The metabolism of C2 compounds in micro-organisms. 5. Biosynthesis of cell materials from acetate in Escherichia coli

Abstract

In brief incubation periods, suspensions of 2 strains of E. coli (w and Crooks) growing on acetate as the sole C source incorporated isotope from [1-C14]acetate only into intermediates of the tricarboxylic acid cycle and into amino acids derived therefrom. The distribution of isotope amongst the labelled products was not consistent with the operation of the tricarboxylic acid cycle as the sole route of acetate metabolism, but showed that acetate entered the cycle at 2 sites, to form citrate at one and malate at the other. Succinate was not labelled at the earliest times. These results indicate that the glyoxylate cycle also operates in these cells, and exclude the primary formation of succinate by oxidative condensation of acetate. Isotope from sodium [C14]bicarbonate was incorporated by E. coli growing on acetate into cellular materials at less than 2% of the rate at which [C14]acetate was utilized. Cell-free extracts of acetate-grown cells, fortified with necessary cofactors catalysed the incorporation of isotope from [C14]acetate into citrate in the presence of oxalo-acetate, and into malate in the presence of either glyoxylate or isocitrate. They also catalysed the net formation of malate from acetyl-coenzyme A and isocitrate. Direct enzymic assay showed that such extracts contained high activities of key enzymes of the glyoxylate cycle. The specific activities (umoles of substrate transformed/mg of soluble protein/hr.) of condensing enzyme averaged 23 for strain w and 14 for the Crooks strain, of malate synthetase 7.0 and 5.5, and of isocitratase 5.3 and 6.3 respectively. The activities of these enzymes, acting in conjunction with other enzymes of the tricarboxylic acid cycle, are adequate to account for the observed rates of growth of the organisms on acetate. Cell-free extracts of E. coli grown on C sources other than acetate contained malate synthetase and isocitratase at less than one-tenth of the specific activities observed with acetate-grown cells. This supports the essential role of the glyoxylate cycle in the growth of E. coli on acetate.