| Under stressful conditions, the Gram negative bacterium Escherichia coli activates a protective response known as stationary phase. The sigma factor RpoS/sigmaS mediates this developmental program by regulating transcription of hundreds of genes that render the cell resistant to many different stresses. This dissertation focuses on the mechanisms of RpoS regulation at the levels of translation and stability.; The first half of the thesis covers general RpoS regulation. The transcription factor LrhA has been shown to reduce RpoS levels. We found that this is because LrhA represses RpoS translation. This repression depends on the sRNA chaperone Hfq and involves the sRNA RprA. In addition, a genetic screen led to the discovery that the Rcs system, involved in sensing cell envelope stress, negatively regulates LrhA synthesis.; SprE (RssB) modulates RpoS stability by bringing it to the ClpX/P protease. Although SprE is most active when cells are growing exponentially, its levels paradoxically rise during stationary phase. We found that this is caused by RpoS-mediated activation of SprE transcription. This loop may serve to maintain RpoS homeostasis; however, SprE has a longer half-life than RpoS which introduces a lag into the feedback system.; The second half of the thesis concerns how RpoS stability increases during carbon starvation. Since SprE shares homology with response regulators, it was assumed that SprE activity was controlled by phosphorylation in response to carbon availability. We disproved this model by mutating the phosphorylation site of SprE and demonstrating that signal transduction with respect to carbon starvation was not abolished. Since RpoS proteolysis changes rapidly after the addition of a carbon source to starved cells, we predicted that the signal could be a metabolite. Degradation was not affected by transport of the carbon source or general macromolecule synthesis. Moreover, using mutants that block various metabolic pathways, we showed that activity of either the citric acid cycle or glycolysis could induce RpoS degradation. Thus we favor a model in which an energy-transducing molecule such as ATP, rather than a specific metabolite, coordinates the metabolic state of the cell with RpoS accumulation. |
