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UBC Theses and Dissertations

Regulation of carbohydrate-catabolizing enzymes in Pseudomonas aeruginosa ATCC 9027 Lynch, William Henry Walter


Although growth with six-carbon carbohydrates, as well as with glycerol or glycerate, has been shown to induce the enzymes of glucose catabolism in Pseudomonas aeruginosa, the actual inducer(s) of these enzymes has not been identified. Low levels of these enzymes are present during growth with acetate, pyruvate, or tricarboxylic acid cycle intermediates. By examining the induction of glucose-catabolizing enzymes in the wild-type and glucose-negative mutants of P. aeruginosa ATCC 9027 by glucose, gluconate, 2-ketogluconate, or glycerol, the complexity of the regulation of these enzymes was demonstrated, i.e. of the seven enzymes examined in detail, no less than four inductive events were required to cause their synthesis. Glucose dehydrogenase was induced by glucose-6-phosphate (and possibly glucose); gluconate dehydrogenase by 2-ketogluconate; glucokinase, and glucose-6-phosphate dehydrogenase by 6-phospho-gluconate; and the Entner-Doudoroff enzymes by 6-phosphogluconate or some derivative thereof. Gluconokinase and 3_phosphoglyceraldehyde dehydrogenase were induced by gluconate. However, a weak induction of both glucono-kinase (possibly by 2-ketogluconate) and 3-phosphoglyceraldehyde dehydrogenase (possibly by 6-phosphogluconate) was also observed and did not appear to involve gluconate. Glycerokinase and L-α-glycerophosphate dehydrogenase were specifically induced by glycerol and not by either pyruvate or glucose metabolism. The importance of the oxidative non-phosphorylated pathway for glucose or gluconate dissimilation in P. aeruginosa was demonstrated. During growth of this organism with glucose or gluconate, the majority of both substrates was rapidly oxidized to 2-ketogluconate prior to being phosphorylated and further catabolized by the Entner-Doudoroff pathway enzymes. Therefore, the major amount of growth, when either glucose or gluconate was added as the carbon source, actually took place with 2-ketogluconate serving as the source of carbon. The mutants of P. aeruginosa, isolated for their inability to grow: with glucose as a carbon source, were also incapable of growth when either gluconate or 2-ketogluconate was the sole source of carbon. Growth of the mutants with pyruvate, acetate, or tricarboxylic acid cycle intermediates was inhibited by the presence of glucose, gluconate or 2-ketogluconate. The inhibition of growth of the glucose-negative mutants of P. aeruginosa with pyruvate was due to an accumulation of high internal concentrations of 6-phosphogluconate from the metabolism of either glucose, gluconate, or 2-ketogluconate. This accumulation of 6-phosphogluconate caused a repression (either direct or indirect) of pyruvic dehydrogenase synthesis and consequently resulted in growth stasis. The examination of these mutants also indicated that 6-phosphogluconate dehydrogenase was absent and a 6-phosphogluconate phosphatase was present in this strain of P. aerug inosa.

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