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UBC Theses and Dissertations

Factors influencing the aerobic respiration of Escherichia coli Rainnie, Donald James


Facultative anaerobic bacteria such as Escherichia coli are capable of obtaining energy from glycolysis, aerobic respiration, and anaerobic respiration. Although a considerable amount of information is available on the production of ATP in E. coli by glycolysis, very little is known about the production of energy by aerobic or anaerobic respiration. Since the properties of aerobic respiration were experimentally more readily attacked, an investigation of the factors influencing the aerobic respiration and respiratory chain-linked energy production of E. coli was undertaken. The principal technique utilized in these investigations was the polarographic determination of oxygen tension. Investigation of the properties of a commercially available, vibrating-reed, oxygen electrode revealed that silver ions were released from the silver anode of the uncoated oxygen electrode into the buffer solution surrounding the electrode. Loss of silver from the electrode was dependent on the buffer concentration and the type of buffer, and was relatively independent of the presence or absence of the polarizing voltage. It is postulated that the release of silver from the anode of the oxygen electrode involved chelation by the buffer ions. This problem was avoided subsequently by using a Clark oxygen electrode. The pH, buffer ion and the buffer concentration of the assay medium were observed to influence the rate of respiration of E. coli. In addition the buffer ion and the pH influenced the linearity of oxygen consumption with time. Glycylglycine-KOH buffer, pH 7.0, at a concentration of 300 mM was determined as "optimal" according to the criteria of: (i) supporting a high respiratory rate; (ii) supporting a constant rate of oxygen utilization; and (iii) maintenance of these characteristics of the E. coli cell suspension for a greater period of time than required to complete the experiment. The applicability of these criteria of classical enzyme kinetics to the determination of "optimal" conditions for the investigation of systems involved in energy conservation is questioned. During the simultaneous measurement of acid production and oxygen consumption, a 15 to 30 second lag in acid production was observed to occur during the transition from aerobic to anaerobic glucose utilization. This, observation is discussed with respect to the information currently available on the regulation of the amphibolic pathways of E. coli. Silver ions inhibited the oxidation of endogenous substrates, glucose, glycerol, D- and L-lactate, acetate, succinate and fumarate by intact-cell suspensions of E. coli. The oxidation of formate was only slightly inhibited under the conditions which resulted in complete inhibition of respiration on the previously indicated substrates. The oxidation of glucose and glycerol was more sensitive to silver ions than that of D- or L-lactate, fumarate or succinate. This was attributed to inhibition of glyceraldehyde-3-phosphate dehydrogenase. Before the onset of the inhibition by silver ions there was a period when respiration was stimulated. This effect was similar to that given by 2,4-dibromophenol. With both compounds the degree of stimulation was larger in iron-sufficient than in iron-deficient cells. It is postulated that silver ions uncouple respiratory chain-linked energy production as well as inhibiting the respiratory chain and glyceraldehyde-3-phosphate dehydrogenase in E. coli. Growth and cell respiration were affected when iron became limiting in batch cultures of E. coli growing on succinate. A decrease occurred in the efficiency with which succinate was converted to cell mass, in the respiratory control ratio, and in the levels of nonheme iron, cytochrome, and NADH and succinate oxidase activities. On addition of ferric citrate to the iron-limited cultures the above components returned at different rates to the levels found in iron-sufficient cells. The concentration of nonheme iron, the respiratory control ratio and the efficiency of conversion of succinate to cell mass recovered more rapidly than.the level of cytochrome b₁ and the oxidase activities. Succinate oxidase activity recovered more rapidly than either succinate dehydrogenase or cytochrome b₁ levels. It is postulated that nonheme iron is involved in respiratory chain-linked energy production in E. coli.

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