Isolation,Properties And Structural Characterization Of VFM,a New Quorum Sensing Signal Of Dickeya Dadantii

Dickeya dadantii,formerly known as Erwinia chrysanthemi 3937,is a gram-negative bacillus that belongs to the family of Enterobacteriaceae,which is a facultative anaerobes.In the natural plant environment,D.dadantii causes plant maladies such as necrosis,blight and “soft rot,” which is a progressive tissue maceration.The host range of D.dadantii continues to expand,and the bacillus commonly infectsdicotyledonous plants including potato,vegetables,and ornamental crops.It was reported that virulence factors produced by D.dadantii include cell wall-degrading enzymes(CWDE),including a set of pectinases,four proteases and a cellulase),lipopolysaccharides,exopolysaccharides,biofilm,sideropHores,mobility and hrp type III secretion system,etc.William Nasser and his colleagues(2013)found that there is a latent period of Dickeya dadantii,and a sharp emergence of plant diseases could occur when the bacterial concentration reaches a certain threshold.Through transposon mutagenesis,they found two mutants with much reduced production of CWDE,which could be restored when the cell-free supernatant from the D.dadantii wild-type strain was added.Characterization of these two mutants led to identification of a 25 kb gene cluster required for D.dadantii CWDE production and pathogenesis,which was named as VFM(Virulence Factor Modulating).however,the structure and pHysical and chemical properties of VFM signals have not yet been deter mined.On the request from the laboratory of the Dr.William Nasser,we agreed to collaborate on purification and characterization of the VFM signal.This research use strain A5243 as the VFM signal reporter,which was obtained by placing the uidA-Km gene cassette under the control of the vfmE promoter.The polarity of VFM signal molecules was deter mined by extraction with ethyl acetate and n-butanol.The crude extract of VFM signal was obtained by ethanol extraction,MCI and Lh-20 Gel separation.The VFM signal was further separated and purified by normal pHase silica gel.The results from this study laid a solid foundation for further purification of VFM signal by hPLC,and its chemical structure characterization by MS and NMR.Meanwhile,this study also revealed some pHysical and chemical properties of VFM signal,such as the stability in acid,alkaline,and in high temperature environments.The VFM signal was found to be a highly polar substance as it can be extracted by n-butanol but not ethyl acetate.In addition,the results of treatments on VFM signal with different reagents showed that the VFM signal was stable in saturated EDTA solution and methanol,but could not coexist with acetone,and could not be degraded by protease K.Deter mination of the structure and properties of VFM signal from D.dadantti may not only facilitate characterization of similar quorum sensing signals produced by other species of Dickeya genus.,but could also lead to unveil of new regulatory mechanisms on bacterial pathogenesis,and additionally,may provide useful clues on bacterial evolution.