DNA Phosphorothioate Modification Plays a Role in Peroxides ...

DNA phosphorothioate modification, in which a non-bridging oxygen in DNA phosphate backbone is replaced by sulfur, exists widely in bacteria ... Articles IvanMijakovic ChalmersUniversityofTechnology,Sweden VladimirBidnenko INRAECentreJouy-en-Josas,France Jing-ShengCheng TianjinUniversity,China Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction MaterialsandMethods Results Discussion AuthorContributions ConflictofInterestStatement Acknowledgments SupplementaryMaterial Abbreviations References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData ORIGINALRESEARCHarticle Front.Microbiol.,31August2016 |https://doi.org/10.3389/fmicb.2016.01380 DNAPhosphorothioateModificationPlaysaRoleinPeroxidesResistanceinStreptomyceslividans DaofengDai,AiqinDu,KangliXiong,TianningPu,XiufenZhou,ZixinDeng,JingdanLiang*†,XinyiHe*†andZhijunWang*† StateKeyLaboratoryofMicrobialMetabolismandSchoolofLifeScienceandBiotechnology,ShanghaiJiaoTongUniversity,Shanghai,China DNAphosphorothioation,conferredbydndgenes,wasoriginallydiscoveredinthesoil-dwellingbacteriumStreptomyceslividans,andthereafterfoundtoexistinvariousbacterialgenera.However,thephysiologicalsignificanceofthissulfurmodificationoftheDNAbackboneremainsunknowninS.lividans.OurstudiesindicatethatDNAphosphorothioationhasamajorroleinresistancetooxidativestressinthestrain.AlthoughStreptomycesspeciesexpressmultiplecatalase/peroxidaseandorganichydroperoxideresistancegenestoprotectthemagainstperoxidedamage,awildtypestrainofS.lividansexhibitedtwo-foldto10-foldhighersurvival,comparedtoadnd−mutant,followingtreatmentwithperoxides.RNA-seqexperimentsrevealedthat,catalaseandorganichydroperoxideresistancegeneexpressionwerenotup-regulatedinthewildtypestrain,suggestingthattheresistancetooxidativestresswasnotduetotheup-regulationofthesegenesbyDNAphosphorothioation.QuantitativeRT-PCRanalysiswasconductedtotracetheexpressionofthecatalaseandtheorganichydroperoxideresistancegenesafterperoxidestreatments.Abunchofthesegeneswereactivatedinthednd−mutantratherthanthewildtypestraininresponsetoperoxides.Moreover,theorganichydroperoxideperaceticacidwasscavengedmorerapidlyinthepresencethanintheabsenceofphosphorothioatemodification,bothinvivoandinvitro.Thedndgeneclustercanbeup-regulatedbythedisulfidestressordiamide.Overall,ourobservationssuggestthatDNAphosphorothioatemodificationfunctionsasaperoxideresistancesysteminS.lividans. Introduction DNAphosphorothioatemodification,inwhichanon-bridgingoxygeninDNAphosphatebackboneisreplacedbysulfur,existswidelyinbacteria.Originally,themodificationwasfoundinthesoil-inhabiting,antibiotic-producingStreptomycesspecies.ThroughtheDND(DNAdegradation)phenotypeandinsilicoanalysis,themodificationwasalsofoundtodistributeinhumanpathogens,includingMycobacteriumabscessus,anemergingrespiratorypathogenofcysticfibrosispatients,Clostridiumdifficile,themainetiologicagentofnosocomialdiarrhea,andSalmonellaenterica,acauseofintestinalinflammation(Heetal.,2007;Wangetal.,2007,2011;Ouetal.,2009). Phosphorothioatemodificationisstereo-specific,withonlytheRconfigurationfoundinbacteria(Wangetal.,2007),anditisalsosequence-selective(Boybeketal.,1998;DysonandEvans,1998;Zhouetal.,2005;Liangetal.,2007;Wangetal.,2011;Caoetal.,2014a).Quantitativeanalysisshowsthatthreediscretegenomicmodificationfrequencies,rangingfromtwotothreemodificationsper103nt,threetoeightper104nt,andtwotosixhundredsper106nt,alongwithawell-conservedtargetsequenceofthreetofivenucleotidesinlength,existindifferentbacterialspecies(Wangetal.,2011).Althoughthemodificationnucleotidesequenceiswellconserved,onlyafractionofthepotentialsitesaremodified.InaplasmidfromS.lividans1326containing14potentialmodificationsites,DNAphosphorothioationoccurredselectivelyonceperplasmid(Liangetal.,2007).ThepartialmodificationwasfurtherconfirmedinplasmidandgenomicDNAfromEscherichiacoliB7A,andestimatedtooccupy12–14%ofallpotentialsites(Caoetal.,2014a). DNAphosphorothioationisdeterminedbytheproductsofafive-memberedgeneclustertermeddnd(Zhouetal.,2005).Fourofthegenes,dndAanddndCDEareessentialforthemodification.TheproductofdndBwasshowntosuppresstheexpressionofdndgenecluster(Liangetal.,2007;Chengetal.,2015;Heetal.,2015). StudieswithchemicallyphosphorothioatedDNArevealedthatthemodificationconfersresistancetonucleasedigestion(Eckstein,2000),suggestingthatthemodificationmightbepartofahostrestrictionsystem(Wangetal.,2007).Indeed,adecreaseintransformationratewasobserved,comparingbetweenaphosphorothioate-modifiedplasmidanditsnon-modifiedcounterpartinSalmonellaenterica(Xuetal.,2010).ADNAdamagingphenotypewasalsoseenwhenthemodificationgeneclusterwasknockedout,andseveralgenesdownstreamofthedndgeneswereinvolvedintheprocess(Caoetal.,2014a,b;Ganetal.,2014).Interestingly,phosphorothioatedDNAfromS.lividanswasspecificallyrecognizedbyarestrictionendonucleaseinitssisterspeciesS.coelicolor.NMRstructuralstudiesdemonstratethatthesulfurin[Rp,Rp]-phosphorothioatedsDNAlocatesinthemajorgroovefacilitatingselectiveinteractionswiththerestrictionendonuclease(Liuetal.,2015;Lanetal.,2016).Aswithmanyotherspecies,thedndgenesofS.lividansarelocatedonagenomicisland,whichisabsentfromS.coelicolor(Liuetal.,2010;Howardetal.,2013). WeshowedpreviouslythatphosphorothioatedDNAhasreducingproperties,reactingwithperoxidesstoichiometrically(Xieetal.,2012).GrowthcurveanalyseswithSalmonellaentericaindicatedthatDNAphosphorothioationprovidedasurvivaladvantageduringexposuretooxidativestress.Usingaveryhighconcentrationofhydrogenperoxide(880mM)totreatthecells,phosphorothioatemodificationinDNAwasalsofoundtobeconsumed.Itwasthereforesuggestedthatthephosphorothioatemodificationfunctionasananti-oxidantinthebacteria(Xieetal.,2012).Salmonellaspp.isafacultativeintracellularbacteriumthatcausesawiderangeofinfectionsinmammals.TheabilityofthepathogentosurviveandproliferatewithinhostcellsreliesonitsarsenalofdetoxifyingenzymestocopewithPhox-mediatedoxidativestress(Ausseletal.,2011).Additional,anti-oxidationcapabilitysuchasDNAphosphorothioationmightnotberedundant.However,Streptomycesspp.isknowntoharbormultiplecatalaseandorganichydroperoxideresistancegenes(Cruz-Moralesetal.,2013),itisintriguingwhetherthemodificationstillfunctionasananti-oxidationsysteminthespecies. Thechemicalresultsofphosphorothioatereactionwithperoxidesaredifferent.Afteroxidizedbyhydrogenperoxide,themajorityofphosphorothioatebondswereconvertedtonormalDNAbackbone,whichshouldnotbeharmfultothebacteriaifthishappensinvivo.However,thephosphorothioatebondcanbeefficientlycleavedbyperaceticacid(PAA),resultingdegradedDNAfragments(Rayetal.,1995;Liangetal.,2007;Xieetal.,2012).Therefore,itisimportanttounderstandwhetherthephosphorothioateDNAmodificationalleviatesoraugmentsoxidativestressexertedparticularlybyPAAonthehost. Inaddition,themodificationfrequencyoftwotosevenhundredsper106nt,whichequalstoaround10μMpercell,appearstoolowforstoichiometriccounteractionagainstperoxidesatmillimolarconcentration.ThisraisesapossibilitythattheremaybebroaderinteractionsbetweenphosphorothioateDNAandthephysiologicalresponsetooxidativestress.Forexample,phosphorothioationmayfunctionasaregulatoryswitchthatcontrolstheexpressionofgenesinvolvedinresistancetooxidation.Indeed,effectsofphosphorothioationonDNAincludedestabilizingtheB-typehelix,andenhancingthestiffnessofthebackbonethroughinteractionswiththebase-pairsteps(Zhangetal.,2012;Chenetal.,2015),whichcouldpotentiallyaltertheinteractionoftranscriptionalfactorswiththemodifiedDNA,suchasthoseobservedinDNAmethylation(MarinusandCasadesus,2009). AlthoughmanyenzymesandsmallmoleculescanscavengeH2O2invitro,theymaynotserveasthesamefunctioninvivo.Itwasthereforesuggestedthatseveralaspectsshouldbeexaminedbeforecomingtotheconclusionoftheiranti-oxidationfunctions.Theseaspectsincludeacharacteristicsensitivitytoperoxideiftherelatedgeneisabsent,catalyticefficiencyofthesmallmoleculesorenzymes,andtheinductionofthegenebyanoxidant(MishraandImlay,2012). Wesoughttoaddressthesequestionsinthisstudy,andshowthatDNAphosphorothioatemodificationisananti-oxidationsysteminS.lividansthatpreferentiallyscavengesorganichydroperoxides,andthatthedndgeneclusterissubjectedtodiamideregulation. MaterialsandMethods Strains,Plasmids,andMedia BacterialstrainsandplasmidsusedinthisworkarelistedinTableS1.StreptomycesstrainswereculturedinrichliquidmediaTSBY(30g/Ltrypticsoybroth,5g/Lyeast,340g/Lsucrose)orminimalM9media(6.8g/LNa2HPO4,3.0g/LKH2PO4,0.5g/LNaCl,1.0g/LNH4Cl)supplementedwith5g/Lw/vglucoseand0.12g/LMgSO4.Antibioticswereaddedtothemediumasneeded(50μg/mlforapramycin). TogeneratethereportervectorPdndB-xylE,whichcontainsxylEunderthecontrolofthedndBpromoter,aDNAfragmentthatincludedthexylEcodingregion,ribosomalbindingsite(RBS),andupstreamregioncontainingstopcodonsinallthreereadingframeswasamplifiedfrompIJ4083(Tuncaetal.,2007)byPCRwithprimers3700Fand3700R(TableS2).ThePCRproductwasdigestedusingXbaI,andligatedintotheXbaI-digestedpSET152,generatingthexylEreportervector,pJTU3700.Next,a760-bpfragmentcontainingtheintergenicregionbetweendndAanddndB(frompositions−387to373withrespecttothedndBtranscriptionalstartsite,determinedinthisstudy)wasamplifiedfromchromosomalDNAofS.lividans1326byPCRwithprimerPdndBFandPdndBR(TableS2).ThePCRproductwasdigestedusingBamHI,andligatedwithBamHI-digestedplasmidpJTU3700,producingthePdndB-xylEreporterplasmidpJTU3707.ThereporterpJTU3707wasthenintroducedintoS.lividans1326viaconjugationusingE.coliET12567(pUZ8002).Thecatechol-2,3-dioxygenaseactivityassaywasperformedasdescribedwithoutmodification(Ingrametal.,1989). TogenerateplasmidpJTU3707-10,the-10regionofthedndBpromoterwasremovedfrompJTU3707usingPCR.Briefly,afterPCRamplificationusingpJTU3707asthetemplate,1μlofDpnIwasaddedtothePCRreactionmixtureandincubatedat37°Cfor6htoremovethetemplateplasmid.ThePCRproductwastransformedintoE.coliDH5α,resultinginplasmidpJTU3707-10. DisruptionofsigRwasperformedbygenereplacementviahomologousrecombination.Forthispurpose,twoDNAfragmentscontainingpartialsigRsequenceanditsflankingregionwereamplifiedfromS.lividans1326genomicDNAbyPCRusingKODplusDNApolymerase(TOYOBO,Japan).A1582bpPCRproduct(primer5505LFand5505LR),anda1552bpPCR(primer5505RFand5505RR)weredigestedwithXbaIandBglII,andEcoRIandBglIIrespectively.TheplasmidpBluescriptIIwasdigestedusingXbaIandEcoRI.ThenthePCRDNAfragmentswereinsertedintopBluescriptII,generatingpJTU3730.Thecassettecontainingapramycin-resistancegeneandOriTwasobtainedfrompIJ773byPCRamplificationusingprimer773Fand773R(TableS2).TheBglII-digestedcassettefrompIJ773wasinsertedintoBglII-digestedpJTU3730,generatingpJTU3731,whichpJTU3731wasthenintroducedintoS.lividans1326viaconjugationmediatedbyE.coliET12567(pUZ8002)(Kieseretal.,2000).TheresultingexconjugantswereculturedonMSplates.SporeswereharvestedandspreadagainonMSplatescontainingapramycin.Aftercultureat30°Cfor3days,apramycinresistance(aprr)colonieswereselectedandscreenedforsigRdisruptionmutantsbyPCR(usingprimer5505TFand5505TR). SurvivalAssay Totestsurvivalfollowingexposuretooxidativeagents,strainsofS.lividans1326andHXY6containingtheplasmidpIJ702wereused(Zhuetal.,2005).Freshsporeswerepre-germinatedaccordingtothestandardprotocolwithoutmodification(Kieseretal.,2000).Briefly,stocksporeswerepelletedbycentrifugationandre-suspendedinTESbuffer(0.05M,pH8.0).Thesporeswereheatshockedat50°Cfor10min,cooledusingcoldtapwater,andthenanequalvolumeofdouble-strengthGerminationMedium(1%Difcoyeastextract,1%Difcocasaminoacids,and0.01MCaCl2)wasadded.Thesporeswerethenincubatedat37°Cwithshakingat300rpmfor2–3h.Approximately1×107germinatedspores(calibratedbyspectrophotometryusingOD450)werewashedtwiceusingdistilledwater,andthendiluted100-foldpriortoperoxidetreatment.FortheH2O2treatment,H2O2wasaddedtoafinalconcentrationof20mM,andsporeswereincubatedat30°Cfor2h.Samplesweretakenat30minintervals,anddiluted100-foldusingdistilledwater.PAAwasaddedtofinalconcentrationsof200,230,260,and290μM,andsporeswereincubatedat30°Cfor30min.Cumenehydroperoxide(CHP)wasaddedtofinalconcentrationsof10,12.5,15,17.5,and20mM,andsporeswereincubatedat30°Cfor1h.Diamidewasaddedtofinalconcentrationsof15,30,and45mM,andsporeswereincubatedat30°Cfor1h.Controlsconsistedofsamplesnotexposedtooxidants.AliquotswereplatedonMMTmedia,incubatedfor4daysat30°C,andthenumberofviablecellswasdeterminedbycolonycounts. InvivoandInvitroScavengingofPAA