PROJECT SUMMARY/ABSTRACT Extracellular polysaccharides play critical roles across all domains of life. Bacterial polysaccharides are a diverse class of macromolecules with multiple biological functions, including mediating interactions with the external environment and preserving cell wall integrity. Bacterial glycosyltransferases are responsible for polysaccharide diversity through their differences in substrate specificity and linkage production. Polysaccharide biosynthesis and elongation can occur by multiple mechanisms; the least understood is processive polymerization. Processivity is elicited from an enzyme’s ability to retain the acceptor through numerous elongation steps. This process reduces the production of short-length polysaccharides, which could be harmful to bacterial fitness. Processivity may represent a common and critical mechanism for polysaccharide biosynthesis and length control. Production of the mycobacterial galactan by galactofuranosyltransferase 2 (GlfT2) was shown to proceed by a processive mechanism. The galactan of Mycobacterium spp. is an essential structural glycan, functioning as a component of the cell wall structure of human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Galactan truncation decreases cell fitness, promotes periplasm thinning, and increases antibiotic susceptibility. Therefore, enzymatic processivity by GlfT2 likely ensures the galactan is of sufficient length. The proposed studies seek to define the mechanism of GlfT2 processivity and the biophysical parameters that dictate product length distributions. This project encompasses training in enzyme production and characterization, enzyme kinetics assays, and enzyme structure determination. The Kiessling group, leaders in chemical glycobiology, and the Department of Chemistry at MIT provide a rich environment to acquire these research skills. The research environment also offers opportunities to engage in science communication, literature analysis, and career development. The results from the investigations proposed are expected to provide a framework for mechanistic analysis of processive glycosyltransferases found in other bacteria and across the different domains of life. New insights into this under-characterized class of enzymes will provide novel targets to combat the modern emergence of antibiotic-resistant bacteria.