| Pseudomonas aeruginosa(P.aeruginosa)belongs to the gram-negative bacillus.It is a kind of common pathogens with multiple drug resistance.In bacteria,FtsZ is considered as a key component of cytokinetic ring.Meantime,it is also probably the source of contractile force.Therefore,FtsZ takes an important role in the process of bacterial division,and is a kind of conservative cytoskeletal protein in prokaryotes.Since FtsZ is a bacterial tubulin homologous analogue,and also have a GTPase activity.In the first part,we studied the biochemical characteristics of PaFtsZ.The biological activities of FtsZ are able to be measured by its GTPase activity and the abilities of forming protofilaments.We adopt a continuous and coupling assay to determine FtsZ GTPase activity.Similar to EcFtsZ,PaFtsZ has a high GTP hydrolytic activity.PaFtsZ GTPase activity is around four GTP per min per FtsZ.We studied the GTPase activities of FtsZ in different buffer with various ions.Different with EcFtsZ,the changes of cation have little effect on PaFtsZ GTPase activity,whereas the changes of anions have the obvious effect.Similar with EcFtsZ,PaFtsZ also polymerize into single filaments.However,different from most straight filaments of EcFtsZ,PaFtsZ assembles into a lot of the moderate bending structures and even forms arc-shaped structures.In the second part,we researched the effects of ZipA from P.aeruginosa on the polymerization of FtsZ.We found that ZipA can not only enhance the polymerization of FtsZ-GTP protofilaments,but also stabilize and strengthen the highly curved conformation of FtsZ-GDP.FtsZ filaments can generate forces through the transition from straight to curve filaments.The force is likely to be the cause of generating the constriction force for contractile ring in bacteria.ZipA can strengthen and stabilize these constiction forces.In the third part,we researched the main regular system of the constriction ring,MinC,MinD and MinE system from P.aeruginosa.In the gene knockout experiment,the absence of MinC/MinD protein destroy the position of Z ring at the time of cell division,similar with the studies in E.coli.These cause that bacteria form the long line or division generates at the two end of cell.In vitro experiments,we adopt light scattering,TEM and co-pelleting assay.We confirmed that MinC is an inhibitor in Min system.Different from EcMinC,PaMinC inhibits clearly the FtsZ polymerization.Also,MinC and MinD form copolymers,which are two-strand straight filaments or bundles.MinE activated the ATP enzyme activity of MinD and depolymerized MinCD copolymers in the presence of ATP.What's more,MinC and MinD assemble into copolymers occur at the stoichiometric ratio of 1:1.The light scattering experiments confirmed that MinD Plays a decisive role in the copolymers of MinC and MinD.MinC-MinD is hard to form copolymers when the concentration of MinD is under 5 ?M.But,we can still observe the copolymers assembly when the concentration of MinC is under 0.5?M if the concentration of MinD is greater than 5 ?M.These correspond to the concentrations of MinC and MinD in vivo.Therefore,we suggest that the copolymers of MinC/MinD play an important role in vivo.The experimental work of this paper studied PaFtsZ and the functions and biochemical properties of its regular proteins which provided us the basis for understanding P.aeruginosa division mechanism,and offered us the new theoretical foundations and research strategies. |
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