Harmaline-resistant mutant of Methanothermobacter thermautotrophicus with a lesion in Na(+)/H(+) antiport.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na(+)/H(+) antiporter inhibitor harmaline was isolated. The Na(+)/H(+) exchange activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Na(+)/H(+) antiport activity of wild-type cells grown in the high Na(+) concentration (125 mmol/l) was significantly increased as compared to the cells grown under low Na(+) concentration (6.25 mmol/l) conditions. In contrast, harmaline resistant mutant showed almost the same Na(+)/H(+) antiport activity under both these conditions. While harmaline profoundly inhibited methanogenesis in the wild-type, increased methanogenesis was observed both in the presence and absence of harmaline in the mutant strain. ATP synthesis driven by methanogenic electron transport was significantly enhanced in the mutant cells. The experimental data revealed the differential expression of A flavoprotein and molybdenum-containing formylmethanofuran dehydrogenase 1 subunit C in harmaline-resistant mutant. The overexpression of these proteins might contribute to harmaline resistance. Taken together the results indicate that harmaline resistance in this mutant has arisen as a consequence of mutation(s) in antiporter gene(s) or protein(s) linked to antiporter activity. Moreover this work provides the evidence that Na(+)/H(+) exchanger deficiency in harmaline-resistant mutant can induce overexpression of several proteins participating in methanogenesis.

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A(0) domain of A(1)A(0)-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 microM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A(1)A(0)-ATP synthase operon (Mth952-Mth961) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A(1)A(0)-ATP synthase subunits were identified - Val(338)Ala in subunit A and Leu(252)Ile and Ser(293)Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A(1)A(0)-ATP synthase operon.

Methanogenesis is Ca2+ dependent in Methanothermobacter thermautotrophicus strain DeltaH.

The effect of Ca2+ ions on methanogenesis and growth of Methanothermobacter thermautotrophicus was investigated. The calcium chelator ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetra-acetic acid, calcium ionophore A23187 and ruthenium red all inhibited growth of this strain. Methane formation was strongly dependent on the external Ca2+ concentration in a resting cell suspension. In addition, methanogenesis of Ca2+ preloaded cells was stimulated by 400%. Inhibitor studies revealed that Co2+ and Ni2+, inorganic antagonists of Ca2+ transport, strongly inhibited methanogenesis in these cells. Interestingly, our findings imply that one of the enzymes of methanogenesis might catalyse a Ca2+ -dependent step and allow a direct activation of methanogenesis by Ca2+ ions.