Gene Expression Analysis of Energy Metabolism Mutants of Desulfovibrio vulgaris Hildenborough Indicates an Important Role for Alcohol Dehydrogenase

Abstract

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 ...

Two distinct heterodisulfide reductase‐like enzymes in the sulfate‐reducing archaeon Archaeoglobus profundus

“…These genes are expressed in D. vulgaris as was shown by macroarray RNA hybridizations [41]. Expression of these genes was found to be downregulated in a Fe-only hydrogenase mutant strain (Dhyd).…”

“…Downregulation was also observed in a strain carrying a deletion of the adh gene, encoding an alcohol dehydrogenase. One possible function discussed for the H 2 -heterodisulfide oxidoreductase complex in D. vulgaris is the formation of H 2 with reducing equivalents generated by the oxidation of ethanol to acetate, as part of a H 2 -cycling system [41]. Thus far, the Mvh:Hdl genetic organization is unique to sulfate reducers.…”

The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

“…In addition to hydrogen and hydrogenases, the unexpectedly high number of formate dehydrogenases, suggests a second system of chemiosmotic energy conservation by the diffusion of an uncharged metabolic intermediate, formate, from the cytosol with subsequent periplasmic oxidation. The contribution of these enzymes to the cellular energy budget can now be explored through mutagenesis, microarray or other gene expression experiments, combined with physiological characterization 26 . The recognition of the importance of this mode of energy conservation in microbial metabolism lags far behind that of fermentation and traditional respiration but may be found almost universally.…”

“…In any case, the hydrogen produced could then be captured by periplasmic hydrogenases to form a proton gradient. Thus, the genome sequence indicates the presence of novel complexes that are involved in energy transduction (e.g.,'hydrogen cycling') for which the exact mechanism remains to be worked out 13,26 .…”

Biodegradation of BTEX and Other Petroleum Hydrocarbons by Enhanced and Controlled Sulfate Reduction

“…The nutrients were added in molar C:N:P ratios of 100:10:2. Na 2 MoO 4 (20 mM MoO 4 2-) was added as a specific inhibitor to sulfate reduction (Oremland and Capone, 1988) and 3 mM nitrite, which is a competitive inhibitor of sulfate reducers (Greene et al, 2003;Haveman et al, 2003Haveman et al, , 2004Jenneman et al, 1986aJenneman et al, , 1986bLondry and Suflita, 1999), were also added to corresponding microcosm treatments. Microcosms were incubated at room temperature and stored in the dark.…”