Bacterial Functions Involved in Cell Growth Control

复杂和快速适应的调节网络使大肠杆菌等细菌能够改变代谢，以优化哺乳动物宿主和宿主外的有氧和厌氧生长和生存，并应对各种压力。在过去的二十年里，小分子非编码rna在所有生物的调控中所起的重要作用已经被认识到。我们的实验室与其他人合作，在大肠杆菌中进行了两次非编码rna的全球搜索，对目前已知的100-200个调控rna做出了重大贡献。大量这些小RNA （sRNAs）与RNA伴侣Hfq紧密结合。我们和其他人已经证明，与Hfq紧密结合的sRNAs通过与多个靶mRNA配对来调节mRNA的稳定性和翻译，无论是积极的还是消极的，尽管其中一些sRNAs还有其他作用。我们的实验室已经详细研究了许多这样的srna。每种sRNA受到不同应激条件的调控，表明sRNA在适应应激中起着重要作用。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在体内的作用，确定它们参与的调节网络及其在这些网络中的作用。使用我们之前开发的方法筛选感兴趣的靶标和调控它们的sRNAs，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成部分，被发现受小RNA ArcZ调节，并且在没有sRNAs的情况下，直接受Hfq调节，这取决于mutS 5'UTR中的位点，这有点令人惊讶。在静止期细胞中，Hfq抑制MutS翻译允许突变，被认为是一种下注对冲形式，因为细胞耗尽了营养。我们正在研究Hfq是否以这种不依赖于srna的方式调节其他基因，使用一组Hfq突变体的全局RNA测序结果，发现Hfq不依赖于srna结合面的变化。在另一个项目中，从编码TCA蛋白的操纵子的3' UTR加工的小RNA被发现可以调节信号分子乙酰磷酸的水平，并通过“醋酸盐开关”改变通量。这项工作证明了以前未被发现的由3' utr制成的srna的重要性。这些小RNA的作用取决于RNA伴侣Hfq，这是一种与真核蛋白Lsm和Sm家族同源的蛋白质，参与RNA剪接和其他功能。Hfq同时与sRNAs和mrna结合，并刺激配对，但它究竟是如何做到这一点的还不清楚。在与G. Storz （NICHD）和S. Woodson （JHU）合作的一系列研究中，我们对Hfq进行了体内解剖，这改变了我们对该蛋白如何与sRNAs作用的理解。我们发现Hfq依赖的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近端表面已知的sRNA结合位点来维持体内稳定性。I类srna在使用时迅速降解，很可能依赖于配对；它们的目标与远端面部结合。II类sRNAs通常比I类sRNAs更稳定，它们的靶标结合在Hfq的边缘位点上。这些结果有助于解释先前观察到的sRNA之间的竞争以及不同hfq等位基因对不同sRNA:mRNA对的差异效应。大肠杆菌Hfq （CTD）的c端是非结构化的，其作用尚不清楚。与S. Woodson合作，我们确定了CTD在体内和体外稳定和释放II类srna的作用。在我们实验室最近的工作中，我们与G. Storz实验室合作，研究了删除Hfq的CTD的整体影响，并发现仅对RNA积累有细微的影响。然而，与Hfq的RNA结合面突变结合，CTD的缺失可以产生协同效应。我们的研究结果确定了CTD的两个独立作用，一个涉及加强Hfq的远端表面RNA结合活性，第二个由Hfq c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。我们确定了干扰RpoS一般应激因子表达的基因变化（突变或过表达），并利用这些基因变化来研究调控途径。在RpoS负调控质粒库的筛选中，我们发现了两个新的调控因子，它们似乎都通过阻断srna激活RpoS翻译的能力来起作用。在第一种情况下，核糖分解代谢操纵子基因的mRNA被发现充当小RNA诱饵；每当细胞遇到核糖时，这种mRNA就会高水平表达。因此，当核糖存在时，RpoS下调。还发现了一种特征不明确的转录因子，可能间接地抵消RpoS的翻译激活。使用新开发的双功能荧光报告，我们已经确定了新的sRNA稳定性和功能调节因子，包括一个新的RNA海绵和两个以前未表征的蛋白质。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，它专门针对srna的一个子集进行降解。它需要多核苷酸磷酸化酶（PNPase）来完成这一过程，这表明它可能在与PNPase的复合物中起作用。其他人的工作证明了这种蛋白质家族的一种新的内切酶活性。另一种新蛋白在过量生产时对基于srna的调控具有全局影响。这可能是由于它与Hfq远端面部的直接相互作用。出乎意料的是，这种蛋白质是广泛的转乙酰化酶家族的成员，在厌氧条件下需要抵抗高水平的镍。我们的结果表明该蛋白具有多种重要的活性。其中每一个都开启了以前未知的sRNA功能调节水平。总的来说，我们已经开发出高效的体内工具来研究srna及其所在的网络。我们越来越关注sRNA在复杂细菌行为中的作用，研究sRNA的功能机制，并剖析调节翻译起始的新机制。我们也回到了我们对影响胶囊合成的调控级联的兴趣，与S. Buchanan和NCATs合作。该级联中的蛋白质还调节细菌对膜应激的反应，是体内建立共生生长所必需的，也是克雷伯氏菌的重要毒力因子。对调控级联各组分相互作用的研究改变了我们对该系统信号转导的理解，表明信号传导的关键负调节因子通过与磷接力蛋白的相互作用起作用，导致我们对该系统信号传导的理解发生重大修订，并为影响相关和广泛的信号系统的一般原理提供了新的见解。最近的研究表明，这种级联的信号也可以独立于最具特征的途径发生。我们已经开发了一种有效的检测方法，用于筛选激活或灭活级联的小分子，并发现了各种抗生素在诱导系统中的作用的证据。这项研究的长期目标是研究通过摄动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 known. A large number 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. 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 our previously developed approaches for screening targets of interest and the sRNAs regulating them, 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. Hfq repression of MutS translation in stationary phase cells allows mutagenesis, considered to be a form of bet-hedging, as cells run out of nutrients. We are investigating whether Hfq regulates other genes in this sRNA-independent fashion, using global RNA seq results in a set of Hfq mutants, to find changes independent of the sRNA-binding face of Hfq. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins was found to regulate levels of the signaling molecule acetyl phosphate and change flux through the "acetate switch". This work demonstrates the importance of 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 not 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 defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In recent work in our lab, 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. We identify genetic changes (mutations or overexpression) that perturb expression of the RpoS general stress factor, and use those to investigate regulatory pathways. In a screen of a plasmid library for negative regulation of RpoS, we identified two novel regulators, both of which appear to act by blocking the ability of sRNAs to activate RpoS translation. In the first case, the mRNA for a gene of the ribose catabolism operon was found to act as a small RNA decoy; this mRNA is expressed at high levels whenever the cell encounters ribose. Thus RpoS is down-regulated when ribose is present. A poorly characterized transcription factor was also found to counteract translational activation of RpoS, likely indirectly. Using a newly developed bi-functional 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 effects on sRNA-based regulation when overproduced. This is likely due to its direct interaction with the distal face of Hfq. Unexpectedly, this protein, a member of the broad transacetylase family, is needed under anaerobic conditions for resistance to high levels of Nickel. Our results suggest multiple important activities for this protein. Each of these opens up previously unknown levels of regulation of sRNA function. 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 perturb *TRUNCATED*