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Alternative Title

Importance of sRNAs in adaptive thermoregulation in uropathogenic Escherichia coli

Publication Date

2016-05-09

Document Type

Honors Project

Department

Biochemistry

Keywords

Non-coding RNA, Body temperature-Regulation, Escherichia coli-Genetics, Escherichia coli-Reproduction-Regulation, sRNA, Regulatory RNA, Thermoregulation, Escherichia, UTI

Abstract

The purpose of this study was to assess the importance of small regulatory RNAs (sRNAs) to thermo-regulation of gene expression in uropathogenic Escherichia coli(UPEC). Bacteria sense environmental cues from the host, i.e. temperature, pHand osmotic pressure, and adjust gene expression in order to adapt. Such environmental regulation contributes to immune evasion and virulence. Temperature change serves as a sentinel cue to trigger physiological changes in bacteria such asUPEC. Further, sRNAs (small, <200 nt RNAs that may serve regulatory roles) provide a rapid and efficient mechanism for post-transcriptional control of gene expression through mRNA stabilization or degradation, and enhancing or suppressing mRNA translation. This study examined sRNAs in UPEC whose known targets of regulation were also temperature-regulated according to RNA-Sequencing (high-throughput RNA sequencing and quantification) experiments. Two temperatures were used throughout this study: 23˚C, ambient room temperature (on account of the prevalence of UPEC infection in hospital settings), and 37˚C, the temperature inside a human host. Half of those sRNAs examined were shown to be thermoregulated themselves by quantitative real-time PCR (qRT-PCR) in the UPEC strain CFT073. One sRNA, OmrA, was selected for a knockout study based on the robust information on its regulation and the importance of its regulatory targets. A single-gene deletion was created via lambda-red mutagenesis. Wild-type CFT073 and omrA∷kan cells were grown in M9 minimal media in exponential phase at 23˚C and then some shifted up to 37˚C and harvested after four hours. RNA from these cells was used to compare the thermoregulation of OmrA’s targets in the wild-type and knockout strains. One target, cusB, showed a significantly lower temperature differential in expression in the omrA::kan strain compared to the wild-type. This study followed previous work done on thermoregulated sRNAs in commensal K-12 E. coli (strain MC4100) and provided support for the hypothesis that some of the observed effect of temperature on mRNA levels may be mediated by sRNAs.

Language

English

Comments

74 pages : color illustrations. Honors project, Smith College, 2016. Includes bibliographical references (pages 68-74)

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