Regulation of Stationary Phase in Escherichia coli

描述（由申请人提供）：备用sigma因子RpoS是大肠杆菌和许多其他变形菌中固定相和一般应激反应的主要调节剂。在快速生长的细胞中，RpoS生成，但它被ClpX/P蛋白酶降解。当缺乏特定的营养物质时，RpoS合成增加，降解停止，活性增加，或者这些作用的某种组合发生。两个新的调控蛋白是本提案的重点，SprE （RssB）和Crl。SprE是一种孤儿反应调节剂，在生长细胞中作为适配器将RpoS引导到ClpX/P蛋白酶。当碳源耗尽时，这种降解就停止了。我们知道SprE磷酸化/去磷酸化与饥饿信号无关。遗传分析表明，饥饿可以通过ATP水平的降低来感知，生化数据表明ClpX和RpoS本身可以感知这种降低。遗传分析进一步揭示了SprE在聚腺苷化和mRNA稳定性控制中的第二种功能，这表明SprE的磷酸化对这种活性很重要。我们将鉴定分离SprE两种功能的突变，并鉴定相关的小分子或激酶。通过质谱分析，我们发现SprE控制Poly(A)聚合酶和Hfq与mRNA降解体的关联，微阵列数据表明SprE具有沉默外源基因的功能。我们将探讨这些新活动的生理意义。我们已经证明，Crl促进了RpoS与核心RNA聚合酶的结合，我们知道这种活性在氮饥饿条件下特别重要。在这些条件下，crl转录增加25倍。然而，Crl水平没有明显的相应变化。我们证明了转录的这种变化允许从嘈杂到更均匀的Crl产生的切换，我们将探索在这两种条件下这些表达模式的生理意义。营养饥饿是细菌面临的最常见的应激，而RpoS可以说是大肠杆菌中最重要的全球调节蛋白。对固定相生理学的更好理解可能为如何对抗细菌感染提供见解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.