摘要
【目的】探索大肠埃希氏菌(Escherichia coli,E.coli)FtsZ(236-245)结构域两性螺旋特性对FtsZ组装和FtsZ-FtsA相互作用的影响。【方法】利用分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化获得相应目标蛋白;通过同源重组和Pl转导构建QN23-QN29菌株;利用活细胞成像观察FtsZ及其突变体的胞内定位特点;膜蛋白分离和Western blot分析FtsZ突变体的膜结合特性变化;非变性胶分离和体外聚合分析检测定点突变对FtsZ单体组装特性的影响;免疫沉淀和Far Western blot实验检测FtsZ/FtsZ~*-FtsA间的相互作用。【结果】FtsZ^(E234A/K)和FtsZ^(E241A/K)突变体的功能活性降低、备突变体在E.coli内不能正确定位和形成功能性Z环;E237A/K和E241A/K位点突变致备突变体聚合能力降低、FtsZ*-FtsA的相互作用减弱和FtsZ的膜结合特性变化。【结论】E237和E241是影响FtsZ(236-245)区域两性螺旋特性和FtsZ组装及FtsZ-FtsA相互作用的重要氨基酸。
[Objective]To study the effect of amphipathic helix characteristics of FtsZ（236-245） domain on FtsZ assembly and interaction of FtsZ with FtsA in Escherichia coli strains.[Methods]We constructed FtsZ and its mutant＇s plasmids by molecular clone and site-directed mutagenesis,and purified targeted proteins using affinity chromatography.QN23-QN29 strains were constructed by linear DNA homologous recombination and P1 transduction.We observed cellular localization patterns of FtsZ and its mutants in E.coli by living cell imaging experiments,examined membrane binding properties of FtsZ mutants by membrane proteins isolation and Western blot analysis,and analyzed interaction of FtsZ/FtsZ^＊ with FtsA by Co-immunoprecipitation and far Western blot.Native gel separation and in vitro polymerization experiments were done to check effects of FtsZ point mutation on FtsZ assembly.[Results]Yfp-labeled FtsZ^（E237A/K） and FtsZ^（E241A/K） mutant proteins failed to localize in E.coli strains,assemble into functional Z-ring structure,and had decreased function of FtsZ（wt）.In vitro experiments showed that E237A/K and E241A/K mutations of FtsZ decreased the polymerization efficiency of FtsZ monomer,weakened FtsZ^＊-FtsA interaction and changed membrane binding properties of FtsZ.[Conclusion]FtsZ E237 and E241 are critical amino acids that affect the amphipathic helix characteristics of FtsZ（236-245） domain,FtsZ assembly and FtsZ-FtsA interaction in E.coli strains.
出处
《微生物学报》
CAS
CSCD
北大核心
2017年第4期513-525,共13页
Acta Microbiologica Sinica
