Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase

The methanogenic archaeon encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F -dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H -CO , whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δ Δ and Δ Δ Δ mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H , which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F dehydrogenase operon were significantly higher in all strains lacking , suggesting a mechanism to sense the redox state of F The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed. Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.