The Escherichia coli O9a and O8 antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. Their O-antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a variable repeat-unit domain, and a non-glycan terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid acceptor, undecaprenyl pyrophosphoryl-β-GlcNAc, due to the sequential activities of two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The work reported in this doctoral thesis establishes a model for biosynthesis of the O8 and O9a antigens using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1,3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat-unit domains. WbdAO9a polymerizes a tetrasaccharide repeat unit containing two α-(1,3)- and two α-(1,2)-linked mannose residues, while WbdAO8 polymerizes trisaccharide repeat units containing single α-(1,3), α-(1,2), and β-(1,2)-mannoses. Consistent with the multifunctional nature of the WbdA mannosyltransferases, two separable domains were identified in WbdAO9a and three in WbdAO8. Results from mutation of the catalytic site motifs of WbdAO9a and in vitro assays with synthetic acceptors demonstrated that the N-terminal domain of WbdAO9a possesses α-(1,2)-mannosyltransferase activity. Therefore, these studies form a framework to investigate the hypothesis that each domain of WbdA is a catalytically active mannosyltransferase module, possessing one of the activities associated with the enzyme. The O8 and O9a systems provide examples where a unique combination of single domain mannosyltransferases, one of which is capable of adding two mannose residues in succession, and a multidomain polymerizing mannosyltransferase is exploited to build a single glycan. The information gained from this project is expected to extend to other bacteria that utilize similar pathways for biogenesis of cell surface glycopolymers. / Natural Sciences and Engineering Research Council of Canada