RESEARCHARTICLE Flipping chromosomes in deep-sea archaea MatteoCossu1,CatherineBadel1,RyanCatchpole1,DanièleGadelle1,EvelyneMarguet1, Vale´rieBarbe2,PatrickForterre1,JacquesOberto1* 1 InstituteforIntegrativeBiologyoftheCell(I2BC),MicrobiologyDepartment,CEA,CNRS,Univ.Paris-Sud, Universite´Paris-Saclay,Gif-sur-Yvette,France,2 Genoscope,LaboratoiredeBiologieMole´culairepour l’EtudedesGe´nomesC.E.A.,InstitutdeGe´nomique-2rueGastonCre´mieux,EVRY,France *[email protected] a1111111111 Abstract a1111111111 a1111111111 Oneofthemajormechanismsdrivingtheevolutionofallorganismsisgenomicrearrange- a1111111111 a1111111111 ment.InhyperthermophilicArchaeaoftheorderThermococcales,largechromosomalinver- sionsoccursofrequentlythatevencloselyrelatedgenomesaredifficulttoalign.Clearlynot resultingfromthenativehomologousrecombinationmachinery,thecausativeagentof theseinversionshasremainedelusive.Wepresentamodelinwhichgenomicinversions arecatalyzedbytheintegraseenzymeencodedbyafamilyofmobilegeneticelements.We OPENACCESS characterizedtheintegrasefromThermococcusnautiliplasmidpTN3andshowedthat Citation:CossuM,BadelC,CatchpoleR,Gadelle besidescanonicalsite-specificreactions,itcatalyzeslowsequencespecificityrecombina- D,MarguetE,BarbeV,etal.(2017)Flipping chromosomesindeep-seaarchaea.PLoSGenet tionreactionswiththesameoutcomeashomologousrecombinationeventsonDNAseg- 13(6):e1006847.https://doi.org/10.1371/journal. mentsasshortas104bpbothinvitroandinvivo,incontrasttootherknowntyrosine pgen.1006847 recombinases.Throughserialculturing,weshowedthattheintegrase-mediateddivergence Editor:LotteSøgaard-Andersen,MaxPlanck ofT.nautilistrainsoccursatanastonishingrate,withatleastfourlarge-scalegenomicinver- InstituteforTerrestrialMicrobiology,GERMANY sionsappearingwithin60generations.OurresultsandtheubiquitousdistributionofpTN3- Received:December16,2016 likeintegratedelementssuggestthatamajormechanismofevolutionofanentireorderof Accepted:June1,2017 Archaearesultsfromtheactivityofaselfishmobilegeneticelement. Published:June19,2017 Copyright:©2017Cossuetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,which Authorsummary permitsunrestricteduse,distribution,and Mobileelements(MEs)suchasviruses,plasmidsandtransposonsinfectmostliving reproductioninanymedium,providedtheoriginal organismsandoftenencoderecombinasespromotingtheirinsertionintocellular authorandsourcearecredited. genomes.Theseinsertionsalterthegenomeoftheirhostaccordingtotwomainmecha- DataAvailabilityStatement:Allrelevantdataare nisms.First,MEsprovidenewfunctionstothecellbyintegratingtheirowngeneticinfor- withinthepaperanditsSupportingInformation mationintotheDNAofthehost,atoneormorelocations.Secondly,cellularhomologous files.TheNCBIdatabaseaccessionfor Thermococcus.sp.5-4genomicsequenceis recombinationwillactuponmultipleintegratedcopiesandproduceavarietyoflarge- CP021848. scalechromosomalrearrangements.Ifsuchmodificationsareadvantageous,theywill spreadintothepopulationbynaturalselection.Typically,enzymesinvolvedincellular Funding:ThisworkwasfundedbytheEuropean ResearchCouncilundertheEuropeanUnion’s homologousrecombinationandtheintegrationofMEsaredistinct.Wedescribeherea SeventhFrameworkProgram(FP/2007-2013)/ novelplasmid-encodedarchaealintegrasewhichinadditiontosite-specificrecombina- ProjectEVOMOBIL-ERCGrantAgreementno. tioncancatalyzelowsequencespecificityrecombinationreactionsakintohomologous 340440(MC,PF).Thefundershadnoroleinstudy recombination. design,datacollectionandanalysis,decisionto