SporesofS.lividans1326andHXY6wereinoculatedintoTSBYmedia.Followinggrowthfor3daysat30°Cwithshakingat220rpm,myceliawerecollectedbycentrifugation.TomeasureinvivoPAA-scavenging,myceliawerewashedtwiceusingM9mediaandre-suspendedinthesamemediatoanOD450of0.2.Aftergrowingfor3h,cultureswerechallengedwith500μMPAA,incubatedat30°Cwithshakingfor3h,andthensampleswerewithdrawnat30minintervals.TheconcentrationofresidualPAAwasmeasuredin96-wellplatesusingGen5(Biotek)withtheHydrogenPeroxideQuantitativeAssayKit(SangonBiotech),whichisbasedontheferric-xylenolorangeassayandwhichcandetectperoxidesinthemicromolarconcentrationrange(GayandGebicki,2002). FortheinvitroPAA-scavengingassay,myceliawerewashedtwiceandre-suspendedinbuffercontaining50mMpotassiumphosphate(pH7.6)(Pbuffer).Cellsweredisruptedbysonicationandthencentrifugedat15000rpmfor20minat4°Ctoremovecelldebris.ProteinconcentrationwasmeasuredusingtheBradfordProteinAssay,andtheproteinconcentrationsofdifferentstrainswereadjustedtothesamelevelbydilutionwithPbuffer.Thetotalproteinextractsweretreatedwith5mMPAAfor2.5h.Sampleswerethentakenat5minintervals,andthePAAconcentrationwasmeasuredasdescribedabove. RT-PCRandQuantitativeRT-PCR S.lividans1326wasculturedinTSBYmediumtoanOD450readingof0.4,andthentreatedwith1mMparaquat,20mMH2O2,200μMPAA,3mMCHP,or3mMdiamide.Sampleswerecollectedbycentrifugationat0,25,50,or75minpostadditionofoxidantstotheculture.Fortheanalysisofperoxidescavenginggenes,sampleswerecollectedat15minintervals.RNAwasthenisolatedandRNAconcentrationwasmeasuredusingtheNanodrop2000Spectrophotometer(ThermoFisherScientificInc.).GenomicDNAwasremovedusingRNase-freeDNaseI,andtheabsenceofDNAcontaminationwasfurtherconfirmedusingPCR.ReversetranscriptionwasperformedusingthecDNASynthesisKit(ThermoFisherScientificInc.),andtheresultingcDNAwasusedasthetemplateforPCRamplification.ThePCRproductfordndBwas365bp,andrrnAencoding16srRNAwasusedastheinternalcontrol.ForwardandreverseprimersarelistedinTableS2.PCRproductswereexaminedbyelectrophoresisona1.2%agarosegelandvisualizedbystainingwithethidiumbromide. Forquantitativereal-timePCR,reactionswereperformedonanABI7500FastRealTimeSystem(AppliedBiosystems).Astandardreactionmixture(25μl)contained1or150ngcDNA(1ngforreferencegene,150ngfortargetgene),12.5μlSYBRGreenqPCRMasterMix(ThermoFisherScientificInc.),and50nMoftheforwardandreverseprimers.ThePCRproductfordndBwas161bp,andtranscriptamountswerenormalizedtotheamountof16srRNA.TheconditionsforPCRamplificationwere95°Cfor10min,followedby40cyclesof95°Cfor15s,60°Cfor1min.Adissociationcurverangingfrom55°Cto90°Cin0.5°Cincrements,withadwelltimeof30s,wasperformedtoassessthespecificityofthereactions. DeterminationoftheTranscriptionalStartSiteofthedndGeneCluster The5′RACESystemKit(ThermoFisher)wasusedtodeterminethetranscriptionalstartsitesofthedndAanddndBgenes,accordingtoprotocolsprovidedbythemanufacturer.RNAwasisolatedfromS.lividans1326growninTSBYmedium.First-strandcDNAwassynthesizedusingSuperScriptreversetranscriptaseandagene-specificprimer,GSP1(A-GSP1fordndA,B-GSP1fordndB).RNAwasremovedbytreatmentwithRNaseMix,andthecDNAwaspurifiedonaS.N.A.PColumn(ThermoFisher).Apoly(dC)tailwasaddedtothe3′-endofthecDNAusingterminaldeoxynucleotidyltransferaseanddCTP.ThefirstPCRamplificationwasperformedusingrTaqpolymerase(TAKARABIOINC.),anested,gene-specificprimer,GSP2(A-GSP2fordndA,B-GSP2fordndB),whichannealstoasitewithinthecDNA,andAAP(AbridgedAnchorPrimer).ThesecondPCRamplificationusedAUAP(AbridgedUniversalAmplificationPrimer)withanothernested,gene-specificprimer,GSP3(A-GSP3fordndA,B-GSP3fordndB),thesequenceofwhichislocatedupstreamofGSP2andwithinthecDNAproduct.TheproductofthesecondPCRwasligatedintopMD18-Tvectorforsequencing. RNA-seqandDataAnalysis SporesofthewildtypeS.lividans1326andtheHXY6weregrowninTSBYmediatoanOD450readingof0.3.ThecultureswerethencollectedbycentrifugationandtotalRNAwasisolatedusingRNAprotectBacteriaReagentandRNeasyMinikits(Qiagen),followedbyfurtherpurificationusingaMICROBExpresskit(Ambion)toenrichthemRNA.TheresultingmRNAwasfragmentedusinganRNAfragmentationkit(Ambion),producingfragmentsofsizesrangingfrom200bpto250bp.Double-strandedcDNAwasgeneratedusingtheSuperScriptDouble-StrandedcDNASynthesisKit(Invitrogen)andpurifiedusingaQiaQuickPCRextractionkit.AnRNA-seqlibrarywasconstructedusingaPairedEndSamplePrepkit(Illumina),andthesequencingwasperformedusingtheHiSeq2000sequencer(Illumina).RawreadswerefilteredtoremovedirtyreadsasrecommendedbyIllumina,andthentheresultingcleanreadswereanalyzedwithTopHat(version2.0.5)software(Langmeadetal.,2009),usingtheannotatedS.lividans1326genomeasreference.WeemployedRpackagesedgeR(version3.3;Robinsonetal.,2010)fordifferentialgeneexpressionanalysisinthisstudy,usingmultiplehypothesistestforthep-valuecorrection(q-value),andtakingintoaccountthefalsediscoveryrate(FDR).Fold-changewascalculatedaccordingtotheFPKM(FragmentsPerKilobaseofexonmodelperMillionmappedreads)value(screeningcriteriaq-value2≤0.05,andFold-change≥2).GeneOntology(GO)enrichmentanalysisofdifferentiallyexpressedgeneswasanalyzedusingtheGOseqRpackage,withgenelengthbiascorrected(Youngetal.,2010).GOtermswithq-value<0.05wereconsideredsignificantlyenrichedbydifferentiallyexpressedgenes. Results Anti-OxidationAdvantageofDNAPhosphorothioateModificationinS.lividans Toinvestigatethepotentialconsequencesforhostcells,germinatedsporesofS.lividans1326wildtype(WT)strainanditsmutantdaughterstrainHXY6,fromwhichthedndgeneclusterwasremoved,weretreatedwithH2O2,PAA,orCHP.PAAandCHPweretestedasrepresentativesoforganichydroperoxides.Aftertreatment,thesporeswerespreadontoagarplates,andcolonieswerecountedtocalculatethesurvivalrates(Figure1).WThadatwo-foldhighersurvivalratethanHXY6followingH2O2treatment,anda10-foldhighersurvivalratethanHXY6followingtreatmentwith20mMCHPor290μMPAA(usingmycelia,similarresultwasobtainedforPAAtreatment,FigureS1).TheseobservationssuggestthatphosphorothioationoftheDNAconfersananti-oxidationsurvivaladvantagetothehost. FIGURE1 Figure1.SurvivalofS.lividansunderoxidativestress.(A)SurvivalrateoftheS.lividans1326wildtypestrainandthephosphorothioatemodificationnegativemutant(dnd−)followingtreatmentwith20mMH2O2(0.5,1,1.5,or2h),CHP(10–20mM),andPAA(200–290μM).