Project Summary The human gut microbiome is inhabited by trillions of bacteria that encode over 150-fold more genes than the human genome itself. The inter-individual differences in microbial composition can be significant, and the factors contributing to this diversity are not well understood. Metagenomic studies suggest that the microbiome might play an important role in determining an individual’s predisposition to disease and responses to treatments. The paucity of understanding how cofactors and other factors influence microbial composition limit strategies to rationally alter it for therapeutic purposes. Much of our current understanding of the factors that shape the gut microbial flora composition derives form studies on how bacteria generate energy, maintain redox balance and acquire carbon and nitrogen. The enzymatic reactions that support these metabolic processes often rely on cofactors that are in short supply. Vitamin B12 is an example of one such cofactor that is essential for many bacteria that are unable to biosynthesize it and lack parallel B12-independent metabolic pathways to circumvent its absence. So, one approach to the targeted manipulation of the gut microbiome is via altering the levels of available corrinoids. In this proposal, I seek to elucidate the corrinoid selectivity of transport systems to provide needed insights into how gut bacteria compete with each other and their hosts for a critical resource in a complex ecosystem. My studies will focus on Bacteroidetes thetaiotaomicron, a common gut bacterium, which lacks the genes required for de novo synthesis of vitamin B12 but encodes multiple B12-dependent enzymes. 5’-Deoxyadenosylcobalamin is the active cofactor form that is utilized by some B12 dependent enzymes and is synthesized by BtuR in B. thetaiotaomicron. The chaperone and catalytic activities are uncharacterized and will be addressed in Aim 1. It also encodes three copies of the outer membrane B12-transporter BtuB with each system displaying a different preference for corrinoid derivatives. The bacterium also possesses additional transport machinery that is not observed in E. coli, a model organism in which studies on B12 transport in gram-negative bacteria have been focused. Using a combination of biochemical and biophysical approaches, I propose to elucidate the mechanism of B12 transport by the B12-uptake (Btu) system in Aim 2. The kinetic and thermodynamic studies in Aims 1 and 2 will define the selectivity of the Btu proteins for cobamides and provide insights into protein-protein interactions. Combined with the structures determined in Aims 1 and 2, my studies will furnish mechanistic insights into how a precious and rare cofactor is relayed from the environment across two layers of bacterial membranes to support the metabolic needs of a common gut bacterium.