publish,orpreparationofthemanuscript. PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 1/27 Thermococcalesgenomeevolution Competinginterests:Theauthorshavedeclared Introduction thatnocompetinginterestsexist. Large-scalegenomicrearrangementsalloworganismstoevolvemuchmorerapidlythan throughrandommutationalone.Rearrangementscanresultinthemovementofgenes withingenomes,changesincodingstranduse,lossofnonessentialfunctionsandtheincor- porationofforeignDNA.Asaresult,theorganization,contentandprocessingofgenetic informationcanbedeeplyaltered.Inallthreedomainsoflife,chromosomalreorganization ismainlypromotedbyrecombinationbetweenhomologoussequences,forexamplebetween redundantribosomaloperons[1,2]orintegratedcopiesofmobileelements(ME)suchas prophages[3,4],transposons[5,6]andinsertionsequences(IS)[7].Suchrecombinationcan resultintheDNAinversionsreadilyobservedincloselyrelatedgenomes[8,9].Inadditionto homologousrecombination,chromosomescanundergorearrangementthroughretrotran- sposon-associatednon-homologousrecombination[10].Otherelementslikeintegrons conferrapidadaptationtobacteriainchangingenvironmentsbyshufflingcassettearrays encodingavarietyoffunctions,aprocessinvolvingasite-specificrecombinaseandtwo typesofattachmentsites[11].Furthergenomicrearrangement/reorganizationcanoccur throughtheacquisitionofnewgeneticmaterial,predominantlybylateralgenetransfer.Such genetransferoccursinallorganismsthroughinfectionbymobileelementssuchasvirusesor plasmids,orthroughtheuptakeoffreeorencapsulatedDNAfromtheenvironment[12,13]. Genomescanacquirenovelgenesinafashionrangingfromtransienttopermanentdepend- ingonthetypeofelementandthephysiologicalconditionsofthehost.WhenMEsucceedin stablyinsertingtheirgenome,theinsertedDNAisthenreplicatedaspartofthehostchro- mosome.ThetransactionsbetweenMEDNAandhostgenomearecatalyzedbyrecombi- nasestypicallyencodedbytheelementsthemselves.Theserecombinasesrankindifferent classesbasedontheirenzymaticactivityandthespecificityoftheirDNAtargets.Thesmall- estMEareinsertionsequences(IS)composedofashortDNAsegmentencodingonlythe enzymesinvolvedintheirtranspositionwhichcanoccuratmanydifferentgenomicloca- tions[14].TherelatedtransposonsarelargerDNAsegmentswhichcanbetransposedby twoflankingISandfrequentlycarryadditionalgenessuchasantibioticresistancedetermi- nants[15].ThemostfrequentISrecombinasesareDDEtransposaseswhichdonotform covalenttransposase-DNAintermediatesduringtransposition[16].Otherandtypically largerMEsuchasplasmidsandvirusesencoderecombinasespromotingDNAtransactions withastrongerDNAsequencespecificity.Suchsite-specificrecombinationisnotonlyused formobileelementintegrationandexcisioninbacteriabutalsointhespreadofantibiotic resistancebytransposableelements,thecontrolofplasmidcopynumber,regulationofgene expressionandtheresolutionofconcatenatedchromosomes[17].Site-specificrecombinases canbecategorizedintotheserinerecombinasesandtyrosinerecombinases(Y-recombi- nases);which,incontrasttoDDEtransposases,formcovalentenzyme-DNAintermediates duringrecombination,albeitwithmarkedlydifferentmechanismsofaction.Beforereliga- tionofthetworecombiningDNAstrands,serinerecombinasesgeneratebreaksinallstrands whileY-recombinasesproducetwosequentialsingle-strandbreaks[17].Asarule,site-spe- cificintegration/excisionreactionspromotedbyY-recombinasesoccurviaasynapticcom- plexcomposedoftwoDNAduplexescarryingthespecificsitesboundbyfourrecombinase protomers[17].Thetwo-recombinasepairsareactivatedsequentially,allowingonestrand