| 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 |
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| 引用本文： | 霍雨佳,卢乔楠,郑晓伟,马远方,卢锋. 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[J]. 微生物学报, 2016, 56(2): 264-274 |
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| 作者姓名： | 霍雨佳 卢乔楠 郑晓伟 马远方 卢锋 |
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| 作者单位： | 河南大学医学院, 抗体药物河南省工程实验室, 河南 开封 475001,西北农林科技大学林学系, 陕西 杨凌 712100,河南大学医学院, 抗体药物河南省工程实验室, 河南 开封 475001,河南大学医学院, 抗体药物河南省工程实验室, 河南 开封 475001,河南大学医学院, 抗体药物河南省工程实验室, 河南 开封 475001 |
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| 基金项目： | 国家自然科学基金(81372147);河南省教育厅自然科学研究项目(2010B320001);河南大学省部共建项目(SBGJ090713) |
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| 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。
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| 关 键 词： | 大肠埃希氏菌 FtsZ 组装 MreB 细胞内定位 |
| 收稿时间： | 2015-05-26 |
| 修稿时间： | 2015-07-08 |
E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction |
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| Yujia Huo,Qiaonan Lu,Xiaowei Zheng,Yuanfang Ma and Feng Lu. E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction[J]. Acta microbiologica Sinica, 2016, 56(2): 264-274 |
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| Authors: | Yujia Huo Qiaonan Lu Xiaowei Zheng Yuanfang Ma Feng Lu |
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| Affiliation: | Henan Engineering Laboratory of Antibody Medicine, Medical School of Henan University, Kaifeng 475001, Henan Province, China,College of Forestry, Northwest A&F University, Yangling 712100, Shaanxi Province, China,Henan Engineering Laboratory of Antibody Medicine, Medical School of Henan University, Kaifeng 475001, Henan Province, China,Henan Engineering Laboratory of Antibody Medicine, Medical School of Henan University, Kaifeng 475001, Henan Province, China and Henan Engineering Laboratory of Antibody Medicine, Medical School of Henan University, Kaifeng 475001, Henan Province, China |
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| Abstract: | [Objective] To explore effects of FtsZ mutants FtsZE75A, FtsZR78G and ftsZD82A 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(ftsZE75A::yfp-cat), QN8(ftsZR78G::yfp-cat) and QN9(ftsZD82A::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 FtsZE75A, FtsZR78G and ftsZD82A 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. |
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| Keywords: | Escherichia coli FtsZ assembly MreB cellular localization |
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