Project 2, Abstract Biotin is a universally essential cofactor for enzymes that perform various carboxylation, decarboxylation, and transcarboxylation reactions. This includes key enzymes in fatty acid biosynthesis, replenishment of metabolites in the tricarboxylic acid cycle, and amino acid metabolism. When available, Mycobacterium tuberculosis (Mtb) can utilize exogenous biotin but Mtb is unable to scavenge biotin from the host. To grow and survive in the host Mtb thus needs to synthesize biotin. Mtb enzymes involved in synthesizing biotin, ligating biotin, or utilizing biotin have been identified by this group and others as attractive drug targets because their inhibition has the potential to shorten TB chemotherapy. However, a better understanding of the biology of biotin in Mtb is required to fully and effectively exploit Mtb’s susceptibility to biotin starvation for drug development. This Project addresses this need by (i) identifying and functionally classifying the genes Mtb requires to synthesize and utilize biotin, (ii) defining activities Mtb requires to import biotin or recycle biotin, and (iii) determining how biotin starvation affects Mtb’s response to host relevant stresses. These activities directly contribute to the overall program goal of understanding key pathways in Mtb biology that are important in adaptation to disease-relevant stressors.