Samuel J. Ajl

Growth of Escherichiacoli on fatty acids: Requirement for coenzyme a transferase activity

Abstract The ability of an Escherichia coli strain (E-26) to grow on butyrate or valerate as the sole source of carbon has previously been correlated with the glyoxylate-short chained fatty acid acyl-CoA ester condensing activities (1). In attempting to confirm and expand these results, several strains of Escherichia coli K12, of previously defined genotype and physiology (2) were employed. From one of them, constitutive for the enzymes of the glyoxylate shunt, a new mutant, V10, has been selected for its ability to grow on butyrate or valerate as the sole source of carbon. The growth of this strain on these fatty acids is rapid and is not preceded by any significant lag when transfered from a glucose medium. This physiological behavior is related to the presence of a constitutive level of an acyl-CoA: acetate transferase activity. This enzymatic reaction, not previously reported in Escherichia coli , is measurable by spectrophotometric assays and a radioisotopic microassay. A second mutant, derived from V10, is simultaneously deficient in the transferase activity and in the ability to grow on butyrate or valerate. The data presented suggest that the required transferase functions as an activating mechanism which permits the organism to utilize these fatty acids as a carbon source for growth.

Multiple Forms of Bacterial NADP-Specific Isocitrate Dehydrogenase

Electrophoretically distinct forms of nicotinamide adenine dinucleotide phosphate-specific isocritrate dehydrogenase have been observed in extracts of Escherichia coli grown under different culture conditions. In glucose-grown cells, two distinct bands of isocitrate dehydrogenase activity were observed on polyacrylamide gels and have been completely resolved by employing ion-exchange chromatography. These multiple forms of the enzyme have been studied and their possible metabolic role is discussed.

Propionate oxidation in Escherichia coli

An investigation was undertaken to determine the principal pathway of propionate oxidation in Escherichia coli E-26. Studies in a number of systems have established that propionate may be metabolized by several different pathways. Since these pathways may be differentiated by the pattern of14CO2 evolution from specifically labelled carbon atoms of propionate, a radiorespirometric experiment was performed in which propionate-adapted E. coli E-26 was used. Under these conditions, both the rate and final cumulative percentage of14CO2 was greatest for 1-14C-propionate, intermediate for 2-14C-propionate, and least for 3-14C-propionate. The observed labelling pattern is consistent with oxidation of propionate to acetate via lactate.

Propionate metabolism: III. Studies on the significance of the α-hydroxyglutarate pathway☆

The characteristics of Escherichia coli E-26 and a derived mutant E-26V during adaptation to propionate have been examined. When transferred from a glucose citrate medium to a mineral salts medium containing propiouate as the sole source of carbon, E-26V initiated growth more quickly and formed a higher activity of α-hydroxyglutarate synthase than did the parent strain. Moreover, in E-26V, the formation of α-hydroxyglutarate synthase immediately preceded growth on propionate, whereas in E-26, enzyme activity was not detected until the mid to late exponential growth phase. In contrast, isocitrate lyase formation immediately preceded growth initiation in both the parent and mutant cultures. It is proposed that the α-hydroxyglutarate pathway is metabolically significant during growth of E. coli E-26V on propionate by providing an auxiliary mechanism for the formation of C4 acids. Such intermediates are essential for the formation of adaptive enzymes involved in the oxidation of propionate to acetate.

Component enzymatic reactions of the tricarboxylic acid cycle in Escherichia coli

Abstract 1. 1. Alumina-ground Escherichia coli extracts have been found to oxidize all members of the tricarboxylic acid cycle with the exception of acetate. 2. 2. Crude extracts contain an aconitase and an isocitric dehydrogenase which convert citrate to α-ketoglutarate and CO 2 . The occurrence of these enzymes in extracts of E. coli suggests the operation of the tricarboxylic acid cycle in this organism. Other cyclic mechanisms for the oxidative removal of acetate or pyruvate are not excluded by this finding. 3. 3. Quantitative experiments with citrate as substrate reveal an additional pathway of citrate breakdown not involving isocitric dehydrogenase. 4. 4. Under conditions which favor CO 2 production from citrate, α-ketoglutarate is not attacked. On the addition of diphosphopyridine nucleotide, however, the latter disappears and CO 2 is evolved. 5. 5. Aconitase in crude extracts is inactivated by dialysis.

