Bacterial Functions Involved in Cell Growth Control

在过去的十年里，小的非编码RNA在调控中的重要作用已经被认识到并开始被研究。我们的实验室与多个小组合作，在全球范围内对大肠杆菌中的非编码RNA进行了两次搜索，对目前发现的80多个非编码RNA做出了重大贡献。使用免疫沉淀和微阵列分析，我们发现至少三分之一的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都是通过与目标mRNAs配对，调节mRNAs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其中的一些小RNA。我们发现，每个小RNA的表达都受到不同胁迫条件的调节，并且小RNA在适应胁迫中起着重要的作用。其中研究最多的是RyhB。RyhB转录被毛皮铁依赖的抑制子抑制，因此当细胞内铁限制时，小RNA被大量合成。当它被制造出来时，它以编码铁结合蛋白的mRNAs为目标进行降解。因此，在弧菌、沙门氏菌、克雷伯氏菌和耶尔森氏菌中也发现了这种小RNA，它重新编程细胞中的铁利用，可能是一些病原体毒力的重要组成部分。另外两个小RNA，现在被称为OmrA和OmrB，已经被发现调节一些外膜蛋白；这些小RNA是在高渗透压下制造的，是OmpR/EnvZ调节子的一部分。对另一种与HfQ结合的RNA RbB的调控研究表明，它依赖于另一种Sigma因子Sigma E进行转录。当错误折叠的外膜蛋白聚集在细胞周质中时，Sigma E变得活跃。RbB可能在合成和活性水平上对Sigma E进行自动调节。RbB活性的证明表明，向外膜的贩运可能比之前所知的更严格的控制。这些RNA是调节细胞表面的调控RNA家族的特征，在感染过程中可能很重要。另一种小RNA，现在被称为SGRS，是当细胞积累葡萄糖-6-磷酸或磷酸化葡萄糖类似物时产生的，并下调细胞的mRNA。葡萄糖特异性转运蛋白，由ptsG编码。SGRS的诱导依赖于一种新的转录调控因子，它由发散基因编码，被我们命名为sgrR。SgrR蛋白是第一个被研究的转录调节蛋白保守家族的成员(以前被错误注释)，它直接与DNA结合，负向自我调节，并可能直接感觉到糖磷酸的积累。当小RNA或转录调节因子突变时，细胞就不能从葡萄糖-磷酸蓄积中恢复过来。以前的研究已经证明了两个小RNA，DSRA和RPRA，在正向调节应激Sigma因子rpos的翻译中的作用。最近，我们已经表明RPRA还有一些其他的mRNA靶标，这些靶标是负调控的；这在以前的DSRA中已经显示出来。新的RPRA目标扩大了RPRA及其调节器RCSC、RCSD和RCSB在控制这种细菌生物膜形成方面的可能作用。对DSRA和RPRA作用机制的研究表明，它们既影响rpos mRNA的稳定性，也影响rpos mRNA的翻译。RPOS也受到蛋白质周转水平的控制。在活跃的生长过程中，RPOS被迅速降解，这一过程需要依赖能量的ClpXP蛋白酶和适配器蛋白RSSB，RSSB是一种可磷酸化的蛋白质，将RPOS呈现给蛋白酶。RPOS在各种应激或饥饿处理后变得稳定；稳定的模式一直是个谜。我们实验室和其他实验室最近的研究表明，在没有磷酸化的情况下，降解具有显著的调节作用。

In the last decade, the important role of small non-coding RNAs in regulation have been recognized and begun to be studied. Our laboratory, in collaborations with a number of groups, have undertaken two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 that are now identified. Using immunoprecipitation and analysis with microarrays, we find that at least one-third 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. The best-studied of these 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 Vibrio, Salmonella, Klebsiella, and Yersinia, reprograms iron use in the cells and may be an important component of virulence for some pathogens. Two other small RNAs, now called OmrA and OmrB, have been found to regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity are part of the OmpR/EnvZ regulon. Studies on the regulation of RybB, another Hfq-binding RNA, have demonstrated that it is dependent on an alternative sigma factor, Sigma E, for transcription. Sigma E becomes active when misfolded outer membrane proteins accumulate in the periplasm of the cell. RybB autoregulates sigma E, possibly at both the level of synthesis and activity. The demonstration of the activity of RybB suggests that trafficking to the outer membrane may be even more tightly regulated than previously known. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. Another small RNA, now named SgrS, is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog, and down-regulates the mRNA for the . glucose-specific transporter, encoded by ptsG. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, named by us sgrR. The SgrR protein, which is the first studied member of a conserved family of transcriptional regulators (previously mis-annotated), directly binds DNA, negatively autoregulates, and may directly sense the accumulation of sugar phosphate. When either the small RNA or the transcriptional regulator are mutant, cells are unable to recover from glucose-phosphate accumulation. 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; this had been previously shown 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 affect both the stability and translation of rpoS mRNA. RpoS is subject to control at the level of protein turnover as well. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization has been a mystery. Recent studies in our labs and others demonstrated significant regulation of degradation in the absence of phosphorylation.