Genetic, biochemical, and molecular characterization of Methanosarcina barkeri mutants lacking three distinct classes of hydrogenase

Abstract

The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-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 H2/CO2, 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 wild-type, produced methane and CO2 in the expected 3:1 ratio, but had a ca. 33% slower growth rate. Thus, hydrogenases play a significant, but non-essential, role during growth on this substrate. As previously observed, mutants lacking Ech fail to grow on methanol/H2 unless supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the {Delta}ech/{Delta}frh and {Delta}ech/{Delta}frh/{Delta}vht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of reducing equivalents needed for biosynthesis. Quantification of methane and CO2 produced from methanol by resting cell suspensions of various mutants supports this conclusion. Based on global transcriptional profiles, none of the hydrogenases are upregulated to compensate for loss of the others. However, transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420. The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.