Browsing by Author "Dong, Sharon"

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    Genetic Manipulation of Bacteroides for Transient Colonization of Germ-free Mice
    (2022-04) Dong, Sharon; Geuking, Markus; Harrison, Joe; Hirota, Simon; Buret, Andre
    Human and murine hosts have a dynamic relationship with the microbes that colonize their bodily surfaces. Shifts in composition of the community of microbes, or their function, are implicated in many chronic and/or inflammatory conditions. There are currently gaps in understanding whether these shifts cause disease phenotypes or are artefacts of changed host conditions due to disease. The ability to tease apart these dynamics by generating bacterial tools that limited bacterial exposure of germ-free mice to a certain time-period was explored in this thesis. I target genes in synthesis pathways of two uniquely-bacterial amino acids, meso-diaminopimelic acid (mDAP) and D-Alanine (D-Ala) in a species of interest, Bacteroides thetaiotaomicron. These amino acids are incorporated into the peptidoglycan, or cell wall, of most bacteria, like B. thetaiotaomicron. This study builds on concepts from a previously developed E. coli strain called HA107, where disruption of these two synthesis pathways generated an auxotrophic mutant that grew if supplemented with said amino acids, however, did not proliferate and persist in germ-free murine gastrointestinal tracts. I designed deletion alleles that would exchange with the functional allele of target genes, and transferred the allelic exchange vector, pExchange, containing the deletion allele into B. thetaiotaomicron through conjugation. The double and triple gene deletion mutants failed to grow when not supplemented with mDAP and D-Ala. This mutation strategy may be adapted to genetically manipulate other notable Bacteroides species. The generated auxotrophic mutants from this study will be useful tools in answering questions regarding how timing or dosage of bacterial exposure affect health or disease outcomes in germ-free mice.