E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction

Abstract:

[Objective] To explore effects of FtsZ mutants FtsZ^E75A, FtsZ^R78G and ftsZ^D82A 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(ftsZ^E75A::yfp-cat), QN8(ftsZ^R78G::yfp-cat) and QN9(ftsZ^D82A::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 FtsZ^E75A, FtsZ^R78G and ftsZ^D82A 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.