Bacterial Functions Involved in Cell Growth Control

参与细胞生长控制的细菌功能

• 批准号: 8552602
• 负责人: SUSAN GOTTESMAN
• 金额: $ 113.81万
• 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/8552602
• 关键词: Aerobic; Affect; Alleles; Bacillus anthracis; Bacteria; Bacteria sigma factor KatF protein; Binding; Calcium; Cell surface; Cells; Characteristics; Collaborations; Enzymes; Equilibrium; Escherichia coli; Essential Amino Acids; Eukaryotic Cell; Failure; Family; Functional RNA; Genes; Genetic Transcription; Genetic Translation; Growth; Homeostasis; In Vitro; Individual; Infection; Iron; Iron-Binding Proteins; Laboratories; Lead; Link; Lipopolysaccharides; Magnesium; Membrane; Membrane Proteins; Messenger RNA; Methods; Microbial Biofilms; Modeling; Modification; Molecular Chaperones; Mutation; Names; National Institute of Child Health and Human Development; Organism; Osmolar Concentration; Physiological; Play; Polymyxins; Polyribonucleotide Nucleotidyltransferase; Production; Proteins; RNA; RNA Binding; RNA Degradation; RNA Splicing; RNA chemical synthesis; RNA degradosome; Regulation; Reporter; Role; Salmonella; Sigma Factor; Site; Small RNA; Stress; Students; System; Transcription Coactivator; Virulence; Work; antimicrobial peptide; cell growth; cell motility; degradosome; endonuclease; genetic analysis; genetic selection; in vivo; insight; interest; loss of function mutation; mutant; novel; overexpression; parallel processing; pathogen; periplasm; response

In the last fifteen years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized and begun to be studied. 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. We have found that expression of each sRNA is regulated by different stress conditions, and 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.The sRNA RyhB is important for iron homeostasis, by down-regulating expression of non-essential iron binding proteins under iron limitation. Two other sRNA remodel the outer membrane under high osmolarity conditions, while another Hfq-binding RNA, is dependent on an alternative sigma factor, Sigma E, for transcription and down-regulates outer membrane proteins. These sRNAs are characteristic of many regulatory RNAs that regulate the cell surface, possibly important during infection. Consistent with the idea that all major regulatory systems may have small RNA components, another Hfq-binding RNA, named MgrR, is regulated by PhoP and PhoQ, a two-component system important for Salmonella virulence. PhoP and PhoQ activate synthesis of the RNA under low Magnesium and low calcium conditions; the small RNA inactivates an enzyme for modification of the cell surface lipopolysaccharide, eptB, affecting the cells sensitivity to antimicrobial peptides such as polymyxin. This is the first example of regulation of an LPS modifying enzyme by sRNAs. In collaborative work, we have demonstrated that the LPS modification is under control of the sRNA. In addition, we find that the gene for the LPS modification enzyme is positively regulated by the specialized sigma factor Sigma E, leading to expression under conditions of periplasmic stress, when this LPS barrier may be particularly important. In addition, a second small RNA regulator of the eptB gene was identified, linking regulation to a switch between aerobic and anaerobic growth. This work as well as work in other labs underscores the variety of regulatory networks that sRNAs participate in. In addition to regulation of LPS and outer membrane proteins, we have now shown 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. We have focused on the role of DsrA, a small RNA first identified in this lab and known to positively regulate the stress sigma factor RpoS and negatively regulate the H-NS repressor. Overexpression of DsrA increases biofilm production, and this is dependent on regulation of H-NS. Deletion of dsrA decreases biofilm production, although our results suggest this may reflect multiple effects of the sRNA. 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. Using methods developed in the lab for rapidly creating translational fusions to genes of interest, we have screened multiple other transcriptional regulators for sRNA regulation. We find that only a subset of regulators are subject to sRNA effects, and we are investigating the physiological significance of this extra level of regulation. 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. Hfq is a hexamer of identical subunits. While many mutations have been created in Hfq, these have generally been studied in vitro with purified mutant protein and a very narrow set of sRNAs and model mRNAs. In collaboration with G. Storz, NICHD, interesting hfq alleles have now been studied with multiple sRNA:mRNA reporters in vivo; the results demonstrate that some mutants are defective only for some sRNA/mRNA pairs, suggesting that there are multiple modes for Hfq to bind and act to stimulate pairing. In addition, the role of individual subunits in the hexamer had not been examined. We have created genes encoding covalently linked multimers of Hfq, allowing us to place mutations in given subunits. Initial studies suggest that some sites within Hfq need only be present on alternating subunits for full function, while others are needed on all subunits. In order to determine if factors other than Hfq are necessary for the action of these sRNAs, a genetic selection was developed to select for failure of two sRNAs to act. Among the mutations isolated were changes in conserved and essential amino acids in hfq and loss of function mutations in pnp, encoding polynucleotide phosphorylase. Polynucleotide phosphorylase (PNPase) is a 3 to 5 endonuclease that associates with the RNA degradosome, an RNAse known to be involved in degradation of sRNAs as well as their target mRNAs. pnp mutations lead to increased instability and decreased levels of multiple sRNAs, and this decreased accumulation may be sufficient to explain their failure to act. Our genetic analysis suggests that PNPase may play an unexpected role in protecting sRNAs from degradation, probably by regulating the activity of the RNA degradosome. This proposal has now been confirmed by in vitro work from B. Luisi and students at the U. of Cambridge, and we are collaborating with them to further dissect how PNPase, Hfq, and the degradosome interact.

