Overall, Abstract Despite the availability of therapy, Mycobacterium tuberculosis (Mtb) is the major infectious killer of adults. Finding more effective therapeutic strategies will require a better understanding of the fundamental biology of bacterial growth, metabolism and interaction with the host. This program proposes to understand key pathways in Mtb biology using advanced genetic and biochemical methods, with a particular focus on complex pathways important in adaptation to disease-relevant stressors. “Pathways” is defined broadly, including biochemical, structural and genetic interactions. In many cases, traditional genetic approaches to discover and understand these pathways are limited, but many of the most critical aspects of Mtb biology are mediated by such complex genetic mechanisms. This program will leverage new methodology and resources that the collaborators have developed in the last few years, to discover the constituents of key pathways, attribute functions to new pathways, and understand the mechanisms which underlie the ability of bacteria to survive and grow under a variety of conditions, focusing on those encountered during infection. They will also take advantage of the close collaborations among the investigators who have worked well together for several years. The work will be organized into four projects and four cores, but considerable crossover is expected, which will enrich each component. Project 1. Metabolic adaptations required for growth and virulence of Mtb at acidic pH. Project 2. Acquisition, synthesis and importance of biotin in Mtb. Project 3. Defining the RNA processing and degradation pathways of Mtb. Project 4. Defining cell division pathways in Mtb. Core A. Metabolomics and lipidomics. Core B. Biochemistry and enzymology. Core C. Bioinformatics and data sharing. Core D. Administration. All of this work will be facilitated by a highly collaborative group of investigators, almost all of whom have worked together for many years on a large variety of projects. Both project and core investigators will serve as full- fledged intellectual contributors, enabling the team to bring a variety of approaches and thinking to bear on complex biological systems.