Hfq依赖的调控型sRNAs对假交替单胞菌R3群体感应调控的机理研究

Quorum sensing is a cell-to-cell communication process that microbes use to monitor changes in cell-population density and execute collective behaviours for physiological metabolic activity. It has been demonstrated that the production of many important bioactive substances in typical marine bacteria Pseudoalteromonas spp. is regulated by quorum sensing, but the regulatory mechanisms of quorum sensing in Pseudoalteromonas are not clear yet. . This project aims to functionally determine the small non-coding RNA chaperone Hfq-dependent RNAs (Qrr sRNAs) in Pseudoalteromonas sp. R3 and their roles in regulating quorum sensing. Firstly, upon genome sequencing, the quorum sensing-associated functional genes will be annotated. Then, the composition and characteristics of quorum sensing can be predicted. With these, the biological basis of quorum sensing will be revealed. Based on the changes in physiological metabolic activity, the behavior characteristics of quorum sensing and its regulatory role on physiological metabolism can be elucidated. Secondly, northern blots will be used to determine the effect of Hfq on the stability of quorum sensing transcriptional activator gene luxR mRNA. Therefore, the requirement and involvement of Hfq in quorum sensing circuit could be characterized. Moreover, the regulatory roles of Hfq in quorum sensing will be determined. Thirdly, after analysis of Qrr sRNAs sequence and structure characteristics, their correlation will be predicted. After determination of the interaction between Qrr sRNAs and Hfq, the dependence of Hfq on regulation of quorum sensing by Qrr sRNAs will be elucidated. Based on the action information of Qrr sRNAs on quorum sensing behavior, the regulatory mechanisms of quorum sensing by Hfq-dependent Qrr sRNAs will be revealed. With all these in hand, the correlation within molecular basis, behavior characteristics and regulatory mechanisms of quorum sensing in Pseudoalteromonas sp. R3 can be revealed. . We believe that this study would help us to achieve a better understanding of the adaption of Pseudoalteromonas sp. R3 in marine niches through change of collective behaviors controlled by quorum sensing. In addition, it would help us to understand the regulatory mechanisms of quorum sensing across bacteria.

群体感应是微生物调控群体生理代谢活动的一种重要机制。研究已表明海洋典型细菌假交替单胞菌生物活性物质的产生受群体感应调控，但对调控群体感应行为的机制不清。项目旨在研究RNA分子伴侣Hfq依赖的调控型小RNAs（Qrr sRNAs）调控假交替单胞菌R3群体感应的机理。通过全基因组测序，注释功能基因，探明群体感应系统和分子基础；考察Hfq调控群体感应的特征，研究Hfq对转录激活子基因luxR的mRNA稳定性的影响，分析Hfq作用luxR的方式，阐明Hfq参与群体感应调控的规律；表征Qrr sRNAs序列和结构特点，探明Qrr sRNAs与Hfq的互作关系，研究Qrr sRNAs调控群体感应的规律，揭示Qrr sRNAs协同Hfq调控群体感应的机理；同时考察生理代谢的变化，阐明群体感应分子基础、行为特征和Qrr sRNAs调控之间的关系。研究将有助于认识Qrr sRNAs调控细菌群体感应的机制。

群体感应是细菌调控群体生理代谢活动的一种极为重要机制。尽管研究已发现假交替单胞菌的生理代谢活动受群体感应调控，但目前假交替单胞菌群体感应调控机制还不清。本项目以假交替单胞菌R3为研究对象，通过全基因组测序和分析，发现菌株R3存在新颖的LuxI-LuxR型群体感应系统，被命名为YasI-YasR系统；基因敲除和异源表达证实YasI负责信号分子合成，转录调节子YasR是信号分子感应蛋白；薄层层析发现菌株R3产3-OH-C8-HSL和C8-HSL。yasI和yasR在基因组上相邻，但各自转录；5’RACE和启动子截短实验鉴定了yasI启动子区的lux-box，说明YasI和YasR组成正反馈系统；分析yasI与yasR的互作关系，发现它们在转录水平上相互影响，YasR仅对C8-HSL敏感；表型实验发现YasI-YasR调控蛋白酶产生、运动能力、耐药性等。RNA分子伴侣蛋白编码基因hfq缺失导致yasI和yasR相对表达量显著下降，信号分子不再产生；融合实验发现Hfq通过识别yasR的5’UTR区调控YasR的表达，进而间接调控yasI的表达和信号分子的产生；半衰期实验发现Hfq对yasR mRNA稳定性有正调控作用。预测分析了sRNAs，从中选取结合自由能小的sRNA进行分析，发现sRNA00029、sRNA00059和sRNA00210在Dhfq中相对表达量显著下降；进一步， sRNA00059和sRNA00210在DyasR中相对表达量显著下降，说明这两个sRNAs对YasI-YasR型群体感应有调控作用；RNAfold分析发现sRNA00059和sRNA00210与yasR的5’UTR存在结合区域；而且sRNA00059或sRNA00210的缺失导致yasI mRNA和yasR mRNA显著下降，信号分子显著减少，证明sRNA00059和sRNA00210对群体感应具有正调控作用。本研究首次证实假交替单胞菌中Hfq依赖的两个调控性sRNA通过与yasR的5’UTR结合提高yasR mRNA稳定性和翻译，进而提高YasR的表达；YasR通过识别lux-box，促进yasI的表达，增加信号分子的产生，进而调控细胞的生理代谢。研究将有利于了解菌株R3调控群体行为适应环境变化的机制。