Establishing A Hplc Method For Detection Of Bacterial Putrescine And Functional Analyses Of Polyamine Metabolism-Related Genes In Pectoacterium Carotovorum

Pectobacterium carotovorum subsp.carotovorum(Pcc)has a wide range of hosts causing severe soft rot in many crops and ornamental plants,and cannot be effectively controlled,resulting in severe economic losses.In recent years,for the vast developments of molecular biology,it has been maken a rapid progress in undersdanting the mechanisms of pathogenicity of Pcc,which would make many improvements in the methods and strategies for this diseae controlling.Polyamines widely exist in plants,animals and microganisms.They are natural aliphatic amines based on the carbon skeleton,which are necessary for cell normally growth.At physiological pH,positively charged polyamines mainly interact with ATP,enzymes,nucleic acids and other biological macromolecules thus participate in a variety of physiological processes,such as transcription and translation of genes,DNA stability,signal transduction,cell growth and reproduction and membrane.Spermidine/putrescine ABC transporter system is the specific polyamines transporter in microorganisms,which include potABCD system and potFGHI system.High-performance liquid chromatography(HPLC)is widely in substance separation,analysis and purification and other research fields due to its superiorities,such as high efficiency,high sensitivity,wide range of applications,rapid analysis,etc.The utilization of HPLC for analysis polyamines has been popularized in animals,plants,the environment,food and so on,but it was fewer in bacteria.In this work,the method of HPLC for detecting polyamines in Pcc was established after sets of tests inh:spreparation of samples,selection and adjustment of the ratio between mobile and stationary phase,choice of detection wavelength,adjustment of column temperature and the flow rate,etc.The final results showed that the combine of Agilent C18 column(150 mm × 4.6 mm,5 ?m),column temperature at 30 ?,benzoyl chloride as the derivative reagent at the volume of 100 ?L,methanol and water as the mobile phase by gradient elution and the wavelength at 254 nm was the best.In order to elucidate the functions of the genes related to polyamine metabolism.In this work,three gene deletion mutants(?potA,?potD and AspeE)were constructed by homologous recombination.The results of bioassay of hpathogenicity,activities of the extracellur hydrolases,growth ability and production of carbapenem exhibited no significant difference in these mutants comparing with the wild type.So,these genes correlated with polyamines are not important to pathogencity of Pcc.ApotG,one of the single gene deletion mutants of the genes encoding subunits of potFGHI system in Pcc showed significant attenuant virulence in our previous work.In present work,the quantities of intracellular putresine in the strains of wild type and in the strains derived from PccS1 were analyzed by the newly established HPLC method.The results showed that intracellular levels of putrescine in the mutants(?potF,?potG,?potH and ?potG)did not differ significantly compared to that in the wild type.It suggests that the putrescine translocation system of potFGHI is not indispensable in Pcc putrescine metabolism.And there might be some iso-translocating systems of putrescine in PccS1 remainding undiscovered.