PROJECT ABSTRACT Colonization factors that facilitate establishment in the complex and competitive environment of the gastrointestinal (GI) microbiome are still relatively unknown, but knowledge of these factors will be crucial for understanding the formation of this ecosystem and reveal novel therapeutic avenues to manipulate this community. Serving as potential colonization factors, Diversity Generating Retroelements (DGRs) are genetic elements found in bacteria, archaea, and their viruses that are capable of accelerating evolution by rapidly diversifying ligand binding proteins to alter their ligand recognition. The GI microbiome is the most enriched ecosystem for DGRs known to date, but the role of DGRs within the microbiome remains completely unexplored. Therefore, the overarching goal of this proposal is to understand how DGR-driven genotypic variation contributes to adaptive bacterial phenotypes in the GI microbiome, especially in response to dynamic shifts in the environment, such as during colonization or from perturbations to the community. The candidate will use five carefully selected strains of Bacteroides, each of which contains a similar but non-identical DGR that diversifies either a pilus tip adhesin or a periplasmic protein. In Aim 1, he will uncover factors that control DGR activity in Bacteroides spp. In Aim 2, he will characterize the in vivo roles of the diversified proteins and identify other proteins that functionally interact with these diversified proteins. Lastly, in Aim 3, the candidate will determine the selective fitness advantages conferred by DGR-directed accelerated protein evolution. These aims require the application of genetic systems to manipulate Bacteroides genomes, RT-qPCR, genome-wide Tn-insertion sequencing, tandem mass spectroscopy, deep sequencing, computational methods to measure single nucleotide variation, and gnotobiotic mouse models. Practical implications of this work include the identification of DGR-encoded and host-encoded factors that control DGR activity in Bacteroides, characterization of the functions of Bacteroides diversified proteins, and an understanding of how diversification can be utilized to create selective fitness advantages in complex microbial communities. Insights derived from this proposal will ultimately be developed into a toolkit for engineering adaptative colonization systems in beneficial microbes that will facilitate their efficient engraftment into disrupted microbiomes to reverse the dysbiotic states that are often associated with diseases such as obesity, inflammatory bowel disease, and cardiovascular disease. Included in this proposal is a detailed career development plan that outlines a five-year timeline for the candidate that includes hands-on and didactic training in structural biology, bioinformatics, and ecology and evolution. It also details a diverse, multidisciplinary, and complementary advisory team, including an experienced primary mentor, who will guide t...