Integrating Cell Envelope and Starvation Stress: Regulation of Sigma(E) by ppGpp

描述(申请人提供)：E细胞包膜感应通路是细菌用来维持细胞包膜完整性的关键调控系统。？E是几种重要细菌病原体生存所必需的，也是其他细菌致病所必需的。？E活性受两个主要信号系统的调节，这两个系统整合了不同的信号，即细胞被膜胁迫和营养限制。作为对包膜压力的反应，？E通过调节其专用的抗Sigma因子的蛋白分解而被激活。在营养限制期间，E被警报蛋白ppGpp激活，在没有明显包膜压力的情况下，提前为生存准备包膜。每个系统的激活程度用来设置细胞内E的整体活性，使t与细胞需求相匹配。除了激活？E外，ppGpp还激活其他替代的sigma因子和应激途径，同时抑制快速生长所需的基因。实际上，ppGpp介导了从快速生长的转录程序到压力生存优化程序的转换。PpGpp调节E活性的机制尚不清楚。这些知识对于我们理解这种重要且高度保守的替代西格玛因子如何响应变化的环境以保持细胞包膜的完整性至关重要。这个建议的目的是确定全球调节因子ppGpp调节E活性的机制(S)，从而协调被膜和细胞质对饥饿的反应。本研究的中心假设是ppGpp通过直接激活E全酶(E？E)来控制E的活性，并通过影响Sigma因子与核心RNA聚合酶(RNAP)之间的竞争而间接地通过增加E？E的量来控制E的活性。这一假设将通过三个具体目标加以阐述。(1)确定E？E转录启动的生化特性E？E是一类高度保守、分布广泛的选择性西格玛因子，它缺乏几个已知对其他西格玛因子启动转录的特定步骤具有重要作用的保守区和氨基酸。这些差异如何改变转录启动还没有被研究过。在评估Pgp的影响之前，必须确定E？E启动转录的基本参数。(2)阐明ppGpp和Dks A对E？E活性的调节机制。由于ppGpp/DksA对E？70和替代的sigma因子的激活还不是很清楚，因此拟议的工作结果将对这些全球调控因子转录激活的生化基础的整体理解产生强烈的积极影响。(3)确定ppGpp如何独立于Dks A调节？E活性。初步的工作表明，存在一种新的ppGpp调节？E的机制，这一机制不能用现有的模型来解释，这些模型需要Dks A与ppGpp共同作用。我们将探索这一新颖的监管策略。由于？E和ppGpp系统对重要细菌病原体的毒力都是至关重要的，更好地了解这些反应的交集可能会为药物发现和疫苗设计带来新的途径。

DESCRIPTION (provided by applicant): The ?E cell envelope-sensing pathway is a key regulatory system used by bacteria to maintain cell envelope integrity. ?E is essential for viabiliy of several important bacterial pathogens and required for virulence of others. ?E activity is regulated by two major signaling systems that integrate distinct signals, cell envelope stress and nutrient limitation. In response to envelope stress, ?E is activated by regulated proteolysis of it dedicated antisigma factor. During nutrient limitation ?E is activated by the alarmone ppGpp, in the absence of apparent envelope stress, preparing the envelope for survival in advance. The extent of activation by each system serves to set the overall activity of ?E in the cell, matching t to cellular needs. In addition to activating ?E, ppGpp activates other alternative sigma factors and stress pathways, while repressing genes required for rapid growth. In effect, ppGpp mediates a switch from a transcriptional program for rapid growth to one optimized for stress survival. The mechanism by which ppGpp regulates ?E activity is not wel understood. Such knowledge is critical for our understanding of how this important and highly conserved alternative sigma factor responds to changing environments to maintain cell envelope integrity. The objective of this proposal is to determine the mechanism(s) by which ?E activity is regulated by the global regulator, ppGpp, thereby coordinating envelope and cytoplasmic responses to starvation. The central hypothesis of the proposed research is that ppGpp, in conjunction with the coregulator DksA and potentialy other factors, controls ?E activity by directly activating ?E holoenzyme (E?E) and indirectly by increasing the amount of E?E through its influence on the competition among sigma factors for core RNA polymerase (RNAP). This hypothesis will be addressed through three specific aims. (1) Define the biochemical properties of transcription initiation by E?E. ?E is a member of a highly conserved, widely distributed class of alternative sigma factors that lack several conserved regions and amino acids known to be important for specific steps in transcription initiation by other sigma factors. How these differences alter transcription initiation has not been investigated. The fundamental parameters of transcription initiation by E?E must be established before the influence of pGp can be assessed. (2) Elucidate the mechanism by which ppGpp and DksA regulate E?E activity. Since activation of E?70 and alternative sigma factors by ppGpp/DksA is not well understood, results from the proposed work will have a strong positive impact on the overall understanding of the biochemical basis of transcriptional activation by these global regulators. (3) Determine how ppGpp regulates ?E activity independently of DksA. Preliminary work suggests that a novel mechanism of regulation of ?E by ppGpp exists that cannot be explained by current models, which require DksA to act with ppGpp. This novel regulatory strategy will be explored. Because both the ?E and ppGpp systems are critical for virulence in important bacterial pathogens, a better understanding of the intersection of these responses may lead to new avenues for drug discovery and vaccine design.