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ATPase was shown to be present on the cytoplasmic membrane of the methanogenic bacterium strain Gol. The enzyme was identified by an immunoelectron microscopic technique by using polyclonal antiserum directed against the j subunit of Escherichia coli FOF,-ATPase. Negatively stained membrane vesicles exhibited a dense population of stalked particles similar in dimensions and fine structure to typical FOF,-ATPase particles.The presence of ATPases has been demonstrated in a variety of methanogenic bacteria (4,6,10,16). Based on results obtained with Methanosarcina barkeri (1-3), it is assumed that the terminal step in methanogenesis, the 2-(methylthio)ethanesulfonic acid (methyl coenzyme M) methylreductase reaction (14,17), is coupled to the generation of an electrochemical proton gradient across the cytoplasmic membrane. This gradient is then taken advantage of for ATP synthesis (3).Recently, a methylotrophic methanogenic bacterium, strain Gol, has been isolated from which protoplasts can easily be obtained (8). Membrane vesicles and fragments derived from these protoplasts could be used in an electron microscopic approach aimed at the physical demonstration of the presence of ATPase. The enzyme has not been purified from this microorganism, and its identity was expected to be proven by an immunoelectron microscopic technique with polyclonal antiserum directed against the ,B subunit of the F1-ATPase particle of Escherichia coli. This serum has recently been shown to cross-react with membrane vesicle preparations obtained from strain Gol (unpublished data).Strain Gol, obtained from the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, was grown with methanol as the energy and carbon source, as described previously (8). Protoplasts were prepared by pronase treatment of whole cells (8). Inside-out and right side-out membrane vesicles and fragments were produced from protoplasts by disruption in a French pressure cell (11) operated at 65 MPa, using buffer (pH 7.0) containing 0.2 M sodium maleinate, 0.05 M N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate (TES), 0.25 M sucrose, 5 mM MgSO4, titanium citrate (19), resazurin, and DNase I, by repeated freezing and thawing (12) or by osmotic lysis (9).Experiments aimed at the identification of ATPase particles assumed to be attached to membrane vesicles and fragments were performed as follows. Samples (0.5 ,ul) of the vesicle suspension were applied to Formvar-coated 300-mesh nickel grids and kept at room temperature for 1 h. The * Corresponding author. samples were incubated overnight at 4°C on drops containing rabbit antibodies specific for the 1B subunit of the E. coli ATPase (1.4 mg/ml). The grids were then rinsed with phosphate-buffered saline (0.05 M phosphate buffer, 0.9% sodium chloride [pH 6.9]) before incubation with anti-rabbit antibody-gold complexes for 2 h at room temperature. Subsequently, the samples were rinsed with phosphate-buffered saline and distilled water. Colloidal gold was prepared by the method of Slot and Geuze (15) b...

The methanogenic bacterium strain G61 harbors a high-molecular-weight enzyme complex containing methyl coenzyme M methylreductase as revealed by immunoelectron microscopy. This Methanogenic bacteria utilize substrates such as H2-CO2, methanol, monomethylamine, dimethylamine, trimethylamine, and acetate (2,12,18,22) which are converted to a common intermediate, (2-methylthio)ethanesulfonic acid (methyl-CoM) (34). This compound is then reduced to methane and 2-mercaptoethanesulfonic acid in a reaction catalyzed by the enzyme methyl-CoM methylreductase (11,28,30). Studies of the effects of N,N'-dicyclohexylcarbodiimide and 3,5,4',5'-tetrachlorosalicylanilide [3,5-dichloro-N-(4,5-dichlorophenyl)-2-hydroxybenzamide] on methanogenesis, the transmembrane electrical gradient, and the cellular ATP level strongly indicate that ATP is synthesized by a chemiosmotic mechanism and that the methylreductase system is involved in proton translocation (3,4,6,7,22).Various models have been proposed for the generation of an electrochemical transmembrane gradient of protons in methanogenic bacteria (5, 10); one assumes that methylreductase might be membrane associated; one includes the view that a redox carrier might mediate between membrane and (cytoplasmic) methylreductase. Indications have accumulated that the methylreductase system is a multienzyme complex (3,17,23).

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