Study On The Characteristics Of The Novel Small Molecule Regulatory Protein ZapAL(PA5407) And Related Proteins In Pseudomonas Aeruginosa

Bacterial cell division is initiated by the assembly of the contraction ring(Z-ring),which consists of the self-assembled FtsZ protofilaments and dozens of other associate proteins.The FtsZ protein,an eukaryotic tubulin homologue,is the first protein known to be located at the site of division,and one of the most studied proteins involved in cell division.The division of bacteria is strictly regulated in time and space by various regulatory proteins,including positive regulator proteins and negative regulatory proteins.For these negative regulatory proteins Min C,Slm A and Mip Z,which have been studied in vitro,they play a regulatory role by influencing the stability of the FtsZ polymer and thus the formation of Z rings.Positive regulatory proteins,such as Zip A,ZapA,ZapC,etc.,stabilize the Z ring by facilitating the transverse assembly of FtsZ filament fibers and forming bundle-like or flake structures.ZapA,a regulatory protein found in almost all bacteria,stabilizes FtsZ protofilaments to form bundles and enhances the Z-ring condensation.In Pseudomonas aeruginosa,ZapA promotes the polymerization of FtsZ into bundles by forming dimers that have a stoichiometric ratio of approximately 1∶1 to the FtsZ molecule.Here,we reported that another small protein from P.aeruginosa,ZapA-Like protein(ZapAL;PA5407),is a new FtsZ associated protein.ZapAL exists in many Pseudomonas species and shares only 20%sequence identity to ZapA.ZapAL interacts with FtsZ and induces FtsZ to form long straight double filaments;in comparison,ZapA promotes long bundles with multiple FtsZ filaments,arranged in parallel by multiple filament fibers.ZapAL has only a mild effect on GTPase activity of FtsZ,which is reduced by around 26% when 10 μM ZapAL is added in solution.However,to study their assembly dynamics using light-scattering assay,we found that FtsZZapAL double filament is stable and no depolymerization process was observed,which is different from ZapA.Further research found that ZapA and ZapAL are likely to form heterodimers.The bundling structure formed by the assembly of the mixture of FtsZ-ZapAZapAL will depolymerize after GTP is hydrolyzed.Consistent with ZapAL interaction with FtsZ in vitro,the expression of ZapAL-gfp was observed as a narrow band or spots in the middle of the cells,suggesting that it is a component of bacterial division machinery.ZapAL has a similar characteristic to ZapA,and neither is also not essential for bacterial cell division,and when one gene was deleted,it does not affect the division of bacterial cells.Little changes were observed when zap AL gene was deleted,or overexpressed under normal conditions;however,overexpression of ZapAL caused zap A-deficient cells to grow approximately two times longer,showing a mild bacterial division defect.Meanwhile,overexpression of ZapA in PAO1 strain and zap AL deletion has no obvious effect on bacterial division and growth under normal conditions.By retrieving genes in the database,it was found that zap AL(PA5407)shares a promoter with PA5402-5406 to form a gene cluster,so we also studied these genes.Through a series of experiments,it was found that the gene of the PA5402-5407 gene cluster are not essential for bacterial growth,because the growth and division of bacterial cells are not affected when the entire gene cluster is knocked out.Microscopic observations of the gene PA5406 near zap AL on the gene cluster found that it was also part of the Z ring of the shrinkage ring and participated in the co-localization of the Z ring.Electron microscopic negative staining observations revealed that both PA5405 and PA5406 may interact with FtsZ and regulate the shape of FtsZ-ZapAL polymerization and how much it forms.Although we still do not know the exact physiological roles of ZapAL,our results suggest that ZapAL is a novel Z-ring associate protein,which may work together with ZapA to stabilize the FtsZ protofilament and Z-ring structure.Therefore,we speculate that these genes may only have a regulatory effect under certain specific conditions(such as special environments)and thus effectively act on the Z ring and affect cell division.But what the specific conditions are we are still studying.The fine regulation process of FtsZ filament within the Z ring can be more clearly understood by studying other small molecule proteins on gene clusters.But more specific immediate physiological functions such as how they act on FtsZ to regulate cell division in bacteria,and whether other proteins on gene clusters are also localized in the middle of the cell and stabilize the structure of the Z ring,repeated experiments and further experiments are needed to further demonstrate their function.