Screening Of Mciz Mutant Polypeptides With Inhibitory Effect On Bacterial Division Of Escherichia Coli By Random Mutation Technique

This project was divided into two independent parts.The main part was to screen MciZ mutant polypeptides have significant effects on cell division of Escherichia coli by random mutation.The second minor part was to explore the application of MinC-MinD copolymers.FtsZ,the cytoskeleton protein of bacterial cell division,is the main substance controlling bacterial cell division,which exists in almost all prokaryotes and has GTPase activity.FtsZ bound to GTP,can self-polymerize rapidly to form single protofilaments,which located at the middle of the cell,forming a discontinuous highly dynamic Z-ring.A variety of proteins involved in cell division are recruited on the Z-ring timely and spatially.They can regulate the Z-ring precisely.MciZ is a small peptide with 40 amino acids found in B.subtilis.It can bind to the C-terminal of FtsZ.Studies have shown that MciZ inhibited the growth of B.subtilis by terminating the FtsZ protofilaments.It can shorten FtsZ protofilaments,increase the GTPase activity and critical concentration of polymerization of FtsZ.In order to explore whether MciZ can inhibit the growth of other bacterias,we selected gram-negative bacteria E.coli.Since MciZ had a weak inhibitory effect on E.coli,MciZ was used as a template for random mutation and screened by plate spotting method.We investigated the effects of MciZ and its mutant polypeptides on the growth of E.coli by growth curve measurement,Single plate-serial dilution spotting（SP-SDS）,monoclonal counting,morphology and length statistics of bacterias,and GTPase activity measurement of FtsZ.In this part,6 mutant peptides were obtained,two mutant peptides P7LA16T and W17RI19N were found to inhibit the growth of E.coli stronger than that of MciZ by growth curve measurement and monoclonal counting.Compared with MciZ,the both peptides had two amino acids change;in P7LA16T,the 7th position was changed from Pro to Leu,and the16th position was changed from Ala to Thr;in W17RI19N,the 17th position was changed from Trp to Arg,and the 19th position was changed from Ile to Asn.Furthermore,the two peptides were explored by observing the morphology,length of bacteria and measuring the GTPase activity of FtsZ.The results showed that the P7LA16T had stronger inhibitory effect on E.coli than MciZ.Under the influence of P7LA16T,the average length of bacteria was3.31μm,0.88μm longer than that of MciZ,and the length of some bacteria even longer than10μm,which showed that it had inhibition on bacteria growth.In order to further explore the effects of MciZ and its mutant polypeptides on Ec FtsZ.The GTPase activity of Ec FtsZ was measured,and the results showed that P7LA16T could significantly improve the GTPase activity of FtsZ.Although the mutant polypeptide P7LA16T did not completely inhibit the growth of E.coli,it had obvious inhibitory effect.The effects of MciZ and P7LA16T were not obvious on the critical concentration of FtsZ polymerization.We considered that the new complex FtsZ-MciZ was difficult to form stable protofilaments,the depolymerization rate of FtsZ was accelerated,prevented the formation of Z-ring and inhibited the cell division.In addition,we speculated that MciZ may also influence the FtsZ recruited other downstream proteins,which may also be a mechanism to inhibit bacterial division.In the following studies,more ideal mutant peptides will be screened to produce more significant inhibition on bacterial cell division.Pathogens will be selected for further exploration.It will provide experimental basis for the research and development of polypeptide antibacterial drugs.In the appendix,MinC and MinD proteins were found in the Min system.Under the high MinD concentration,MinC and MinD could form the alternating copolymeric of MinC₂-MinD₂-MinC₂-MinD₂ in a ratio of 1:1.Multienzyme complex consisted of several enzymes linked to each other by non-covalent bonds.In order to explore whether the enzymatic reaction can be accelerated by shorting the distance between enzymes.MinC-MinD copolymeric filaments was used as the skeleton,and enzymes of multienzyme complex as the“passengers”,RhlA,RhlB and RhlC related to the synthesis of rhamnolipid were selected for in vivo experiments.Replenishing bacteria were constructed by gene knockout and gene repair technology respectively.Pseudomonas aeruginosa PAO1 and△rhl Arhl B△rhl C knockout bacteria were used as controls,and the rhamnolipid production was measured by orcinol-sulfuric acid method.The results showed that there were no significant differences in rhamnolipid production between the strains with or without MinC^C,and were all lower than that of PAO1.We speculated that the short distance between MinC^C and RhlA/RhlB/RhlC proteins might hinder the polymerization of MinC^C and MinD,or because the promoter was replaced,the expression of the proteins were affected.