Date of Award

Spring 1998

Document Type

Restricted Thesis

Terms of Use

© 1998 Marianna B. Ruzinova. 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

First Advisor

Amy Cheng Vollmer

Abstract

The expression of the uspA gene is induced in response to virtually any stress or starvation conditions, and thus, the Universal Stress Protein (UspA) appears to be a general, non-specific responder to deviations from balanced growth in Escherichia coli. Since others have established that the uspA mutant displays an abnormal acetate utilization pattern, the pta and ackA genes, which encode enzymes involved in metabolizing acetate, were hypothesized to be the targets of UspA control. In order to determine whether UspA controls the expression of these enzymes, the promoter region for the pta/ackA operon was cloned by PCR and fused to the Vibrio fischeri luxCDABE reporter. The reporter plasmid containing the promoter::lux fusion was introduced into uspA⁺ and uspA- isogenic strains and the amount of pta/ackA transcription was measured by monitoring luminescence. The analysis of bioluminescent data has shown that pta/ackA genes exhibit dose-responsive induction by ethanol in both uspA⁺ and uspA⁻ backgrounds. Experiments also indicate that one of the functions of UspA might be the down-regulation of pta/ackA transcriptional activity, since the uspA⁻ transformants showed induced transcription levels that were 2-10 times greater than those detected in the clones of the isogenic parental strain. Since uspA is a member of fatty acid metabolism regulon and is expressed concomitantly with fad genes during the entry into stationary phase, these experiments suggest that UspA may have a role in optimizing the flow of acetyl-CoA through the central metabolic pathways. Restriction of the conversion of acetyl-CoA to acetate through the PTA-ACK pathway may be beneficial for the cells experiencing starvation or stress conditions. In addition, further characterization of uspA was accomplished by studying the extent of its evolutionary conservation using PCR and Southern blot analysis. Most of the bacterial species from the Enterobacteriaceae family indicated the presence of a uspA homologue. Furthermore, the evolutionary conservation of uspA was suggested in a number of gram-negative species, especially in the gamma subdivision of Proteobacteria, as well as in some gram-positive species.

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