Zn2+ could replace Mg2e to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A protonmotive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thernophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms. Energization of various membrane-bound processes, such as active transport of neutral and ionic

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... solutes and ATP synthesis via the membrane-bound proton-translocating ATP synthetase (ATPase), is brought about by chemiosmotic coupling (5, 11). The properties of the ATP synthetase complex of microorganisms have been described for both aerobic (1) and anaerobic microorganisms (14) . The methane-producing bacteria are phylogenetically very old organisms (2), and it seerned interesting to investigate the properties of the ATP synthetase complex. The necessity of ATP for methane production in extracts of methanogenic bacteria has been reported (13, 15, 16) . Both hydrolysis of ATP by cell extracts (15, 16) and a rise in ATP concentration in whole cells during methane production (16) have been demonstrated, but the ATPase has not been studied further. Recently Thauer et al. (18) reviewed energy conservation mechanisms in anaerobic microorganisms. These authors suggest that ATP production in methanogenic bacteria is coupled to electron transport, resulting in the reduction of C02 to CH4. In this paper we describe the properties of the membrane-bound ATPase of Methanobacterium thermoautotrophicum. Also, the synthesis of ATP using an artificially induced proton-motive force is demonstrated in M. thermoautotrophicum, Methanobacterium formicicum, and Methanobacterium strain MoH. The function of the ATP synthetase in the physiology of methanogenic bacteria is discussed. MATERIALS AND METHODS Organisms. M. thermoautotrophicum strain AH, M. formicicum, and Methanobacterium strain MoH were gifts from R. S. Wolfe, Urbana, Ill. The organisms were grown in a 12-liter fermentor at 600C (M. thermoautotrophicum) or 37°C-in a pH 7.1 medium containing, in millimolar concentrations: NaHCO3, 40; K2HPO4, 15; KH2PO4, 7.5; NaCl, 12; cysteine hydrochloride, 1.5; Na2S, 1; (NH4)2SO4, 0.1; NH4CI, 0.08; and MgSO4, CaCl2, and (NH4)2Fe(S04)2, 0.02. The medium was supplemented with resazurin (0.001%) and trace elements (19); M. formicicum and Methanobacterium strain MoH were cultured by the method of Ferry and Wolfe (4). As a carbon and energy source an H2-C02 (80 and 20% [vol/vol], respectively) gas mixture was used. For ATP hydrolysis tests, the cells were harvested anaerobically at the end of the exponential growth phase with a Sharples continuous centrifuge. The yield was about 3 g (wt weight) per liter. The cells were washed twice in 20 mM anaerobic tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.5, and stored at -90°C under an atmosphere of H2 in samples of about 5 g. For ATP synthesis in whole cells, samples were taken during exponential growth of the culture. After the cells were collected, no precautions were taken to maintain an anaerobic environment, because the ATP synthetase complex appeared insensitive to oxygen. Cells were washed twice in a 0.