Bacterial Functions Involved in Cell Growth Control

复杂且适应性强的调控网络使得大肠杆菌等细菌能够在体内生长。大肠杆菌改变代谢，以优化生长和生存，在哺乳动物宿主和宿主外，并响应各种压力。在过去的二十年中，小的非编码RNA在所有生物体中的调节中的重要作用已经被认识到。本实验室与其他实验室合作，在全球范围内对大肠杆菌中的非编码RNA进行了两次搜索。大肠杆菌中，对现在已知的100-200种调控RNA做出了重大贡献。这些小RNA（sRNA）中的大多数与RNA伴侣Hfq紧密结合。我们和其他人已经表明，与Hfq紧密结合的sRNAs通过与多个靶mRNA配对来发挥作用，积极或消极地调节mRNA的稳定性和翻译，尽管其中一些sRNAs也有其他作用。我们的实验室已经详细研究了许多这样的sRNAs。每个sRNA受不同胁迫条件的调节，表明sRNA在适应胁迫中起着重要作用。我们还研究了Hfq允许sRNA发挥作用的机制，使用Hfq的RNA结合面突变的体内研究。在与G. Storz（NICHD）和S. Woodson（JHU），我们已经对Hfq进行了体内和体外解剖，这改变了我们对这种蛋白质如何与sRNA作用的理解。这项工作表明，Hfq依赖性sRNA分为两类，根据它们在不同Hfq突变体中的行为来定义。I类sRNA在使用时迅速降解，最有可能依赖于配对;它们的靶标结合到远端面。II类sRNA通常比I类sRNA更稳定，并且它们的靶标结合到Hfq中的边缘位点。这些结果有助于解释先前观察到的sRNA之间的竞争，不同的hfq等位基因对不同的sRNA：mRNA对的差异效应，并提供对调控网络的见解。E. coli Hfq（CTD）是非结构化的，其作用尚不清楚。与S合作。Woodson，我们定义了CTD在II类sRNA的稳定和释放中的体内和体外作用。与G. Storz，我们已经检查了删除Hfq的CTD的整体效果，并且发现对RNA积累的影响很小。然而，与Hfq的RNA结合面上的突变组合，CTD的丧失可具有协同效应。我们的研究结果定义了CTD的两个独立的作用，一个涉及加强Hfq的远端面RNA结合活性，第二个由Hfq的C-末端尖端的突变定义。这些不同的作用有助于解释为什么以前的研究对CTD的作用得出了相互矛盾的结论。在使用最近开发的双功能荧光报告基因筛选，我们已经确定了新的调节sRNA的稳定性和功能，包括一个新的RNA海绵和两个以前未知的蛋白质。其中一种新蛋白质是细菌中高度保守的蛋白质家族的创始成员，它特异性地靶向降解sRNA的一个子集。它需要多核苷酸磷酸化酶（PNIPs）来完成这一过程，这表明它可能与PNIPs形成复合物。其他人的工作证明了这种蛋白质家族的新型核酸内切酶活性。另一种新蛋白质在过量生产时对基于sRNA的调节具有全局而非sRNA特异性影响。出乎意料的是，这种蛋白质，广泛的转乙酰酶家族的成员。我们的研究结果表明，这种蛋白质，现在命名为HqbA，直接与Hfq的远端面相互作用，有助于RNA结合的质量控制。由于这种效应不依赖于乙酰化，因此结果表明这是一种双功能蛋白质，它的发现表明可能存在其他这种以前未知的调节剂。总的来说，我们已经开发了高效的体内工具来研究sRNA及其所在的网络。我们的重点是越来越多的sRNA在复杂的细菌行为中的作用，调查sRNA的功能机制，并解剖新的机制，调节翻译起始。我们也回到了我们的兴趣，在监管级联影响胶囊合成，在与S。布坎南和NCAT该级联中的蛋白质还调节细菌对膜应激的反应的各个方面，是体内建立细菌生长所需的，并且是克雷伯氏菌中重要的毒力因子。对调节级联的组分的相互作用的研究改变了我们对通过该系统的信号转导的理解，表明信号传导的关键负调节剂通过与磷酸化蛋白的相互作用起作用，导致我们对该系统中信号传导的理解的重大修订，并对影响相关和广泛的信号传导系统的一般原则提供了新的见解。最近的工作表明，信号传导到这个级联也可以独立于最好的表征途径。我们已经开发了一种有效的检测方法，用于筛选激活或抑制级联反应的小分子，并发现了各种抗生素诱导该系统的作用的证据。其长期目标是研究通过干扰这一重要调节子来发挥作用的新型抗生素的发展。

Complex and rapidly adaptable regulatory networks allow bacteria such as E. coli to change metabolism to optimize growth and survival, in mammalian hosts and outside of the host and in response to a variety of stresses. In the last twenty years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized. Our laboratory, in collaboration with others, undertook two global searches for non-coding RNAs in E. coli, contributing significantly to the 100-200 regulatory RNAs that are now known. The majority of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that sRNAs that bind tightly to Hfq act by pairing with multiple target mRNAs, regulating stability and translation of the mRNA, either positively or negatively, although some of these sRNAs also have additional roles. Our lab has studied many of these sRNAs in detail. Each sRNA is regulated by different stress conditions, suggesting that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act, using in vivo studies of mutations in the RNA binding faces of Hfq. In a series of studies, in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out in vivo and in vitro dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. This work demonstrated that Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. Class I sRNAs are rapidly degraded when used, most likely dependent upon pairing; their targets bind to the distal face. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites in Hfq. These results help to explain previously observed competition between sRNAs, differential effects of different hfq alleles on different sRNA:mRNA pairs and provide insights into regulatory networks. The C-terminus of E. coli Hfq (CTD) is unstructured, and its role has been unclear. In collaboration with S. Woodson, we defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In collaboration with the lab of G. Storz, we have examined the global effect of deleting the CTD of Hfq, and find only subtle effects on RNA accumulation. However, in combination with mutations on the RNA binding faces of Hfq, loss of the CTD can have synergistic effects. Our results define two independent roles for the CTD, one involved in reinforcing the distal face RNA binding activity of Hfq and the second defined by mutations at the C-terminal tip of Hfq. These different roles help to explain why previous studies came to conflicting conclusions about the role of the CTD. In a genetic screen using a recently developed bifunctional fluorescent reporter, we have identified novel regulators of sRNA stability and function, including a new RNA sponge and two previously uncharacterized proteins. One of the new proteins, the founding member of a family of proteins highly conserved in bacteria, specifically targets a subset of sRNAs for degradation. It requires polynucleotide phosphorylase (PNPase) to do this, suggesting that it may work in a complex with the PNPase. Work by others demonstrated a novel endonuclease activity for this family of proteins. The other new protein has global rather than sRNA-specific effects on sRNA-based regulation when overproduced.. Unexpectedly, this protein, a member of the broad transacetylase family. Our results show that this protein, now named HqbA, directly interacts with the distal face of Hfq,helping in the quality control of RNA binding. Because this effect is independent of acetylation, the results suggest this is a bifunctional protein and its discovery suggests that yet other such previously unknown regulators may exist. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. Our focus is increasingly on the role of the sRNAs in complex bacterial behavior, investigations into the mechanism of sRNA function, and dissecting of novel mechanisms for regulating translation initiation. We have also returned to our interest in the regulatory cascade affecting capsule synthesis, in a collaboration with S. Buchanan and NCATs. The proteins in this cascade also regulate aspects of the bacterial response to membrane stress, are needed for in vivo establishment of commensal growth, and are important virulence factors in Klebsiella. Studies on the Interactions of the components of the regulatory cascade have changed our understanding of signal transduction through this system, demonstrating that a critical negative regulator of signaling acts by interaction with a phosphorelay protein, leading to a major revision in our understanding of signaling in this system and providing new insight into the general principles affecting related and widespread signaling systems. Recent work has demonstrated that signaling to this cascade can also take place independently of the best-characterized pathway. We have developed an efficient assay for screening for small molecules that activate or inactivate the cascade and have found evidence for effects of a variety of antibiotics in inducing the system. The long-term goal of this is to investigate the development of novel antibiotics that act by perturbing this important regulon.