| Starved Escherichia coli cells mount impressive stress responses that are mediated by the sigma factor RpoS/sigmaS. Advances in recent years have refined our understanding of the glucose starvation network that influences RpoS by regulated proteolysis. Results presented in this dissertation identify ammonia starvation as a condition in which growth is arrested by starvation without a dramatic increase in RpoS stability or levels, suggesting that ammonia-starved RpoS is regulated at the level of protein activity. Further dissection of phosphate starvation has revealed additional differences compared to ammonia starvation and glucose starvation, as phosphate-starved RpoS concentrations are elevated owing to increased synthesis. Preliminary approaches to identify novel regulatory RNAs that act during phosphate starvation are discussed. Together, these studies have led to reevaluation of the hypothesis that a single response is initiated in response to limitation for any nutrient, and as a result the physiological signals that indicate starvation must also be unique in each case for carbon, nitrogen, and phosphorus. Work on RpoS activity led to the discovery that Crl, which enhances RpoS activity, is redox-regulated. During stationary phase, Crl forms disulfide-linked homodimers in the reducing environment of the cytoplasm. Investigation of this phenomenon revealed that oxidation of Crl was enhanced in the absence of RpoS or the absence of cytoplasmic redox regulators. Crl protein in which the cysteines have been mutated is no longer active to enhance RpoS activity, strongly suggesting a link between these oxidation phenotypes and regulation of RpoS. A model is presented whereby Crl senses reactive oxygen and/or nitrogen species, and oxidized (active) Crl encourages antioxidant responses through RpoS. In vitro studies with purified Crl protein have identified Crl's mechanism of action: Crl promotes EsigmaS holoenzyme assembly. This explains how a transcription factor that does not bind DNA can affect RpoS activity on a global scale, and this "pro-sigma factor" activity represents a novel mechanism for prokaryotic transcriptional activation. Future work will elucidate the mechanism by which redox sensing affects Crl's ability to stimulate EsigmaS assembly. |
