Isolation and characterization of a neomycin-resistant mutant of Methanobacterium thermoautotrophicum with a lesion in Na+-translocating ATPase (synthase).

A mutant of Methanobacterium thermoautotrophicum with a lesion in membrane Na+-translocating ATPase (synthase) was isolated. The total ATPase activity in permeabilized cells of this mutant was elevated three-fold as compared with the wild-type strain. In contrast to wild-type cells, mutant ATPase was neither inhibited by DCCD nor stimulated by Na+ ions. The methane formation orate of the mutant cells at pH 7.5 under non-growing conditions was nearly twice that of the wild-type strain and was stimulated by sodium ions. On the other hand, the ATP synthesis driven by methanogenesis under the same conditions was lower that of the wild-type under the same conditions, and contrary to the wild-type was not stimulated by Na+ ions. ATP synthesis driven by a potassium diffusion potential in the presence of sodium ions was markedly diminished in the mutant cells. The membrane potential values of the wild-type and the mutant cells in the presence of 10 mM NaCl at pH 7.0 were comparable at energized conditions (-223 mV and -230 mV respectively). The Mg2+-dependent ATPase activity of the 10(5) x g supernatant of broken cells from the mutant cells was 30% higher than in the wild-type. On the other hand, two bands with Mg2+-dependent ATPase activity were identified by native PAGE in this fraction in both wild-type as well as in mutant. These data suggest that the binding of Na+-translocating ATPase (synthase) to the membrane spanning part is changed in the mutant strain.