Biochemical Characteristics Of Bacterial Tubulin Homlogue FtsZ And CetZ Of Halophilic Archaea And The Effects Of ZapL,A Novel Division Regulatory Protein Of Pseudomonas Aeruginosa

Bacterial contraction ring is an important structure for bacteria to complete cell division.It is composed of more than ten kinds of proteins,which are highly dynamic.The most important protein is bacterial skeleton protein-FtsZ.FtsZ exists in almost all bacteria and archaea.In vitro,FtsZ can self-polymerize to form highly dynamic fibrils,and in vivo,it can assemble into highly dynamic bacterial contraction rings.In the later stage of bacterial division,the contraction ring will continue to contract,thus completing the cell division of bacteria.Since bacterial division is an essential process of bacterial growth,it is of great scientific significance to elucidate the mechanism of bacterial division.Compared with eubacteria,the FtsZ protein family of archaea is very special.It contains two different proteins: FtsZ and CetZ,and has multiple copies,which is completely different from bacteria,in general bacteria only have a single copy of FtsZ protein.We studied the in vitro biochemical characteristics of FtsZ1,FtsZ2,CetZ1,CetZ2 in Haloferax volcanii superfamily and observed their interactions.In this study,we used light scattering signal changes to study their kinetic properties,and compared the morphological changes of protein-polymerized fibrils and the changes of GTP hydrolysis activity by electron microscopy.The results showed that both FtsZ1 and FtsZ2 in the FtsZ family could be polymerized to form fibrils in the presence of GTP,except that FtsZ1 formed a single structure and FtsZ2 could form a large bundle structure.When FtsZ1 was added at different ratios in FtsZ2,the light scattering signal of the copolymer polymerization decreased with the increase of FtsZ1,indicating that FtsZ1 and FtsZ2 could be copolymerized;Ts Z2 can also aggregate to form singlestructured fibrils in the presence of GDP.The GTP hydrolysis rate of FtsZ1 is larger than that of FtsZ2,and the GTP hydrolysis rate of FtsZ1 and FtsZ2 copolymers is the sum of the two.The results further indicate that FtsZ1 and FtsZ2 can be copolymerized.CetZ1 and CetZ2 in CetZ family can be polymerized to form protofilament fibers in the presence of GTP,except that CetZ2 forms a single structure and CetZ1 can form a large bundle structure.When CetZ1 is added at different ratios in CetZ2,the kinetics of copolymer polymerization decreases with the increase of CetZ1,indicating that CetZ1 and CetZ2 can be copolymerized;the GTP hydrolysis rate of CetZ1 is higher than that of CetZ2 is much larger,and the GTP hydrolysis rate of CetZ1 and CetZ2 copolymers is also the sum of the two.The results further show that CetZ1 and CetZ2 can be copolymerized.We determined the GTPase activity of the mixture of FtsZ and CetZ,and the results showed that FtsZ and CetZ may be mutually inhibited.The bacterial contraction rings were co-localized in different forms of archaea,and the in vivo experiments were also needed to verify the clearer results.In the second part of the paper,we studied ZapL,a possible regulatory protein of the contraction loop of Pseudomonas aeruginosa.It is a good model organism for further study of cell division mechanism.We studied the biochemical characteristics and functions of ZapL by comparing with ZapA.The results showed that ZapL could enhance the polymerization kinetics of FtsZ,but did not exhibit the disaggregation kinetics compared with ZapA.After labeling with GFP,the localization of ZapL-GFP in cells was rare,only tended to be after cell division was completed.Then we overexpress the zap L,the bacterium morphology became significantly longer in mutant ?zap L?zap A.Our results preliminarily speculate that ZapL may be a negative regulator of cell division mechanism,and more precise experimental results are still in progress.