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，无论是有氧和厌氧，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，对现在鉴定的100-200种调控RNA做出了重大贡献。大量的这些小RNA（sRNA）与RNA分子伴侣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还具有其他作用。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。该实验室继续研究小RNA的体内作用，确定它们参与的调控网络及其在这些网络中的作用。使用方法筛选感兴趣的目标和调节它们的sRNAs，以前在实验室开发的，我们继续研究sRNAs的调控途径。mutS编码错配修复系统的一个组分，被发现受小RNA ArcZ调节，并且，有些令人惊讶的是，在不存在sRNA的情况下，直接受Hfq调节，这取决于mutS 5 'UTR中的位点。这些位点的突变导致稳定期细胞中MutS蛋白水平增加和诱变减少，证明了转录后调节在细胞耗尽营养物时允许诱变的作用。在另一个项目中，已经发现从编码TCA蛋白的操纵子的3' UTR加工的小RNA调节信号分子乙酰磷酸的水平并通过“乙酸开关”改变通量。从该项目中获得的经验表明，许多其他以前未被重视的sRNA由3'UTR制成的重要性。这些小RNA的作用取决于RNA伴侣Hfq，一种与参与RNA剪接和其他功能的真核蛋白质的Lsm和Sm家族具有同源性的蛋白质。Hfq与sRNA和mRNA结合，并刺激配对，但它究竟是如何做到这一点的已经很清楚了。在与G. Storz（NICHD）和S. Woodson（JHU），我们已经对Hfq进行了体内解剖，这改变了我们对这种蛋白质如何与sRNA作用的理解。我们已经发现，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。所有这些sRNA都依赖于Hfq近端面上的已知sRNA结合位点来实现体内稳定性。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之间的竞争和不同hfq等位基因对不同sRNA：mRNA对的差异效应。E. coli Hfq（CTD）是非结构化的，其作用尚不清楚。与S合作。Woodson，我们已经定义了CTD在II类sRNA的稳定和释放中的体内和体外作用。在我们实验室最近的工作中，我们与G。Storz等人检查了删除Hfq的CTD的整体效应，并且仅发现对RNA积累的细微影响。然而，与Hfq的RNA结合面上的突变相结合，CTD的丧失可以具有协同效应，这应该为其作用提供新的见解。使用新开发的双功能荧光报告，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海绵和以前未知的蛋白质。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。对调节级联各组分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。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。在其他实验中，我们正在解剖信号级联，确定一个重要的负调节和磷酸化蛋白之间的意想不到的相互作用，导致我们在这个系统中的信号的理解的重大修订，并提供新的见解影响相关和广泛的信号系统的一般原则。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, both aerobically and anaerobically, 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 identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that sRNAs that binds 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. The lab continues to investigate the in vivo roles of small RNAs, identifying the regulatory networks they participate in and their roles in those networks. Using approaches for screening targets of interest and the sRNAs regulating them, previously developed in the laboratory, we continue to investigate regulatory pathways for sRNAs. mutS, encoding a component of the mismatch repair system, was found to be regulated by a small RNA, ArcZ, and, somewhat surprisingly, directly by Hfq in the absence of sRNAs, dependent upon sites in the mutS 5'UTR. Mutation of these sites leads to increased levels of MutS protein in stationary phase cells and decreased mutagenesis, demonstrating the role of post-transcriptional regulation in allowing mutagenesis as cells run out of nutrients. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins has been found to regulate levels of the signaling molecule acetyl phosphate and change flux through the "acetate switch". Lessons learned from this project suggest the importance of many other previously unappreciated sRNAs made from 3' UTRs. The action of these small RNAs depends on the RNA chaperone Hfq, a protein with homology to the Lsm and Sm families of eukaryotic proteins involved in RNA splicing and other functions. Hfq binds both to sRNAs and to mRNAs, and stimulates pairing, but exactly how it does this has been clear. In a series of studies, in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out an in vivo dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. We have found that the Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. All of these sRNAs depend on the known sRNA binding site on the proximal face of Hfq for in vivo stability. 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 and differential effects of different hfq alleles on different sRNA:mRNA pairs. The C-terminus of E. coli Hfq (CTD) is unstructured, and its role has been unclear. In collaboration with S. Woodson, we have defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In recent work in our lab, we have, in collaboration with G. Storz, 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 that should give new insight into its role. Using a newly developed bi-functional fluorescent reporter we have identified novel regulators of sRNA stability and function, including a new RNA sponge and previously uncharacterized proteins. 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. 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. In other experiments, we are dissecting the signaling cascade, identifying unexpected interactions between an essential negative regulator and 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. The long-term goal of this is to investigate the development of novel antibiotics that act by perturbing this important regulon.