Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens 英文参考文献.docVIP

FunctionalAnalysisoftheVirSRPhosphorelayfrom Clostridiumperfringens JackieK.Cheung.,MilenaM.Awad.,SheenaMcGowan¤,JulianI.Rood* DepartmentofMicrobiology,MonashUniversity,Clayton,Victoria,Australia Abstract Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidationofthegenescontrolledbythisnetwork;thereislittleinformationregardingthephosphorelaycascadethatis the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosageandphosphorylationinvivo.Wehaveusedrandomandsite-directedmutagenesistoidentifyresiduesinVirSthat are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essentialforfunction.InvitrophosphorylationstudiesshowedthatVirSc,atruncatedVirSproteinthatlackedtheN-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate responseregulator,VirR.ConservedresiduesofbothVirSandVirR,includingtheD57residueofVirR,wereshowntobe essentialforthisprocess.ByuseofTargetrontechnology,wewereabletointroduceasinglecopyofvirRorvirRD57N onto thechromosomeofavirRmutantofC.perfringens.Theresultsshowedthatinvivo,whenvirRwaspresentinsinglecopy,the productionofwild-typelevelsofperfringolysinOwasdependentonthepresenceofvirSandanunalteredD57residuein VirR.TheseresultsprovidegoodevidencethatphosphorylationiscriticalforVirRfunction. Citation:CheungJK,AwadMM,McGowanS,RoodJI(2009)FunctionalAnalysisoftheVirSRPhosphorelayfromClostridiumperfringens.PLoSONE4(6):e5849. doi:10.1371/journal.pone.0005849 Editor:HolgerBruggemann,MaxPlanckInstituteforInfectionBiology,Germany ReceivedApril7,2009;AcceptedMay11,2009;Publish