Normal function of the animal gut depends on metabolic and regulatory interactions with resident, symbiotic microorganisms. This community of microbes, also called the gut microbiota, facilitates processing and assimilation of nutrients by its host, and impacts the development and function of the host immune system. To fully understand human nutrition and health we must ascertain the principals that underlie assembly, dynamics and function of host-associated microbial communities. The central goal of this project is to construct a defined gut microbiota in germ-free Drosophila, and then to utilize this model system to investigate the mechanistic basis of gut microbial community assembly and function. The first aim is to determine the effect of microbiota species composition on gut community assembly. The four dominant gut bacterial species of Drosophila will be reintroduced into germ-free flies (re-associated) singly and in pairs, and the colonization level and localization of each species determined. This analysis will identify interactions between microbes that impact gut microbiota structure, and will generate fly lines with defined gut microbial communities. The second aim will test if defined microbiota can restore normal functions to germ-free flies, which show a reliably altered nutrient profile and delayed development compared to conventionally-reared flies. Re-associated fly lines generated through Aim 1 will be compared to germ-free and conventionally reared controls for these phenotypes, and profiled for indicators of immune and metabolic function. To elucidate the mechanistic basis of symbiosis, the final aim will identify specific genes and metabolic pathways required for the persistence of a prominent bacterial species in the Drosophila gut using transposon mutagenesis and in vivo selection in re-associated flies. The results of these studies will generate a comprehensive and detailed model for how members of the Drosophila microbiota assemble and interact. Future research will test this model to further investigate how host and microbiota integrate to form a functional partnership in the gut. The completion of this project will yield fundamental insights into how gut microbial communities assemble and function, with relevance to human nutrition and a range of diseases including obesity, antibiotic-associated diarrhea, and inflammatory bowel disease.