黏质沙雷氏菌磷脂酶A_(1)辅助蛋白PlaS对宿主菌细胞膜的影响及亚细胞定位  

Effect of Phospholipase A1 Auxiliary Protein PlaS of Serratia marcescens on the Cell Membrane of Host Bacteria and Its Subcellular Localization

作　　者：周杰 吴政浩 李闯 刘庆涛 张会敏 王洲[1] 刘艳[1] 薛正莲[1] ZHOU Jie;WU Zhenghao;LI Chuang;LIU Qingtao;ZHANG Huimin;WANG Zhou;LIU Yan;XUE Zhenglian(Anhui Engineering Laboratory for Industrial Microbiology Molecular Beeding,Anhui Engineering Technology Research Center of Microbial Fermentation,College of Biological and Food Engineering,Anhui Polytechnic University,Wuhu 241000,China)

机构地区：[1]安徽工程大学生物与食品工程学院,安徽省工业微生物分子育种工程实验室,微生物发酵安徽省工程研究中心,安徽芜湖241000

出　　处：《食品科学》2022年第24期138-144,共7页Food Science

基　　金：国家自然科学基金面上项目(31471615)。

摘　　要：为证明黏质沙雷氏菌磷脂酶A_(1)辅助蛋白PlaS在大肠杆菌BL21(DE3)异源表达中对细胞膜有明显的破坏作用,探究了细胞膜特性的变化,并采用气相色谱-质谱联用技术鉴定了其对细胞膜脂肪酸的影响,通过激光共聚焦显微镜验证了亚细胞水平定位。结果显示:PlaS异源表达后,宿主菌内外膜通透性明显提高、细胞膜流动性与表面疏水性严重下降,表明细胞膜功能已经丧失;脂肪酸饱和程度下降,即直链饱和脂肪酸和反式单不饱和脂肪酸相对含量降低,以及顺式单不饱和脂肪酸相对含量增加、膜刚性增强、流动性减少,细胞膜组分与结构表现异常导致细胞易死亡。激光共聚焦显微镜显示,在宿主细胞膜上发现明显荧光。综上所述,PlaS属于膜蛋白,在大肠杆菌异源表达中会破坏宿主菌细胞膜的结构与功能,表现生长抑制现象,这将为进一步PlaS抑制机理的研究与其功能开发提供一定支持。To prove that the phospholipase A_(1)accessory protein PlaS of Serratia marcescens has a significantly disruptive effect on the cell membrane of Escherichia coli BL21(DE3) during heterologous expression,changes in cell membrane properties were explored,their effect on cell membrane fatty acids was identified by gas chromatography-mass spectrometry(GC-MS),and subcellular localization was observed by laser confocal microscopy.The results showed that after the heterologous expression of PlaS,the inner and outer membrane permeability of the host bacterium was significantly increased,and cell membrane fluidity and surface hydrophobicity were severely decreased,indicating loss of cell membrane functions;the saturation degree of fatty acids was decreased,that is,the relative contents of straight-chain saturated fatty acids and trans-monounsaturated fatty acids in fatty acids were reduced,and the relative contents of cis-monounsaturated fatty acids were increased.Moreover,membrane rigidity was enhanced,membrane fluidity was reduced,and the cell membrane components and structure were abnormal,so that the cells could be susceptible to death.Laser confocal microscopy showed that significant fluorescence was observed in the host cell membrane.In summary,the heterologous expression of the membrane protein PlaS in E.coli disrupts the structure and function of the host cell membrane and exhibits growth inhibition,which will provide support for further research on the mechanism of Pla S inhibition and its functional development.

关 键 词：磷脂酶A_(1)辅助蛋白PlaS 细胞膜 脂肪酸 亚细胞定位 

分 类 号：Q939.9[生物学—微生物学]