Regulation and biosynthesis of unipolar polysaccharide (UPP) in Agrobacterium tumefaciens

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) at single cell poles that contact surfaces during adhesion, but not in planktonic cells or in colonies on standard solid media. UPP is a polar adhesin required for surface attachment and biofilm formation. Studying the regulation and biosynthesis of UPP is important for understanding the surface sensing, motile-to-sessile transition and biofilm initiation.;First of all, my studies revealed that bacteria with elevated cyclic diguanosine monophosphate (c-di-GMP) extrude the UPP independently of surface contact, suggesting that surface contact stimulates c-di-GMP production and thus UPP elaboration. Genetic screens and further investigation also identified that central flagellar regulators VisN and VisR negatively regulated UPP and cellulose production in part through three diguanylate cyclase proteins that were involved in c-di-GMP production. However, flagellar regulators Rem and FlbT that were under the transcriptional control of VisN and VisR positively regulated UPP production, an effect opposite to that of VisN-VisR. In-frame deletion mutants of rem or flbT also exhibited decreased intracellular c-di-GMP levels. Taken together, these data reflect a deep integration of UPP regulation with swimming motility.;Secondly, several genetic screens were devised to systematically explore the UPP biosynthetic machinery. Further analysis defined a Wzy dependent polysaccharide biosynthetic pathway for UPP production and revealed a functional redundancy of the Wzy type genes. Staining for the UPP with several fluorescent lectins in the wild type strain and different UPP biosynthetic mutants suggests that more than one polysaccharide may be localized to the pole, and that the UPP may be composed of multiple distinct polysaccharides. We also identified a UPP biosynthetic protein UppG with a putative c-di-GMP binding domain. At last, our study revealed a conditional functional redundancy of Atu0102 to the UPP biosynthetic gene uppE under the conditions of limited phosphorus (Pi), illustrating the influence of Pi availability on A. tumefaciens surface colonization.