(B)Platesshowingcoloniesbeforeandafterthetreatmentwith290μMPAAfor30min.ThechemicalstructuresofPAAandCHPareshown.Threereplicateswereperformed. GlobalTranscriptionalResponseofS.lividansFollowingLossofDNAPhosphorothioation TheglobalchangeingeneexpressionanalysisinresponsetophosphorothioationmodificationwasconductedtoinvestigatewhetherDNAphosphorothioationup-regulatesgenesthatexpressperoxidescavengersandthatcontributetotheanti-oxidativepropertiesassociatedwiththemodification.ThemRNAlevelsofeachgenewerecomparedbetweenWTandthednd−mutantHXY6.Atotalof206geneshadsignificantlydifferentexpression,asmeasuredbya2-fold(log2-ratio.1,p-value=0.05)orgreaterdifferencebetweenthetwostrains,with146genesup-regulated(TableS3)and60genesdown-regulated(TableS4)inthemutant(Figure2).GeneOntologyenrichmentanalysisshowsthatthedifferentiallyexpressedgenesscatteredtoseventeenpathways(ormolecularfunctions),andnoneofthemhasp-valuelowerthan0.05andatthesametimecontainsmorethan5genes(Figure3).Althoughabunchoftranscriptionalregulatorswereseeninthedifferentialexpressedgenes(TablesS3,S4),homologyanalysisofthesegenesindicatedthatthesegenesarescatteredtomultipledifferentpathways.GeneOntologyenrichmentanalysisalsoindicatedthatnoneoftheseregulatorgeneshasp-valuelowerthan0.05andconcentrateinpathwaycontainingmorethan5genes.TheseobservationssuggestedthattheglobalRNAtranscriptionsofthetwostrainsareessentiallysimilar.Particularly,differencesofanti-oxidativegenes(includingcatalase,alkylhydroperoxidereductaseCD,andorganichydroperoxideresistancegenes)betweenthetwostrainswerenotseen.TheresistancetohydrogenperoxideconferredbyDNAphosphorothioationmightnotinvolveup-regulationoftheseanti-oxidationgenesinadvance. FIGURE2 Figure2.GeneexpressiondifferencesbetweentheWTanddnd−strains.ThenumbersofdifferentiallyexpressedgenesbetweenWTandthednd−mutantareindicated.GenesweredeterminedusingthepubliclyavailableS.lividans1326genome.Whencomparedtothecontrol,geneswithalog2foldchange>1.0,andFDR-adjustedp-values≤0.05,wereconsideredtobedifferentiallyexpressed. FIGURE3 Figure3.GOenrichmentanalysisofdifferentiallyexpressedgenes.GOtermswithq-value<0.05wereconsideredsignificantlyenrichedbydifferentiallyexpressedgenes. TranscriptionalResponseofCatalaseandHydroperoxideResistanceGenesFollowingPeroxideTreatments QuantitativeRT-PCRanalysiswasconductedtocharacterizethetranscriptionalresponseofperoxidescavenginggenesfollowingthelossofDNAphosphorothioation.Therearefivecatalase(SLI_RS01475,SLI_RS02375,SLI_RS02905,SLI_RS31365,andSLI_RS37155),twoalkylhydroperoxidasereductases(SLI_RS25315andSLI_RS25320),andthreeorganichydroperoxideresistancegenes(SLI_RS15855,SLI_RS12925,andSLI_RS32700)inthegenome.Transcriptionalchangesofthesegenesweretracedin45minafterperoxidetreatments(Figure4,andFigureS2fortheremovalofhydrogenperoxide). FIGURE4 Figure4.QuantitativeRT-PCRanalysisofperoxidescavenginggeneresponsetoperoxides.S.lividanscellsfromdnd+ordnd−strainsweregrowntoanOD450of0.3beforeadditionof20mMH2O2(A),or200μMPAA(B).Attheindicatedtimeintervals,cellswerecollectedandthetranscriptionofcatalase,alkylhydroperoxidasereductase,andhydroperoxideresistancegenesweremeasuredusingquantitativeRT-PCR(thereferencevalueforfoldchangeistimezero).TherrnAgenewassetastheinternalstandard.Threereplicateswereperformed. Figure4Ashowsthatthefivecatalase,andtwoalkylhydroperoxidasereductasegeneswereactivatedby20mMhydrogenperoxide,reachingpeaksin15minafterthetreatment.Twoorganichydroperoxideresistancegenes(SLI_RS12925andSLI_RS32700)didnotresponsetothehydrogenperoxidetreatment.Notably,Sli_RS15855,thehomologofohrinS.coelicolor,wasup-regulatedhundredsoffoldsin15min.Theeightresponsivegenesweremoreactivatedinthednd−mutantthanthewildtypestrain.SimilarresponseswerealsoobservedforthePAAtreatment(Figure4B,andFigureS2fortheremovalofPAA). DNAPhosphorothioateModificationAsaPeroxide-ScavengingSysteminS.lividans Theobservationthatcatalaseandorganichydroperoxideresistancegenes(thiol-dependentoxidase;Cussioletal.,2003,2010),weremoreinducedinthednd−strainbyH2O2andPAAtreatments,reachinglevelshigherthanthewildtype,suggestedthattheDNAphosphorothioationsystemcompensatedfortheincreasedcatalaseandperoxidaseactivitypresentinthemutant. PAA,whichisanorganichydroperoxide,wasusedtotestwhethertheWTstraincanscavengetheperoxidedirectly.ResultsshowedthatPAAwasscavengedmorerapidlybyWTthanbythednd−mutant,bothinvivo(500μM)andinvitro(5mM)immediatelyafterperoxideexposure(Figure5),supportingthesuggestionthatthemodificationsystemconstitutesanewtypeoforganichydroperoxideresistancesysteminS.lividans. FIGURE5 Figure5.PAAscavengingactivityofphosphorothioatemodification.Thecurvesshowtheremoval(scavenging)ofPAAbywildtypeandthednd−mutantcells(invivo),orthecorrespondingwholecelllysates(invitro).Concentrationsof500μMand5mMwereusedfortheinvivoandinvitroscavengingreactions,respectively.Threereplicateswereperformed. DeterminationofthedndPromoter Determinationofthetranscriptionalresponseofthedndpromotertooxidantsmightprovideadditionalevidenceforitsphysiologicalfunctionasanantioxidationsystem. 5′RACEwasconductedfirsttodeterminethetranscriptionalstartsiteofthedndgenecluster.GenespecificprimersweredesignedtoamplifyfragmentsofcDNAofdndAanddndB.Aftertheadditionofpoly(dC)tails,PCRwasperformed,andthesecondroundofnestedPCRgeneratedclearamplificationproducts(Figure6A).Thefragmentswerethenclonedandsequenced,obtainingthetranscriptionalstartsitesofdndAanddndB.The-10regionsofdndAanddndBpromotersarequitesimilar,containingtheconsensussequenceTA(t/g)CGT.Twokindsofrepeatsequences,theRrepeat(R1-R2-R3)andtherrepeat(r1-r2)werealsofoundinthe-35region(Figure6B). FIGURE6 Figure6.Identificationofthedndpromoter.(A)Thetranscriptionalstartsites(tss)ofdndAanddndB,whicharedivergentlytranscribedgenes,weredeterminedusing5′RACE.ThepromoterswereanalyzedusingtworoundsofPCR,andA1andA2,andB1andB2,indicatetheproductsfromthefirstandsecondroundofPCRforthedndAanddndBgenes,respectively.(B)Schematicdiagramofthedndpromoterregion.TherepeatsR1,R2,andR3aremarkedbydarkarrows,andthedirectrepeatsr1andr2aremarkedbylightgrayarrows.The-10regionsareinboldandunderlined,andthetssareindicatedbyverticalarrows.PromotersofdndAanddndBwerealignedwiththeconsensussequenceforthe-10region,whichismarkedbydots.(C)DiagramofpJTU3707showingthestructureofthexylEreporterplasmid.