Abstract: Sulfation is a common extracellular signaling modification used by higher eukaryotes in communication between cells. A small but expanding number of bacteria are now known to communicate with eukaryotic hosts through sulfate-modified molecules. The mycobacterial genus synthesizes unique sulfated metabolites that are found at the host-pathogen interface. This thesis describes the elucidation of biosynthetic pathways in mycobacteria that produce sulfated small molecules, specifically, the Mycobacterium tuberculosis virulence-associated molecule Sulfolipid-1. A long-term goal is to determine their significance in pathogenesis. Chapter 1 describes the sulfate modification and provides the historical background that serves as a framework for the rest of the thesis. The decades of work on mycobacterial sulfation is detailed with special attention given to the more recent advances in our knowledge of the sulfate modification. Chapter 2 details our work toward identifying novel sulfated metabolites in the opportunistic human pathogen, M. avium. In the course of this work, we uncovered a unique sulfate assimilation pathway and developed methodologies for improved metabolite detection and enrichment. The next three chapters delve into the biosynthesis of Sulfolipid-1, beginning with the discovery and characterization of two acyltransferases, PapA1 and PapA2. As described in Chapter 4, we probed for biochemical intermediates and virulence defects in Sulfolipid-1-deficient mutants. Chapter 5 introduces a new membrane protein, termed Gap1, involved in the transport and synthesis of Sulfolipid-1. Finally, Chapter 6 introduces our work toward defining the biosynthesis of polyacyltrehalose, a glycolipid structurally related to Sulfolipid-1 that is produced by virulent M. tuberculosis.