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Abstract
The bacterial cell wall is largely comprised of peptidoglycan (PG), a bacteria-specific molecule that is sturdy and protective, yet fluid enough to allow for cellular growth and division. PG structure is highly conserved, though there are examples of natural variation. Elucidating the mechanisms of this PG variation will inform our understanding of antibiotic resistance, PG signaling within hosts, and the evolution of the bacterial cell wall. We are interested in PG structure in Vibrio fischeri, because of PG’s role in the model symbiosis between V. fischeri and the Hawaiian bobtail squid. Specifically, PG monomers released from V. fischeri trigger normal host development of the symbiotic organ. In this dissertation, I altered V. fischeri’s PG biosynthesis via isolation of spontaneous suppressor mutants from strains that were previously auxotrophic for PG-specific D-amino acids. First, I detail the characterization of a D-glutamate suppressor with a novel biosynthesis pathway to wildtype PG structure. Fusion of two proteins buries the secretion signal of BsrF, a periplasmic broad-spectrum racemase. When relegated to the cytoplasm, BsrF produces the D-glu and D-ala necessary biosynthesis of wild-type PG structure. Next, I describe a set of D-glu suppressors that have mutations in gltS, which encodes a sodium:glutamate symporter. These mutations lead to increased D-glu transport and permissive transport of toxic glutamate structural analogues. I then characterize D-ala auxotrophy in V. fischeri, and the extent to which it can be suppressed. Finally, I describe our work to develop a novel method for transiently colonized Euprymna scolopes through use of a D-ala auxotrophic strain. Together, these discoveries and insights broaden our understanding of D-amino acids and the evolution of PG structure.