I-016. Biofilm Formation as a Selectable Phenotype for Functionality of Universal Stress Protein A in Escherichia coli

M. E. Bolles, A. C. Vollmer;
Swarthmore College, Swarthmore, PA.

Background: Most bacterial stress responses are highly specific to the nature of the stress. Universal Stress Protein A of Escherichia coli, encoded by the gene uspA, is unusual in its ubiquitous expression to a broad range of stresses. The protein is phosphorylated at a serine or threonine under conditions of stasis. One obstacle in the genetic analysis to elucidate a specific function of UspA has been the lack of a strong differential phenotype. We have previously investigated the ability of our strains to form biofilms in microtiter plates. In these preliminary studies, biofilm formation was shown to have promise as a selectable phenotype for uspA functionality since mutants appear to form a less robust biofilm. Methods: A Quikchange© protocol was used for site-specific mutagenesis of uspA in an attempt to determine the site(s) and necessity of phosphorylation for protein function. The biofilm assay is a modification of the O’Toole and Kolter (1998) assay in a 96-well polystyrene plate, stained with crystal violet and visualized spectrophotometrically at 600nm. Results: Using Quikchange©, four site-specific mutations have been created and confirmed by sequencing, and each shows a diminished capacity to form biofilms. The uspA- deletion strain shows a significant phenotype differential from wild type (W3110) at 24 hours growth in LB, with the assay giving mutant to W3110 ratios of 0.70. Using this assay we have begun to conduct a suppressor screen looking for secondary mutations that restore biofilm formation to wild type levels. In addition, early studies of paralog uspC, D, and E- deletion strains show varying strengths of biofilm formation. Conclusions: The 24 hour biofilm assay shows a reliable and significant differential between wild type E. coli and uspA mutants, making it suitable for several paths of investigation. The paralog mutants, uspC-, D-, and E-, have varying effects on biofilms, while four site-specific mutations of the uspA gene result in a significantly impaired biofilm formation phenotype.