生防绿针假单胞菌HT66中脂肽的鉴定与功能

摘要

[目的]脂肽(Lipopeptide,LP)是微生物合成的一类重要的生物表面活性剂,不仅影响细菌的生物学功能,还对多种植物和人类病原菌具有广谱的拮抗作用.然而至今未见绿针假单胞菌(Pseudomonas chlororaphis)中脂肽产物的报道.[方法]通过生物信息学手段预测绿针假单胞菌HT66中脂肽的氨基酸组成及顺序,构建脂肽合成基因缺失突变株HT66△clp,根据突变株缺失代谢产物的UPLC/QTOF-MS信息验证预测结果,并研究了脂肽对该菌株的生长、吩嗪-1-甲酰胺(PCN)合成、生物膜形成和群集运动性的影响.[结果]预测菌株HT66的脂肽氨基酸顺序为L-Leu-D-Glu-D-allo-Thr-D-Val-L-Leu-D-Ser-L-Leu-D-Ser-L-Ile,通过比对野生型和突变株代谢产物的质谱信息确定该产物为黏液菌素(Viscosin);脂肽合成基因缺失后,菌株HT66的生长无明显变化,但其PCN合成、生物膜形成和群集运动性均有不同程度地下降.[结论]菌株HT66的脂肽产物为黏液菌素,对菌株的代谢、生物膜形成和运动性等生物学功能具有重要的调控作用.研究报道了绿针假单胞菌中一种脂肽分子的结构与功能,为研究其合成和调控机制及开发和应用奠定了基础.%[Objective] Lipopeptides (LPs) are important biosurfactants produced by microorganisms and have significant influences on bacterial biological functions and broad-spectrum antagonistic abilities towards many botanic and human pathogens.LPs produced by Pseudomonas chlororaphis are not reported as yet.[Methods] This study predicted the composition and sequence of the amino acids within the LP produced by P.chlororaphis HT66.The LP biosynthetic genes deletion strain HT66△clp was constructed,and subsequently the LP produced by strain HT66 was analyzed by the UPLC/QTOF-MS according to the lost metabolite in the mutant.At last,the roles of the LP on bacterial growth,phenazine-1-carboxamide (PCN) production,biofilm formation and swarming motility were studied.[Results] The amino acid sequence of the LP produced by strain HT66 was predicted to be L-Leu-D-Glu-D-allo-Thr-D-Val-L-Leu-D-Ser-L-Leu-D-Ser-L-Ile and the LP was verified to be viscosin via comparing the mass spectra data of the metabolites in the wild type and the mutant.Deficiency of LP was found to have no distinct effect on bacterial growth of strain HT66,however,the reduction in PCN production,biofilm formation and swarming motility were observed in the mutant.[Conclusion] The LP produced by strain HT66 is viscosin and it regulates several biological functions including metabolism,biofilm formation and motility.This study reported the structure and function of an LP produced by P.chlororaphis,which may be helpful in learning its synthesis and regulation mechanism and lay a foundation for its development and application.