生防假单胞菌2P24中RNA结合蛋白RsmA/E对抗生素合成的差异调控

The Gac/Rsm system in bacteria is responsible for sensing and responding to environmental stimuli and regulating a wide variety of secondary metabolites. In Pseudomonas, the two component system GacS/GacA was used to regulate the transcription of a set of small RNAs, which subsequently bind to RsmA/RsmE proteins and sequester them away from their mRNA targets. RsmA and RsmE in P. fluorescens 2P24 have redundant regulatory roles on most of the known biocontrol characters, including the production of antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). We hypothesized that RsmA and RsmE only show the different regulation of secondary metabolism under certain adverse conditions. The aim of this study is to find out the environmental conditions under which RsmA and RsmE differentially affect 2,4-DAPG production, and to reveal the regulatory mechanism of the RsmA and RsmE under these conditions. A high-throughput screening system combining the Phenotype Microarray System (Biolog Inc) with a chromogenic reporter of 2,4-DAPG biosynthesis has been established and used to screen the cultural conditions. Molecular genetic and biochemical experiments have been designed to clarify the regulatory mechanisms of RsmA and RsmE on 2,4-DAPG production, including quantification of transcriptional and translational levels of rsmA and rsmE genes, and identification of RsmA/RsmE-binding sites at 5’-UTR of 2,4-DAPG biosynthetic gene transcripts. Further analysis using a novel CLIP-seq technique will show the differences between RsmA- and RsmE-binding transcripts at the whole transcriptome level. The predicted results not only help us to understand the regulation mechanism of antibiotic production in a biocontrol Pseudomonas strain, but also provide us novel cues to understand the roles of redundant regulatory elements in the bacterial adaptive capacity upon environmental stresses.

假单胞菌的RsmA家族蛋白转录后调控次生代谢的表达。菌株2P24中该家族蛋白RsmA和RsmE对大多数生防性状的调控作用相似，表现出冗余性。推测RsmA和RsmE仅在特定逆境条件下才表现出对某些次生代谢的差异调控。本研究基于上述假设设计高通量的筛选方法，筛选RsmA和RsmE差异调控下游基因的环境条件并解析作用机制。利用Biolog表型组试剂盒提供的大量培养条件，筛选到RsmA和RsmE差异调控抗生素2,4-DAPG的条件，通过分子遗传学和生化等手段阐明差异调控机制，包括RsmA和RsmE在转录和翻译水平的表达差异、与靶基因mRNA的结合位点和亲和性等，并进一步通过新的RNA组学技术（CLIP-seq）在整个转录组水平上分析RsmA和RsmE靶位点的差异。研究结果对理解细菌冗余性调控和环境适应机制有重要参考意义。

细菌的RsmA家族蛋白转录后调控次生代谢的表达。假单胞菌2P24中该家族蛋白RsmA和同源蛋白RsmE对大多数生防性状的调控表现冗余性，二者是否在特定条件下差异表达及其差异表达机制尚未知。本课题通过高通量技术筛选RsmA和RsmE差异调控的环境条件和表型，并利用遗传学、生化和表观遗传组学方法研究RsmA和RsmE差异调控的分子机制。首先利用Biolog表型组试剂盒提供的大量培养条件，筛选并试验确认在pH 5.7 KBG条件下RsmA和RsmE差异调控抗生素2,4-diacetyphloroglucinol（2,4-DAPG）合成。再通过分子遗传学和生化等手段，发现酸性条件下（pH 5.7）RsmA和RsmE通过差异结合2,4-DAPG合成基因的mRNA，影响2,4-DAPG合成基因phlA和phlD的翻译，最终表现为2,4-DAPG产量差异。利用荧光RNA凝胶迁移试验首次确认RsmA和RsmE对phlA和phlD mRNA的结合位点。最后进一步利用RIP-seq在全转录组水平分析RsmA和RsmE 靶标差异，探究面对酸刺激（pH 5.7）时RsmA和RsmE的功能变化，发现胞内pH稳态受RsmE影响而不是RsmA。本项目对理解细菌冗余性调控和环境适应机制有重要参考意义。