Semester
Spring
Date of Graduation
1999
Document Type
Dissertation
Degree Type
PhD
College
School of Medicine
Department
Microbiology, Immunology, and Cell Biology
Committee Chair
Tom Elliott.
Abstract
RpoS is an alternative sigma factor which is required for the expression of genes that help bacteria cope with environmental stress. The regulation of RpoS is multifactorial and occurs at the levels of synthesis and protein turnover. Our lab has previously identified a rpoS translation enhancing factor in Salmonella typhimurium, the RNA-binding protein HF-I. An antisense element consisting of rpoS mRNA secondary structure is predicted to sequester the rpoS ribosome binding site and inhibit translation. HF-I may interact with this element to increase translation. Using rpoS genetic fusions, we have shown that native rpoS promoters can be substituted with tac or lacUV5 promoters without altering the normal HF-I mediated rpoS regulation. A long deletion from the 5 ' end of the rpoS transcript, which still contains the antisense element, was no longer regulated by HF-I. This suggests that HF-I may not simply interact with the secondary structure to enhance rpoS translation.;DsrA is a small untranslated RNA which has been shown to stimulate rpoS translation. We have shown that DsrA RNA interacts directly with rpoS mRNA. Mutational analysis has identified which RNA bases are important for the pairing between DsrA and rpoS mRNA that enhances rpoS translation.;The protein stability of RpoS is controlled by a putative two-component regulatory system. This system contains a protease, ClpXP, and a putative response regulator, MviA in S. typhimurium. We have tested several models which may explain MviA action and have confirmed that RpoS protein stability in S. typhimurium is dependent on the same genes as in Escherichia coli. We have shown that phosphorylation of MviA is important for its activity and that the source of this phosphate is probably not acetyl phosphate or the PTS system. Additionally, we observed an increase of RpoS during exponential growth on acetate as the sole carbon and energy source. Surprisingly, this RpoS increase during growth on acetate is independent of HF-I action.
Recommended Citation
Cunning, Christofer Lee, "Regulation of the synthesis and protein stability of the alternative sigma factor RpoS in Salmonella typhimurium" (1999). Graduate Theses, Dissertations, and Problem Reports. 3125.
https://researchrepository.wvu.edu/etd/3125