Bacterial Functions Involved in Cell Growth Control

在过去的二十年里，小非编码 RNA 在所有生物体调节中的重要作用已得到认识。我们的实验室与其他实验室合作，对大肠杆菌中的非编码 RNA 进行了两次全球搜索，对目前已鉴定的 80 多种调节 RNA 做出了重大贡献。大量这些小 RNA (sRNA) 与 RNA 伴侣 Hfq 紧密结合。我们和其他人已经证明，每一种与 Hfq 紧密结合的 RNA 都通过与靶 mRNA 配对来发挥作用，积极或消极地调节 mRNA 的稳定性和翻译。我们的实验室详细研究了其中一些 sRNA。每个sRNA都受到不同应激条件的调节，这表明sRNA在适应应激中发挥着重要作用。我们还研究了 Hfq 使 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以及它们在这些网络中的作用。 sRNA 的许多靶点都受到不止一种 sRNA 的调节，我们早期的方法不允许我们识别调节给定靶点的所有 sRNA。此外，如果低水平或特殊条件下表达的调节蛋白的转录物不够丰富，则可能无法将其识别为给定 sRNA 的靶标。我们已经开发并利用了一种方法来处理这些限制。可以轻松地为可能的目标基因创建翻译融合，根据其 mRNA 结合 Hfq 的能力等标准进行识别，并且可以使用质粒库快速筛选融合，每个质粒都表达不同的 sRNA。可以类似地筛选细菌行为。我们已将这种方法应用于细菌运动和生物膜形成的研究，并且还检查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 sRNA。我们发现多种 sRNA 调节细菌运动，其中许多是通过调节鞭毛合成的关键转录激活因子 flhDC 来调节的。两种 sRNA 正向调节运动性，而至少四种下调运动性。这些为深入研究的鞭毛合成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。毫不奇怪，我们发现 sRNA 在生物膜形成中也发挥着重要作用。我们的结果表明，运动性的中央调节因子 flhDC 和编码应激 sigma 因子的 rpoS 都充当多个 sRNA 调节的节点。在我们对 sRNA 调控的转录调控因子的筛选中，我们发现只有一部分调控因子（包括 Lrp 和 SoxS）受到 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，发现一种小 RNA 对 tolC 产生负调节，tolC 是大肠杆菌中多个药物外排泵的核心。 mutS 是错配修复系统的一个组成部分，受小 RNA ArcZ 调节并直接受 Hfq 调节；任何一个的缺失都会影响诱变的水平。这些小RNA的作用取决于RNA伴侣Hfq，这是一种与参与RNA剪接和其他功能的真核蛋白Lsm和Sm家族同源的蛋白。 Hfq 既能与 sRNA 又能与 mRNA 结合，并刺激配对，但具体如何做到这一点尚不完全清楚。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，这改变了我们对这种蛋白质如何与 sRNA 相互作用的理解。我们发现 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 的体内稳定性都依赖于 Hfq 近端面上已知的 sRNA 结合位点。较大的一组称为 I 类，在使用时会迅速降解，很可能取决于配对。 Hfq 远端面的突变会破坏靶标 mRNA 的结合，稳定 I 类 sRNA，而 Hfq 边缘的突变则与配对有关，会破坏 sRNA 的稳定性。 II类sRNA通常比I类sRNA更稳定，不会因边缘突变体而不稳定，但会因远端位点突变体而不稳定。这些结果证明了 sRNA 与 Hfq 结合的至少两种不同模式；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，并表明 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 对的差异影响。我们认为 I 类和 II 类 sRNA 在细胞中的作用有些不同，反映了它们不同的稳定性，并通过创建改变 sRNA 行为的突变体和嵌合 sRNA 来测试这一点。我们的翻译调控研究的一个意想不到的额外方向来自丙酮酸脱氢酶突变体（aceE 对 RpoS 表达的影响的研究，作为我们蛋白水解项目的一部分进行讨论。除了稳定 RpoS 之外，aceE 突变体还大大增加了 RpoS 的合成，独立于启动子和已知的 sRNA 调节因子。RpoS 翻译激活的增加可能表明代谢应激下以前未知的选择性翻译模式。在另一个项目中，由已发现编码 TCA 蛋白的操纵子的 3' UTR 可以调节信号分子乙酰磷酸的水平。从该项目中吸取的教训表明，许多其他以前未受重视的由 3' UTR 制成的 sRNA 的重要性在与 E. Nudler 的合作中，描述了 sRNA 通过控制转录终止因子 Rho 与 mRNA 的接触来调节转录延伸的新作用。 sRNA 及其所在的网络。我们的重点越来越多地集中在 sRNA 在复杂细菌行为中的作用、研究 sRNA 功能机制以及剖析调节翻译起始的新机制。我们还回到了对影响荚膜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，因为该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.