H₂-independent growth of the hydrogenotrophic methanogen Methanococcus maripaludis

Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H₂ oxidation by the hydrogenase Eha provides these electrons, consistent with an H₂ requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that 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 H₂ production via an F₄₂₀-dependent formate:H₂ lyase activity shifted markedly toward H₂ compared to the wild type. Hydrogenotrophic methanogens are thought to require H₂ as a substrate for growth and methanogenesis. here we show alternative pathways in methanogenic metabolism that alleviate this H₂ requirement 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.