Title

Regulation of surface polysaccharides in Pantoea stewartii subsp. stewartii

Date of Completion

January 2008

Keywords

Biology, Microbiology|Agriculture, Plant Pathology

Degree

Ph.D.

Abstract

Pantoea stewartii subsp. stewartii is a xylem-limited bacterial pathogen of maize. Successful invasion of host vascular tissues relies on the cell density dependent expression of stewartan capsular/exopolysaccharide (CPS/EPS) and the formation of biofllms within the host's xylem. The EsaI/EsaR quorum sensing (QS) system represses production of EPS at low cell density. In this dissertation research, I used genetic and biochemical methods to (1) define the molecular mechanism by which the EsaR QS regulator represses the transcription of the stewartan EPS biosynthetic genes. Specifically, I showed that EsaR represses the expression of rcsA, which encodes the essential RcsA co-activator of the stewartan wce biosynthetic gene system. Specifically, I showed by promoter mapping and DNA footprinting experiments that EsaR binds to a site between a downstream low-level constitutive and upstream RcsA/RcsB inducible promoter of the rcsA gene. This creates a positive feedback regulatory loop that is governed by the EsaR QS system. (2) Genome sequence analysis and genetic studies revealed the presence of previously unrecognized stewartan EPS biosynthetic genes that are coordinately regulated by the EsaI/EsaR QS, RcsA/RcsB-regulatory cascade. (3) I showed for the first time that P. stewartii synthesizes coordinately stewartan EPS and cell-bound LPS, and that both forms of this polymer are present at high cell density. (4) I describe a stewartan degradation enzyme, encoded by the P. stewartii wce I gene cluster for EPS biosynthesis. We show that degradation of cell-bound capsular stewartan is essential to provide a level of protection against EPS-dependent bacteriophages. These data together suggest that quorum sensing in P. stewartii may serve as a switching mechanism between different surface glycopolymers, which satisfy specific cellular needs at defined stages of growth and biofilm development. ^