Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain

Abstract

Methanogenesis-driven ATP synthesis in a neomycin-resistant mutant of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain ΔH) was strongly inhibited at both pH 6.8 and pH 8.5 by the uncoupler 3,3′,4′,5 -tetrachorosalicylanilide (TCS) in the presence of either 1 or 10 mM NaCl. The generation of a membrane potential in the mutant cells at pH 6.8 was also strongly inhibited by TCS in the presence of 1 or 10 mM NaCl. On the other hand, at pH 8.5 in the presence of 10 mM NaCl, a protonophore-resistant membrane potential of approximately 150 mV was found. These results indicate that in the mutant cells the process of energy transduction between methanogenesis and membrane potential generation is not impaired. In contrast to the wild-type strain, ATP synthesis in the mutant cells was driven by an electrochemical gradient of H + under alkaline conditions. Unlike wild-type cells, the mutant lacks the capacity to transduce an uncoupler-resistant membrane potential energy at pH 8.5 into ATP synthesis. Na +/H + exchange was comparable in the wild type and the mutant cells. Western blots of sub-cellular fractions with polyclonal antiserum reactive to the B-subunit of the halobacterial A-type H +-translocating ATPase confirmed the presence of A-type ATP synthase in the mutant cells. Furthermore, in the mutant cells a protein band of molecular mass about 45 kDa is absent but there was an abundant protein band at about 67 kDa. Based on the observed bioenergetic features of the mutant cells, neither the A 1A o ATP synthase alone nor together with the Na +/H + antiporter seems to be responsible for ATP synthesis driven by sodium motive force. Rather, some other links between neomycin-resistance and failure of sodium motive force-dependent ATP synthesis in the neomycin resistant mutant are discussed.