Defense Date

5-22-2014

Graduation Date

Summer 2014

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Nancy Trun

Committee Member

Joseph McCormick

Committee Member

Valerie Oke

Committee Member

Jana Patton-Vogt

Keywords

Cold-shock proteins, gene expression, nucleic acid-binding proteins, qRT-PCR, RNA chaperones, stress response

Abstract

Cold-shock proteins (CSPs) are a family of small nucleic acid-binding proteins. In Escherichia coli K-12, there are 9 homologous csp genes named A-I. Aside from microarray studies, transcript levels from all nine csp genes have never been assayed using the same technique. The objectives of this study were to determine when these genes are expressed, if homologous pairs are expressed the same, how many are cold-shock inducible, and if there is a ranking of importance for these genes. To answer these questions, I determined csp mRNA expression patterns for all nine csp genes during normal growth at 37°C, and after cold-shock, using quantitative real-time PCR. To uncover functional redundancies, deletion mutants were analyzed for their colony-forming ability after cold-shock, and after exposure to the chromosome decondensing agent, camphor. The results show that mRNA levels for each csp gene change with growth phase, and growth rate, and that homologous pairs are not expressed the same. The six most highly expressed csp genes in E. coli K-12 at 37°C are cspA, -B, -C, -E, -G, and -I. Transcription of cspA, -B, -G, and -I was induced after a shift to 15°C in defined minimal medium. Of the six quintuple csp deletion mutants that were created, one shows the most severe macroscopic phenotypes (TC155, cspABCEG). TC155 is cold-sensitive, and camphor-sensitive on both defined rich and defined minimal agar and only overexpression of CspC, CspE, CspG, or CspI can rescue the growth of this mutant. Taken together, these results indicate that there is some functional redundancy among the nine CSPs in E. coli K-12, but there is also a ranking of importance for these proteins in the cell.

Format

PDF

Language

English

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