Regulation of Stationary Phase in Escherichia coli

描述(由申请人提供)：交替的西格玛因子rpos是大肠杆菌和许多其他蛋白质细菌中固定相和一般应激反应的主要调节者。在快速生长的细胞中，会产生rpos，但它会被ClpX/P酶降解。当缺乏特定的营养时，RPOS的合成增加，降解停止，活性增加，或者这些影响的某种组合发生。两个新的调节蛋白是这一提议的焦点，SprE(RSSB)和CRL。SprE是一种孤儿反应调节因子，其功能是将RPO定向到生长细胞中的ClpX/P蛋白水解酶。当碳源耗尽时，这种降解就会停止。我们知道SprE的磷酸化/去磷酸化与饥饿信号无关。遗传分析表明，饥饿是通过ATP水平的下降来感知的，而生化数据表明，这种下降是由ClpX和rpos本身感知的。遗传分析进一步揭示了SprE在多聚腺苷化和控制mRNA稳定性方面的第二个功能，并提示SprE的磷酸化对这一活性是重要的。我们将确定将SprE的两个功能分开的突变，并确定相关的小分子或激酶。利用质谱仪，我们发现SprE控制着Poly(A)聚合酶和HfQ与mRNA降解体的联系，来自微阵列的数据表明SprE具有沉默外源基因的功能。我们将探索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，我们知道这一活性在氮饥饿条件下尤其重要。在此条件下，CRL转录增加了25倍。然而，CRL水平没有明显的相应变化。我们证明，转录的这种变化允许从嘈杂的CRL产生切换到更均匀的CRL产生，我们将探索这些表达模式在这两种条件下的生理意义。营养饥饿是细菌面临的最常见的压力，而rpos可以说是大肠杆菌中最重要的全球调节蛋白。对静止期生理学的更好理解可能会为如何抗击细菌感染提供见解。 与公共卫生相关：营养缺乏是细菌面临的最常见的压力，而rpos可以说是许多革兰氏阴性生物中最重要的全球调节蛋白。对静止相生理学的更好理解可能会为如何一方面抗击细菌感染，另一方面刺激正常菌群提供见解。

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.