Bacterial Functions Involved in Cell Growth Control

复杂和快速适应的调节网络允许大肠杆菌等细菌改变代谢，以优化哺乳动物宿主、宿主外和各种压力下的生长和生存。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，对大肠杆菌中的非编码rna进行了多次全球搜索，对目前鉴定的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，并需要Hfq来发挥其功能。我们和其他人已经证明，与Hfq结合的sRNAs通过与多个靶mrna配对，调节mrna的稳定性和翻译，无论是积极的还是消极的。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。利用先前在实验室开发的筛选感兴趣靶点和调控它们的sRNAs的方法，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，被发现由一个小RNA ArcZ调节，并且出乎意料地，在没有sRNAs的情况下，直接由Hfq调节，依赖于mutS 5'UTR中的位点。这些位点的突变导致静止期细胞中MutS蛋白水平的增加和诱变的减少，证明了转录后调控在细胞营养耗尽时允许诱变的作用。我们现在正在研究在没有sRNAs的情况下，鉴定由Hfq调节的其他mrna的方法。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已被发现调节信号分子乙酰磷酸的水平，并通过“醋酸开关”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srna和mrna结合，并刺激配对，但它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Hfq的远端表面结合。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。Hfq的主要活性依赖于该蛋白高度保守的核心。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。在随后的工作中，我们的实验室与G. Storz的实验室合作，我们检查了删除Hfq的CTD的整体影响，并发现仅对RNA积累有细微的影响。然而，结合Hfq的RNA结合面突变，CTD的缺失可以产生协同效应，这使得我们定义了该区域的两个独立功能。核心以外的氨基酸似乎有助于Hfq远端表面的活性，而靠近c端尖端的序列和Hfq CTD的上游连接物与Hfq边缘协同作用，影响sRNA的积累和功能。这些不同的功能有助于解释出版文献中的一些矛盾。在与Storz实验室的另一项合作中，我们将新开发的用于sRNA-mRNA对全局鉴定的RIL-Seq方法与选定的Hfq突变等位基因结合在一起，剖析了各种Hfq RNA结合表面在配对中的作用。这进一步使我们能够更多地了解I类和II类srna的不同之处。使用新开发的双功能荧光报告，我们已经确定了sRNA稳定性和功能的新调节因子，包括一种新的RNA海绵和两种以前未表征的蛋白质，其中一种对基于sRNA的调节具有全局影响，另一种对特定sRNA的稳定性具有影响。特异性调节因子似乎作为主要核糖核酸酶的独特适配器起作用。这些新功能中的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许多参与膜应激反应的细菌基因，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。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扰乱这一重要规则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 cComplex and rapidly adaptable regulatory networks allow bacteria such as E. coli to change metabolism to optimize growth and survival in mammalian hosts, 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 multiple 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 and require Hfq for their function. We and others have shown that sRNAs that bind to Hfq act by pairing with multiple target mRNAs, regulating stability and translation of the mRNAs, either positively or negatively. 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 that were 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, unexpectedly, 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. We are now investigating approaches to identifying other mRNAs regulated by Hfq in the absence of sRNAs. 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 of Hfq. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites on Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on different sRNA:mRNA pairs. The major activities of Hfq are dependent upon the highly conserved core of the protein. 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 subsequent work in our laboratory in collaboration with the laboratory of G. Storz, we examined the global effect of deleting the CTD of Hfq, and found 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, leading us to define two independent functions for this region. Amino acids right beyond the core appear to contribute to the activity of the distal face of Hfq, while sequences near the C-terminal tip and upstream linker of the Hfq CTD synergize with the rim of Hfq, affecting sRNA accumulation and function. These separate functions help to explain some of the contradictions in the published literature. In another collaboration with the Storz lab, we combined the newly developed RIL-Seq method for global identification of sRNA-mRNA pairs with selected Hfq mutant alleles to dissect the roles of the various Hfq RNA binding surfaces on pairing. This further allows us to understand more about how Class I and Class II sRNAs differ. 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 which has global effects on sRNA-based regulation and another of which has effects on the stability of specific sRNAs. The specific regulator appears to function as a unique adaptor for a major ribonuclease. Each of these novel functions 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. 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 many bacterial genes involved in the 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. 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.