Project Summary/Abstract Despite the tremendous amount of data that has been generated over the last decade regarding the human intestinal microbiota, we still know little about energy generation processes of most of the abundant members of this ecosystem. This gap in our fundamental knowledge of the gut microbiota hinders our understanding of how the Bacteroides interact with other community members, the conditions in the gut environment that contribute to microbial compositional changes, and how we may appropriately alter the composition of the ecosystem to improve human health. Bacteroides is the most abundant and stable genus of the human intestinal microbiota with strains colonizing their hosts for decades. Currently, Bacteroides are predicted to have a primitive anaerobic respiration pathway that would produce little energy to power cellular processes. In this application, based on strong supporting data, we will test our premise that Bacteroides have a more complex respiration pathway that creates a much larger amount of energy including the direct generation of both H+ and Na+ gradients to power cellular processes. Central to this new paradigm is our prediction that Bacteroides have a complete aerobic respiration pathway that allows them to utilize oxygen at the mucus layer of the colonic epithelium, which we predict significantly contributes to their fitness in the gut. In addition, we will test our hypothesis that aerobic respiration by the Bacteroides has community-wide effects, significantly contributing to the low oxygen environment of the colon, allowing oxygen intolerant members to colonize the gut, especially near the mucus layer. The objectives of this application will be addressed in three aims, taking advantage of the diverse strengths of three co-PIs. In Aim 1, we will use genetics, functional assays and biochemistry to completely elucidate both the aerobic and anaerobic pathways of Bacteroides. In Aim 2, we will focus on the ion gradients that the respiration pathway creates and the cellular processes that they energize. In Aim 3, we will use gnotobiotic mouse models to test which enzymes of the respiration pathway are critical for in vivo fitness, the role of aerobic respiration in conferring a fitness advantage to the Bacteroides, and the community wide effects of aerobic respiration on the gut microbiota and colonization of enteric pathogens. We predict this project will reveal new paradigms that will alter our thinking about Bacteroides, their ecological effects in the gut microbiota, and the conventional wisdom that these bacteria are strict anaerobes.