Bacterial Functions Involved in Cell Growth Control

近十年来，小分子非编码rna在所有生物调控中的重要作用已被认识并开始研究。我们的实验室与其他人合作，已经完成了两项非编码rna在&lt；I&gt；E中的全球搜索。coli</I>，对目前已鉴定的80多种调控rna有重要贡献。大量这些RNA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小rna。我们发现，每一种小RNA的表达都受到不同胁迫条件的调控，并且小RNA在适应胁迫中起着重要的作用。</ < >< &gt；RyhB的转录受到依赖于铁的抑制因子的抑制，因此当细胞内铁受到限制时，小RNA就会大量产生。当它被制造出来时，它的目标是编码铁结合蛋白的mrna，以便降解。因此，这种小RNA，也存在于&lt；I> Vibrio&lt；/I中。沙门氏菌& &gt；；克雷伯菌& &gt；；和耶尔森菌& &gt；；对细胞中的铁使用进行了重新编程，并且可能是某些病原体毒性的重要组成部分。& >；另外两种小rna，现在称为OmrA和OmrB，调节许多外膜蛋白；这些小rna是作为OmpR/EnvZ调控的一部分在高渗透压下生成的，以前以其调节主要的外膜孔蛋白而闻名。此外，OmrA和OmrB也调节它们自己的转录调节因子，提供一个负反馈循环，可能是这些调节系统的一个常见组成部分。RybB是另一种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rna是一个不断增长的调节rna家族的特征，它们调节细胞表面，可能在感染期间很重要。另一种外膜蛋白OmpX现在已被证明受CyaR调节，CyaR也是一种hfq结合RNA，受环AMP和CRP正向调节。除了对细胞粘附起重要作用的OmpX外，CyaR还下调其他蛋白质的合成，包括LuxS，一种群体感应分子的合成酶，据信对许多物种都有效。CyaR似乎可能帮助细胞下调在营养不良条件下（低糖）从生物膜中逃脱的重要功能。与所有主要调控系统都可能具有小RNA成分的观点一致，另一种hfq结合RNA，称为MgrR，由PhoP和PhoQ调节，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在低镁条件下激活RNA的合成；小RNA使修饰细胞表面脂多糖的酶失活，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。</P><P> <P&gt；先前的研究已经证明了两种小rna DsrA和RprA在正向调节应激sigma因子RpoS的翻译中的作用。最近，我们已经证明RprA也有许多其他的mRNA靶点，这些靶点是负调控的；这些目标与DsrA的目标不同。新的RprA靶点扩大了RprA及其调控因子RcsC、RcsD和RcsB在控制该细菌生物膜形成中的可能作用。对DsrA和RprA作用机制的研究表明，它们增加了&lt；I>rpoS</I&gt； mRNA的稳定性和翻译，保护其免受RNAse E的降解。</P><P&gt；在其他实验中，我们选择了没有已知小RNA调节因子的基因，与它们建立了翻译融合，并使用遗传筛选来鉴定sRNA翻译调节因子。特别是，我们发现&lt；I>dpiAB</I&gt；基因受先前未表征的hfq结合RNA RybC调控。由于DpiAB参与了帮助细胞在低水平抗生素存在下存活，因此小RNA可能参与了这种反应。用于研究&lt；I>dpiAB</I&gt；的方法已经发展成为一套非常灵活的菌株，允许快速分析许多目标，已经用于许多实验室项目。因为我们相信基本上所有依赖hfq的sRNAs现在都已经被鉴定出来，每个sRNAs的表达质粒库已经被制作出来，现在可以用来快速筛选给定目标融合的调节。在对RpoS和其他sigma因子的翻译调控工作的扩展中，发现了第三个sRNA RyhA通过直接配对强烈刺激RpoS翻译。RyhA在从有氧到厌氧生长的转换过程中受到调节。一组其他的sRNAs已经被发现适度地刺激RpoS的表达，可能是通过对三种已知的直接调节因子的影响；还有一些基因负调控RpoS，可能是通过竞争Hfq蛋白来实现的。</ < >< &gt；为了确定Hfq以外的因素对这些sRNAs的作用是否必要，研究人员开发了一种遗传选择来选择两个sRNAs的失败作用。在分离的突变中，hfq中的保守氨基酸和必需氨基酸发生了变化，编码多核苷酸磷酸化酶的pnp和编码多聚a聚合酶的pcnB发生了功能突变。pnp突变导致一些sRNAs水平下降，这种减少的积累可能足以解释它们的失效。然而，缺乏pnp如何导致这种表型仍有待研究。我们的工作，结合其他实验室对这一调控家族的研究，表明细菌中的大量基因将受到这种转录后调控。</P><P> <P&gt；在与G. Storz博士的合作下，我们正在使用&lt；I&gt；E。此外，在合作工作中，炭疽芽孢杆菌中的小rna正在被鉴定和表征，以及该生物体中多个Hfq物种的作用。这为大肠杆菌的研究提供了一个有用的比较。

<P>In the last decade, the important role of small non-coding RNAs in regulation in all organisms have been recognized and begun to be studied. Our laboratory, in collaboration with others, have undertaken two completed global searches for non-coding RNAs in <I>E. coli</I>, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these RNAs 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 small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress.</P><P>One of the first studied small RNAs is RyhB. RyhB transcription is repressed by the Fur iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets mRNAs that encode iron-binding proteins for degradation. Therefore, this small RNA, which is also found in <I> Vibrio</I., <I>Salmonella</I>, <I>Klebsiella</I>, and <I>Yersinia</I>, reprograms iron use in the cells and may be an important component of virulence for some pathogens.</P><P>Two other small RNAs, now called OmrA and OmrB, regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity as part of the OmpR/EnvZ regulon, previously known for its regulation of major outer membrane porins. In addition, OmrA and OmrB also regulate their own transcriptional regulators, providing a negative feedback loop that may be a frequent component of these regulatory systems. RybB, another Hfq-binding RNA, is dependent on an alternative sigma factor, Sigma E, for transcription and down-regulates outer membrane proteins. