| Prokaryotic cell division is mediated by a series of proteins complex known as divisome.Assembly of this machinery initiated with the polymerization of the tubulin homolog FtsZ into a ring-like structure,the contractile ring(Z-ring),at the future division site.FtsZ,a bacterial tubulin homolog,binds GTP and polymerizes into filaments as a scaffold for the recruitment of downstream division proteins to develop the Z-ring,and also might generate the inward force to initiate the bacterial cell division.In Escherichia coli,the Z-ring contains more than 20 associate proteins,which include both negative regulators and positive regulators.The negative regulators,including MinC and SulA,could inhibit FtsZ polymerization directly and regulate the Z-ring formation.Meanwhile,the positive regulators,including ZipA(FtsZ interacting protein A)and ZapA(FtsZ associated protein A),would enhance FtsZ assembly in vitro,promote their lateral contact to form bundles and stabilize the Z-ring structure in vivo.In Escherichia coli,the primary assembly of the first protein complex,the proto-ring,involves the interaction of FtsZ with two other proteins,ZipA and FtsA,which tether and stabilize the Z-ring onto the inner membrane.Although nonessential for the division formation,the Zap family proteins may be considered as regulation components of the proto-ring to stabilize the Z-ring.In Bacillus subtilis,since there is no ZipA protein,it is reported that divisome proteins FtsA,SepF,ZapA and EzrA involved in the early proto-ring assembly.Previously studies mostly focused on E.coli FtsZ,ZipA and Zap A proteins.In the current study,we have studied the in vitro dynamic properties of Pseudomonas aeruginosa(Pa)FtsZ,and the effects of PaZapA and PaZipA on PaFtsZ.First of all the FtsZ,ZipA and ZapA protein was overexpressed in E.coli which was then isolated and purified by the process of His-tag purification method.The polymer formation and disassembly were checked by the light scattering assay,while GTPase assay was used for the measurement of FtsZ activity and finally for visualization of polymer and structure the electron microscopy was performed.We determine that PaFtsZ not only assembled into straight protofilaments but also to curved filaments.The GTPase activity of PaFtsZ is very high and the assembly was very fast.More curved filaments observed in the MEK buffer.The EM image shows that Pa ZipA only induces FtsZ bundling formation in MMK buffer at pH 6.5.In HMK buffer which is close to physiological condition at pH 7.5,the PaFtsZ is mostly assembling into single protofilaments.The combination of PaFtsZ-PaZipA can assemble into curved filaments in HEK buffer.It is recommended that ZipA can enhance FtsZ assembly and promote FtsZ curved conformation in pH 7.5 buffer.Similar to E.coli ZipA,PaFtsZ-ZipA assembles into huge tight bundles at pH 6.5.We observed that single or double FtsZ filaments make a tight bundle structure through the lateral contact or crosslinked by Zip A protein.PaZipA has little effects on PaFtsZ assembly in HMK buffer,both GTPase and assembly kinetics and in MMK buffer the GTPase activity of PaFtsZ is decreased significantly when ZipA is added.Unlike ZipA,the Zap A induced PaFtsZ to form large sheets and bundles,it is surprising that ZapA has only a little effect on the PaFtsZ GTPase activity in HMK.To get more information,the assembly kinetics of PaFtsZ protofilaments with or without ZipA or ZapA proteins is measured by the light-scattering assay.We observed that ZipA has little effect on kinetic of PaFtsZ in HMK buffer which is consistent with EM image observation that the size of PaFtsZ filaments has no changes with or without ZipA.While the ZapA induces bundling formation,the light scattering signal was very stronger and slower,it took 200 sec to finish.The light-scattering measurements confirm that in MMK buffer,all of FtsZ,FtsZ plusZipA and FtsZ plusZap A can assemble into large bundles.However,their bundle’s formation shows different properties.Finally,we concluded that in physiological conditions,FtsZ not only assemble into straight filaments but also into curved filaments which are the unique properties of FtsZ,and the ZipA enhances FtsZ assembly,but not bundles.Also,the ZipA can stabilize curved conformation.However,ZapA induces FtsZ bundles formation.The Zap A dimer or tetramer interacts with FtsZ filaments,stabilizes their structure and form straight bundles.It suggests that Zap A stabilize Z-ring. |
