The Effect Of FtsZ^P74R?FtsZ^G77D And FtsZ^A81R On The Function And Cellular Self-assembly Of FtsZ Protein In Escherichia Coli Strains

Background In recent years, the emergence of new pathogens and multiple drug-resistant bacteria lead to a serious threat to human health. It is urgent to develop and screen antibiotics with potential new targets,chemical structures and novel mechanisms. FtsZ?filamentous temperature-sensitive protein Z?, a bacterial protein essential for cell division and is highly in pathogens, such as Mycobacterium tuberculosis,Pseudomonas aeruginosa and Staphylococcus aureus. In addition, the FtsZ is also tubulin prokaryotic analogs for the similar three-dimensional structure with tubulin. In bacterial cytokinesis, FtsZ monomers assembles into linear protofilaments by longitudinal interaction, then the protofilaments further assemble into bundles through lateral bonds, and finally develop into a macromolecular ploymers with a Z Ring at the center of the cell. Stable Z ring needs the participation of Zip A, Zap A, Fts A, Zap B, Zap C and other proteins. The Z ring plays skeleton roles and forms proteins complex divisome at the centre of deviding cell via recruiting downstream division proteins related to cell division to regulate the division of becteria cell.And, the mechanisms underlying how the FtsZ monomers inside the Z ring structure assemble and Which amino acids are involoved into the side interaction between FtsZ monomers and Z ring stability are still not entirely clear, which limits the development and utilization of new FtsZ-targeted antibiotics.E. coli, one of Single-celled organisms, is a common gram-negative bacillus and a model organism for functional genomics research due to its simple structure and easily culture in vitro, and genome operation is relatively mature. We speculated that P74, G77 and A81 are the important amino acids that can affect the function and assembly of FtsZ. We found that the amino acid including P74?G77and A81 in the domain of H3 in E.coli FtsZ is highly conserved and participate in gender helical structure formation of local area by analysis the three-dimensional structure and FtsZ homology relationship of E.coli with Mycobacterium tuberculosis?Staphylococcus aureus?Pseudomonas aeruginosa?Helicobacter pylori and other pathogens. We made a preliminary exploration about P74, G77 and A81 amino acid on FtsZ function, intracellular localization and assembly significance and molecule mechanism by site-directed mutagenesis and related research, which has an important guiding significance to design new FtsZ-targeted drugs aiming to specific amino acid sites or areas.Objective To investigate the self-assembly and cellular localization patterns of filamentous temperature-sensitive protein Z? FtsZ? in Escherichia coli? E.coli? strains and its molecular mechanisms by using FtsZ^P74R?FtsZ^G77D and FtsZ^A81R mutants.Methods His or YFP-tagged FtsZ proteins and the plasmids of FtsZ mutants were constructed by using molecular clone and site-directed mutagenesis methods. The targeted proteins were purified by affinity chromatography. FL37? ?fts Z-Cat? strains were constructed via linear DNA homologous recombination.Living cell imaging were performed to observe the cellular localization patterns of FtsZ protein and its mutants in E.coli strains. The interactions among FtsZ-FtsZ / FtsZ mutants were examined by co-immunoprecipitation assay. The activities of GTPase were monitored by using high performance liquid chromatography. The polymerization properties of FtsZ mutants were analyzed by Light scattering.Results 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 FtsZ^P74R,FtsZ^G77D and FtsZ^A81R mutants failed to assemble into functional Z-ring structure in E. coli strains. The interactions between FtsZ protein and its mutants were weakened or completely disappeared. In addition,in vitro experiments showed that P74 R, G77 D and A81 R mutations caused a decrease in the polymerization efficiency of FtsZ monomer. The activity of GTPase was significantly decreased in the FtsZ^A81R mutant.Conclusions 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.