[E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction].
[E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction].
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发表时间：
2016-02
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通讯作者：
Yujia Huo;Qiaonan Lu;Xiaowei Zheng;Yuanfang Ma;Feng Lu
中科院分区：
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作者：
Yujia Huo;Qiaonan Lu;Xiaowei Zheng;Yuanfang Ma;Feng Lu
目的探讨FtsZ突变体FtsZ（E75 A）、FtsZ（R78 G）和FtsZ（D82 A）对大肠杆菌FtsZ自组装及其与MreB相互作用的影响。方法：采用分子克隆和定点突变的方法构建FtsZ及其突变体的质粒，用亲和层析法纯化目的蛋白。QN6（ftsZ：：yfp-cat）、QN7（tsZ：：yfp-cat）、QN8（ftsZ（R78G）：：yfp-cat）和QN9（ftsZ（D82A）：：yfp-cat）菌株。我们观察了FtsZ及其突变体在大肠杆菌中的细胞定位模式。大肠杆菌活细胞成像实验，免疫共沉淀法和细菌双杂交法检测FtsZ/FtsZ*-FtsZ* 和FtsZ/FtsZ*-MreB的相互作用，光散射法分析FtsZ突变体的组装特征。结果）Yfp标记的FtsZ（E75 A）、FtsZ（R78 G）和FtsZ（D82 A）突变体蛋白在E.大肠杆菌菌株。FtsZ与其突变体的相互作用，或FtsZ*-FtsZ* 和FtsZ*-MreB相互作用减弱或完全消失。此外，体外实验表明E75 A、R78 G和D82 A突变降低了FtsZ单体的聚合效率。结论FtsZ E75、R78和D82是FtsZ蛋白组装、功能及FtsZ与MreB相互作用的关键氨基酸。大肠杆菌菌株。
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(tsZ::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 Coimmunoprecipitation 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.