Plague Toxin: its Effect in vitro and in vivo.

The murine toxin of Pasteurella pestis inhibited the respiration of heart mitochondria from the rat and the mouse but had little or no effect on the respiration of mitochondria from the rabbit, chimpanzee, dog, and monkey. Alterations occurred in tile S-T segments of the electrocardiogramus recorded corded from rats injected with � to 10 LD50 of toxin, but not in those from rats dying of hemorrhagic shock, hypoxia, intoxication with glucose, or Escherichia coli endotoxin. No abnormalities were observed in electrocardiograms from rabbits injected with large amounts of toxin.

Propionate metabolism: V. The physiological significance of isocitrate lyase during growth of E. coli on propionate☆

Abstract The role of isocitrate lyase during growth on propionate was investigated using three strains (E-26, K-12, and W) of Escherichia coli . The activity of this enzyme during growth on propionate varied with respect to the strain employed, the concentration of propionate added to the mineral salts culture medium, and the phase of growth at which cells were harvested. The growth behavior of E-26 and of K-12 mutants indicates that in these strains of E. coli , the formation of C 4 acids from propionate can be accomplished either by the phosphoenolpyruvate (PEP) carboxylase pathway or by the glyoxylate bypass. In the early phase of growth, pyruvate formed from the oxidation of propionate is metabolized primarily via the PEP carboxylase pathway. In such cultures, the activity of isocitrate lyase and malate synthase is low. As growth proceeds, there occurs a shift in metabolism, such that the carboxylase pathway is inhibited and the decarboxylation of pyruvate to form acetyl-CoA is stimulated. In such cultures, the formation of the enzymes of glyoxylate bypass is derepressed. In contrast to E-26 and K-12, the formation of isocitrate lyase is not derepressed in E. coli W cultured on propionate.

Selection 0f mutants constitutive for several glyoxylatecondensing enzymes during growth on valeric acid

Abstract An investigation was undertaken to assess the formation of several glyoxylate condensing enzymes during growth on a series of short-chain fatty acids. When Escherichia coli was grown on acetate, high malate synthase and low β- n -propylmalate synthase activities were observed. Cells grown on either propionate or butyrate possessed high malate synthase activity, and in addition formed low levels of α-hydroxyglutarate, β-ethylmalate and β- n -propylmalate synthase activity. In contrast, growth on valerate effected high activity of all these glyoxylate condensing enzymes. Evidence is presented that valerate does not act to induce or derepress enzyme formation, but rather acts to select a mutant population which is constitutive for the formation of these enzymes. Such mutants also are able to grow with a much shorter lag on propionate, butyrate, valerate, caproate and heptanoate. The possible relationship between formation of these enzymes and growth on short-chain fatty acids is discussed.

An isotopic method for assaying the condensation of glyoxylate with acetyl-CoA and other short-chain fatty acid acyl-CoA derivatives

1. 1. An isotopic method has been described for assaying the malate synthetase reaction. This procedure is based on measuring the disappearance of 1-C14-glyoxylate as a function of acetyl-CoA. 2. 2. This assay is applicable for the determination of α-hydroxyglutarate, β-ethylmalate, and β-n-propylmalate synthetase activity. 3. 3. The method deseribed is specific and sensitive to changes in glyoxylate concentration of less than 0.001 μmole. 4. 4. This assay provides a sensitive and rapid procedure which can be used for investigating the enzymic condensations of glyoxylate with other short-ehain fatty acid acyl-CoA derivatives.

Propionate metabolism: IV. Significance of carboxylation reactions during adaptation to propionate☆

Abstract The data presented are concerned with factors regulating adaptation of Escherichia coli to growth on propionate. A mutant strain of E. coli E-26 was employed; this mutant grows on acetate and on a number of other substrates, but is unable to initiate growth when propionate is the sole source of carbon. The mutant possesses a functional tricarboxylic acid cycle and glyoxylate bypass; is able to oxidize propionate to acetate; but is unable to effect sufficient formation of C 4 acids required for initiation of growth on propionate. This deficiency can be overcome by the addition of either succinate or HCO 3 − to propionate media. It is proposed that the mutant is deficient in one or more carboxylation reactions, since such cells possess a markedly decreased capacity to incorporate 14 CO 2 into cell material. It is further suggested that carboxylation reactions are most active during initiation of growth on propionate and decline as growth proceeds.