| Summary: | The utilisation of acetate by Rhodopseudomonas spheroides grown aerobically in the dark was investigated. Evidence was obtained which strongly suggested that the organism, grown on acetate plus CO2, oxidised acetate via the tricarboxylic acid cycle. However, extracts were completely devoid of isocitrate lyase activity, though they did contain malate synthase activity. Evidence obtained from the short-term studies of [2-14C] acetate incorporation by cultures growing on acetate indicated that the glyoxylate cycle did not operate. No alternative to isocitrate as a source of glyoxylate was found. During aerobic, dark growth on acetate, a CO2 requirement in the initial stages of growth was observed. This became much less marked if a heavy inoculum of the exponentially growing organism was used. The short-term studies of 14CO2 incorporation by cultures growing on acetate showed relatively little net CO2 fixation. No evidence was obtained either from short-term 14C incorporation experiments or from Incubations with cell extracts for the formation of pyruvate from acetyl-CoA and CO2. Rps. spheroides was shown to possess an acetyl-CoA- dependent pyruvate carboxylase and to be devoid of PEP synthase and PEP carboxylase activities. A mutant strain devoid of pyruvate carboxylase yet still able to grow both aerobically in the dark and anacrobically in the light on acetate plus CO2, provided evidence that Rps. spheroides replenishes TCA cycle intermediates from acetate plus CO2 by a pathway not involving pyruvate carboxylase and hence not the reductive carboxylic acid cycle. With this same mutant pyruvate was shown to inhibit growth on acetate plus CO2 in a manner expected for the repression of the synthesis of one or more enzymes of the anaplerotlc pathway. The complete and unequivocal degradation of [14C] glutamate isolated from the ethanol-soluble fraction of cells growing aerobically in the dark on 14C labelled acetate and CO2 revealed a novel distribution pattern. Carboxyl carbon atoms of acetate were incorporated into C-2 and C-3 of glutamate as well as the expected (for the synthesis via the TCA cycle) incorporation into C-1 and C-5. Decarboxylation of [14C] aspartate isolated from the same fractions supported these findings. Comparison of these results with those obtained from similar degradation studies with E.coli W and Rps.palustris (organisms known to operate a glyoxylate cycle) indicated that in Rps. spheroides there was a novel pathway for the replenishment of TCA cycle intermediates during growth on acetate. A mutant strain of spheroides, unable to grow on acetate plus CO2 and having all the characteristics of being blocked in the anaplerotic pathway, was also isolated.
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