The stationary phase-specific expression of many genes in free living bacteria such as Escherichia coli is controlled at the level of transcription by the alternative sigma factor RpoS encoded by the rpoS gene. This central regulator of E coli's stationary phase regulon (and several stress response regulons) is known to be required for the induction of over 30 proteins in stationary phase cultures and proteins induced in response to environmental stresses such as carbon starvation or osmotic upshift. To date, several RpoS-dependent genes have been identified in the literature. However, since no single inducer exists to which all members of the regulon respond, identifying RpoS-dependent genes based on phenotypic screening (e.g. carbon starvation inducible genes) methods may not provide us with a complete enumeration of the regulon. The present study is a continuation of previous work done with a previously-generated bank of 5,000 promoter- lacZ operon fusion mutants which were screened for RpoS-dependence by introducing an rpoS null allele into these strains and scoring for reduced ß-galactosidase activity. The identities of several of these RpoS-dependent promoter-lacZ fusions were determined by DNA sequencing and subsequent sequence analysis using the BLAST algorithm. The RpoS- and growth-phase-dependence of several of the genes identified in this study was verified by Northern blot analysis. The genes identified here fall in into three groups: (i) genes previously shown to be RpoS-dependent; (ii) genes of known function that werenot previously known to be RpoS-dependent; and (iii) ORFs not previously known to be RpoS-dependent.
Expression and activity of RpoS itself is subject to regulation that occurs at the levels of transcription, translation, and protein stability. How these different levels of control interact to affect the activation of RpoS and the RpoS regulon is only partially elucidated. This study identifies BarA as the first two-component transcriptional regulator required for the activity of rpoS and provides evidence that signal(s) may be present in exponentially growing cultures that lead to early exponential phase stimulation of rpoS and subsequently, the RpoS regulon. An E coli strain with a mutation in barA exhibits a hydrogen peroxide sensitive phenotype resulting from reduced levels of HPI and HPII catalase (which is under the control of RpoS). The reduction in HPII activity is a result of a reduction in the levels of katE message (encoding HPII) in a harA strain. Western blots probed with anti-RpoS antisera and Northern blots probed with an rpoS- specific probe demonstrate that this deficiency for the HPII catalase is caused by a decrease in the levels of the regulator, RpoS, present in the harA strain. Northern analysis and promoter-lacZ fusion expression data provide evidence for a model of early exponential phase expression of the RpoS regulon. Signal(s) responsible for this induction may be present in early exponential phase cultures and may ultimately lead to RpoS-dependent gene expression in stationary phase. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/29448 |
| Date | January 1998 |
| Creators | Audia, Jonathon P. |
| Contributors | Schellhorn, H. E., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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