Qingsu Ma, Huijuan Zhang, Xiaowei Zheng, Yujia Huo, Feng Lu
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引用次数: 0
摘要
目的:研究FtsZ(236-245)结构域两亲螺旋特性对大肠杆菌FtsZ组装及FtsZ与FtsA相互作用的影响。方法:采用分子克隆和定点诱变技术构建FtsZ及其突变体质粒，并利用亲和层析技术纯化靶蛋白。采用线性DNA同源重组和P1转导构建QN23-QN29菌株。我们通过活细胞成像实验观察了FtsZ及其突变体在大肠杆菌中的细胞定位模式，通过膜蛋白分离和Western blot分析了FtsZ突变体的膜结合特性，通过共免疫沉淀和far Western blot分析了FtsZ/FtsZ*与FtsA的相互作用。通过天然凝胶分离和体外聚合实验，考察FtsZ点突变对FtsZ组装的影响。结果:yfp标记的FtsZE237A/K和FtsZE241A/K突变蛋白无法在大肠杆菌中定位，组装成功能性z环结构，FtsZ功能降低(wt)。体外实验表明，FtsZ的E237A/K和E241A/K突变降低了FtsZ单体的聚合效率，减弱了FtsZ*-FtsA的相互作用，改变了FtsZ的膜结合性能。结论:FtsZ E237和E241是影响大肠杆菌FtsZ(236-245)结构域两亲螺旋特征、FtsZ组装和FtsZ- ftsa相互作用的关键氨基酸。
[Effect of amphipathic helix characteristics of FtsZ (236-245) domain on FtsZ assembly and its function in Escherichia coli strains].
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 FtsZE237A/K and FtsZE241A/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.
期刊介绍：
Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal
publishes original papers,reviews in microbiological science,and short communications describing unusual observations.
Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database
(CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical
Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.
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