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Dynamics of genetic adaptation in Escherichia coli K12 MG1655

Abstract

Bacteria replicate quickly enough that adaptive changes can be observed on a timescale of weeks to months. Laboratory adaptive evolution experiments with bacteria, especially of Escherichia coli, have been used to study population dynamics and evolutionary processes. Recent advances in DNA sequencing now make it possible to identify all of the acquired mutations and interrogate the adaptive process at the molecular level. The first two research studies in this dissertation analyze mutations acquired by E. coli during prolonged growth on glycerol minimal media. In the first study I measured fitness differences between independently-evolved lineages and between mutations acquired by the same gene in different lineages by head-to-head competitions. This allowed small fitness differences to be detected between independently- evolved strains and between mutations to the same genes. This also detected epistatic interactions between several mutations. Overall these results provide the basis for understanding how the mutations individually and cooperatively affect fitness. The second study examines adaptive mutations to glycerol kinase (GlpK), which were acquired by almost all glycerol-adapted lineages. The relative ability of the GlpK mutations to increase fitness was used to identify the enzyme function responsible for improving phenotype, which was found to be sensitivity to the inhibitor fructose-1,6-bisphosphate. However, the GlpK mutants also induce autocatabolite repression which reduces glpK transcription, attenuating the ability of the mutations to increase glycerol metabolism. This likely occurs to prevent methylglyoxal toxicity. Additionally, the GlpK mutations altered growth on several sugars, but it is unclear what mechanism could be involved suggesting it is a novel function. This study demonstrates how adaptive mutations can be studied to identify constraints on expressed phenotype and to identify novel protein interactions. The goal of the third study was to integrate genes for cellulose-degrading enzymes into the E. coli genome, and to use adaptive evolution to select for strains that improve use of the genes that allow growth on cellulose. This was both a metabolic engineering and basic research project, to produce a potentially useful strain and to study the molecular dynamics involved in integrating a novel metabolic function into the existing metabolic and regulatory network

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