Characterization of phosphoenolpyruvate synthase mutants in Salmonella typhimurium

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Abstract
The enteric bacteria are able to grow by utilizing three-carbon compounds (pyruvate, lactate, and alanine) as sole carbon sources only if they have a functional phosphoenolpyruvate synthase (PEP synthase). PEP synthase catalyzes the phosphorylation of pyruvate to PEP with the hydrolysis of ATP to AMP. This anaplerotic reaction is needed for the synthesis of carbohydrates and citric acid cycle intermediates that are essential for continued cell growth. Insertion mutants were isolated in Salmonella typhimurium that specifically lack the ability to grow on three-carbon compounds. These mutants also fail to utilize acetate as a sole carbon source. Enzyme assays were performed and the results showed that these mutants contain no PEP synthase activity. By using bacteriophage P22, the pps mutations isolated in this study were found to be contransducible with genetic markers in both the aroD and btuC genes. Three-factor crosses pinpointed the order of these genes and their distances with respect to each other. One of the mutants carries a pps::lac operon fusion. This fusion was used to explore the transcriptional regulation of the pps gene. A functional copy of the pps gene is required for its own induction. The pps gene is also under catabolite repression, but the addition of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) to cells grown in the presence of glucose does not relieve this repression. These results indicate that the synthesis of PEP synthase is regulated in a more complex manner than has been previously recognized.