ROLE OF ALGB AND P AERUGINOSA ALGINATE GENE EXPRESSION

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Most patients with the genetic disease cystic fibrosis (CF) become colonized and infected with mucoid strains of Pseudomonas aeruginosa. These strains are mucoid due to the overproduction of a polysaccharide called alginate, which plays a role in the pathogenesis of P. aeruginosa in the lungs of CF patients. This viscous polymer impairs lung function and makes it almost impossible to eradicate the organism from the CF respiratory tract, making P. aeruginosa infections the major cause of death in CF patients. Therefore, the long-term objective of this proposal is to understand the molecular basis for the overproduction of alginate by strains of P. aeruginosa which infect cystic fibrosis patients. The alginate biosynthetic genes are controlled by a complex, multigenic regulatory network. The research proposed here will focus on the role of a AlgB, a key alginate regulatory protein, in the control of alginate biosynthesis. In order to understand this, two critical questions which constitute the aims of this proposal will be addressed. The first goal is to identify the molecular targets of AlgB. This will be accomplished by DNA binding studies, a deletion analysis of algD, genetic characterization of algB suppressor strains, and the use of transcriptional fusions to isolate genes under AlgB control. Another aim of this proposal is to determine the mechanism of algB transcriptional activation. In particular, the roles of the alginate regulator AlgT and the DNA binding/bending protein integration host factor in algB expression will be investigated. The algB promoter(s) responsive to these proteins will be characterized by ribonuclease protection mapping and primerextension analysis. The combination of biochemical and genetic approaches utilized in these studies should provide insights into the complex regulation of this important virulence factor with the ultimate goal of an improved quality of lite for CF patients colonized with alginate-producing strains of P. aeruginosa.