Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H₂ metabolism between closely related species

A mutation in the mch gene, encoding the enzyme 5,10-methenyl tetrahydromethanopterin (H₄MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H ₂/CO₂, methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H₂/CO₂, demonstrating its ability to utilize H₂ as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H₂/CO₂ is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H₂/CO ₂, could not be made in wild-type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H₂. M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H₂ oxidation. Interestingly, the ech-dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H₂-dependent methyl reduction.