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. An additional outer membrane protein, OmpX, has now been shown to be regulated by CyaR, also an Hfq-binding RNA that is positively regulated by cyclic AMP and CRP. In addition to OmpX, believed to be important for cell adhesion, CyaR down-regulates the synthesis of other proteins, including LuxS, the synthase for a quorum-sensing molecule believed to work for a broad range of species. It seems possible that CyaR helps the cell down-regulate functions important in escape from biofilms under poor nutrient conditions (low glucose).</P><P> 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 conditions; the small RNA inactivates an enzyme for modification of the cell surface lipopolysaccharide, affecting the cells sensitivity to antimicrobial peptides such as polymyxin. This is the first example of regulation of an LPS modifying enzyme by sRNAs.</P><P>Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated; these targets differ from those for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they increase both the stability and translation of <I>rpoS</I> mRNA, protecting it from degradation by RNAse E.</P><P>In other experiments, we have chosen genes with no known small RNA regulators, created translational fusions to them, and used genetic screens to identify sRNA translational regulators. In particular, we found that the <I>dpiAB</I> genes are regulated by a previously uncharacterized Hfq-binding RNA, RybC. Because DpiAB are involved in helping cells survive in the presence of low levels of antibiotic, it is possible the small RNA is involved in this response. The approach used to study <I>dpiAB</I> has been developed into a very flexible set of strains to allow the rapid analysis of many targets, already being used for many lab projects. Because we believe that essentially all of the Hfq-dependent sRNAs have now been identified, a library of expression plasmids for each of these sRNAs has been made and now can be used to rapidly screen for regulation of a given target fusion. Extending the work on translational regulation of RpoS and other sigma factors, a third sRNA, RyhA, was found to strongly stimulate RpoS translation by direct pairing. RyhA is regulated in response to switches from aerobic to anaerobic growth. A set of other sRNAs have been found to modestly stimulate RpoS expression, possibly by effects on the three known direct regulators; yet others negatively regulate RpoS, possibly by competing for the Hfq protein.</P><P>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, and in pcnB, encoding polyA polymerase. pnp mutations lead to decreased levels of some sRNAs, and this decreased accumulation may be sufficient to explain their failure to act. However, how lack of pnp leads to this phenotype remains to be investigated.</P><P>Our work, combined with work from other labs on this family of regulators, suggests that a large number of genes in bacteria will be subject to this post-transcriptional regulation.</P><P>Tiling arrays allow a detailed examination of the RNA transcripts in the cell. In collaboration with Dr. G. Storz, we are using an <I>E. coli</I> tiling array to examine whether any additional small RNAs are present in E. coli and to define possible mRNA target RNAs.</P></P>In addition, in collaborative work, small RNAs in B. anthracis are being identified and characterized, as is the role of the multiple Hfq species in that organism. This provides a useful comparison to the E. coli work.</P>