Bacterial attachment is a critical first step in the formation of complex, matrix-enmeshed, microbial communities called biofilms. In Agrobacterium tumefaciens, secretion of the unipolar polysaccharide (UPP) adhesin is necessary for stable polar attachment that drives biofilm formation on a variety of surfaces, but this UPP is neither produced by cells grown in dispersed culture nor by bacterial colonies grown on solid media. Elevation of intracellular levels of the second messenger cyclic diguanosine monophosphate (cdGMP) increases UPP production and bypasses the surface contact-dependence of UPP synthesis, thereby promoting UPP secretion by planktonic cells and in bacterial colonies. Multiple genes involved in UPP biosynthesis have been identified and their products suggest that they constitute a Wzx/Wzy-type polysaccharide biosynthesis pathway. Preliminary data suggest that the UPP structure may be composed of at least two distinct polysaccharides, with one polysaccharide containing N-acetylglucosamine (GlcN) and the other containing N-acetyl galactosamine (GalN) as determined by fluorescent short-term binding of UPP with the lectins Wheat Germ Agglutinin (WGA; specific to GlcN) and Dolichos biflorus Agglutinin (DBA; specific to GalN). Wzy-type polymerase homologs UppY and UppW appear to specify the DBA- and WGA-binding polysaccharides (UPPGalN and UPPGlcN) respectively. Two Wzx-type flippase homologs, UppX and UppV are likely to catalyze the transfer of polysaccharide subunits attached to a lipid carrier on the intracellular leaflet of the cytoplasmic membrane across the bilayer to the periplasmic leaflet where they can be polymerized in the periplasm by the action of the Wzy-type polymerase homologs. In addition to UPP, A. tumefaciens also secretes four other exopolysaccharides (EPS): cellulose (C), curdlan (D), Beta-1,2-glucan (G), and succinoglycan (S), all of which contribute in varying degrees to biofilm formation. In my dissertation studies, I built upon previous work that identified the UppY and UppW polymerase homologs and their specificity for the different UPP polysaccharides. Initial genetic analysis of UPP biosynthesis, to which I contributed, had identified a large number of genes required for UPP production, defining a large and complex UPP pathway. To further delineate the dual polysaccharide pathways, I engineered independent single deletion mutants of uppY and uppW that have elevated levels of cdGMP and therefore generate colonies that pigment dark red on solid media containing the polysaccharide-reactive dye Congo-red. Transposon mutagenesis was used to identify mutants that had decreased Congo-red phenotypes, and en masse sequencing was used to map the transposon insertions. This analysis revealed that many of the requisite steps of the UppY- and UppW-dependent pathways share the same biosynthetic components to produce the two discrete polysaccharides. One striking exception is the UppG glycosyl hydrolase which only impacts the UppW-dependent UPPGlcN polysaccharide. The mechanism by which these pathways diverge may to be gated by the activity of UppG.