdenum regulation of formate dehydrogenase expression in Desulfovibrio vulgaris Hildenborough.

Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. In this work, we studied the effect of both metals on the levels of the three formate dehydrogenases encoded in the genome of Desulfovibrio vulgaris Hildenborough, with lactate, formate, or hydrogen as electron donors. Using Western blot analysis, quantitative real-time PCR, activity-stained gels, and protein purification, we show that a metal-dependent regulatory mechanism is present, resulting in the dimeric FdhAB protein being the main enzyme present in cells grown in the presence of tungsten and the trimeric FdhABC3 protein being the main enzyme in cells grown in the presence of molybdenum. The putatively membrane-associated formate dehydrogenase is detected only at low levels after growth with tungsten. Purification of the three enzymes and metal analysis shows that FdhABC3 specifically incorporates Mo, whereas FdhAB can incorporate both metals. The FdhAB enzyme has a much higher catalytic efficiency than the other two. Since sulfate reducers are likely to experience high sulfide concentrations that may result in low Mo bioavailability, the ability to use W is likely to constitute a selective advantage.

Energy metabolism in Desulfovibrio vulgaris Hildenborough: insights from transcriptome analysis.

Sulphate-reducing bacteria are important players in the global sulphur and carbon cycles, with considerable economical and ecological impact. However, the process of sulphate respiration is still incompletely understood. Several mechanisms of energy conservation have been proposed, but it is unclear how the different strategies contribute to the overall process. In order to obtain a deeper insight into the energy metabolism of sulphate-reducers whole-genome microarrays were used to compare the transcriptional response of Desulfovibrio vulgaris Hildenborough grown with hydrogen/sulphate, pyruvate/sulphate, pyruvate with limiting sulphate, and lactate/thiosulphate, relative to growth in lactate/sulphate. Growth with hydrogen/sulphate showed the largest number of differentially expressed genes and the largest changes in transcript levels. In this condition the most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase. The results also provide evidence for the involvement of formate cycling and the recently proposed ethanol pathway during growth in hydrogen. The pathway involving CO cycling is relevant during growth on lactate and pyruvate, but not during growth in hydrogen as the most down-regulated genes were those coding for the CO-induced hydrogenase. Growth on lactate/thiosulphate reveals a down-regulation of several energy metabolism genes similar to what was observed in the presence of nitrite. This study identifies the role of several proteins involved in the energy metabolism of D. vulgaris and highlights several novel genes related to this process, revealing a more complex bioenergetic metabolism than previously considered.

Hydrogen as an energy source for the human pathogen Bilophila wadsworthia.

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H(2), formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide.

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide. Nevertheless, the large subunit has a conserved four residue lipobox and its synthesis is sensitive to the signal peptidase II inhibitor globomycin. The D. vulgaris [NiFeSe] hydrogenase is the first example of a bacterial lipoprotein translocated through the Tat pathway.

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.

Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c3 family.

Resonance Raman (RR) spectroscopy was used to investigate conformational characteristics of the hemes of several ferricytochromes of the cytochrome c3 family, electron transfer proteins isolated from the periplasm and membranes of sulfate-reducing bacteria. Our analysis concentrated on the low-frequency region of the RR spectra, a fingerprint region that includes vibrations for heme-protein C-S bonds [nu(C(a)S)]. It has been proposed that these bonds are directly involved in the electron transfer process. The three groups of tetraheme cytochrome c3 analyzed, namely Type I cytochrome c (3) (TpIc (3)s), Type II cytochrome c (3) (TpIIc (3)s) and Desulfomicrobium cytochromes c3, display different frequency separations for the two nu(C(a)S) lines that are similar among members of each group. These spectral differences correlate with differences in protein structure observed among the three groups of cytochromes c3. Two larger cytochromes of the cytochrome c3 family display RR spectral characteristics for the nu(C(a)S) lines that are closer to TpIIc3 than to TpIc3. Two other multiheme cytochromes from Desulfovibrio that do not belong to the cytochrome c3 family display nu(C(a)S) lines with reverse relative areas in comparison with the latter family. This RR study shows that the small differences in protein structure observed among these cytochrome c3 correlate to differences on the heme-protein bonds, which are likely to have an impact upon the protein function, making RR spectroscopy a sensitive and useful tool for characterizing these cytochromes.

Hydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenase.

The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H(2) production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S](2+/1+) cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe](1) Hase, with the Type I cytochrome c (3) as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.

A novel membrane-bound Ech [NiFe] hydrogenase in Desulfovibrio gigas.

In the present study, we report the identification of an operon with six coding regions for a multisubunit membrane-bound [NiFe] hydrogenase in the genome of Desulfovibrio gigas. Sequence analysis of the deduced polypeptides reveals a high similarity to subunits of proteins belonging to the family of Ech hydrogenases. The operon is organised similarly to the operon coding for the Ech hydrogenase from Methanosarcina barkeri, suggesting that both encode very similar hydrogenases. Expression of the operon was detected by Northern blot and RT-PCR analyses, and the presence of the encoded proteins was examined by Western blotting. The possible role of this hydrogenase is discussed, relating it with a potential function in the H(2) cycling as a mechanism for energy conservation in D. gigas. The present study provides therefore valuable insights into the open question of the energy conserving mechanism in D. gigas.

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer.

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.

Reclassification of the only species of the genus Desulfomonas, Desulfomonas pigra, as Desulfovibrio piger comb. nov.

The growth characteristics, DNA G+C content and sequences of 16S rDNA and the transcribed 16S-23S rDNA internal spacer were determined for Desulfomonas pigra ATCC 29098T, Desulfovibrio desulfuricans subsp. desulfuricans strains Essex 6T (= ATCC 29577T) and MB (= ATCC 27774) and 'Desulfovibrio fairfieldensis' ATCC 700045. Despite phenotypic differences (shape and motility) between Desulfomonas pigra and Desulfovibrio strains, the molecular analysis suggests that Desulfomonas pigra should be reclassified within the genus Desulfovibrio. Thus, the reclassification is proposed of Desulfomonas pigra, the type and only species of the genus, as Desulfovibrio piger comb. nov., which implies the emendation of the description of the genus Desulfovibrio.