Project Summary The microbial community in the human gut plays a key role in complex disease states, with less diverse microbiomes observed in obesity, inflammatory bowel disease, and even mental health. However, the specific interspecies interactions that drive gut community structure, leading to beneficial or deleterious microbiomes, remain poorly understood. There is, therefore, an urgent need to map these microbial interspecies interactions, in order to identify key nutrients that could be leveraged to shape microbiomes towards beneficial health outcomes. Nevertheless, direct study of these communities is difficult: in vivo models of the human microbiome in mice remain close analogues, but their low-throughput, complex, and expensive nature prevents systematic dissection of individual components of an in vivo microbiome. In contrast, pure culture studies have illuminated a wide variety of nutritional requirements and pinpointed potential interspecies interactions, such as those involving the Vitamin B12 family of cofactors known as corrinoids. Nevertheless, many bacteria have not been successfully isolated because a required nutrient remains unknown, and pure culture studies remove the native community nutrients that can alter growth and metabolism. Enrichment cultures, which are simplified microbial communities that grow out of inoculation, represent an attractive stepping stone that combine the high-throughput capabilities of pure culture methods with medium complexity. Some bacteria readily grow in these enrichment cultures but are unable to be isolated axenically in the culture medium, suggesting that many natural interspecies interactions remain. The overarching goal of this proposal is to elicit a comprehensive understanding of microbial interactions in the gut by exploiting the potential of in vitro derived gut enrichment communities for use in high-throughput discovery. In the first approach, I will determine the impact that corrinoid producer growth has on community assembly and response to nutritional perturbation (Aim 1) . Second, I will perform a high-throughput isolation campaign to identify growth factors for `unculturable' bacteria that readily grow in the enrichment culture, and identify the community members that produce these growth factors (Aim 2). Finally, I propose to directly map physical interactions in enrichment cultures by exploiting the high-specificity recognition capability of nucleic acids to perform targeted pulldowns of a given species from the enrichment culture (Aim 3). Successful completion of these approaches will identify nutritional strategies to perturb the microbiome that may be leveraged for the treatment of microbiome-related disease.