H2-Independent Growth of the Hydrogenotrophic Methanogen Methanococcus maripaludis

ABSTRACTHydrogenotrophic methanogenic Archaearequire reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H2oxidation by the hydrogenase Eha provides these electrons, consistent with an H2requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludisthat operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H2-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F420, should be abundant. Indeed, when F420-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H2production via an F420-dependent formate:H2lyase activity shifted markedly toward H2compared to the wild type.IMPORTANCEHydrogenotrophic methanogens are thought to require H2as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H2requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H2. The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.