Lawrence Berkeley National Laboratory

One objective of the Virtual Institute for Microbial Stress and Survival (VIMSS) and the Environmental Stress Pathway Project (ESPP) is to determine the genetic and physiological basis for cooperative and competitive interactions among environmental microbial populations of relevance to the DOE. The ESPP Applied Environmental Core (AEC) and Functional Genomics Core (FGC) have identified a number of genes that may participate in cooperative interactions between sulfate reducers and methanogens under low sulfate conditions. Specifically, the gram-negative Deltaproteobacterium D. vulgaris is able to grow in the absence of an electron acceptor via syntrophic growth with hydrogenotrophic organisms. Despite decades of research, energy conservation in D. vulgaris is not well understood. The presence of multiple hydrogenases, including those located in the periplasm in all studied Desulfovibrio strains - and the observation that hydrogen is produced and then consumed during growth of D. vulgaris Miyazaki with lactate and sulfate (Tsuji &Yagi, 1980) - lead to the formulation of the hydrogen cycling hypothesis as a mechanism for energy conservation (Odom & Peck, 1981). The availability of a completed genome sequence of D. vulgaris Hildenborough has since revealed genes for at least six different hydrogenases: four periplasmic and two cytoplasmic. Although several have been partially characterized biochemically and genetically, their roles in D. vulgaris under different growth conditions is not well understood. We examined the growth and metabolite production of an echA (DVU0434) D vulgaris Hidenborough mutant under three different growth conditions: i) in medium amended with lactate and sulfate and ii) in medium amended with acetate, hydrogen and sulfate, and iii) in coculture the hydrogenotrophic methanogen M. maripaludis, lacking an electron acceptor. On lactate, the mutant demonstrated a comparable growth rate and yield to the wild type strain, but evolved more hydrogen as measured by its accumulation in the headspace during growth in batch culture. In a medium containing 5 mM acetate and an atmosphere of H2/CO2 (80:20), growth of the mutant was severely impaired relative to the wild type. A coculture consisting of the mutant strain and a hydrogenotrophic methanogen (M. maripaludis) demonstrated only slightly reduced growth rate and biomass relative to the wild type. Although this suggested some role in energy conservation, the more obvious phenotype was its greatly limited growth in monoculture with acetate, hydrogen and sulfate. Thus, the available data suggest that the primary role of the Ech Hydrogenase is oxidation of hydrogen during sulfate respiration, possibly also contributing to the production of reduced ferredoxin required for conversion of Acetyl CoA to pyruvate by pyruvate oxidoreductase, as was previously demonstrated for the homologous hydrogenases in M. barkeri and M. maripaludis (Meuer et al., 2002; Porat et al., 2006).