Essentially all living systems produce cell surface structures to rigidify cells, form protective coats, or facilitate cell adhesion and migration. Microbial ‘cell walls’ usually perform protective functions for survival under detrimental conditions, to reduce the efficacy of their host’s innate immune response, or to form 3-dimensional meshworks, called biofilms. Common building materials for these extracellular structures are polysaccharides that either function on their own or are integrated with other polymers into elaborate composite materials. Capsular polysaccharides (CPS) are abundant among Gram-negative and –positive bacteria. The polymers form dense extracellular structures that limit diffusion, aid in osmoregulation, and form thick protective coats around the cell. Some CPS mimic host glycans, thereby disguising potent pathogens under an immunologically invisible coat. The polymers are synthesized and deposited on the cell surface by two fundamentally different pathways. One assembles the polymer in the periplasm from short lipid-linked precursors and translocates it across the outer membrane (OM) concomitantly. In the ABC transporter- dependent pathway, however, the CPS is synthesized intracellularly on a lipid anchor and transported after its completion through a secretion system that spans the inner and the OM. The molecular and mechanistic mechanisms of both pathways remain poorly understood. To aid the development of novel antibiotic strategies, we seek to establish a detail structure-function analysis of the abundant ABC transporter-dependent CPS biosynthesis pathway. Our approach is two-pronged. First, we seek to establish a robust genetically tractable model system for CPS secretion (Aim 1A and B). Second, we will complement our functional analyses with detailed structural insights into the CPS ABC transporter (Aim 2), thereby providing the molecular basis for substrate recognition, CPS translocation, as well as interaction with periplasmic and OM transporter components. To this end, we engineered a standard E. coli laboratory strain to produce a polysaccharide capsule from plasmid-encoded components. The expressed operon contains 9 genes and each can be removed from its expression plasmid by standard restriction enzyme digestion. Further, we also developed a molecular probe enabling the detection of the synthesized capsule on the cell surface, thereby correlating CPS production with the expression of the biosynthetic machinery. To integrate our functional analyses with a 3D structure of the CPS ABC transporter, we purified a stable transporter in complex with its periplasmic subunit that likely stabilizes interactions with the OM pore. We will use cryo electron microscopy to determine the transporter’s structure in different nucleotide-bound states. Combined, our proposed research will provide the molecular basis for CPS secretion and lay the foundation for structure-guided drug development.