Electron transfer pathways of formate-driven H2 production in Desulfovibrio.

The potential of sulfate-reducing bacteria (SRB) as biocatalysts for H2 production from formate was recently demonstrated, but the electron transfer pathways involved were not described. In the present work, we analyzed the H2 production capacity of five Desulfovibrio strains: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio alaskensis, Desulfovibrio fructosivorans, and Desulfovibrio gigas. D. vulgaris showed the highest H2 productivity (865 mL Lmedium (-1)), and D. gigas the lowest one (374 mL Lmedium (-1) of H2). The electron transfer pathways involved in formate-driven H2 production by these two organisms were further investigated through the study of deletion mutants of hydrogenases (Hases) and formate dehydrogenases (Fdhs). In D. vulgaris, the periplasmic FdhAB is the key enzyme for formate oxidation and two pathways are apparently involved in the production of H2 from formate: a direct one only involving periplasmic enzymes and a second one that involves transmembrane electron transfer and may allow energy conservation. In the presence of selenium, the Hys [NiFeSe] Hase is the main periplasmic enzyme responsible for H2 production, and the cytoplasmic Coo Hase is apparently involved in the ability of D. vulgaris to grow by converting formate to H2, in sparging conditions. Contrary to D. vulgaris, H2 production in D. gigas occurs exclusively by the direct periplasmic route and does not involve the single cytoplasmic Hase, Ech. This is the first report of the metabolic pathways involved in formate metabolism in the absence of sulfate in SRB, revealing that the electron transfer pathways are species-specific.

Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus.

Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

Roles of HynAB and Ech, the only two hydrogenases found in the model sulfate reducer Desulfovibrio gigas.

Sulfate-reducing bacteria are characterized by a high number of hydrogenases, which have been proposed to contribute to the overall energy metabolism of the cell, but exactly in what role is not clear. Desulfovibrio spp. can produce or consume H2 when growing on organic or inorganic substrates in the presence or absence of sulfate. Because of the presence of only two hydrogenases encoded in its genome, the periplasmic HynAB and cytoplasmic Ech hydrogenases, Desulfovibrio gigas is an excellent model organism for investigation of the specific function of each of these enzymes during growth. In this study, we analyzed the physiological response to the deletion of the genes that encode the two hydrogenases in D. gigas, through the generation of ΔechBC and ΔhynAB single mutant strains. These strains were analyzed for the ability to grow on different substrates, such as lactate, pyruvate, and hydrogen, under respiratory and fermentative conditions. Furthermore, the expression of both hydrogenase genes in the three strains studied was assessed through quantitative reverse transcription-PCR. The results demonstrate that neither hydrogenase is essential for growth on lactate-sulfate, indicating that hydrogen cycling is not indispensable. In addition, the periplasmic HynAB enzyme has a bifunctional activity and is required for growth on H2 or by fermentation of pyruvate. Therefore, this enzyme seems to play a dominant role in D. gigas hydrogen metabolism.

Role of NorR-like transcriptional regulators under nitrosative stress of the δ-proteobacterium, Desulfovibrio gigas.

NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.