Methane synthesis by membrane vesicles and a cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum

Abstract

M. thermoautotrophicum when grown on ordinary culture medium has a tough cell wall which is lysozyme-resistant and difficult to disrupt by physical means. The cell wall, however, can be weakened by the addition of D-sorbitol to the growth medium and the organisms form protoplasts after lysozyme addition. This technique allowed the isolation of 2 types of intracellular small vesicles: (a) isolated by disruption of the total cell population (lysozyme-sensitive and lysozyme-resistant cells) by ultrafrequency sound and (b) isolated by osmotic lysis of protoplasts. For the 1st time, a small vesicle fraction isolated as in (a) was capable of synthesizing methane from CO2 and H2 without cytoplasm. There was, however, an absolute requirement for a small, heat-stable, O2-sensitive cofactor which was isolated from the cytoplasm. Methane synthesis with this vesicle fraction was inhibited by the detergent deoxycholate, and by the protonophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Mg2+-ATPase appeared to be located on the outer or cytoplasmic surface of the small vesicle fraction isolated as in (b). Evidently, the interior of the small intracellular vesicles becomes acidic during methane synthesis.