铜绿假单胞菌小RNA调控RpoS表达的机理研究

RpoS was originally identified in Escherichia coli as an alternative σ factor that activates gene expression in stationary phase when cells are experiencing nutrient starvation. RpoS is considered as a master stress response regulator important for adaptation to a variety of conditions including heat, osmotic and oxidative stress and plays as a global regulatory effecter. Due to its important role, regulation of RpoS expression occurs strictly at the levels of transcription, translation and protein stability, which is one of the most complex regulatory systems in bacteria. In E. coli, the translational control of RpoS largely depends upon the 5' untranslated region (UTR) of rpoS mRNA and small non-coding RNAs (sRNAs). However, these sRNAs are absent in Pseudomonas aeruginosa. Previous research has revealed that the 5' UTR of rpoS mRNA and the sRNA chaperone Hfq are involved in the regulation of RpoS translation in P. aeruginosa, suggesting that there is (are) novel sRNA(s) which has(ve) regulatory role on RpoS expression. In this project, we will first construct the sRNAs over-expression library and identify novel sRNAs which have the regulatory effect on RpoS expression at post-transcriptional level in P. aeruginosa. The complementary regions between sRNA and rpoS mRNA will then be investigated. And experiments will be performed to analyze the roles of Hfq and RNases in this regulatory process. The molecular regulatory mechanism of sRNAs will be identified. Our final goal is to systematically analyze the translational regulatory mechanism of RpoS and provide clues to the virulence regulation in P. aeruginosa.

细菌RpoS调控压力耐受及毒力基因的转录，具有全局调控效应，因此胞内的RpoS在转录、翻译及蛋白稳定性水平上均受到严格调控，是细菌中最为复杂的调控机制之一。大肠杆菌主要通过小RNA改变rpoS mRNA 二级结构在翻译水平调控RpoS表达，但这些小RNA并不存在于铜绿假单胞菌中。铜绿假单胞菌rpoS mRNA的非编码区参与自身调控，且有报道小RNA伴侣蛋白Hfq影响RpoS蛋白水平，但并不改变rpoS mRNA水平，预示铜绿假单胞菌中存在小RNA在转录后水平调控RpoS表达。本项目拟通过构建铜绿假单胞菌中小RNA的过表达文库，筛选调控RpoS表达的小RNA，分析小RNA与rpoS mRNA互补位点，检测小RNA对RpoS表达的调控与Hfq、RNA降解酶等因子的关系，分析其中的调控分子机制，为系统阐述RpoS翻译水平调控机理及铜绿假单胞菌毒力调控奠定基础。

RpoS调控细菌压力耐受及毒力基因的转录，具有全局调控效应。本项目研究了铜绿假单胞菌调控RpoS表达的小RNA RgsA的调控机理，取得了以下结果：. 1. 建立了铜绿假单胞菌sRNA过表达文库，并构建rpoS-lacZ转录后融合表达载体，在该文库中筛选到3个sRNA抑制rpoS-lacZ表达，分别是RgsA、pant255及pant313，其中仅RgsA抑制胞内RpoS表达，其转录依赖于RpoS。. 2. RgsA在指数期抑制RpoS表达，在转录后水平影响rpoS mRNA稳定性，而在稳定期无调控效应，说明RgsA调控RpoS表达具有生长时期依赖性，而此效应依赖于RpoS自身。Hfq作为sRNA伴侣蛋白参与RgsA调控RpoS，体外EMSA证明RgsA直接结合rpoS mRNA，并鉴定出RgsA-rpoS结合的关键区域。. 3. 发现RgsA调控铜绿假单胞菌毒力，进一步对RgsA调控靶基因进行鉴定，证明RgsA在Hfq辅助下通过直接互补配对调控全局性转录调控蛋白Fis及酰基转运蛋白AcpP的表达，并且RgsA在假单胞菌属的高度保守区对其调控靶基因表达至关重要。RgsA通过与fis mRNA形成11+11bp二聚体抑制其表达，并且需要互补配对区下游35 nt区域参与调控。发现fis mRNA具有两个起始密码子，并且均受RgsA调控。. 本项目对RgsA调控RpoS表达的机理进行了解析，首次通过sRNA将全局性转录调控因子Fis与RpoS联系起来，揭示了新的sRNA-转录调控蛋白反馈调控网络。部分研究结果已发表在微生物学领域权威刊物(Lu et al, 2016, Mol Microbiol)。