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PROJECT SUMMARY / ABSTRACTThe composition of bacterial communities is paramount to their function. Intense competitive interactionsbetween bacteria can influence community composition by altering assembly or stability through the productionof anti-bacterial molecules. One such ecosystem rife with competitive interactions is the human gastrointestinalmicrobiota, which harbors an abundance of bacteria from the order Bacteroidales. These bacteria encode thetype VI secretion system (T6SS), a contact-dependent toxin delivery pathway shown to mediate potent inter-species competition. Bacteria that encode the T6SS also encode immunity proteins which bind to andspecifically inactivate toxins. I have found that Bacteroidales genomes possess arrays of ?orphan immunity?genes in the absence of the T6SS or corresponding toxin. These polyimmunity arrays are found associatedwith xerD-like (PAX) recombinase genes that suggest an active mechanism of recruitment. I hypothesize thatgenes within PAX clusters represent selective pressure from past episodes of direct interbacterial antagonism.Therefore, characterization of the function, activity, and biogenesis of PAX clusters offers an avenue for theidentification of direct bacterial interactions in vivo and an understanding of their ecological consequences.This proposal aims to augment my interdisciplinary background in cell biology, evolutionary biology, andgenetics with new training in germfree mouse experimentation to investigate this new mechanism of adaptationto T6SS-mediated interbacterial antagonism within Bacteroidales and understand its impact on gut microbiomeassembly and dynamics. In Aim 1, I propose to characterize the function of genes within PAX clusters throughin vitro expression experiments in E. coli and growth competition experiments in vitro and in vivo underconditions that promote contact-dependent interbacterial antagonism. In Aim 2, I will test the hypothesis thatthe PAX recombinase mediates gene acquisition and excision from PAX clusters via tyrosine recombinaseactivity in a manner analogous to integrons. In Aim 3, I will extend my characterization of PAX clusters to theiruse as a new tool for deciphering interbacterial interactions by sequencing new gene insertions after exposureof a PAX-containing strain to different bacterial communities in vivo. I will train with Dr. Lynn Hajjar at theUniversity of Washington Gnotobiotic Animal Core facility to gain experience in using germfree mice to studythe role of PAX clusters in the gut microbiota in vivo. Dr. Andrew Goodman at Yale University will collaborateand provide advice and intellectual support. The proposed experiments will lay the foundation for myindependent research program, in which bioinformatics, bacterial genetics, and in vivo experimentation will becombined to yield insight into direct interactions within the gut microbiota and the implications of theseinteractions for human health and disease.

Ross, Benjamin
University of Washington
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