extracellular dna provides structural integrity to a micrococcus luteus biofilm jamie t. blakeman资料.pdf

该【extracellular dna provides structural integrity to a micrococcus luteus biofilm jamie t. blakeman资料 】是由【赖大文档】上传分享，文档一共【8】页，该文档可以免费在线阅读，需要了解更多关于【extracellular dna provides structural integrity to a micrococcus luteus biofilm jamie t. blakeman资料 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。ArticleCiteThis:LangmuirXXXX,XXX,XXX?XXXpubs./LangmuirusluteusBio?lm???§∥?,-García,*,,JoyMukherjee,KlausGori,,??,andMarkGeoghegan?DepartmentofPhysicsandAstronomy,TheUniversityofShe?eld,Houns?eldRoad,She?eldS37RH,.?ProcterandGambleNewcastleInnovationCentre,Longbenton,NewcastleuponTyneNE129TS,.§DepartmentofChemicalandBiologicalEngineering,TheUniversityofShe?eld,MappinStreet,She?eldS13JD,.∥NovozymesA/S,Krogsh?jvej36,Bagsv?rd2880,Denmark*SSupportingInformationABSTRACT:ForcespectroscopywasusedtoshowthatextracellularDNA(eDNA)hasapre-eminentstructuralroleinabio?(erephthalate),amodelhydrophobicsurface,wasmeasuredinresponsetotheirdegradationbyhydrolyticenzymesknownfortheirbio?lmdispersionpotential:DNaseI,protease,cellulase,?cantlyusluteuswiththesurface,pletelyeliminatedanycomponentsofthebio?lmmaintainingtheadhesion,establishingakeystructuralroleforeDNA.■INTRODUCTIONofacid?base,Lifshitz?vanderWaals,andspeci?cforces,eDNAformsameshinwhichitisboundtoextracellularMicrobiallifedepositsonsurfacesthroughpolymer-encased?harides,formingdual?brils,1012andmatrixpro-assembliesknownasbio??teins,1318andispostulatedtoprovidesomesupportfortheounter-?(EPSs)?lmdispersalinasurroundthemprovideresistancetobiocides,antibiotics,?widervarietyofGram-positive,10,11,20Gram-negative,2123andation,UVdamage,shearforces,,25anismsthanthoseforwhichcleavageofproteinssettings,theirdispersalisparamount,suchasinbiolm-related?infections(.,cystic?brosis,1dentalplaque,2andwounds3),,andpathogen-riddensurfaces,ponentsintheEPS,treatmentwithonlyDNaseIise?ective,particularlyinyoungwherefailuretoremovethemcouldcausemorbidityand??,bio?lmformationcausestremen-biolms,ponentin?26DownloadedviaUNIVAUTONOMADECOAHUILAonApril18,2019at06:55:52(UTC).,targetingeDNAcoulddouseconomicdetriment,suchasinthecontaminationof?Seehttps://pubs./,textilestaining?andmalodorinlaundry,6biofoulingofshiphulls,7andTheremovalofunwantedbiolmsrequiresanabilitytoharboringfood-,which?isalreadyinnatetobio??lmsrestsonThus,thereisaneedtodevelopbiolmdispersantsthatare?aturewhichallowthemtopolymers,ideallyinasustainablemanner,thatis,withlowrespondtoenvironmentalcues,suchasincreasedshearforcesenergyinputandproductsoflowtoxicityandhighandstarvation,bymodifyingtheirshape,mechanicalproper-27??llthisgoal,andties,?lmsonsurfaces,,whereasminorrearrangementswouldleaveExtracellularDNA(eDNA)ponentintheprimarystructureintactandonlymodifytheperipheralbio??nessandcharge,9itactsasbothabridgebetweenbacteriaandsurfaces,Received:January31,2019toestablishinitialbio?lms,andalsobetweenbacteriaRevised:April4,2019themselves,?XXXXAmericanChemicalSocietyADOI:.9b00297LangmuirXXXX,XXX,XXX?,,arebothintegraltotheadaptabilityofbio?.A1%PETsolutionin2:1HFIP/2CPwasspunneededtotargetthekeystructuralelementsthatwouldtriggerontosiliconwafersat2500rpmfor60sandannealedfor12hat200°.??(4698)wereHere,theeectofdierenthydrolyticenzymesonthestoredat?80°Cin50%glycerolstocksandpropagatedontoTSAadhesionforceofalivebio?lmwithamodelhydrophobicagar,incubatingat30°°?lmsoftheGram-positiveTiplesssiliconnitrideatomicforcemicroscopecantileverswere?sterilizedin70%ethanolfor10min,andapolycationiclayerwasusluteusareintermittentlyattachedtoatμsurfacesofpoly(erephthalate)(PET),andtheirattachedontothembyadding200Lof1%poly-L-lysinehydrobromideaqueoussolution(w/w%),protease,mannanase,°%mensal,whichisknowntoenhancestrengthTSBandincubatedat30°(AFM)experiment,theassembledprobeswerepolymicrobialbio??er(videinfra),30abilitytoutilizeanumberofGlassslideswerepreparedasthebioprobeswere,usinga1%poly-L-lysinehydrobromidesolutioncarbonsources,31abilitytoresuscitatefromdormancy,3233(w/w%),-depthinimagingbu?ercontainingtherelevantenzyme(controlslidesusedponentsareinterwovenandofonlyimagingbu?er),ponenthasonthetheslideswerestainedwithBacLight(SYTO9stainwithexcitationatadhesivepro?leofthebio?lmareneededtoimprovebio?lm497nmandemissionat543nmandpropidiumiodidestainwithdispersalstrategies,34andforthisreason,forcespectroscopy,aexcitationat571nmandemissionat638nm)andrinsedgentlybeforeimaginginaLeicaSP8confocalmicroscope,employingimagesnanoscopictechniquethatelucidatesthebehaviorofsinglewitha10×/-,(Bitplane,Belfast,UK)usedtostudythee?ectofenzymesonbacteriaandsoftwareusingthesurfacecreationwizard,withtheupperandlowerbio????erentenzymes,?providesstructuralintegritytothebio?(AsylumResearch,MFP-3D),usingatiplessharideshavingasecondary,nonstructuralfunction,?cantileverwithanominalspringconstantof60pN/?eratroomtemperature,usingthermal?uctuation,38andallwere■EXPERIMENTALSECTIONfoundtobewithinthemanufacturer’×10forcemapswithascanrateof1Hz,dwellPETpelletsofanumberaveragemolarmassMn=,andretractionrateof2μm/?:(1)determinationoftheSemicor,-Chloropentane(2CP)wasobtainedfrominitialconditions(.,forceacquisitionunderimagingbu?er),(2)aFluorochem(Had?eld,UK).1,1,1,3,3,3-Hexa?uoro-2-propanol(hex-40minincubationinenzyme-containingimagingbu?erorfresha?uoroisopropanol,HFIP),glycerol,trypticsoybroth(TSB),tricine,imagingbu?er(control),and(3)measurementaftertheincubationsodiumchloride,magnesiumchloride,calciumchloride,(TSA),andpoly-L-lysinehydrobromidewithamolarmassoffreshbioprobe,andhenceineachcaseauniquebio??70kg/molwerepurchasedfromSigma-Aldrich(Gillingham,Consequently,thethree-stepprotocolhadtobeconductedeveryUK).Live/DeadBacLightwaspurchasedfromThermoFishertime,tomeasuretherelativechangesininteractionforce,asanScienti?c(Loughborough,UK).,(NP-O10)(Camarillo,CA,USA).Savinase16L(protease),paredusingattestwith95%con?(cellulase),Mannaway25L(mannanase),andDNaseIwereofforceandextensionforthesecondaryadhesioneventswereobtainedfromNovozymesA/S(Bagsv?rd,Denmark).DNaseI()isendogenoustoAspergillusoryzae,anditsaminoacidsequenceisdescribedinpatentWO2015/155350.■RESULTSImagingBu?????-bueredsolutionofMilli-,luteuswereculturedfor18honglassslides,andtheirdispersalsupplementedwith200mg/LofNa+,+,+,and250mg/LCl?.Thesesaltconcentrationscloselymatchthoseofuponenzymetreatmentwasimagedusingconfocalmicroscopy(Figure1).After40minincubation,DNaseIwasabletothe10%strengthTSBtominimizeanyenvironmentalshockon?bacteriacausedbytheimagingbu?eruponremovalfromthegrowthremovethemajorityofthebiolmswithonlytallspire-(DNaseI,protease,mannanase,andcellulase)structuresremaining,suggestingthattheseweretheoldest27weretestedatconcentrationsinimagingbu??,chosensothatabovethisvaluetheenzymebehaviorwasthatDNasesarelesse?ectiveagainstolderbio?lms,26,39whichBDOI:.9b00297LangmuirXXXX,XXX,XXX?XXXLangmuirArticlecomponentsorthateDNAissupplantedbyothermacro-??lmwasalsostudiedusingcontactanglemeasurements(TableS1).Thereductioninthehydrophobicityofthebio?lmsaftertreatmentwasrecorded,-binedwithbindingassays,isawidelyusedtechniquethatcangiveimportantinformationabouttheponentsofabio?,40,,toassessademonstrableroleforthesemolecules,itisnecessarytointeractwiththebio?lminadi?,forcespectroscopywasusedtospecifythee??lmsandPETsurfacesandthustodemonstratetheimportanceofeDNAtothebio?,whileimmersedinenzyme-freeimagingbu??separationcurveswereacquired,andtheiradhesionforce(theordinateoftheprimarycontactinFigure2)?separationcurvebetweenabio?lmandasurface,showingtheprimarycontact,fromwhichtheadhesionforceisextracted,,protease,mannanase,andcellulaseon18correspondtothesecondaryadhesionevents.(Inset)Experimentalhbio??lmswereculturedonglass,,andvisualizedusingconfocalmicroscopy,takingz-,inthesecondstep,theimagingbu?erwasrep