Bacterial Functions Involved in Cell Growth Control

参与细胞生长控制的细菌功能

• 批准号: 9779570
• 负责人: SUSAN GOTTESMAN
• 金额: $ 160.74万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9779570
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acetates; Aerobic; Affect; Alleles; Anaerobic Bacteria; Antibiotics; Area; Bacteria; Bacteria sigma factor KatF protein; Behavior; Binding; Binding Sites; Biological Assay; Carbon; Cell Communication; Cells; Collaborations; Complex; Development; Dissection; Distal; Equilibrium; Escherichia coli; Eukaryotic Cell; Face; Family; Genetic Transcription; Genetic Translation; Goals; Growth; In Vitro; Investigation; Klebsiella; Laboratories; Mediating; Membrane; Messenger RNA; Metabolism; Mismatch Repair; Molecular Chaperones; Mutagenesis; Mutation; National Institute of Child Health and Human Development; Nutrient; Operon; Organism; Pathway interactions; Phase; Physiological; Play; Porifera; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Binding; RNA Splicing; RNA Stability; Regulation; Regulatory Pathway; Regulon; Reporter; Resistance; Role; Running; Series; Sigma Factor; Signal Transduction; Signaling Molecule; Site; Small RNA; Stress; System; Translation Initiation; Untranslated RNA; Untranslated Regions; Virulence Factors; Work; acetyl phosphate; biological adaptation to stress; capsule; cell behavior; cell growth; experimental study; falls; genetic regulatory protein; in vivo; insight; interest; mutant; novel; pathogen; response; screening; small molecule; symbiont; tool; transcription factor

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.

复杂和快速适应的调节网络允许细菌，如大肠杆菌，改变新陈代谢，以优化生长和生存，在哺乳动物宿主内和宿主外，并对各种压力做出反应。在过去的二十年里，小的非编码RNA在调节所有生物体中的重要作用已经被认识到。我们的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，对现在发现的100-200个调控RNA做出了重大贡献。其中大量的小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mRNAs配对，积极或消极地调节mRNA的稳定性和翻译，尽管其中一些sRNA也有额外的作用。我们的实验室已经详细研究了其中的许多sRNA。每个sRNA受不同的胁迫条件调节，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在体内的作用，确定它们参与的调控网络以及它们在这些网络中的角色。使用以前在实验室中开发的筛选感兴趣的靶标和调节它们的sRNA的方法，我们继续研究sRNA的调控途径。MutS编码错配修复系统的一个组成部分，被发现受到一个小RNA ArcZ的调控，有些令人惊讶的是，在没有sRNA的情况下，MutS直接由Hfq调控，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蛋白水平的增加和突变的减少，表明转录后调控在细胞耗尽营养物质时允许突变的作用。在另一个项目中，从编码TCA蛋白的操纵子的3‘UTR加工而来的小RNA被发现通过“醋酸盐开关”调节信号分子乙酰磷酸的水平和改变通量。从这个项目中吸取的经验教训表明，许多其他以前没有得到重视的由3‘UTRs组成的sRNA的重要性。这些小RNA的作用依赖于RNA伴侣Hfq，Hfq是一种与真核蛋白质LSM和Sm家族同源的蛋白质，参与RNA剪接和其他功能。Hfq既与sRNA结合，又与mRNA结合，并刺激配对，但它到底是如何做到这一点的一直是清楚的。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，改变了我们对这种蛋白质与sRNA作用的理解。我们发现依赖于Hfq的sRNA分为两类，根据它们在不同Hfq突变体中的行为定义。所有这些sRNA都依赖于Hfq近端已知的sRNA结合部位来保证体内的稳定性。I类sRNA在使用时会迅速降解，很可能依赖于配对；它们的靶标结合到远端面部。第二类sRNA通常比第一类sRNA更稳定，它们的靶标与Hfq中的RIM位点结合。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的Hfq等位基因对不同的sRNA：mRNA对的不同影响。大肠杆菌Hfq(CTD)的C末端是非结构化的，其作用尚不清楚。在与S.Woodson的合作中，我们确定了CTD在体内和体外稳定和释放第二类sRNAs的作用。在我们实验室最近的工作中，我们与G.Storz合作，检查了删除Hfq的CTD的全球影响，并发现对RNA积累只有微小的影响。然而，结合Hfq RNA结合面的突变，CTD的丢失可以产生协同效应，这应该会让我们对其作用有新的认识。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，包括新的RNA海绵和以前未描述的蛋白质。总体而言，我们已经开发了高效的活体工具来研究sRNA及其所在的网络。我们的重点越来越多地放在sRNA在复杂的细菌行为中的作用，对sRNA功能机制的研究，以及对调控翻译启动的新机制的剖析。我们还与S.Buchanan和NCATS合作，重新对影响胶囊合成的调节级联反应感兴趣。这种级联蛋白还调节细菌对膜胁迫的反应，是在体内建立共生生长所必需的，也是克雷伯氏菌重要的毒力因子。对调控级联系统各组成部分相互作用的研究改变了我们对通过该系统进行信号转导的理解。我们已经开发了一种有效的方法来筛选激活或灭活级联反应的小分子，并发现了各种抗生素在诱导该系统中的作用的证据。在其他实验中，我们正在剖析信号级联，确定必要的负调控因子和磷酸中继蛋白之间的意外相互作用，导致我们对这个系统中的信号传递的理解发生重大修订，并对影响相关和广泛的信号传递系统的一般原理提供新的见解。这项研究的长期目标是研究通过干扰这一重要调控而发挥作用的新型抗生素的开发。