Polymerization Of MinC~C And MinD Regulatory Proteins In Pseudomonas Aeruginosa

In the process of bacterial division,the Z-ring is located in the middle of the bacteria to make it divide equally.There are two negative regulatory systems in the regulation of Z-ring in gram-negative bacteria:Min system and nucleoid exclusion.The Min system includes the inhibitory protein MinC of FtsZ,the membrane linked ATPase MinD,and a topological factor Min E.The N-terminal domain(MinC~N)and C-terminal domain(MinC~C)of MinC have different functions.The N-terminal"releases"FtsZ,which is the main inhibitor of FtsZ assembly;the C-terminal combines with MinD and the conserved C-terminal domain of FtsZ to"capture"FtsZ.MinC is a weak inhibitory protein of FtsZ,and the concentration of MinC in vivo is low.So how does MinC inhibit FtsZ?Our previous studies suggested that the copolymerization of MinC and MinD played an important role.Therefore,we propose a"capture-release"model to explain the regulatory effect of MinC-MinD on the localization of contractile rings.MinC-MinD captures FtsZ fibrils to prevent FtsZ from spreading to both ends of cells,and releases depolymerized FtsZ subunits through the inhibition of MinC~N.In order to further understand the polymerization characteristics of MinC-MinD copolymer,the C-terminal of MinC(MinC~C)of Pseudomonas aeruginosa was used as the main research object in this experiment.The polymerization characteristics of MinC~C and MinD,and the binding characteristics of MinC~C and FtsZ were studied by electron microscope negative staining technology and light scattering technology;and through the observation of bacterial morphology,determination of growth curve and fluorescence localization and other in vivo experiments to explore the ability of MinC~C to"capture and release"FtsZ.The results showed that the electron microscopic negative staining of MinC~C and MinD produced fascicular structure in vitro.After adding FtsZ,the bundle structure of MinC~C-MinD became thicker,which was consistent with the results of MinC of Pseudomonas aeruginosa,indicating that MinC~C could combine with FtsZ.In order to enhance its binding ability with FtsZ,we constructed Zap A-MinC~C.In vivo experiments,knockout of min C or min D resulted in the failure of Z-ring localization,and the mycelium was filamentous and produced microbacteria.The mycelium was still filamentous after the supplement of min C~C and zap A-min C~C to?min C,the length of mycelium was 6.63±3.64?m and 6.84±3.76?m respectively,which was longer than that of wild type(2.31±0.56?m)and shorter than that of?min C(12.22±6.68?m),the length of the bacteria of the recharge of min C~C and zap A-min C~C was not obvious,and the recovery of min C was basically normal.Knockout min C inhibited the division of bacteria,and the growth was the slowest.The growth rate of min C~C and zap A-min C~C was faster than that of the?min C and slower than that of the wild type,but the growth rates of the two strains were almost the same.The localization of Z-ring was observed by fluorescence.It was found that Z-ring might still be located at the two poles of min C~C.In vitro,MinC~C can combine with MinD to form MinC~C-MinD copolymer,which can combine with FtsZ.This may be due to the interaction between CCTPs of FtsZ precursor fiber and MinC~C in MinC~C-MinD copolymer.In vivo experiments,although the function of compensation min C~C is improved,it does not make?min C return to normal,which indicates that MinC~C may have some functions,the ability of"capturing"FtsZ may be limited,and it may also be important to"release"FtsZ in the process of Z-ring positioning.Because MinC~C-MinD can form copolymers,we explored the application of the complex.Multienzyme complexes are crucial in cell biosynthesis.It is found that the shorter distance between enzymes can accelerate the chemical reaction rate.We take the cascade enzymes of menadione synthesis,Men D?Men H and Men F,as the research object,and fuse various cascade enzymes with MinC~C to form Men D-MinC~C,Men H-MinC~C and Men F-MinC~C.By reacting with MinD,we explore whether Men D-MinC~Ccan react with MinD to form multi enzyme complexes and accelerate the enzymatic reaction rate.The results of electron microscopy showed that Men D-MinC~C,Men H-MinC~C and Men F-MinC~C could form fascicular structure with mind respectively,which indicated that they might form multienzyme complexes.More detailed experimental results are in progress.