E75、R78和D82是影响大肠埃希氏菌FtsZ自身组装及其与MreB相互作用的重要氨基酸

E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction

摘要:

[目的] 探索大肠埃希氏菌Escherichia coli FtsZ突变体FtsZ^E75A、FtsZ^R78G和ftsZ^D82A对FtsZ自身组装和FtsZ-MreB相互作用的影响。[方法] 利用常规分子克隆和定点突变技术,构建FtsZ及其突变体表达载体,亲和纯化得到相应的目标蛋白;通过同源重组构建QN6 (ftsZ::yfp-cat)、QN7 (ftsZ^E75A::yfp-cat)、QN8 (ftsZ^R78G::yfp-cat)和QN9 (ftsZ^D82A::yfp-cat)菌株;利用活细胞成像技术观察FtsZ及其突变体的胞内定位模式;免疫沉淀和细菌双杂交实验检测FtsZ/FtsZ^*-FtsZ^*或FtsZ/FtsZ^*-MreB间的相互作用;光扫描检测定点突变对FtsZ组装特性的影响。[结果] FtsZ^E75A、FtsZ^R78G和ftsZ^D82A突变体的功能活性降低、各突变体在E. coli内不能正确的定位和形成功能性Z环;FtsZ/FtsZ^*-FtsZ^*单体间的相互作用减弱或消失,FtsZ^*-MreB相互作用破坏;FtsZ突变体体外聚合效率降低。[结论] FtsZ E75、R78和D82是影响FtsZ正确组装和功能及FtsZ-MreB相互作用的重要氨基酸。

关键词:大肠埃希氏菌;FtsZ 组装;MreB;细胞内定位

Abstract:

[Objective] To explore effects of FtsZ mutants FtsZ^E75A, FtsZ^R78G and ftsZ^D82A on FtsZ self-assembly and interaction of FtsZ with MreB in Escherichia coli strains. [Methods] We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis methods, and purified targeted proteins by affinity chromatography. QN6(ftsZ::yfp-cat), QN7(ftsZ^E75A::yfp-cat), QN8(ftsZ^R78G::yfp-cat) and QN9(ftsZ^D82A::yfp-cat) strains were constructed by linear DNA homologous recombination. We observed cellular localization pattern of FtsZ and its mutants in E. coli by living cell imaging experiments, examined interaction of FtsZ/FtsZ^*-FtsZ^* and FtsZ/FtsZ^*-MreB by Co-immunoprecipita-tion and bacteria two hybrid, and analyzed assembly characteristics of FtsZ mutants by Light scattering. [Results] The Yfp-labeled FtsZ^E75A, FtsZ^R78G and ftsZ^D82A mutant proteins failed to assemble into functional Z-ring structure and localize correctly in E. coli strains. Interaction of FtsZ with its mutants, or FtsZ^*-FtsZ^* and FtsZ^*-MreB interaction were weakened or completely disappeared. In addition, in vitro experiments show that E75A, R78G and D82A mutations decreased the polymerization efficiency of FtsZ monomer. [Conclusion] FtsZ E75, R78 and D82 are critical amino acids in the assembly, function of FtsZ protein and FtsZ-MreB interaction in E. coli strains.

Key words:Escherichia coli;FtsZ assembly;MreB;cellular localization