(D)Thecatechol2,3-dioxygenaseactivityassaywasusedtomonitortheeffectofthedeletionofthe-10regiononactivityfromthedndBpromoter. ThelocationofthedndpromoterwasfurthercharacterizedusingthexylEreportergene.ADNAfragmentcontainingtheregionfrom−387to373bpwithrespecttothedndtranscriptionalstartsitewasinsertedinfrontofthexylEgene(Figure6C),andtomeasurexylEexpressionlevels,totalproteinwasextractedandcatechol2,3-dioxygenaseactivitywasassayed.Twostrainswerecompared,theWTandamutantfromwhichthe-10regionofthedndBpromoterwasremovedfromupstreamofxylE.AsshowninFigure6D,withthedeletionofthe-10regionfromthedndBpromoter,thecatechol2,3-dioxygenaseactivitydroppedsharplycomparedtothewildtypecontrol. Up-RegulationofthedndGeneTranscriptionbyDiamide RNAwasextractedandRT-PCRwasperformedtomonitorthetranscriptionofthedndgenecluster.AsshowninFigures7A,B,thedndgeneclusteristranscribedearlyduringgrowth,correspondingtologphasegrowthofthestrain.TheearlyexpressionofthedndgeneclusterwasfurtherconfirmedusingthexylEreporterconstruct(Figures6B,C,7B). FIGURE7 Figure7.Transcriptionalresponsesofdndtooxidants.(A)Growthcurveofthewildtypestrain.(B)Thetimecourseoftranscriptionofthedndgeneclusteranalyzedusingsemi-quantitativeRT-PCRandthecatecholdioxygenaseactivityassay.Forthesemi-quantitativeRT-PCR,thewildtypestrainwasusedandforthecatecholdioxygenaseactivityassay,thewildtypestraincontainingpJTU3707wasused.(C)QuantitativeRT-PCRanalysisofdndBtranscriptionfollowingtreatmentwith1mMparaquat,20mMH2O2,3mMdiamide,3mMCHP,or200μMPAA(thereferencevalueforfoldchangeistimezero).TherrnAgenewassetastheinternalcontrolforevaluatingtranscriptionalactivityfromthedndBpromoter. Atthemid-logphaseofgrowth,thewildtypestrainwastreatedusing20mMH2O2,1mMparaquat,3mMCHP,3mMPAA,and3mMdiamide,theconcentrationsforwhichwerechosenbasedonpublishedstudies(Leeetal.,1993;Kallifidasetal.,2010).Thesearemodelcompoundsthatcaninducetheexpressionofsuperoxidedismutase,catalase,andorganichydroperoxideresistancegenes.Diamideisspecialforitscapabilitythatcaninducetheexpressionofbothsuperoxidedismutase,andcatalasegenesviathiol-basedswitches(Privalleetal.,1993;Zhengetal.,1998;AntelmannandHelmann,2011).RNAwasthenisolatedandquantitativeRT-PCRwasconductedtodeterminewhetherthedndtranscriptionissubjectedtooxidantregulation.Transcriptionfromthedndgeneclusterdecreasedslightlyafter1mMparaquattreatment(Figure7C),butwithH2O2,CHP,andPAAtreatment,therewasnoobviouschangeintranscriptionwithinthesametimeperiodof75min(lowerconcentrationsfrom100μMto1mMwerealsotried,withsimilarresultsobserved).However,withdiamidetreatment,transcriptionincreasedabout2.5foldin50min. Sincediamideinducedtheexpressionofdndgenes,wetestedthesurvivalofthestrainsunderdiamidestress.Figure8showsthattherearenÕtsignificantdifferencesbetweenWTandthednd−mutant. FIGURE8 Figure8.SurvivalofS.lividansunderdiamidestress.SurvivalrateoftheS.lividans1326wildtypestrainandthednd−mutantfollowingtreatmentupto45mMdiamideforanhour.Threereplicateswereperformed. InS.coelicolor,asigmaR-RsrAregulationsystemcomposingofasigmafactorandaredoxsensoranti-sigmafactor(Paget,1998;Kangetal.,1999),respondstodiamidetreatment.WedisruptedsigRgene,andthentestedwhetherdndtranscriptionissubjectedtoSigRregulation.Figures9A,BshowsthatthereisnoobviousdndgeneexpressiondifferencebetweenWTandthesigR−mutantafterdiamidetreatment.Therefore,theresponsetodiamideisnotmediatedbySigR. FIGURE9 Figure9.Potentialregulatorsofdndgeneclusterinresponsetodiamide.(A)ThesigRgenewasdisruptedusinganapramycinresistancegenecassette,confirmedbyPCR.Transcriptionalresponseofdndgeneclustertodiamide(3mM)wascomparedbetweenthewildtypeandsigR−mutant(B).ForcomparisonbetweenthewildtypeanddndB−mutant,sampleswerecollectedat45min(C). DndBistherepressorofdndgenecluster.ThepossibilityofDndBÕsresponsetodiamidewasexplored.Figure9CshowsthatindndBin-framedeletionmutant,theresponsetodiamidestimulationislost.DndBmightthereforeparticipateinthediamideinductionprocess. Discussion Streptomycesspeciesarehighlyadaptedtolivinginsoil,anenvironmentwithanextremelydiversemicrobialcomposition,andwherecompetitionbetweenmicrobesfororganiccomponentsisexpected.Sincereactiveoxygenspecies(ROS),suchassuperoxide(O2−),H2O2,andthehydroxylradical(OH•)candamagelipids,proteins,andDNA,andcausevariouscellularlesions(Imlay,2013),theirdestructivepropertieshavebeenharnessedbyorganismsasaweaponagainstotherspecies(GeisztandLeto,2004;Lambeth,2004).Forexample,NADPHoxidase,whichgeneratesROS,ishighlyconservedacrossvirtuallyallmulticellularlife(LambethandNeish,2014).Additionally,lacticacidbacteriasuppressthegrowthofcompetingmicrobesbyusingpyruvateandlactateoxidasestoexcretelargedosesofH2O2(Imlay,2008;MartínandSuárez,2010).Recently,itwasshownthatactinorhodin,anaturalproductsynthesizedbyS.coelicolor,cancatalyzetheproductionoftoxiclevelsofH2O2,andinhibitbacterialgrowthviathegeneratedH2O2(Nishiyamaetal.,2014).Therefore,itseemsthatthesoilenvironmentisabattlefieldwhereROSisaformidableweapon. Interestingly,anti-oxidationsystemsarealsoapparentlyabundantinStreptomyces.Therearemultiplecatalases,thiolperoxidases,alkylhydroperoxidereductases(Ahp),andorganichydroperoxideresistanceproteinhomologsinS.lividans,whichareexpectedtohaveantioxidationactivities.ItissuspectedthatDNAphosphorothioatemodificationalsofunctionsasananti-oxidationsysteminS.lividans,and,inthiswork,weprovidedseverallinesofevidencesupportingthisrole.First,theabsenceofDNAphosphorothioationincreasessensitivitytotheperoxidesH2O2,PAA,andCHP(Figure1).Secondly,thereisadistinctivedifferenceinthePAA-scavengingactivitybetweenthewildtypeanddnd−mutantstrains,andthirdly,thedndpromoterissubjecttooxidative(diamide)regulation. Hydrogenperoxideandorganichydroperoxidesaredifferenttypesofoxidants.Physiologically,organichydroperoxides,suchaslipidhydroperoxide,areprominentnon-radicalproductsgeneratedintheprocessofunsaturatedfattyacid-initiatedlipidperoxidationunderoxidativestress(Girotti,1998).Thehydroperoxidesarescavengedbyorganichydroperoxideresistanceproteinsinbacteriausingthiolorlipoylgroupasthereductant(Cussioletal.,2003,2010).Althoughbacteriasynthesizeonlysaturatedormonounsaturatedfattyacids,manybacteriawilltakeupexogenouspolyunsaturatedfattyacidsiftheyarepresentedintheculturemedium(Watanabeetal.,1994).TheexistenceoforganichydroperoxideresistancegenessuggeststhatthestressexistsinS.lividans. Thespecialanti-oxidativeadvantageconferredbyphosphorothioatemodificationmaybetheorganichydroperoxideresistance.Theviewpointissupportedbytheobservationofthe10-folddifferenceinsurvivalbetweenthewildtypestrainthathasDNAphosphorothioatemodificationandthednd−mutant,uponPAAorCHPtreatment(Figure1).AnotherclueisthatSLI_RS15855wasinducedhighlyinthednd−mutantfollowing20mMH2O2orPAAtreatment(Figure4).ThehigherinductionofohrAinthednd−mutantsuggestedthatmoreorganichydroperoxidesaccumulatedinthemutantthanthewildtype. Twosignificantquestionsremaintobeaddressed:firstly,howthescarcelyphosphorothioatedsitescounteracttheperoxidesbyordersoffolds;secondly,whythemodifiedDNAbondswerenotcleavedbyperoxides,especiallybyPAAinvivo.Theturning-overmodel(recyclingofthephosphorothioatesulfur),assuggestedpreviously(Xieetal.,2012),couldexplainthefirstquestionverywell,butnotthesecondonesincetheresultofreactionwithPAAofthebondinvitro,isDNAdegradation,whichshouldbelethaltothebacteria.Analternativesuggestionisthattheperoxide-scavengingbehaviorofthephosphorothioatemightbeawell-controlledcatalyticprocess,implyingtheinvolvementofenzymes.ThepotentialenzymesareDndproteins:theproteinthemselvesortheprotein-phosphorothioateDNAcomplexmightscavengetheperoxides. ThedndBCDEgeneclusterconstitutesapolycistronictranscriptionalunit(Xuetal.,2009).Theclusterisactivelytranscribedintheearlystageofexponentialgrowth,possiblybecauseDNAisreplicatedinthisstage.Interestingly,thedndgeneclusterwasinducedbydiamide,whichcancausedisulfidestressincells.Synthesizedphosphorothioatecanformdisulfidebond(WuandOrgel,1991).Thesurvivalrateofthewildtypeandthednd−mutantstraintreatedbydiamideexhibitednodifference(Figure8),suggestingthatDNAphosphorothioatemodificationmightnotscavengedisulfidebondsinvivo.Inductionofthedndgeneclusterbydiamidesuggeststhattheunderlyingactivationmechanismofthegeneclustermightbetheformationofdisulfidebonds,whichisreminiscentofOxyR(Zhengetal.,1998),aregulatorofoxidativegenesincludingcatalases(Imlay,2008).Similarly,inS.coelicolor,transcriptionalregulationofcatAbyCatR,isalsoRedox-dependent,withtheinvolvementofthioloxidationinredoxmodulationofCatR(Hahnetal.,2000). WithS.coelicolor,thecellsrespondedtodisulfidestressthroughasigmaR-RsrAregulationsystem,composedofasigmafactorandaredoxsensoranti-sigmafactor(Paget,1998;Kangetal.,1999).SigmaRregulatespromoterscontainingtheconservedsequencesGGAATandGTTGatthe−35and−10regions,respectively,(Paget,1998;Kangetal.,1999),butthesetwosequenceswerenotobservedinthedndpromoterofS.lividans.Indeed,significantdifferencesoftranscriptionalresponsetodiamidewerenotobservedbetweenasigRmutantandwildtype(Figures9A,B).Apotentialdisulfide-responsiveregulatorofdndgenesmightbeDndB,whichisanegativeregulatorofDNAphosphorothioation(Chengetal.,2015;Heetal.,2015).QuantitativeRT-PCRanalysisshowsthatinadndBdeletionmutant,thetranscriptionalactivationbydiamideofdndgeneclusterwaslost(Figure9C).ThepossiblemechanismmightbethatDiamideinduceddisulfideinactivateDndBandallowmoretranscriptionofthedndgenecluster. Overall,theobservationssuggestedthatDNAphosphorothioatemodificationinS.lividansisaperoxideresistancesystem. AuthorContributions DD,AD,KX,andTPdidtheexperiments.DD,AD,TP,XZ,ZD,JL,XH,andZWanalyzedthedata.DD,XZ,ZD,JL,XH,andZWwrotethemainmanuscripttext.DD,JL,andZWpreparedfigures.Allauthorsreviewedthemanuscript. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments ThisworkwassupportedbytheTangBerkeleyScholarship,theMinistryofScienceandTechnology(9732015CB554203,and2012CB721004),theNationalScienceFoundationofChina(31470830and31121064),theNaturalScienceFoundationofShanghai(14ZR1421500),theNationalProgramofDevelopmentofTransgenicNewSpeciesofChina,ShanghaiPujiangProgram(14PJD019),andtheInternationalCooperationandExchangeProgramofShanghaiJiaoTongUniversity. SupplementaryMaterial TheSupplementaryMaterialforthisarticlecanbefoundonlineat:https://www.frontiersin.org/article/10.3389/fmicb.2016.01380 FigureS1.SurvivalrateofPAAtreatment.MyceliaofS.lividansWTandthednd−mutantweretreatedusingincreasingconcentrationofPAA.Thecellsweretreatedfor1h.Thenthecolonieswerecountedtocalculatethesurvivalrates.Theexperimentswererepeatedthreetimes. FigureS2.RemovalofH2O2andPAA.MyceliaofS.lividansWTandthednd-mutantweretreatedusing20mMH2O2or200μMconcentrationofPAA.Atindicatedintervals,theremainsoftheperoxidesweremonitored. TableS1.Strainsandplasmidsusedinthestudy. TableS2.Primersusedinthisstudy. TableS3.Up-regulatedgenesinthemutant. TableS4.Down-regulatedgenesinthemutant. Abbreviations PAA,peraceticacid;CHP,cumenehydroperoxide;OD,opticaldensity. References Antelmann,H.,andHelmann,J.D.(2011).Thiol-basedredoxswitchesandgeneregulation.Antioxid.RedoxSignal.14,1049–1063.doi:10.1089/ars.2010.3400 PubMedAbstract|CrossRefFullText|GoogleScholar Aussel,L.,Zhao,W.,Hébrard,M.,Guilhon,A.-A.,Viala,J.P.M.,Henri,S.,etal.(2011).Salmonelladetoxifyingenzymesaresufficienttocopewiththehostoxidativeburst.Mol.Microbiol.80,628–640.doi:10.1111/j.1365-2958.2011.07611.x PubMedAbstract|CrossRefFullText|GoogleScholar Boybek,A.,Ray,T.D.,Evans,M.C.,andDyson,P.J.(1998).Novelsite-specificDNAmodificationinStreptomyces:analysisofpreferredintragenicmodificationsitespresentina5.7kbamplifiedDNAsequence.NucleicAcidsRes.26,3364–3371.doi:10.1093/nar/26.14.3364 PubMedAbstract|CrossRefFullText|GoogleScholar Cao,B.,Chen,C.,DeMott,M.S.,Cheng,Q.,Clark,T.A.,Xiong,X.,etal.(2014a).Genomicmappingofphosphorothioatesrevealspartialmodificationofshortconsensussequences.Nat.Commun.5:3951.doi:10.1038/ncomms4951 PubMedAbstract|CrossRefFullText|GoogleScholar Cao,B.,Cheng,Q.,Gu,C.,Yao,F.,DeMott,M.S.,Zheng,X.,etal.(2014b).PathologicalphenotypesandinvivoDNAcleavagebyunrestrainedactivityofaphosphorothioate-basedrestrictionsysteminSalmonella.Mol.Microbiol.93,776–785.doi:10.1111/mmi.12692 PubMedAbstract|CrossRefFullText|GoogleScholar Chen,L.,Wang,X.-L.,Shi,T.,Wu,T.,Deng,Z.,andZhao,Y.-L.(2015).TheoreticalstudyontherelationshipbetweenRp-Phosphorothioationandbase-stepinS-DNA:basedonenergeticandstructuralanalysis.J.Phys.Chem.B119,474–481.doi:10.1021/jp511359e PubMedAbstract|CrossRefFullText|GoogleScholar Cheng,Q.,Cao,B.,Yao,F.,Li,J.,Deng,Z.,andYou,D.