Study On The Characteristics Of A New Bacterial Contractile Loop Binding Protein ZapL In Pseudomonas Aeruginosa And Escherichia Coli

A series of protein complexes called divisome mediate the division of prokaryotic cells.The assembly of this complex is started by the polymerization of the tubulin homolog FtsZ into a ring structure at the division site,that is,the contractile ring(Z ring).As the main cytoskeleton protein,FtsZ plays a key role in cell division.The contractile ring is the main part of the bacterial cell division.The FtsZ protofilament is adjusted by the combination and hydrolysis of GTP to regulate the polymerization and depolymerization cycle.By hydrolyzing GTP,it converts chemical energy into mechanical energy and then into inward contraction force,reducing its own diameter,and pull the septum invaginated until the end of cell division,forming two new cells.In E.coli,the Z ring contains more than 20 related proteins,including both negative and positive regulators.The negative regulatory proteins MinC and Sul A can directly inhibit FtsZ polymerization and regulate the formation of Z ring.At the same time,positive regulatory proteins including ZipA and ZapA can enhance the assembly of FtsZ in vitro,promote its lateral contact to form a bundling structure,and stabilize the Z-ring structure in vivo.ZapA is a small molecule regulator protein,which can stabilize the relatively weak side bonds between FtsZ monomers.In the searching of Pseudomonas aeruginosa sequencing,we found that there is another protein also be marked as ZapA by some sequencing data.PaZapA is encoded by the gene pa5227,and this new protein we are now exploring is encoded by the gene pa5407.Through sequence comparison,we found that the sequences of these two proteins are quite different.In this study,we called this new protein as ZapL(ZapA-like).The interaction between PaZapA and PaZapL and PaFtsZ is compared to study the function and biochemical properties of PaZapL.Since the genes pa5405-pa5407 are located in a gene cluster,the study of these genes can also enable us to further understand the function of PaZapL.There is also a small molecule protein in Escherichia coli that has been labeled as EcZapA by some sequencing results.But it is also very different from EcZapA protein.By searching for this protein sequence,we found that this protein also exists in ?-phage,Shigella and some other phages.Because the sequence contained in the phage is very limited,the biochemical properties and function of this protein are worth exploring.it's the sequence similarity between EcZapL and PaZapL is not high.However,our research showed that they may have similar characteristics.So we also called this new protein in E.coli as EcZapL(EcZapA-like).From the negative staining results of electron microscopy,it can be seen that PaZapL can induce PaFtsZ to form double parallel protofilament structures.In the presence of EcZapL,its conformation changes,and some closed monomeric fibril structures are also formed.By detecting the effects of PaZapA,PaZapL,and EcZapL on the GTPase activity of PaFtsZ and EcFtsZ,respectively,it was found that when the concentration of PaZapA,PaZapL,and EcZapL in the system was continuously increased,the hydrolysis rate of the corresponding FtsZ GTPase decreased accordingly However,all three have only slight effects on the GTPase activity of FtsZ.Through the detection of light scattering signal,After mixing PaZapA and PaFtsZ,it is detected that it can produce a strong light scattering signal.There is a short delay and it is almost impossible to observe.The light scattering signal drops rapidly after reaching the maximum value in about 50 s.Detecting the light scattering signal of the PaZapL and PaFtsZ mixture found that the light scattering signal rose rapidly before about 200 s,and then remained in a flat state,and there was no downward trend.When detecting the light scattering signal of the mixture of EcZapL and EcFtsZ,it can be seen that after a short delay,the light scattering signal shows a continuous and stable upward trend,and there is no rapid rise stage,and no depolymerization kinetics.The original filament fiber The structure is relatively stable.The mixtures of PaZapA,PaZapL,EcZapL and the corresponding FtsZ respectively showed strong light scattering signals,but they showed different dynamic characteristics.We used a fluorescent inverted microscope to observe the localization of PaZapL and EcZapL in Pseudomonas aeruginosa and E.coli,respectively.The results showed that both GFP-PaZapL and GFP-EcZapL appeared in the middle of the cell.We suggest that ZapL is a component of the division machinery.However,ZapL is not a necessary protein for the cell divison.Knockout the ZapL gene has little effect on the cell division.It can be seen from the negative staining results of the electron microscope after Pa5405,Pa5406,PaZapL and PaFtsZ that PaFtsZ formed a large bundle-like structure of the original filament.It is worth noting that when Pa5405,PaZapL,and PaFtsZ are mixed,there are some closed miniring and highly curved conformations,and the two parallel fibril structures formed by PaZapL and PaFtsZ are not observed.This research has investigated the functional and biochemical properties of a new bacterial contractile ring binding protein ZapL.Based on the results,we preliminarily speculate that PaZapL and EcZapL may be positive regulators of the cell division mechanism.More accurate experimental results are still needed.The experiment is in progress.