Disruption of the operon encoding Ehb hydrogenase limits anabolic CO2 assimilation in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H, or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ebb. To determine the role of Ebb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessan, to restore growth to nearly wild-type levels, suggesting that Ebb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ebb in anabolic CO2 assimilation in methanococci.