To Explore Molecular Mechanism Of FtsZ Mutant Affecting Cellular Locolization Of MreB

Background:The problem of bacterial resistance is increasingly serious and more and more bacteria becomeresistant to existing antibiotics.. Natural antibiotics is a secondary metabolite produced by bacteria and achemistry material used to gainst other microorganisms to protect their own security. And theantimicrobial resistance when microorganisms exposed the material is mainly happed by changing theirmetabolic pathways or producing corresponding inactivated substance. The emergence of bacterialresistance and bacterial infections of drug-resistant often makes it difficult to clinical work and brings greatchallenges to the clinical anti-infective therapy. It is urgent to develop new targets for antibiotics. FtsZprotein is the key to mediated bacterial cell division,and homologous to human tubulin but have bigdifference with human tubulin. If it is possible to design an inhibitors to have selective effect to FtsZwithout interfering to bacterial host cells, FtsZ protein may have hope to become a new target forantimicrobial drug research.There are two basic cytoskeletal protein of Escherichia coli:FtsZ and MreB. FtsZ is a highly conservedGTP enzyme and homologous to eukaryotic tubulin. FtsZ can form a cyclic structure in the intermediateposition of the cell.It have a function as scaffold in process of plastid division and forms a protein complexto take charge a variety of cell division.In bacteria, FtsZ is considered to be the first kind of proteinpositioned in the split parts.A ring structure in Escherichia coli FtsZ (Z ring) and other adsorbed divisionsprotein.They form a bacterial cell divides in the neutral position. It is essential to the formation of diaphragmcomplex and cell division. Bacterial cell division is controlled by tubulin analogue FtsZ while cell elongation is controlled by theactin analogue MreB.MreB is a similar actin-ATP enzyme,maintain typically rod cells and a basic polarityenzyme in E. coli cells. MreB also have relation with chromosomes split, the cell membrane organellepositioning and synthesis biologically relevant substances coordinating cell division. Because MreB andFtsZ involves a series of basic cellular function, they will ultimately lead to cell death when their functionis inhibited.A dynamic network of proteins include protein raise and transport, and cell movement and split.Cellinternal procedures in coordination with the role of actin and microtubule protein homologue are inseparable.In eukaryotes, the function of microtubule protein to actin is mainly carry actin microfilament to make itswing. FtsZ and MreB can form macromolecular structure and have the vital significance to the maintain ofcell division and cell shape respectively.How to coordinate the growth of bacteria and divide also remainsunclear in the current study. So further study of the functions of cytoskeleton, understand coordinating rolebetween actin and tubulin family is very important.Reports have pointed out that direct interaction between FtsZ and MreB can promote bacteria celldivision and cell growth.We use the live cell imaging and observed FtsZ and MreB of E. coli intracellularcolocalization positioning mode. By the three-dimensional structure and amino acid sequence analysis ofFtsZ,We select several important amino acid as the mutation target sites point to make them mutate,Pull-Down and western blot analysis found that the interaction between MreB and FtsZ is an importantcondition for the positioning of the spiral MreB cells. The results of this study demonstrate the importanceof a strong protein responsible for cell morphology and cell division protein responsible for thecoordination of the functions for further research on how to coordinate the molecular mechanisms ofbacterial division and growth and maintain normal morphology provides experimental support. Purpose：1.Study MreB and FtsZ positioning mode in the cell.2.FtsZ part of the mutations on the MreB cells spiral body positioning.3. The molecular mechanisms of FtsZ affect MreB intracellular localizationMethod：1Knockout MreB gene from E. coli genome by homologous recombination.2Constructed plasmids expressing different FtsZ-YFP, YFP-FtsZ, yfp-MreB fusion protein, usedfluorescence microscopy to study MreB and FtsZ positioning mode in the cell.3Constructed different mutants using overlapping PCR FtsZ, explore the E. coli FtsZ mutantswhether or not affect the positioning MreB and molecular mechanisms.Result1. FtsZ formed ring structure in E. coli.2.MreB formed spiral structure in E.coli.3.FtsZ and MreB interaction with in cell, and in a certain phase of the corresponding portionpositioned in E.coli.4.FtsZ mutant MreB fluorescence molecular mechanisms affecting the positioning mode is mainlyaffected the interaction between FtsZ-MreB.ConclusionFtsZ interaction with MreB is an important condition MreB intracellular localization of the spiral.