在过去的十五年中，已经认识到并开始研究所有生物体中小型非编码RNA在调节中的重要作用。 我们的实验室与他人合作，在大肠杆菌中对非编码RNA进行了两次全球搜索，这对现在已经确定的80多种监管RNA做出了重大贡献。 大量这些小RNA（SRNA）与RNA伴侣HFQ紧密结合。 我们和其他人表明，每一个与HFQ紧密结合的RNA通过与靶mRNA配对，调节mRNA的稳定性和翻译，无论是积极还是负面的。 我们的实验室已经详细研究了许多此类SRNA。 我们发现每个SRNA的表达受不同的应力条件调节，而SRNA在适应应力方面起着重要作用。我们还研究了HFQ允许SRNA作用的机制。 该实验室继续研究小RNA的体内作用，鉴定其参与的调节网络及其在这些网络中的作用。SRNA RYHB对铁限制在铁限制下的非必需铁结合蛋白的表达对于铁稳态而言至关重要。 在高渗透压条件下，其他两个SRNA重塑了外膜，而另一种HFQ结合RNA取决于替代的Sigma因子Sigma E，用于转录和下调外膜蛋白。这些SRNA是许多调节细胞表面的调节RNA的特征，可能在感染过程中很重要。 与所有主要调节系统都具有小的RNA组件的想法一致，另一个名为MGRR的HFQ结合RNA受PHOP和PHOQ调节，Phop和Phoq是一种对沙门氏菌毒力很重要的两组分系统。 PHOP和PHOQ在低镁和低钙条件下激活RNA的合成；小的RNA使酶失活以修饰细胞表面脂多糖EPTB，从而影响细胞对抗菌肽（如多聚霉素）的敏感性。 这是通过SRNA调节LPS修饰酶的第一个例子。 在协作工作中，我们证明了LPS的修改受到SRNA的控制。 此外，我们发现LPS修饰酶的基因受专门的Sigma因子Sigma E的积极调节，当该LPS屏障可能特别重要时，导致表达在周质应激条件下。 此外，确定了EPTB基因的第二个小RNA调节剂，将调节与有氧和厌氧生长之间的切换联系起来。这项工作以及其他实验室的工作强调了SRNA参与的各种调节网络。除了调节LPS和外膜蛋白外，我们现在表明，多个SRNA调节细菌运动能力，其中许多通过调节鞭毛合成的关键转录激活剂，FLHDC。 两个SRNA积极地调节运动性，而至少四个下调运动。 这些为鞭毛合成的良好调节提供了意外的新输入。 在某些情况下，细菌（例如大肠杆菌）在某些情况下是运动，但是在某些生长条件下形成了非运动生物膜。 毫不奇怪，我们发现SRNA在生物膜形成中也起着重要作用。 我们专注于DSRA的作用，DSRA是在该实验室中首先鉴定出的小RNA，并已知可以积极调节应力Sigma因子RPOS，并对H-NS阻遏物进行负面调节。 DSRA的过表达增加了生物膜的产生，这取决于H-NS的调节。 DSRA的缺失降低了生物膜的产生，尽管我们的结果表明这可能反映了SRNA的多种影响。 我们的结果表明，编码应力sigma因子的运动中心调节剂FLHDC是通过多个SRNA调节的节点。 使用实验室中开发的方法来快速创建对感兴趣基因的转化融合，我们筛选了多个其他转录调节因子以进行SRNA调控。 我们发现，只有一部分调节剂受到SRNA效应的影响，我们正在研究这种额外的调节水平的生理意义。 这些小RNA的作用取决于RNA伴侣HFQ，这是一种与LSM同源的蛋白质，以及参与RNA剪接和其他功能的真核蛋白的SM家族。 HFQ均与SRNA和mRNA结合，并刺激配对，但确切的工作方式尚不完全清楚。 HFQ是相同亚基的六聚体。 尽管在HFQ中创建了许多突变，但通常在体外研究了这些突变，并通过纯化的突变蛋白和非常狭窄的SRNA和模型mRNA进行了研究。 现已与G. Storz，NICHD合作，有趣的HFQ等位基因已与多个SRNA：Vivo中的MRNA记者进行了研究；结果表明，某些突变体仅对某些SRNA/mRNA对有缺陷，这表明HFQ有多种模式可以结合并作用以刺激配对。 此外，尚未检查单个亚基在六聚体中的作用。 我们创建了编码共价链接的HFQ多聚体的基因，从而使我们能够将突变放在给定的亚基中。 最初的研究表明，HFQ中的某些站点仅在交替的亚基上以进行完整功能，而其他亚基则需要。 为了确定HFQ以外的其他因素对于这些SRNA的作用是否必要，开发了遗传选择以选择两个SRNA作用的失败。 在分离的突变中，HFQ中保守和必需氨基酸的变化以及PNP中功能突变的丧失，编码多核苷酸磷酸化酶。 聚核苷酸磷酸化酶（PNPase）是与RNA降解体相关的3至5个核酸酶，该核酸酶是一种已知参与SRNAS降解及其靶标mRNA的RNase。 PNP突变会导致不稳定和多个SRNA水平降低，并且这种累积减少可能足以解释其无法采取的行动。 我们的遗传分析表明，PNPase可能通过调节RNA降解体的活性来保护SRNA免受降解发挥意外作用。现在，该提案已通过B. Luisi和剑桥美国学生的体外工作证实，我们正在与他们合作，进一步剖析PNPase，HFQ和Degradosome相互作用。