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Abstract
During adaptive laboratory evolution, an organism is evolved for several hundred to several thousand generations. Usually, these studies yield multiple mutations in any selected isolate. Mutations that occur in uncharacterized genes, or those with no clear role in the adaptive process may be challenging to investigate yet may provide the most novel information. In this dissertation, I present my analysis of an evolved ADP1-derived strain selected for growth on guaiacol, a lignin-derived aromatic compound. Guaiacol is abundant in many types of lignin streams. Improving the bioconversion of guaiacol represents an important step in the overall goal of producing valuable products from lignin. My analysis revealed an unexpected role of a regulatory protein in the adaptation to growth on guaiacol. Ribosomal silencing factor proteins (RsfA), are known to inhibit protein synthesis during stationary phase and in nutrient-poor conditions. Through modified competition assays, I show that lack of RsfA provides cells with a competitive growth advantage during the transition from stationary phase to fresh medium and only when guaiacol is the carbon source. This advantage is presumably due to derepressed protein synthesis in mutant cells relative to the wild-type cells, enabling the former to remain in a state of continued protein synthesis. RsfA has not been previously characterized in ADP1 and
previous reports in E. coli have shown that rsfA mutants are always outcompeted by wild-type cells. To our knowledge, this is the first report of conditions under which lack of RsfA is beneficial. RsfA is involved in the conserved process of ribosome hibernation. The results presented here may inform the design of future laboratory evolution experiments. During batch cultures, serial transfers may be done after cells enter stationary phase, and strains that lack RsfA at the onset of evolution may be advantageous. In addition, other previously uncharacterized genes are suggested as targets for future investigative efforts. Finally, preliminary results are presented on development of genetic methods and suggested modified culture conditions for laboratory evolution. Overall, my results have implications that extend beyond the metabolism of lignin-derived aromatic compounds to broader areas such as experimental design and global regulation.
previous reports in E. coli have shown that rsfA mutants are always outcompeted by wild-type cells. To our knowledge, this is the first report of conditions under which lack of RsfA is beneficial. RsfA is involved in the conserved process of ribosome hibernation. The results presented here may inform the design of future laboratory evolution experiments. During batch cultures, serial transfers may be done after cells enter stationary phase, and strains that lack RsfA at the onset of evolution may be advantageous. In addition, other previously uncharacterized genes are suggested as targets for future investigative efforts. Finally, preliminary results are presented on development of genetic methods and suggested modified culture conditions for laboratory evolution. Overall, my results have implications that extend beyond the metabolism of lignin-derived aromatic compounds to broader areas such as experimental design and global regulation.