Title

Characterization of the EsaI/EsaR quorum sensing system: A model for quorum sensing repression

Date of Completion

January 2006

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Gram-negative bacteria utilize the conserved quorum sensing regulatory strategy for the population dependent control of gene expression. In the Gram-negative bacterium Pantoea stewartii subsp. stewartii, the EsaI/EsaR quorum sensing regulatory pair governs the cell density dependent expression of an exopolysaccharide virulence factor, which is essential for Stewart's wilt disease in maize. The EsaI protein is a conserved acyl-homoserine lactone signal synthase; EsaR is the cognate transcription factor. This dissertation research focuses on the characterization of the EsaI and EsaR proteins, and the mechanism for EsaI/EsaR quorum sensing regulation of exopolysaccharide biosynthesis: Prior genetic data indicate that EsaR functions as a transcriptional repressor that is responsive to inducing aryl-homoserine lactone concentrations for derepression. This dissertation provides additional genetic and extensive biochemical evidence to confirm the repressor function of EsaR in the control of its own regulation and the repression of the rcsA gene. This research further defines two 20-base pair imperfect palindromes, which serve as specific EsaR DNA binding sites. The position of these two elements within the regulated promoters is consistent with an EsaR repressor role, in that, bound EsaR sterically inhibits RNA-polymerase/promoter interactions under acyl-homoserine lactone limiting conditions. Inducing signal concentrations neutralize EsaR repressor activity and permit RNA polymerase to initiate transcription. Accordingly, this research provides unequivocal proof for a quorum sensing regulatory model by transcriptional repression. ^ This dissertation further establishes that the mechanism for quorum sensing regulation of exopolysaccharide synthesis in Pantoea stewartii follows a hierarchical pathway. EsaR represses the rcsA gene under signal-limiting conditions, while allowing expression at inducing concentrations. The rcsA gene encodes the essential RcsA co-activator, which, in complex with the RcsB protein, activates the exopolysaccharide biosynthesis pathway. More significantly, this study provides new information indicating that quorum sensing regulation may serve as an important switch to direct the expression of different surface polymers depending on the cell-density or the developmental state of the bacterial population. This dissertation research therefore achieves the goal to characterize essential elements for the EsaI/EsaR quorum sensing system, and provides a foundation for research aimed at defining the biological role of quorum sensing regulation in Pantoea stewartii. ^