The protonmotive force and ATP synthesis/membrane-fixed-charge relationships.

tic cis-benzenediol was present on both plates, with R, values of 0.21 and 0.24 respectively. Another portion of the thoroughly dried ether extract was treated with Bis (trimethylsily1)trifluoracetamide and trimethylchlorosilane for 30min at 90° C and the trimethylsilyl derivative injected into a Pye 104 chromatograph fitted with a glass column packed with 4% OV 255 on Chromosorb WHP and a flame-ionization detector. A programming control system with an initial oven temperature of 100°C increasing by 6OC/min and N, (30ml/min.) as carrier gas was used. A peak with a retention time of 9.2min, identical with that given in this system by authentic trimethylsilyl ester of cis-benzenediol. was obtained. Pseudomonas putida (strain 39/D) cells were grown in glucose/mineral-salts medium and adapted to metabolize toluene, as described by Gibson et al. (1968). These were incubated with IU-'*C lbenzene (SOpCi; specific radioactivity 100mCi/mmol) in phosphate buffer (2OOml,0.05 M, pH 7.2) and the U.V. spectrogram of the culture fluid monitored. When the maximum amount of cis-benzenediol and catechol had been formed, the culture was worked up; it afforded labelled samples of these metabolites, which were separated and purified by t.1.c. They were also checked by g.1.c. and their radioactivities measured in a Philips liquid-scintillation counter. These biologically prepared specimens of ci~-IU-'~C1benzenediol ( 1 SpCi) and catechol (20pCi) were introduced into separate catecholmetabolizing methanogenic cultures. Samples of culture fluid (250ml) were withdrawn periodically (after 3 ,6 and 12 h) and their neutral and acidic ether extracts examined by t.1.c. and g.1.c. (using a 504 Radiogas Detector; ESI Nuclear, Redhill, Surrey, U.K.). Radioactive phenol was formed in both cultures and then disappeared with time. A control experiment, where radioactive samples of cis-benzenediol and catechol were incubated separately in autoclaved cultures under similar conditions, showed that no labelled phenol was contained in the neutral ether extracts. In view of the known facile conversion of cis-benzenediol into phenol under acidic conditions, this result is of crucial importance. A catechol-adapted methanogenic culture metabolized phenol without a lag period. IU-*4C1Phenol (30pCi: specific radioactivity 35mCi/mmol) was added to such a culture (2 litres, 37OC) and samples of culture fluid (250ml) withdrawn after 3 , 6 and 12h. Their neutral ether extracts on examination by g.1.c. gave labelled cyclohexanone and 2-hydroxycyclohexanone; the identity of these were confirmed by t.1.c. of their 2,4-dinitrophenylhydrazones. G.1.c. of the methylated acidic ether extracts (diazomethane) gave peaks corresponding to the methyl esters of adipate, succinate, propionate and acetate. These were confirmed by radioautography of t.1.c. plates after chromatography of the acids and their hydroxamates, as described by Balba & Evans (1977). These results provide unequivocal evidence for the participation of cis-benzenediol in the methanogenic fermentation pathway of catechol through phenol and the reductive route, as illustrated in Scheme 1. We had previously shown the dehydroxylation of the monohydroxybenzoates to benzoate in their methanogenic cultures (Balba et al., 1979). Elimination of hydroxyl substituents from the aromatic nucleus appears to be a frequent reaction in microbial systems under anaerobic conditions-probably through the action of a reductase followed by a dehydratase. It was first observed during the mammalian metabolism of certain plant phenolics by Scheline et al. (1960) through the action of the intestinal flora. Subsequently, Perez-Silva et al. (1966) showed that pseudomonad-type bacteria from rat faeces dehydroxylated caffeate anaerobically to give m-hydroxyphenylpropionate and mcoumarate.