Abstract Decades of research have revealed that the composition of the human gut microbiome directly impacts human health, yet we still do not understand the mechanisms underlying formation and maintenance of this complex community. Counterintuitively, increasing evidence suggests that competitive interactions play a role in maintaining community stability. While genes encoding components of diverse systems involved in interbacterial antagonism are widespread in human gut metagenomes, the specific antagonistic interactions occuring in situ remain elusive. One of the pathways mediating interbacterial antagonism is the Esx secretion system, which is conserved in two prominent gut phyla, Firmicutes and Actinobacteria. While this pathway has been shown to mediate contact-dependent interbacterial antagonism by a limited number of Gram-positive species in vitro, many questions remain about its physiological function. Antibacterial toxins secreted by the Esx machinery are present throughout human gut metagenomes, suggesting that Esx-mediated antagonism could occur in this community. These findings led me to propose to identify the targets of Esx-mediated interbacterial antagonism in a model natural community, the murine gut microbiome. To accomplish this goal, I will take advantage of the fact that bacteria express cognate immunity genes to prevent self-intoxication by secreted toxins. In Aim 1 of my proposed studies, I will use bioinformatics and in vitro functional assays to identify Esx toxin and immunity genes encoded in a model murine gut metagenome derived from wild-caught mice. My preliminary findings indicate at least two antibacterial Esx toxins are encoded in this natural gut microbiome. In Aim 2, I describe an in situ conjugation strategy to deliver the immunity genes identified in Aim 1 to all members of the murine gut community. I hypothesize that expression of immunity genes in bacteria targeted by specific Esx toxins will increase their abundance in a community. Therefore, by identifying changes to community composition that depend on immunity genes, I can determine the targets of Esx-mediated antagonism in situ. This unique approach to examine interbacterial antagonism under physiological conditions will significantly improve our understanding of the physiological function of the Esx system in gut bacterial species. It will also provide the first direct characterization of antagonism between gut microbiome constituents. The methods I propose to develop may additionally later be applied broadly to the characterization of diverse antagonistic pathways present in gut bacterial species, an essential next step needed to define the mechanisms shaping gut microbiome composition and ultimately human health. By working in the lab of Dr. Joseph Mougous at the University of Washington and with my experienced collaborators, I will be able to learn diverse techniques, practice effective mentoring strategies, and hone my scientific communication skill...