Lack Of Phosphatidylcholine Affects PseE/PseF Protein Coupling With Bacterial Cell Membranes In Type ? Efflux System

Phosphatidyl choline(PC)as one of membrane phospholipids,with other phospholipids and membrane protein composition constitute the bacterial biofilm system.Previous studies in this laboratory have found that the PC play an important role in pathogenic bacteria.Such as pseudomonas syringae pv.syringae van Hall CFCC 1336pcs-mutant lack of PC almost lost the ability to cause disease,which is related to the reduced amount of syringomycin and syringopeptin.While the secretion of syringomycin and syringopeptin mainly go through TypeI Secretion System(T1SS)to extracellular.T1SS consists of three parts:the outer membrane protein(OMP),cytoplasmic membrane fusion protein(MFP),ABC transporter.In order to further investigate whether the PC influence the protein expression of TypeI Secretion System(TISS)in the cytoplasm or machine components on the membrane assembly,resulting in abnormal work of T1SS machine,which then affects the secretion of syringopeptin.The main research works are as following:1.Cloning,exogenous expression and purification of pseudomonas syringae pv.syringae van Hall CFCC 1336 PseE/PseF efflux system component proteins.According to the psyr2617(pseE),psyr2618(pseF)gene sequence of Pseudomonas syringae pv.Syringae B728a,design specific primers to amplify pseE and pseF genes from pseudomonas syringae pv.syringae van Hall CFCC 1336 genome.Analyze the sequence of pseE,pseF and the homology between pseudomonas syringae 1336 and pseudomonas syringae B728a reached 95%,96%respectively.pseE and pseF were respectively inserted into the expression vector pET23a,pET28a,then putting the reorganization carrier into E.coli BL21(DE3)plysS,inducing it to express protein.PseE and PseF purified proteins are purified by the column of Co2+ affinity chromatography.2.Preparation of anti-PseE,anti-PseF protein polyclonal antibody.purified proteins are injected into rabbit,Collecting serum to get polyclonal antibody.3.Research on whether PC affect the PseE,PseF protein expression in the cytoplasm.To extract 1336 wild type,1336pcs-mutant total proteins of cytoplasm respectively,the prepared polyclonal antibody is used for Western blot reaction.The results show that:lack of PC will not affect the translation level of pseE,pseF.4.Research on whether PC affect the PseE,PseF protein on the membrane assembly.To extract 1336 wild type and 1336pcs-mutant total membrane proteins respectively,the prepared polyclonal antibody is used for Western blot reaction.The results show that:lacking of PC has a gerat influence on the PseE,PseF proteins on the membrane assembly,the bacteria lack of PC,PseE,PseF protein cannot be effectively assembled in bacterial cell membranes.5.Research on whether the PC affect transcription level of pseE and pseF gene.Specific primers for Reverse transcription PCR(RT-PCR)were respectively designed:pseE-rt-F,pseE-rt-R,pseF-rt-F,pseF-rt-R.To extract total RNA of 1336 wild type,1336pcs-mutation respectively,cDNA is obtained by reverse transcription reaction.cDNA is as the template,with the design of rt-pcr primers for RT-PCR.The results show that:lack of PC will not affect the normal transcription level of pseE,pseF gene.6.Research on whether PC affect synthesis of syringopeptin related gene transcription.Using the RT-PCR technology to analyze the transcription level of sypA?sypB?sypC?salA?pseA?pseB?pseC?syrBl?syrC?syrE?syrF?syrG between 1336 wild type and 1336pcs-mutation,the results show that:lack of PC will not affect the synthesis of syringopeptin related gene transcription.Conclusion:The lack of phosphatidyl choline in the bacterial membrane phospholipids neither influence synthesis of syringopeptin related gene transcription nor affect the pseE,pseF gene transcription and translation level,but affect PseE,PseF protein effectively assembled in bacterial cell membranes.pseudomonas syringae pv.syrzngae van Hall CFCC 1336pcs-mutant,which lack of PC can't effectively secrete syringopeptin is due to the PseE and PseF proteins in PseE/PseF efflux system can not normally assemble in the bacterial membrane.