Date of Graduation
1997
Document Type
Dissertation/Thesis
Abstract
We have found that Host Factor I (HF-I), the product of the hfq gene, is required for efficient expression of the RpoS stationary phase sigma factor in Salmonella typhimurium. HF-I is an RNA-binding protein originally characterized for its role in replication of phage Q{dollar}\\beta{dollar} of E. coli. Its role in the uninfected bacterial cell has previously been unknown. Assays of {dollar}\\beta{dollar}-galactosidase in strains with rpoS-lac fusions, Western blot analysis, and pulse-labeling and immunoprecipitation of both fusion proteins and native RpoS show that an hfq mutant has a four- to seven-fold reduction in the translation of rpoS. Suppressor mutations were isolated that map to the region directly upstream of rpoS. These mutations increase rpoS-lac expression in the absence of HF-I and also confer substantial independence of HF-I. DNA sequence analysis of the mutants suggests a model in which the RNA secondary structure near the ribosome binding site of the rpoS mRNA plays an important role to limit expression in wild type. Genetic tests of the model confirm its predictions, at least in part. It seems likely that the mutations analyzed here activate a suppression pathway to bypass the normal HF-I dependent route of rpoS expression, however, it is also possible that some of them identify a sequence element with an inhibitory function that is directly counteracted by HF-I. In stationary phase cultures, the HF-I dependence of a 5{dollar}\\sp\\prime{dollar} deletion to the rpoS KpnI site that removes the native rpoS promoters is similar to wild type, confirming earlier findings that HF-I affects rpoS at the translational level. As the size of the deletion increases, the HF-I dependence is gradually reduced, suggesting that HF-I may act at more than one site on the rpoS RNA. Osmotic induction of these deletion fusions is reduced. Since the 5{dollar}\\sp\\prime{dollar} region of rpoS was required for osmotic induction but not for stationary phase expression, different mechanisms may induce rpoS in response to different stimuli.
Recommended Citation
Brown, Larissa Carol, "Regulation of the RpoS sigma factor in Salmonella typhimurium by host factor I, a small RNA binding protein encoded by thehfq gene." (1997). Graduate Theses, Dissertations, and Problem Reports. 8541.
https://researchrepository.wvu.edu/etd/8541