功能鉴定 Serratia plymuthica 硝吡咯菌素生物合成基因簇及其调控

The antibiotic pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chloro-phenyl) pyrrole, Prn] is a secondary metabolite derived from tryptophan which was ?rst isolated from Pseudomonas pyrrocinia. Prn has known to be produced mainly by a narrow of strains of Gram-negative bacteria Burkholderia, Pseudomonas, and Serratia. Prn displays broad-spectrum antifungal activity due to an inhibitory effect on the electron transport system of fungi, and has been used as a clinical antifungal agent for the treatment of skin mycoses. Additionally, natural products of Prn can serve as synthetic lead compounds of novel agricultural fungicides (fludioxonil and fenpiclonil) as promising alternatives to synthetic pesticides. The Prn biosynthesis by rhizobacteria presumably also has a key role in their life strategies as antibiotics are hypothesized to confer a selective advantage to the producer when competition is important to microbial fitness.Although the cloning and characterization of a prnABCD gene cluster has been first described from Pseudomonas fluorescens BL915 in 1997,the functions of the prnABCD operon from Serratia species and its regulation remains poorly understood so far..We have previously shown the potential of Serratia plymuthica as promosing biocontrol agents with synergistic multiple mechanisms, and identified several global regulatory systems such as Qurum sensing,post-transcriptional Rsm system and the RNA-binding protein Hfq etc.In this proposal, we will use the endophytic strain G3 of S. plymuthica as a model organism,and tomato or bean with the grey mould Botrytis cinerea as plant-pathogen system for cloning,characterization and phylogenetic analysis of the Prn biosynthetic gene cluster prnABCD by integrating state of the art technologies and strategies of phytopathology, molecular genetics and bioinformatics etc. For example, construction of a IPTG-inducible mutant and lux or lacZ-based reporter gene fusions, mutational analysis of biocontrol-related phenotypes in vitro and in greenhouse, with an emphasis on comparative proteomics to unravel the role of the Prn in bacterial physiology, especially in the beneficial interactions between Serratia and host plant beyond Prn as antibiotic; Confocal laser scanning microscopy(CLSM)observation of GFP or DsRed-labelled bacteria will be used to investigate the impact of the Prn on biofilm architecture and colonization in planta.Further investigation on noval regulatory elements involved in Prn biosynthetic pathway will help in better understanding the Prn regulatory networks in Serratia to obtain Prn-overproducing strains. The results obtained from this multidisciplinary project will pave the way for developing environmental-friendly biopesticides by manipulation Prn regulatory networks to enhance biocontrol efficiency and stability for higher crop yields.These results will also be extrapolated to more distant fields such as closely related bacteria in medicine, environment and food industry.

硝吡咯菌素Prn是仅由少数细菌产生的广谱抗生素。本项目在前期研究基础上，拟以内生菌Serratia plymuthica G3为模式细菌，以菜豆/番茄-灰霉菌Botrytis cinerea为植物-病原系统，组合应用植物病理学、分子遗传学的实验方法和显微技术等，对菌株G3 Prn 生物合成基因簇prnABCD进行分子克隆、系统进化分析，并鉴定其功能。通过构建可诱导突变体、基于lux/lacZ报告基因融合等策略，应用蛋白质组学的系统学方法，重点解析Prn在细菌生理、尤其是植物与微生物有益互作过程中的作用；侧重结合生防相关表型分析和温室试验等揭示Prn对病菌抑制、诱导系统抗性、以及植物生长发育的影响，为系统揭示Prn的生物学功能提供依据。通过突变体库筛选进一步完善Prn生物合成调控网络的知识，并构建Prn-高产工程菌株。旨在为改善生防菌的效率和稳定性、研发环境友好的多功能生防菌提供新思路。

由色氨酸作为前体派生的细菌次生代谢产物硝吡咯菌素pyrrolnitrin (PRN)，已知是仅由少数几种革兰氏阴性细菌如假单胞菌Pseudomonas,布氏杆菌Burkholderia 和沙雷氏菌 Serratia产生的广谱抑菌性抗生素，作为关键生防因子之一发挥至关重要作用；同时也赋予根际细菌选择优势，增强其环境竞争力和生存能力。PRN已经被用作临床杀菌剂和新型生物农药前导化合物在生物农药、临床医学和环境等诸多领域具有广阔的开发应用前景。然而相比其它抗生素，目前对于PRN生物合成调控的研究还非常有限。前期研究我们已经报道PRN 生物合成受细菌群体感应（Quorum sensing,QS）信号乙酰基高丝氨酸内酯（N-acylhomoserin lactones,AHLs）和RNA 分子伴侣Hfq 正向调控，在此基础上本项研究以小麦内生菌Serratia plymuthica G3 为模式细菌，组合应用生物信息学、植物病理学和分子生物学等实验方法和技术，对S. plymuthica G3 菌株PRN 生物合成基因簇prnABCD 进行了分子克隆和系统进化分析，并在大肠杆菌E.coli中成功异源表达，并展示出较强的抑菌活性。进一步通过同源重组和基因替换策略分别构建了prnA突变株和IPTG可诱导的条件突变菌株prnind，通过TLC分析和离体抑菌实验证实了2种类型突变株均丧失了PRN产生能力和抑菌活性；结合表型分析首次观察到PRN除了作为抗生素具有广谱抑菌性，还参与调节细菌生长和泳动运动性等多种生理功能，暗示它有作为扩散性信号分子调节基因表达的潜能，尽管还有待进一步研究和验证。最后组合突变体筛选和lux/lacZ 报告基因融合等策略，鉴定了几个新的PRN生物合成调控子（如LysR型和TyrR型转录因子；转录后Rsm系统和GGDEF—EAL域蛋白）整合形成复杂调控网络，依赖生长阶段和环境条件精细调节PRN的生物合成水平。综上所述，本项研究丰富了对PRN生物学功能的了解，以及PRN生物合成调控网络的知识，为遗传改良筛选PRN高产工程菌，改善生防菌的效率和稳定性、研发环境友好的多功能生防菌提供了新思路；为更好地开发应用PRN提供了理论依据。