(2015).RegulationofDNAphosphorothioatemodificationsbythetranscriptionalregulatorDptBinSalmonella.Mol.Microbiol.97,1186–1194.doi:10.1111/mmi.13096 PubMedAbstract|CrossRefFullText|GoogleScholar Cruz-Morales,P.,Vijgenboom,E.,Iruegas-Bocardo,F.,Girard,G.,Yáñez-Guerra,L.A.,Ramos-Aboites,H.E.,etal.(2013).ThegenomesequenceofStreptomyceslividans66revealsanoveltRNA-dependentpeptidebiosyntheticsystemwithinametal-relatedgenomicisland.GenomeBiol.Evol.5,1165–1175.doi:10.1093/gbe/evt082 PubMedAbstract|CrossRefFullText|GoogleScholar Cussiol,J.R.R.,Alegria,T.G.P.,Szweda,L.I.,andNetto,L.E.S.(2010).Ohr(organichydroperoxideresistanceprotein)possessesapreviouslyundescribedactivity,lipoyl-dependentperoxidase.J.Biol.Chem.285,21943–21950.doi:10.1074/jbc.M110.117283 PubMedAbstract|CrossRefFullText|GoogleScholar Cussiol,J.R.R.,Alves,S.V.,deOliveira,M.A.,andNetto,L.E.S.(2003).Organichydroperoxideresistancegeneencodesathiol-dependentperoxidase.J.Biol.Chem.278,11570–11578.doi:10.1074/jbc.M300252200 PubMedAbstract|CrossRefFullText|GoogleScholar Dyson,P.,andEvans,M.(1998).Novelpost-replicativeDNAmodificationinStreptomyces:analysisofthepreferredmodificationsiteofplasmidpIJ101.NucleicAcidsRes.26,1248–1253.doi:10.1093/nar/26.5.1248 PubMedAbstract|CrossRefFullText|GoogleScholar Eckstein,F.(2000).Phosphorothioateoligodeoxynucleotides:whatistheiroriginandwhatisuniqueaboutthem?AntisenseNucleicAcidDrugDev.10,117–121.doi:10.1089/oli.1.2000.10.117 PubMedAbstract|CrossRefFullText|GoogleScholar Gan,R.,Wu,X.,He,W.,Liu,Z.,Wu,S.,Chen,C.,etal.(2014).DNAphosphorothioatemodificationsinfluencetheglobaltranscriptionalresponseandprotectDNAfromdouble-strandedbreaks.Sci.Rep.4:6642.doi:10.1038/srep06642 PubMedAbstract|CrossRefFullText|GoogleScholar Gay,C.A.,andGebicki,J.M.(2002).Perchloricacidenhancessensitivityandreproducibilityoftheferric–xylenolorangeperoxideassay.Anal.Biochem.304,42–46.doi:10.1006/abio.2001.5566 PubMedAbstract|CrossRefFullText|GoogleScholar Geiszt,M.,andLeto,T.L.(2004).TheNoxfamilyofNAD(P)Hoxidases:hostdefenseandbeyond.J.Biol.Chem.279,51715–51718.doi:10.1074/jbc.R400024200 PubMedAbstract|CrossRefFullText|GoogleScholar Girotti,A.W.(1998).Lipidhydroperoxidegeneration,turnover,andeffectoractioninbiologicalsystems.J.LipidRes.39,1529–1542. PubMedAbstract Hahn,J.S.,Oh,S.Y.,Chater,K.F.,Cho,Y.H.,andRoe,J.H.(2000).H2O2-sensitivefur-likerepressorCatRregulatingthemajorcatalasegeneinStreptomycescoelicolor.J.Biol.Chem.275,38254–38260.doi:10.1074/jbc.M006079200 PubMedAbstract|CrossRefFullText|GoogleScholar He,W.,Huang,T.,Tang,Y.,Liu,Y.,Wu,X.,Chen,S.,etal.(2015).RegulationofDNAphosphorothioatemodificationinSalmonellaentericabyDndB.Sci.Rep.5,12368.doi:10.1038/srep12368 PubMedAbstract|CrossRefFullText|GoogleScholar He,X.,Ou,H.-Y.,Yu,Q.,Zhou,X.,Wu,J.,Liang,J.,etal.(2007).AnalysisofagenomicislandhousinggenesforDNAS-modificationsysteminStreptomyceslividans66anditscounterpartsinotherdistantlyrelatedbacteria.Mol.Microbiol.65,1034–1048.doi:10.1111/j.1365-2958.2007.05846.x PubMedAbstract|CrossRefFullText|GoogleScholar Howard,S.T.,Newman,K.L.,McNulty,S.,Brown-Elliott,B.A.,Vasireddy,R.,Bridge,L.,etal.(2013).InsertionsiteanddistributionofagenomicislandconferringDNAphosphorothioationintheMycobacteriumabscessuscomplex.Microbiology159,2323–2332.doi:10.1099/mic.0.070318-0 PubMedAbstract|CrossRefFullText|GoogleScholar Imlay,J.A.(2008).Cellulardefensesagainstsuperoxideandhydrogenperoxide.Annu.Rev.Biochem.77,755–776.doi:10.1146/annurev.biochem.77.061606.161055 PubMedAbstract|CrossRefFullText|GoogleScholar Imlay,J.A.(2013).Themolecularmechanismsandphysiologicalconsequencesofoxidativestress:lessonsfromamodelbacterium.Nat.Rev.Microbiol.11,443–454.doi:10.1038/nrmicro3032 PubMedAbstract|CrossRefFullText|GoogleScholar Ingram,C.,Brawner,M.,Youngman,P.,andWestpheling,J.(1989).xylEfunctionsasanefficientreportergeneinStreptomycesspp.:useforthestudyofgalP1,acatabolite-controlledpromoter.J.Bacteriol.171,6617–6624. PubMedAbstract|GoogleScholar Kallifidas,D.,Thomas,D.,Doughty,P.,andPaget,M.S.B.(2010).ThesigmaRregulonofStreptomycescoelicolorA32revealsakeyroleinproteinqualitycontrolduringdisulphidestress.Microbiology156,1661–1672.doi:10.1099/mic.0.037804-0 PubMedAbstract|CrossRefFullText|GoogleScholar Kang,J.G.,Paget,M.S.,Seok,Y.J.,Hahn,M.Y.,Bae,J.B.,Hahn,J.S.,etal.(1999).RsrA,ananti-sigmafactorregulatedbyredoxchange.EMBOJ.18,4292–4298.doi:10.1093/emboj/18.15.4292 PubMedAbstract|CrossRefFullText|GoogleScholar Kieser,T.,Bibb,M.J.,Buttner,M.J.,Chater,K.F.,andHopwood,D.A.(2000).PracticalStreptomycesGenetics.Norwich:JohnInnesFoundation. GoogleScholar Lambeth,J.D.(2004).NOXenzymesandthebiologyofreactiveoxygen.Nat.Rev.Immunol.4,181–189.doi:10.1038/nri1312 PubMedAbstract|CrossRefFullText|GoogleScholar Lambeth,J.D.,andNeish,A.S.(2014).Noxenzymesandnewthinkingonreactiveoxygen:adouble-edgedswordrevisited.Annu.Rev.Pathol.Mech.Dis.9,119–145.doi:10.1146/annurev-pathol-012513-104651 PubMedAbstract|CrossRefFullText|GoogleScholar Lan,W.,Hu,Z.,Shen,J.,Wang,C.,Jiang,F.,Liu,H.,etal.(2016).StructuralinvestigationintophysiologicalDNAphosphorothioatemodification.Sci.Rep.6:25737.doi:10.1038/srep25737 PubMedAbstract|CrossRefFullText|GoogleScholar Langmead,B.,Trapnell,C.,Pop,M.,andSalzberg,S.L.(2009).Ultrafastandmemory-efficientalignmentofshortDNAsequencestothehumangenome.GenomeBiol.10:R25.doi:10.1186/gb-2009-10-3-r25 PubMedAbstract|CrossRefFullText|GoogleScholar Lee,J.S.,Hah,Y.C.,andRoe,J.H.(1993).TheinductionofoxidativeenzymesinStreptomycescoelicoloruponhydrogenperoxidetreatment.J.Gen.Microbiol.139,1013–1018.doi:10.1099/00221287-139-5-1013 CrossRefFullText|GoogleScholar Liang,J.,Wang,Z.,He,X.,Li,J.,Zhou,X.,andDeng,Z.(2007).DNAmodificationbysulfur:analysisofthesequencerecognitionspecificitysurroundingthemodificationsites.NucleicAcidsRes.35,2944–2954.doi:10.1093/nar/gkm176 PubMedAbstract|CrossRefFullText|GoogleScholar Liu,G.,Ou,H.-Y.,Wang,T.,Li,L.,Tan,H.,Zhou,X.,etal.(2010).CleavageofphosphorothioatedDNAandmethylatedDNAbythetypeIVrestrictionendonucleaseScoMcrA.PLoSGenet.6:e1001253.doi:10.1371/journal.pgen.1001253 PubMedAbstract|CrossRefFullText|GoogleScholar Liu,G.,Zhang,Z.,Zhao,G.,Deng,Z.,Wu,G.,andHe,X.