Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science in Cell and Molecular Biology

Department

Cell & Molecular Biology

First Advisor

Paul S. Cohen

Abstract

Previous studies in this laboratory have indicated that E. coli Nissle 1917 can act as a probiotic and is able to prevent the pathogenic E. coli EDL933 from growing to high numbers in the mouse large intestine. This study evaluated the influence of gluconeogenesis in E. coli Nissle 1917 and E. coli EDL933 to compete against each other and for their maintenance in the intestine. Knockout mutants of both strains were created for the analysis of the importance of gluconeogenesis in colonization experiments in the mouse large intestine. To knock out the ability to use gluconeogenesis and therefore metabolizing substrates such as pyruvate, TCA cycle intermediates and amino acids, simultaneously both genes pckA, encoding for phosphoenolpyruvate carboxykinase, and ppsA, encoding for phosphoenolpyruvate synthetase, were replaced by a non-functional chloramphenicol cassette.

It was shown that E. coli Nissle 1917 uses glycolysis and gluconeogenesis concurrently throughout colonization of the mouse large intestine. E. coli EDL933 utilized glycolysis exclusively when it was the only E. coli strain present, but switched to gluconeogenesis when it was in the presence of a competing E. coli strain. Knocking out the ability to use gluconeogenesis in E. coli EDL933, led to a severe disability to colonize the mouse large intestine when it competed against E. coli Nissle 1917 which used up most of the glycolytic substrates. By implication E. coli Nissle 1917 seems to use up most of the glycolytic substrates and some of the gluconeogenic substrates, E. coli EDL933 depends on utilizing gluconeogenic substrates to be able to maintain itself in the mouse large intestine. Phenotypic characterization of the usage of different substrates in E. coli Nissle 1917 and E. coli EDL933 strains revealed several differences between the strains.

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