Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs

Abstract

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated "restricted facultative' methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Regulation of citrate synthase activity in methylotrophs by reduced nicotinamide-adenine dinucleotide, adenine nucleotides and 2-oxoglutarate

“…1). Weitzman & Jones (1968) found that Gram-negative bacteria could be divided into two groups depending on whether or not AMP relieved the NADH-mediated Colby & Zatman (1972, 1975 inhibition of their citrate synthase, and that this division followed established taxonomic divisions. In this respect, bcaterium 5B1 and Pseudomonas 3A2 resemble other bacteria of the Pseudomonadaceae, Azotobacteriaceae and Neisseriaceae but differ from bacteria of the Enterobacteriaceae.…”

“…Organisms 4B6, C2AI, W3A1, 5B1, Pseudomonas 3A2 and Bacillus PM6 were grown in 8-litre batches ofmineral base E (Owens & Keddie, 1969), containing 0.2% trimethylamine hydrochloride and vitamins where required (Colby & Zatman, 1973, 1975. The culture vessels were as described by Colby & Zatman (1974), except that additional aeration was achieved by bubbling filtered air through the cultures by means of a glass tube immersed in the culture medium.…”

“…All further procedures were done at 0-40C. Crude sonic homogenates of organisms 4B6, C2A1, W3A1, 5B1, Pseudomonas 3A2 and Bacillus PM6 were prepared as described by Colby & Zatman (1973 (Colby & Zatman, 1972, 1975 (1963). Assay mixtures contained, in 1 ml: 100,umol of Tris-HCl buffer, pH 8.0; 0.15,umol of acetyl-CoA; 0.1gmol of 5,5'-dithiobis-(2-nitrobenzoic acid); extract; 0.2,umol of oxaloacetic acid.…”

“…Weitzman & Jones (1968) considered this to be feedback inhibition when NADH is regarded as an end product of the catabolic function of the tricarboxylic acid cycle. Such a control mechanism would not be expected to operate in obligate methylotrophs and restricted facultative methylotrophs, because the low or zero specific activities of some tricarboxylic acidcycle enzymes found under all growth conditions (Colby & Zatman, 1972, 1975Dahl et al;Davey et al, 1972;Patel et al, 1969) suggests that the cycle plays an anabolic rather than an 'amphibolic role in these organisms. Feedback inhibition ofcitrate synthase by NADH would, however, be expected in Gram-negative typical facultative methylotrophs such as bacterium 5B1 and Pseudomonas 3A2, which contain high activities of all the tricarboxylic acid-cycle enzymes when growing on some non-Cl compounds (Colby & Zatman, 1972, 1975; under the latter conditions the cycle is presumably important for NADH generation.…”

Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis

“…The enzyme from the restricted facultative methylotroph, bacterium W3A1 (Colby & Zatman, 1975), has been isolated and shown to have a mol.wt. of 147000 (Steenkamp & Mallinson, 1976) and to contain a single tetrameric iron-sulphur centre (Hill et al, 1977), as well as an unusual yellow chromophore (Steenkamp & Singer, 1976).…”