To Analyze The Important Amino Acids Of Affecting The E.coli FtsZ Assembly Itself And Its Interaction With MreB

IntroductionDue to extensive use of antibiotics, bacterial resistance has become a common clinical problem,resulting in many pathogenic bacteria resistance to antibiotics. The overuse of antibiotics not only caused a waste of drug resources, but also increases the economy burden of patients, more important is to make the human body flora imbalance decreased immunity and promote the generation of drug-resistant strains.So looking for new antimicrobial drug targets, developing a new generation of antibiotics drug is imminent.In the formation of the sub-division and cell division of Bacteria process, need a lot of protein to participate in. Those protein which plays essential role in bacterial division for bacterial survival and proliferation is indispensable and that such protein is conservative in the form and difficult variation, thus becoming a promising new targets to anti-bacterial drug development. Division and the maintenance of a specific form of bacterial cells is regulated by various proteins, such as FtsZ, FtsA, ZapA, MreB etc, in which tubulin analogue FtsZ and actin analogue MreB play a central role, but the exact mechanism is not entirely clear, which limits the FtsZ as targets for the development and utilization of a new generation of antibiotics.FtsZ is E. coli and other bacterial necessary proteins for division, it is highly conserved in prokaryotes.FtsZ monomer by longitudinal and transverse(side) polymer interact to form a bundle, and finally forming Z ring in the middle of the cells.The stability of the Z rings need Fts A, ZipA, Zap A, ZapB and other proteins, which ultimately played a role in skeleton, through the recruitment of downstream cell division associated proteins, formed divisome(divisome) in the middle of the forthcoming division of bacterial cell,regulation of bacterial division. Divisome shrinkage makes division protein complex formation diaphragm,and finally the average bacterial cell into two to form two identical daughter cells.MreB is currently recognized as the main protein to control E.coli morphology, in eukaryotes, shape and movement of organisms is through the periodic adjusteent of actin cytoskeleton network polymerization and depolymerization to complete, and in bacteria, MreB is actin analogues, it can be polymerized into filaments at the cell periphery, and to coordinate bacterial cell wall synthesis. Geneknockout can affect the synthesis of cell wall, which can lead to E.coli bacteria into the rod-shaped spherical, but when high levels expression of FtsZ in bacteria vivo, inhibit MreB defects caused by morphology change. In recent years, researchers have found that FtsZ special site amino acid mutations can cause bacteria horseshoe-shaped, crescent-shaped and antlers, etc; In addition, researchers have found some of MreB mutant can cause abnormal division of bacteria, described FtsZ and there is some coordination between MreB, but the relevant mechanism is rarely reported.To further investigate whether MreB and FtsZ common control bacterial growth and division, through analysis of different bacteria ftsz, we found that they are highly homologous amino acid sequence.In this study was use Escherichia coli as a model organism, through analyzing the three-dimensional and two-dimensional structure of FtsZ,we found E75, R78 and D82 amino acids are located in H3 domain structure of FtsZ side, and may participate in the formation of FtsZ(74-82) helical structure and FtsZ monomer assembly on the side of Ftsz, in addition, E75, R78 and D82 amino acids are located on the same side of the amphipathic helix structure. Speculate E75, R78 and D82 may important amino acid to affect H3 cell structure and internal FtsZ assembly. We through mutated the E75, R78 and D82 specific amino acid positions to observe the influence of FtsZ intracellular assembly and location, to observe the influence of FtsZ-MreB interaction, preliminary exploration related molecular mechanisms, in order to provide some help to a new generation of antibiotics research and development which use FtsZ as target. In our experiments, live cell imaging techniques, immunoprecipitation and bacterial two-hybrid and other methods to detect both together may play a role in the regulation of normal bacterial growth and division.ObjectiveTo explore FtsZ E75 A, FtsZ R78 G and FtsZ D82 A of E.coli FtsZ mutant effect on FtsZ assembly itself and FtsZ-MreB interaction.Methods1. The use of conventional molecular cloning and site-directed mutagenesis techniques to construct FtsZ and its mutant expression vector, affinity purification to get the corresponding target protein.2. By homologous recombination mechanism construct strains of QN6(ftsZ :: yfp-cat), QN7(ftsZE75A :: yfp-cat),QN8(ftsZR78G :: yfp-cat) and QN9(ftsZD82A :: yfp-cat).3. The use of live cell imaging techniques to observe intracellular localization pattern of FtsZ and itsmutan.4. Through immunoprecipitation and bacterial two-hybrid assay to detect FtsZ / FtsZ *-FtsZ * or FtsZ/ FtsZ *-MreB interactions.5. Detecting FtsZ assembly property effection of site-directed mutagenesis by optical scanning.Results1. Mutations of FtsZE75 A, FtsZR78 G and FtsZD82 A function and activity reduce, each mutant can not correctly positioned and the formation of functional Z ring in E.coli.2. FtsZ / FtsZ *-FtsZ * interactions between monomers weakened or disappeared, FtsZ *-MreB interaction destruction.3. FtsZ mutants in vitro polymerization efficiency lower.ConclusionsFtsZ E75, R78 and D82 are mportant amino acids to affect FtsZ properly assembled and FtsZ-MreB interactions.