Evidence for an internal electrochemical proton gradient in Methanobacterium thermoautotrophicum

At pH > 7.5 cells of Methanobacterium thermoautotrophicum , when synthesizing CH sub(4) from CO sub(2) and H sub(2), are interior acid with respect to the buffer. If the buffer pH is lowered below pH 6.5 with HCl, the pH gradient has the usual orientation, i.e. the cells become interior alkaline. Flow dialysis experiments carried out at pH > 7.5 with permeant base methylamine indicated that these cells establish an inverse pH gradient. With pH > 7.5 the polarity of the protonmotive force, across the cytoplasmic membrane therefore is reversed and would not serve to generate cellular ATP. Results with different uncoupling reagents suggest that in M. thermoautotrophicum) the synthesis of ATP and CH sub(4) is carried out by intracellular vesicles and that the cytoplasmic membrane probably is not the site of energy transduction. Carbonyl cyanide m-chlorophenylhydrazone, partially dissipates the pH gradient and at low concentrations partially inhibits CH sub(4) or ATP synthesis. Chloroform or 2,4-dinitrophenol, when added after carbonyl cyanide m-chlorophenylhydrazone, gives an additional and significant release of acid from these cells and thereby completes the discharge of the pH gradient.