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/H-2/CO2, demonstrating its ability to utilize H-2 as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H-2/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 H-2/CO2, 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 H-2. 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 H-2 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 H-2-dependent methyl reduction.