Regulation of the Heat Shock Response in E. Coli

描述（由申请人提供）：维持细胞稳态和对抗压力的有效机制对所有生物体都至关重要。我们研究两个问题的基本所有活细胞：如何构建一个有效的反应，热应力（？32-定向热休克反应; HSR），以及如何维持细胞离散区室的稳态（该？E-定向包络应力响应）。我们未来的工作将解决3个关键问题：1）内稳态反馈控制是如何实现的？32完成？HSR的活性通过稳态反馈控制系统与细胞折叠状态耦合。令人惊讶的是，我们的新研究涉及FtsY，信号识别颗粒（SRP）的受体（SR），这是将蛋白质插入内膜所必需的。我们将研究SR/SRP蛋白在反馈调节中的作用，并测试挑衅性的假设，膜定位？32在这个过程中起着调节作用;可能是通过放大时间的长度？32不能用于结合RNA聚合酶。如果这些研究暗示SR/SRP机器在细胞控制中，它们也将为这些普遍保守的GTP酶的范式改变提供基础。2)如何整合包络应力信号以激活？E回应？包膜应力控制RseA的降解速率，RseA是负调节的跨膜抗σ因子。E，从而耦合？E活性与细胞包膜状态的关系。以前，我们认为这一途径只是由未组装的孔蛋白激活，但我们最近的工作表明，激活需要两个额外的信号。我们将识别这些信号，确定三个信号之间的关系，并在体外重建这一调控通路。这些研究将为两个细胞区室如何通信提供重要的理解。3)sRNAs在体内有什么影响？E（和？32)介导的应激反应我们最近的工作和其他人的工作表明，一个主要方面的？E反应是产生下调外膜孔蛋白的sRNA。我们会确认剩下的？E sRNA和测试的假设，它们的功能，以提供全面的监督外膜孔蛋白的内容和关键控制的性质的压力反应。此外，我们的初步证据表明，sRNA控制DnaK，一个调节？32.因此，我们还将确定？32-控制sRNA并筛选具有调节作用的sRNA。公共卫生相关性：微生物占世界生物量的整整一半，对地球上的生命至关重要，原因多种多样，包括在我们的生态系统中进行矿物回收，制造对生物技术和环境解毒重要的产品，以及引起疾病。在过去的十年中，已经公布了444种细菌的基因组序列，还有数百种正在进行中。研究所有这些细菌是不可能的，但我们可以对选定的细菌进行深入研究，因为我们开发的范例广泛适用于所有细菌。我们研究了模式生物E.大肠杆菌，在那里尖端的研究是可能的。这些反应是高度保守的，并且两者都涉及相关生物体的发病机制。此外，我们正在定义的战略之一被证明是一种应对模式，可以协调抗生素、毒力和对环境清理至关重要的制剂的生产。最后，我们的研究可能会发现抗生素的新靶点。

DESCRIPTION (provided by applicant): Effective mechanisms to maintain cellular homeostasis and combat stress are vital to all living organisms. We study two issues fundamental to all living cells: how to construct an effective response to thermal stress (the ?32-directed heat shock response; HSR), and how to maintain homeostasis in discrete compartments of the cell (the ?E-directed envelope stress response). Our future work will address 3 critical questions: 1) How is homeostatic feedback control of ?32 accomplished? The activity of the HSR is coupled to cellular folding state by a homeostatic feedback control system. Surprisingly, our new studies implicate FtsY, the receptor (SR) for the Signal Recognition Particle (SRP), which is required for insertion of proteins into the inner membrane in this pathway. We will examine the role of SR/SRP proteins in feedback regulation, and test the provocative hypothesis that membrane localization of ?32 plays a regulatory role in this process; possibly by amplifying the length of time ?32 is unavailable for binding RNA polymerase. If these studies implicate the SR/SRP machine in cellular control, they will also provide the basis for a paradigm changing view of these universally conserved GTPases. 2) How are envelope stress signals integrated to activate the ?E response? Envelope stress controls the rate of degradation of RseA, the membrane spanning antisigma factor that negatively regulates ?E, thereby coupling ?E activity to status of the cell envelope. Previously, we thought that this pathway was activated simply by unassembled porins but our recent work suggests that two additional signals are required for activation. We will identify these signals, determine the relationship between the three signals, and reconstitute this regulatory pathway in vitro. These studies will provide critical understanding of how the two cellular compartments communicate. 3) What is the impact of sRNAs in the ?E (and ?32) mediated stress responses? Our recent work and that of others indicates that a major aspect of the ?E response is production of sRNAs that downregulate outer membrane porins. We will identify the remaining ?E sRNAs and test the hypothesis that they function to provide overall surveillance of outer membrane porin content and critical control of the properties of the stress response. Additionally, our preliminary evidence indicates that an sRNA controls DnaK, a regulator of ?32. Therefore, we will also identify ?32-controlled sRNAs and screen for those with regulatory roles. PUBLIC HEALTH RELEVANCE: Project relevance Microbes account for fully half of the world's biomass and are of immense importance to life on earth, for reasons as diverse as performing mineral recycling in our ecosystem, making products of important for biotechnology and detoxification of the environment, and causing disease. In the last decade, the genomic sequences of 444 bacteria have been published and hundreds more are in progress. It is impossible to study all of these bacteria, but we can perform an intensive study of selected bacteria, as the paradigms we develop are widely applicable to all bacteria. We study the regulation and function of two critical responses to stress, in the model organism E. coli, where cutting edge studies are possible. These responses are highly conserved, and both have been implicated in pathogenesis in related organisms. Moreover, one of the strategies we are defining is proving to be a paradigm for responses that orchestrate production of antibiotics, virulence and agents important in environmental cleanup. Finally, our studies may uncover new targets for antibiotics.