(2015).CrystallizationandpreliminaryX-rayanalysisofthetypeIVrestrictionendonucleaseScoMcrAfromStreptomycescoelicolor,whichcleavesbothDcm-methylatedDNAandphosphorothioatedDNA.ActaCrystallogr.FStruct.Biol.Commun.71(Pt1),57–60.doi:10.1107/S2053230X14025801 PubMedAbstract|CrossRefFullText|GoogleScholar Marinus,M.G.,andCasadesús,J.(2009).RolesofDNAadeninemethylationinhost-pathogeninteractions:mismatchrepair,transcriptionalregulation,andmore.FEMSMicrobiol.Rev.33,488–503.doi:10.1111/j.1574-6976.2008.00159.x PubMedAbstract|CrossRefFullText|GoogleScholar Martín,R.,andSuárez,J.E.(2010).BiosynthesisanddegradationofH2O2byvaginallactobacilli.Appl.Environ.Microbiol.76,400–405.doi:10.1128/AEM.01631-09 PubMedAbstract|CrossRefFullText|GoogleScholar Mishra,S.,andImlay,J.(2012).Whydobacteriausesomanyenzymestoscavengehydrogenperoxide?Arch.Biochem.Biophys.525,145–160.doi:10.1016/j.abb.2012.04.014 PubMedAbstract|CrossRefFullText|GoogleScholar Nishiyama,T.,Hashimoto,Y.,Kusakabe,H.,Kumano,T.,andKobayashi,M.(2014).Naturallow-molecularmassorganiccompoundswithoxidaseactivityasorganocatalysts.Proc.Natl.Acad.Sci.U.S.A.111,17152–17157.doi:10.1073/pnas.1417941111 PubMedAbstract|CrossRefFullText|GoogleScholar Ou,H.-Y.,He,X.,Shao,Y.,Tai,C.,Rajakumar,K.,andDeng,Z.(2009).dndDB:adatabasefocusedonphosphorothioationoftheDNAbackbone.PLoSONE4:e5132.doi:10.1371/journal.pone.0005132 PubMedAbstract|CrossRefFullText|GoogleScholar Paget,M.S.B.(1998).sigmaR,anRNApolymerasesigmafactorthatmodulatesexpressionofthethioredoxinsysteminresponsetooxidativestressinStreptomycescoelicolorA3(2).EMBOJ.17,5776–5782.doi:10.1093/emboj/17.19.5776 PubMedAbstract|CrossRefFullText|GoogleScholar Privalle,C.T.,Kong,S.E.,andFridovich,I.(1993).Inductionofmanganese-containingsuperoxidedismutaseinanaerobicEscherichiacolibydiamideand1,10-phenanthroline:sitesoftranscriptionalregulation.Proc.Natl.Acad.Sci.U.S.A.90,2310–2314.doi:10.1073/pnas.90.6.2310 PubMedAbstract|CrossRefFullText|GoogleScholar Ray,T.,Mills,A.,andDyson,P.(1995).Tris-dependentoxidativeDNAstrandscissionduringelectrophoresis.Electrophoresis16,888–894.doi:10.1002/elps.11501601149 PubMedAbstract|CrossRefFullText|GoogleScholar Robinson,M.D.,McCarthy,D.J.,andSmyth,G.K.(2010).edgeR:aBioconductorpackagefordifferentialexpressionanalysisofdigitalgeneexpressiondata.Bioinformatics26,139–140.doi:10.1093/bioinformatics/btp616 PubMedAbstract|CrossRefFullText|GoogleScholar Tunca,S.,Barreiro,C.,Sola-Landa,A.,Coque,J.J.R.,andMartín,J.F.(2007).TranscriptionalregulationofthedesferrioxaminegeneclusterofStreptomycescoelicolorismediatedbybindingofDmdR1toanironboxinthepromoterofthedesAgene.FEBSJ.274,1110–1122.doi:10.1111/j.1742-4658.2007.05662.x PubMedAbstract|CrossRefFullText|GoogleScholar Wang,L.,Chen,S.,Vergin,K.L.,Giovannoni,S.J.,Chan,S.W.,DeMott,M.S.,etal.(2011).DNAphosphorothioationiswidespreadandquantizedinbacterialgenomes.Proc.Natl.Acad.Sci.U.S.A.108,2963–2968.doi:10.1073/pnas.1017261108 PubMedAbstract|CrossRefFullText|GoogleScholar Wang,L.,Chen,S.,Xu,T.,Taghizadeh,K.,Wishnok,J.S.,Zhou,X.,etal.(2007).PhosphorothioationofDNAinbacteriabydndgenes.Nat.Chem.Biol.3,709–710.doi:10.1038/nchembio.2007.39 PubMedAbstract|CrossRefFullText|GoogleScholar Watanabe,K.,Ishikawa,C.,Inoue,H.,Cenhua,D.,Yazawa,K.,andKondo,K.(1994).Incorporationofexogenousdocosahexaenoicacidintovariousbacterialphospholipids.J.Am.OilChem.Soc.71,325–330.doi:10.1007/BF02638061 CrossRefFullText|GoogleScholar Wu,T.,andOrgel,L.E.(1991).Disulfide-linkedoligonucleotidephosphorothioates:novelanaloguesofnucleicacids.J.Mol.Evol.32,274–277.doi:10.1007/BF02102183 PubMedAbstract|CrossRefFullText|GoogleScholar Xie,X.,Liang,J.,Pu,T.,Xu,F.,Yao,F.,Yang,Y.,etal.(2012).PhosphorothioateDNAasanantioxidantinbacteria.NucleicAcidsRes.40,9115–9124.doi:10.1093/nar/gks650 PubMedAbstract|CrossRefFullText|GoogleScholar Xu,T.,Liang,J.,Chen,S.,Wang,L.,He,X.,You,D.,etal.(2009).DNAphosphorothioationinStreptomyceslividans:mutationalanalysisofthedndlocus.BMCMicrobiol.9:41.doi:10.1186/1471-2180-9-41 PubMedAbstract|CrossRefFullText|GoogleScholar Xu,T.,Yao,F.,Zhou,X.,Deng,Z.,andYou,D.(2010).Anovelhost-specificrestrictionsystemassociatedwithDNAbackboneS-modificationinSalmonella.NucleicAcidsRes.38,7133–7141.doi:10.1093/nar/gkq610 PubMedAbstract|CrossRefFullText|GoogleScholar Young,M.D.,Wakefield,M.J.,Smyth,G.K.,andOshlack,A.(2010).GeneontologyanalysisforRNA-seq:accountingforselectionbias.GenomeBiol.11:R14.doi:10.1186/gb-2010-11-2-r14 PubMedAbstract|CrossRefFullText|GoogleScholar Zhang,Y.-C.,Liang,J.,Lian,P.,Han,Y.,Chen,Y.,Bai,L.,etal.(2012).TheoreticalStudyonStericEffectsofDNAPhosphorothioation:B-HelicalDestabilizationinRp-PhosphorothioatedDNA.J.Phys.Chem.B116,10639–10648.doi:10.1021/jp302494b PubMedAbstract|CrossRefFullText|GoogleScholar Zheng,M.,Aslund,F.,andStorz,G.(1998).ActivationoftheOxyRtranscriptionfactorbyreversibledisulfidebondformation.Science279,1718–1721.doi:10.1126/science.279.5357.1718 PubMedAbstract|CrossRefFullText|GoogleScholar Zhou,X.,He,X.,Liang,J.,Li,A.,Xu,T.,Kieser,T.,etal.(2005).AnovelDNAmodificationbysulphur.Mol.Microbiol.57,1428–1438.doi:10.1111/j.1365-2958.2005.04764.x PubMedAbstract|CrossRefFullText|GoogleScholar Zhu,D.,He,X.,Zhou,X.,andDeng,Z.(2005).ExpressionofthemelCoperoninseveralStreptomycesstrainsispositivelyregulatedbyAdpA,anAraCfamilytranscriptionalregulatorinvolvedinmorphologicaldevelopmentinStreptomycescoelicolor.J.Bacteriol.187,3180–3187.doi:10.1128/JB.187.9.3180-3187.2005 PubMedAbstract|CrossRefFullText|GoogleScholar Keywords:DNAphosphorothioatemodification,organichydroperoxideresistance,antioxidant,catalase Citation:DaiD,DuA,XiongK,PuT,ZhouX,DengZ,LiangJ,HeXandWangZ(2016)DNAPhosphorothioateModificationPlaysaRoleinPeroxidesResistanceinStreptomyceslividans.Front.Microbiol.7:1380.doi:10.3389/fmicb.2016.01380 Received:06June2016;Accepted:22August2016;Published:31August2016. Editedby:IvanMijakovic,ChalmersUniversityofTechnology,Sweden Reviewedby:VladimirBidnenko,CentredeRecherchesdeJouy-en-Josas(INRA),FranceJing-ShengCheng,TianjinUniversity,China Copyright©2016Dai,Du,Xiong,Pu,Zhou,Deng,Liang,HeandWang.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:JingdanLiang,[email protected],[email protected],[email protected] †Theseauthorshavecontributedequallytothiswork. COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>