中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2015年
4期
241-246
,共6页
郑晓伟%卢乔楠%霍雨佳%马远方%卢锋
鄭曉偉%盧喬楠%霍雨佳%馬遠方%盧鋒
정효위%로교남%곽우가%마원방%로봉
大肠埃希菌( E.coli)%FtsZ突变体( FtsZ倡)%细胞内定位
大腸埃希菌( E.coli)%FtsZ突變體( FtsZ倡)%細胞內定位
대장애희균( E.coli)%FtsZ돌변체( FtsZ창)%세포내정위
Escherichia coli ( E.coli)%FtsZ mutant ( FtsZ*)%Cellular localization
目的：探索大肠埃希菌（ Escherichia coli， E．coli） FtsZ 突变体 FtsZP74R、FtsZG77D和FtsZA81R对FtsZ功能和胞内自身组装的影响。方法利用分子克隆和定点突变技术，常规构建带His或YFP标签的FtsZ及其突变体融合表达质粒，亲和纯化得到相应的蛋白；采用同源重组技术构建FL37（△ftsZ-Cat）菌株；活细胞成像观察FtsZ及其突变体在胞内定位模式；免疫共沉淀试验检测FtsZ与其突变体间的相互作用；光扫描检测定点突变对FtsZ组装特性的影响；高压液相色谱法检测FtsZ突变体GTPase酶活性的变化。结果 FtsZP74R、FtsZG77D和FtsZA81R突变体在E．coli内不能正确定位和形成功能性Z环、FtsZ-FtsZ倡单体间的相互作用减弱或消失、突变体体外聚合效率减少及FtsZA81R的GTPase酶活性显著降低。结论 P74、G77和A81是影响FtsZ功能和胞内正确组装的重要氨基酸；A81可同时影响FtsZ的侧面相互作用和GTPase活性。
目的：探索大腸埃希菌（ Escherichia coli， E．coli） FtsZ 突變體 FtsZP74R、FtsZG77D和FtsZA81R對FtsZ功能和胞內自身組裝的影響。方法利用分子剋隆和定點突變技術，常規構建帶His或YFP標籤的FtsZ及其突變體融閤錶達質粒，親和純化得到相應的蛋白；採用同源重組技術構建FL37（△ftsZ-Cat）菌株；活細胞成像觀察FtsZ及其突變體在胞內定位模式；免疫共沉澱試驗檢測FtsZ與其突變體間的相互作用；光掃描檢測定點突變對FtsZ組裝特性的影響；高壓液相色譜法檢測FtsZ突變體GTPase酶活性的變化。結果 FtsZP74R、FtsZG77D和FtsZA81R突變體在E．coli內不能正確定位和形成功能性Z環、FtsZ-FtsZ倡單體間的相互作用減弱或消失、突變體體外聚閤效率減少及FtsZA81R的GTPase酶活性顯著降低。結論 P74、G77和A81是影響FtsZ功能和胞內正確組裝的重要氨基痠；A81可同時影響FtsZ的側麵相互作用和GTPase活性。
목적：탐색대장애희균（ Escherichia coli， E．coli） FtsZ 돌변체 FtsZP74R、FtsZG77D화FtsZA81R대FtsZ공능화포내자신조장적영향。방법이용분자극륭화정점돌변기술，상규구건대His혹YFP표첨적FtsZ급기돌변체융합표체질립，친화순화득도상응적단백；채용동원중조기술구건FL37（△ftsZ-Cat）균주；활세포성상관찰FtsZ급기돌변체재포내정위모식；면역공침정시험검측FtsZ여기돌변체간적상호작용；광소묘검측정점돌변대FtsZ조장특성적영향；고압액상색보법검측FtsZ돌변체GTPase매활성적변화。결과 FtsZP74R、FtsZG77D화FtsZA81R돌변체재E．coli내불능정학정위화형성공능성Z배、FtsZ-FtsZ창단체간적상호작용감약혹소실、돌변체체외취합효솔감소급FtsZA81R적GTPase매활성현저강저。결론 P74、G77화A81시영향FtsZ공능화포내정학조장적중요안기산；A81가동시영향FtsZ적측면상호작용화GTPase활성。
Objective To investigate the self-assembly and cellular localization patterns of fila-mentous temperature-sensitive protein Z (FtsZ) in Escherichia coli (E.coli) strains by using FtsZP74R, FtsZG77D and FtsZA81R mutants.Methods YFP or His labeled FtsZ proteins and the plasmids of FtsZ mu-tants were constructed by using molecular clone and site-directed mutagenesis methods.The targeted proteins were purified by affinity chromatography.FL37(△ftsZ-Cat) strains were constructed via linear DNA homol-ogous recombination.Living cell imaging was performed to observe the cellular localization patterns of FtsZ protein and its mutants in E.coli strains.The interactions between FtsZ-FtsZ/FtsZ mutants were examined by coi-mmunoprecipitation assay . The polymerization properties of FtsZ mutants were analyzed by light scattering.The activities of GTPase were monitored by using high performance liquid chromatography.Re-sults The P74, G77 and A81 amino acids were respectively replaced by different polar amino acids to change the amphipathicity of the helix within the domain of FtsZ ( 74-82 ) .The YFP-labeled FtsZP74R , FtsZG77D and FtsZA81R mutants failed to assemble into functional Z-ring structure in E.coli strains.The inter-actions between FtsZ protein and its mutants were weakened or completely disappeared.In addition, in vitro experiments showed that P74R, G77D and A81R mutations caused a decrease in the polymerization efficien-cy of FtsZ monomer.The activity of GTPase was significantly decreased in the FtsZA81R mutant. Conclusion The P74, G77 and A81 were critical amino acids in the function and assembly of FtsZ protein in E.coli strains.Moreover, A81 amino acid regulated the lateral interaction of FtsZ monomer and the activity of GTPase.
