Analysis of the key amino acids involved in the function and cellular self-assembly of FtsZ protein in Escherichia coli strains
10.3760/cma.j.issn.0254-5101.2015.04.001
- VernacularTitle:影响大肠埃希菌 FtsZ 功能和自身组装的重要氨基酸分析
- Author:
Xiaowei ZHENG
;
Qiaonan LU
;
Yujia HUO
;
Yuanfang MA
;
Feng LU
- Publication Type:Journal Article
- Keywords:
Escherichia coli ( E.coli);
FtsZ mutant ( FtsZ*);
Cellular localization
- From:Chinese Journal of Microbiology and Immunology
2015;(4):241-246
- CountryChina
- Language:Chinese
-
Abstract:
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.
