Date of Award

Spring 2006

Document Type

Restricted Thesis

Terms of Use

© 2006 Meagan Bolles. All rights reserved. Access to this work is restricted to users within the Swarthmore College network and may only be used for non-commercial, educational, and research purposes. Sharing with users outside of the Swarthmore College network is expressly prohibited. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Biology Department

First Advisor

Amy Cheng Vollmer

Abstract

Most bacterial stress responses are highly specific to the nature of the stress. Universal Stress Protein A of Escherichia coli displays activation in response to a wide 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. 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 OToole and Kolter (1998) assay in a 96-well polystyrene plate, staining adherent cells with crystal violet and visualizing the solubilized stain spectrophotometrically at 600nm. 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. In addition, paralog uspC, D, and E- deletion strains show varying strengths of biofilm formation. Four site-specific mutations of uspA have been created and confirmed by sequencing, and each shows biofilm formation comparable to a uspA+ strain. 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, C-, D-, and E-, have varying effects on biofilms, while four site-specific mutations of the uspA gene result in a restored biofilm formation phenotype but severely impaired viability under genotoxic stress.

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