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并开始研究。 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。 我们发现每种sRNA的表达受到不同应激条件的调节，并且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 RyhB通过下调铁离子作用下非必需铁结合蛋白的表达，对铁稳态很重要局限性。 另外两种 sRNA 在高渗透压条件下重塑外膜，而另一种 Hfq 结合 RNA 则依赖于另一种 Sigma 因子 Sigma E 进行转录并下调外膜蛋白。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。 与所有主要调控系统可能都有小 RNA 成分的观点一致，另一种 Hfq 结合 RNA，名为 MgrR，由 PhoP 和 PhoQ 调控，PhoP 和 PhoQ 是对沙门氏菌毒力很重要的双成分系统。 PhoP和PhoQ在低镁和低钙条件下激活RNA的合成；小RNA使修饰细胞表面脂多糖的酶eptB失活，从而影响细胞对多粘菌素等抗菌肽的敏感性。 这是 sRNA 调节 LPS 修饰酶的第一个例子。 在合作工作中，我们已经证明 LPS 修饰受到 sRNA 的控制。 此外，我们发现 LPS 修饰酶的基因受到特殊的 Sigma 因子 Sigma E 的正向调节，导致在周质应激条件下表达，此时 LPS 屏障可能特别重要。 此外，还鉴定了 eptB 基因的第二个小 RNA 调节因子，该调节因子将调节与需氧和厌氧生长之间的转换联系起来。这项工作以及其他实验室的工作强调了 sRNA 参与的多种调控网络。除了调节 LPS 和外膜蛋白外，我们现在还发现多种 sRNA 可以调节细菌运动，其中许多是通过调节关键的转录鞭毛合成激活剂，flhDC。 两种 sRNA 正向调节运动，而至少四种下调运动。 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。 我们重点关注 DsrA 的作用，这是本实验室首次发现的一种小 RNA，已知它可以正向调节应激 sigma 因子 RpoS 并负向调节 H-NS 阻遏蛋白。 DsrA 的过度表达会增加生物膜的产生，这取决于 H-NS 的调节。 dsrA 的缺失会减少生物膜的产生，尽管我们的结果表明这可能反映了 sRNA 的多重效应。 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。 使用实验室开发的快速创建与感兴趣基因的翻译融合的方法，我们筛选了多种其他用于 sRNA 调控的转录调节因子。 我们发现只有一小部分调节因子会受到 sRNA 的影响，并且我们正在研究这种额外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。 Hfq 是相同亚基的六聚体。 虽然 Hfq 中产生了许多突变，但这些突变通常是使用纯化的突变蛋白和一组非常窄的 sRNA 和模型 mRNA 在体外进行研究。 与 NICHD 的 G. Storz 合作，现在已经使用多个 sRNA:mRNA 报告基因在体内研究了有趣的 hfq 等位基因；结果表明，一些突变体仅在某些 sRNA/mRNA 对上存在缺陷，这表明 Hfq 存在多种结合模式并刺激配对。 此外，尚未检查六聚体中各个亚基的作用。 我们已经创建了编码 Hfq 共价连接多聚体的基因，使我们能够在给定的亚基中放置突变。 初步研究表明，Hfq 内的某些位点只需存在于交替亚基上即可实现完整功能，而其他位点则需要存在于所有亚基上。 为了确定这些 sRNA 的作用是否需要 Hfq 以外的因素，开发了遗传选择来选择两个 sRNA 无法发挥作用。 分离出的突变包括 hfq 中保守氨基酸和必需氨基酸的变化以及编码多核苷酸磷酸化酶的 pnp 中功能缺失突变。 多核苷酸磷酸化酶 (PNPase) 是一种与 RNA 降解体相关的 3 至 5 核酸内切酶，RNA 降解体是一种已知参与 sRNA 及其靶 mRNA 降解的 RNA 酶。 pnp 突变导致多种 sRNA 的不稳定性增加和水平降低，这种积累的减少可能足以解释它们无法发挥作用。 我们的遗传分析表明，PNPase 可能通过调节 RNA 降解体的活性，在保护 sRNA 免遭降解方面发挥意想不到的作用。这一提议现已得到 B. Luisi 和剑桥大学学生的体外研究的证实，我们正在与他们合作，进一步剖析 PNPase、Hfq 和降解体如何相互作用。