Bacterial Functions Involved in Cell Growth Control

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，为目前鉴定的80多种调节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，每个与Hfq紧密结合的RNA都通过与靶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。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。许多sRNA的靶标是由多个sRNA调控的，我们早期的方法不能让我们识别所有调控给定靶标的sRNA。此外，在低水平或特殊条件下表达的调节蛋白，如果其转录本不够丰富，可能无法被确定为特定sRNA的靶标。我们已经发展并利用了一种处理这些限制的方法。翻译融合可以很容易地创建到可能的目标基因，根据其mRNA结合Hfq的能力以及其他标准进行鉴定，并且可以用质粒库快速筛选融合，每个质粒表达不同的sRNA。细菌的行为同样可以被筛选。我们已经将这种方法应用于细菌运动和生物膜形成的研究，并且还研究了调节一系列重要转录调节因子和参与应激反应的蛋白质的sRNAs。我们发现多种sRNAs调节细菌运动，其中许多通过调节鞭毛合成的关键转录激活因子flhDC。两个sRNAs正调控运动，而至少四个sRNAs下调运动。这些为鞭毛合成的调控提供了意想不到的新输入。大肠杆菌等细菌在某些情况下是活动的，但在某些生长条件下形成非活动的生物膜。不出所料，我们发现sRNAs在生物膜形成中也起着重要作用。我们的研究结果表明，运动的中央调节剂flhDC和编码应激sigma因子的rpoS都是多个sRNAs调节的节点。在我们对sRNA调控的转录调控因子的筛选中，我们发现只有一小部分调控因子，包括Lrp和SoxS，受sRNA调控，并且我们已经确定了这些sRNA的作用方式。在另一项研究中，发现了一种小RNA负调控tolC，这是大肠杆菌中多种药物外排泵的核心。mutS是错配修复系统的一个组成部分，由小RNA ArcZ和Hfq直接调控；任何一个基因的缺失都会影响突变的水平。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完全清楚。在与G. Storz （NICHD）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。较大的组，称为Class I，在使用时迅速退化，很可能依赖于配对。Hfq远端表面的突变破坏了目标mRNA的结合，稳定了I类sRNAs，而Hfq边缘的突变涉及配对，破坏了sRNAs的稳定。II类sRNAs通常比I类sRNAs更稳定，不会被边缘突变破坏，但会被远端突变破坏。这些结果表明至少有两种不同的sRNA与Hfq结合的模式；这些不同的sRNA结合模式也决定了I类和II类sRNA的mrna的不同结合模式，这表明II类sRNA可能会排除I类靶标与Hfq的结合。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。我们认为第一类和第二类sRNAs在细胞中的作用有所不同，反映了它们不同的稳定性，并通过创建改变sRNAs行为的突变体和嵌合sRNAs来测试这一点。我们研究翻译调控的一个意想不到的额外方向来自丙酮酸脱氢酶（aceE）突变体对RpoS表达的影响的研究，这是我们蛋白水解项目的一部分。除了稳定RpoS外，aceE突变体还大大增加了RpoS的合成，不依赖于启动子和已知的sRNA调节因子。RpoS翻译激活的增加可能暗示了代谢应激下一种以前未知的选择性翻译模式。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。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。在与E. Nudler的合作中，描述了sRNAs通过控制转录终止因子Rho对mrna的访问来调节转录延伸的新作用。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节级联的兴趣，与S. Buchanan合作定义了一些调节蛋白的结构并开发了小分子筛选程序，因为这种级联是克雷伯氏菌病原体的毒力因子。

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 more than 80 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 every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number 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. Many targets of sRNAs are regulated by more than one sRNA, and our earlier approaches have not allowed us to identify all of the sRNAs regulating a given target. In addition, regulatory proteins expressed at low levels or under special conditions may not be identified as targets for a given sRNA if their transcripts are not reasonably abundant. We have developed and made use of an approach to dealing with these limitations. Translational fusions can easily be created to possible target genes, identified based on the ability of their mRNA to bind Hfq, among other criteria, and the fusions can be rapidly screened with a library of plasmids, each expressing a different sRNA. Bacterial behavior can similarly be screened. We have applied this approach to the study of bacterial motility and biofilm formation, and have also examined the sRNAs regulating a set of important transcriptional regulators and proteins involved in stress responses. We find that multiple sRNAs regulate bacterial motility, many of them by regulating a critical transcriptional activator of flagellar synthesis, flhDC. Two sRNAs positively regulate motility, while at least four down-regulate motility. These provide unexpected new inputs to the well-studied regulation of flagellar synthesis. Bacteria such as E. coli are motile under some circumstances, but in some growth conditions form non-motile biofilms. Not surprisingly, we find that sRNAs play important roles in biofilm formation as well. Our results suggest that both flhDC, the central regulator of motility, and rpoS, encoding the stress sigma factor, act as nodes for regulation by multiple sRNAs. In our screen of transcriptional regulators for sRNA regulation, we find that only a subset of regulators, including Lrp and SoxS, are subject to sRNA regulation, and we have identified how these sRNAs act. In another study, a small RNA was found to negatively regulate tolC, the core of multiple drug efflux pumps in E. coli. mutS, a component of the mismatch repair system, is regulated by a small RNA, ArcZ and directly by Hfq; deletion of either affects the level of mutagenesis. 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 is not entirely clear. In a series of studies, in collaboration with G. Storz (NICHD), 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. The larger group, called Class I, is rapidly degraded when used, most likely dependent upon pairing. Mutations in the distal face of Hfq, which disrupt target mRNA binding, stabilize the Class I sRNAs, and mutations in the rim of Hfq, implicated in pairing, destabilize the sRNAs. Class II sRNAs are generally more stable than Class I sRNAs, are not destabilized by the rim mutants, but are by the distal site mutants. These results demonstrate at least two distinct modes of sRNA binding to Hfq; these different modes of sRNA binding also dictate different modes of binding of the mRNAs for Class I and Class II sRNAs, and suggest that Class II sRNAs are likely to exclude Class I targets from binding to Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on different sRNA:mRNA pairs. We suggest that the roles of Class I and Class II sRNAs in the cell are somewhat different, reflecting their different stabilities, and are testing this by creating mutants and chimeric sRNAs that alter the behavior of the sRNAs. An unexpected additional direction for our study of translational regulation came from studies of the effect of mutants in pyruvate dehydrogenase (aceE on expression of RpoS, discussed as part of our proteolysis project. In addition to stabilizing RpoS, the aceE mutants greatly increase synthesis of RpoS, independent of the promoter and known sRNA regulators. This increased in translational activation of RpoS may suggest a previously unknown mode of selective translation under metabolic stress. 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. Lessons learned from this project suggest the importance of many other previously unappreciated sRNAs made from 3' UTRs. In a collaboration with E. Nudler, a novel role of sRNAs in regulating transcription elongation by controlling the access of a transcription termination factor Rho to mRNAs was described. 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 to define structure of some of the regulatory proteins and develop small molecule screening procedures, since this cascade is a virulence factor in Klebsiella pathogens.