Bacterial Functions Involved in Cell Growth Control

在过去的十五年中，小分子非编码rna在所有生物调控中的重要作用已经被认识并开始研究。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。我们发现每种sRNA的表达受到不同胁迫条件的调控，并且sRNA在适应胁迫中起着重要的作用。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。sRNA RyhB在铁限制下下调非必需铁结合蛋白的表达，对铁稳态很重要。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依赖于另一种sigma因子sigma E进行转录并下调外膜蛋白。这些sRNAs是许多调节细胞表面的调控rna的特征，可能在感染期间很重要。与所有主要调控系统都可能含有小RNA成分的观点一致，另一种名为MgrR的hfq结合RNA受到PhoP和PhoQ的调控，PhoP和PhoQ是一个对沙门氏菌毒力很重要的双组分系统。PhoP和PhoQ在低镁低钙条件下激活RNA的合成；小RNA使细胞表面脂多糖（eptB）修饰酶失活，影响细胞对抗菌肽（如多粘菌素）的敏感性。这是第一个由sRNAs调控LPS修饰酶的例子。在合作工作中，我们已经证明LPS修饰是受sRNA控制的。此外，我们发现LPS修饰酶的基因受到专门的sigma因子sigma E的正调控，导致在质周应激条件下表达，此时这种LPS屏障可能特别重要。此外，还发现了eptB基因的第二个小RNA调节因子，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LPS和外膜蛋白的调控外，我们现在已经证明了多种sRNAs调节细菌的运动，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。我们关注的是DsrA的作用，DsrA是本实验室首次发现的一种小RNA，已知可以正向调节应激sigma因子RpoS，并负向调节H-NS抑制因子。DsrA的过表达增加了生物膜的生成，这依赖于H-NS的调节。虽然我们的研究结果表明这可能反映了sRNA的多重作用，但缺失dsrA会减少生物膜的生成。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。使用实验室开发的方法快速创建感兴趣基因的翻译融合，我们筛选了多种其他转录调节因子用于sRNA调节。我们发现只有一小部分调控因子受到sRNA的影响，我们正在研究这种额外水平调控的生理意义。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。Hfq是相同亚基的六聚体。虽然在Hfq中已经产生了许多突变，但这些突变通常是用纯化的突变蛋白和一组非常狭窄的sRNAs和模型mrna在体外研究的。在与G. Storz， NICHD的合作下，有趣的hfq等位基因现在已经在体内用多个sRNA:mRNA报告基因进行了研究；结果表明，一些突变体仅对某些sRNA/mRNA对存在缺陷，这表明Hfq存在多种结合模式和刺激配对的作用。此外，个体亚基在六聚体中的作用尚未得到检验。我们已经创造了编码共价连接的Hfq多聚体的基因，使我们能够在给定的亚基中放置突变。最初的研究表明，Hfq中的一些位点只需要存在于交替的亚基上即可发挥全部功能，而其他位点则需要存在于所有亚基上。为了确定除Hfq以外的其他因素是否对这些sRNAs的作用是必要的，研究人员开发了一种遗传选择来选择两种sRNAs的失效。在分离的突变中，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功能突变的缺失。多核苷酸磷酸化酶（PNPase）是一种与RNA降解体相关的3 - 5内切酶，是一种已知参与sRNAs及其靶mrna降解的RNA酶。pnp突变导致不稳定性增加和多个sRNAs水平降低，这种减少的积累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sRNAs免受降解方面发挥了意想不到的作用。这一建议现在已经被B. Luisi和剑桥大学学生的体外研究证实，我们正在与他们合作进一步分析PNPase、Hfq和降解体是如何相互作用的。

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.