Quorum Sensing (QS) is a conserved mechanism of population density-dependent gene regulation. In Gram-negative bacteria, this generally involves the release and perception of self-produced acyl-homoserine lactone (AHL) signals. A grant has been awarded to study the mechanism of quorum sensing control of capsular polysaccharide (CPS) synthesis in the Stewart's wilt pathogen, Pantoea stewartii. subsp. stewartii. The critical regulatory components of quorum sensing in P. stewartii are the EsaI acyl-homoserine lactone synthase and the cognate EsaR response regulator. Disruption of the esaI gene leads to CPS deficiency, while the inactivation of the esaR gene leads to CPS overproduction, or hypermucoidy. Both conditions disrupt the normal development of Stewart's wilt disease in maize. EsaR, a LuxR homologue, functions by gene repression and AHL-dependent derepression in the control of its own expression. Presumably, quorum sensing by repression plays a role also in the control of CPS synthesis. The current model predicts that EsaR represses CPS synthesis by direct repression of genes encoded by the cps regulon or other biosynthetic functions that may contribute to the hypermucoid phenotype. Alternatively, EsaR may govern CPS synthesis indirectly through intermediary transcription factors. The first objective of this project is to define relevant transcript start sites upstream and within the cps gene system to identify potential EsaR contact sites. A consensus EsaR binding sequence will be established to aid the localization of such sites in absence of defined lux box-like DNA targets. Second, a comparative analysis of the fatty acid and exopolymer composition of the wild type and hypermucoid strains will be used to determine whether deregulated CPS synthesis is primarily a function of the cps gene system and/or alternate biosynthetic pathways. Third, exhaustive transposon mutagenesis of the hypermucoid mutant strain using a Tn5-based transposon with a promoterless green fluorescent protein reporter function will be used to isolate nonmucoid, fluorescent strains. Strains showing enhanced or depressed fluorescence after introduction of a functional esaR gene will be analyzed and characterized by reverse genetics to identify structural and/or regulatory genes critical for capsule synthesis and control by EsaR. Finally, two-dimensional gel electrophoresis will be employed to profile EsaR-controlled protein products. The expected results will generate a comprehensive understanding of a seemingly unconventional quorum sensing control mechanism and contribute to a novel perspective on the role of surface polymers in plant and animal pathogenesis.
