The alternate sigma factor RpoS is the master regulator of stationary phase and the general stress response in Escherichia coli and many other proteobacteria. In rapidly growing cells, RpoS is made but it is degraded by the ClpX/P protease. When starved for a particular nutrient, RpoS synthesis increases, degradation ceases, activity is increased, or some combination of these effects occurs. Two novel regulatory proteins are the focus of this proposal, SprE (RssB) and Crl. SprE is an orphan response regulator that functions as an adaptor to direct RpoS to the ClpX/P protease in growing cells. When carbon sources are depleted, this degradation stops. We know that SprE phosphorylation/dephosphorylation is not involved in starvation signaling. Genetic analysis suggests that starvation is sensed as a decrease in ATP levels, and biochemical data suggest that this decrease is sensed by ClpX and RpoS itself. Genetic analysis has further revealed a second function for SprE in polyadenylation and the control of mRNA stability, and it suggests that phosphorylation of SprE is important for this activity. We will identify mutations that separate the two functions of SprE and identify the relevant small molecule or kinase. Using mass spectrometry we have discovered that SprE controls the association of Poly(A) polymerase and Hfq with the mRNA degradosome and data from microarrays suggest that SprE functions to silence foreign genes. We will probe the physiological significance of these novel activities. We have shown that Crl facilitates the association of RpoS with core RNA polymerase, and we know that this activity is especially important under nitrogen starvation conditions. Under these conditions crl transcription increases 25 fold. However, there is no significant corresponding change in Crl levels. We demonstrate that this change in transcription allows a switch from noisy to more uniform Crl production and we will probe the physiological significance of these expression patterns under both conditions. Nutrient starvation is the most common stress that bacteria face and RpoS is arguably the most important global regulatory protein in E. coli. A better understanding of stationary phase physiology may provide insights into how to combat bacterial infections.
Nutrient starvation is the most common stress that bacteria face, and RpoS is arguably the most important global regulatory protein in many Gram-negative organisms. A better understanding of stationary phase physiology may provide insights into how to combat bacterial infections on one hand and stimulate normal flora on the other.
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