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Author

Charlie Joyce

Date of Award

8-2009

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Supervisor

H.E. Schellhorn

Language

English

Abstract

This thesis is composed of two projects. The first project examined stochastic variation in gene expression in the RpoS regulon of Escherichia coli. This variation allows isogenic cells to develop different phenotypes. While this variation is often minimized, variation in stress gene expression may allow sub-populations of stress resistant cells to form within an isogenic population, increasing its fitness level. RpoS is the master stress regulator in E. coli, and so a great deal of variation in the expression of RpoS and its regulon may exist. A protocol was partly developed here for measuring GFP expression based on previous lacZ reporter protocols. Furthermore, construction of a plasmid-borne chromosomally-integratable rpoS-mPlum fusion, allowing determination of the expression of RpoS-dependent gfp fusions and rpoS itself, has begun with the intermediate plasmid pCJ2. These steps will allow a future student to complete this project.

The second project examined the effect of oxidative stress proteins against antibiotics. It was shown that bactericidal antibiotics produce cell death through an ROSmediated pathway, regardless of their primary target. As the ROS that are generated are O2-, H20 2, and 'OH, superoxide dismutases (SOD) and catalases should have an effect on the lethality of these antibiotics. Catalase deletion strains were more sensitive to the majority of bactericidal antibiotics compared to the wild type. Hydroxylamine, a catalase inhibitor, and thiourea, a ∙OH scavenger, both reduced this effect, making the wild type and deletion sensitivities more similar. The loss of SodA or SodB did not increase sensitivity to antibiotics, while a double SOD deletion strain was more sensitive to antibiotics than the wild type. Mutants resistant to bactericidal antibiotics were isolated, and many of these mutants were cross-resistant to paraquat, showing that selection on antibiotics can result in oxidative stress resistance. Therefore ROS scavenging enzymes have an effect on antibiotic sensitivity.

McMaster University Library

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