fromeachduplextobeexchangedatatimeviatwoconsecutiveandsymmetricalHolliday junctions.AnotableexceptionisVibriocholeraephageCTX.Notonlydoesthisphageinte- grateintoitshostgenomeinsinglestrandedformwheretwositesfoldintoahairpinstruc- ture,mimickingarecombinationtargetforthecellularXerCDchromosomeresolvase;but alsoonlyrequiresXerCforintegration[18]. PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 2/27 Thermococcalesgenomeevolution Oneofthebest-studiedY-recombinasesistheintegraseofphageλ.Theprimaryfunction ofthisenzymeistheintegrationofphageDNAintothechromosomeofitsbacterialhost(and itsexcision).Thisfunctionisachievedbypromotingsite-specificrecombinationbetweenthe phageattachmentsiteattPanditschromosomalcounterpartattB[19].Underparticularcir- cumstances,theintegraseofthelambdoidphageHK022iscapableofgeneratinginversions betweenattPandasecondaryattachmentsiteintheHK022leftoperon[3].Similarly,thepri- maryfunctionoftheyeastFLPproteinisthecontrolofthe2μplasmidcopynumber[20] byDNAinversionbetweentwodivergent34bpFRTsiteslocatedontheplasmid[21].FLP recombinaseactivityhasalsobeensuccessfullyusedforintegrationandexcisionofsynthetic DNAinmammaliangenomes[22].TherecombinationactivitiesofbothλintegraseandFLP recombinasearesummarizedasshowninS1Fig.Historically,thisreciprocalandconservative recombinationbetweentwostringentlydefineddouble-strandedDNAsequencesineach chromosomewasdenominatedtheCampbellmodel[23]. ThesequencesofaconsiderablenumberofY-recombinaseshavebeencomparedtoreveal thepositionofconservedresiduesandinferthelocationofthecatalyticactivesite[24].They shareintheirC-terminalmoietyaratherwellconservedregionof~120aminoacidscontain- inguptosixnearlyinvariantaminoacidsR..K..HxxR..[W/H]..Yformingtheactivesite [25,26].AsmallnumberofY-recombinaseshavebeencharacterizedbiochemicallyinArchaea, forexampletheXerArecombinaseofthehyperthermophiliceuryarchaeonPyrococcusabyssi whichexhibitsaperfectactivesiteconsensus[27].Sequencealignmentshaverevealedthat otherarchaealactivesitesdivergeslightlyfromthebacterialconsensusR..HxxR..Y[28].The integrasesofvirusesSSV1isolatedfromthehyperthermophiliccrenarchaeonSulfolobusshiba- tae[29]andSSV2fromSulfolobusislandicus[30]sharetheconsensusR..KxxR..Ywhilethe plasmidicintegraseofSulfolobussp.NOB8H2displaysR..YxxR..Y[28]. Mobileelementsthereforecontributetogenomeevolutionthroughbothsite-specificand homologousrecombination,whichusuallyoperatebydistinctmechanismsandenzymatic activities.Homologousrecombinationisalsoknowntooccurfrequentlybetweenmultiple IScopiesresultinginlargescalearchaealgenomicrearrangements,asobservedinbothCre- narchaeotae.g.Sulfolobusislandicus[31]andEuryarchaotae.g.Pyrococcusabyssi[32].The distributionofarchaealISsispatchynotonlyatthephylumlevelbutalsoatgenuslevel[9]. Interestingly,genomeshufflingoccursinThermococcus[33]evenifISsareseldomfoundin thisgenussuggestingthatalternativerecombinationmechanismsarecapableofproducing large-scalegenomicrearrangements. Ifsite-specificrecombinationonlyrequiresspecificnucleotidesequencestargetedbyaded- icatedrecombinase,homologousrecombinationontheotherhandisamuchmorecomplex process.Inallorganisms,homologousrecombinationconstitutesoneofseveralpathwaysto repairdouble-strandbreaks.InadditiontoDNAsynthesis,itrequiresdedicatedrecombinases andtheiraccessoryfactorswhichactonstretchesofnear-sequence-identicalDNA.Ineukary- oticandbacterialcells,theenzymesandpathwaysinvolvedinhomologousrecombination havebeenextensivelystudied(see[34,35]forreviews),whereasarchaealhomologousrecombi- nationisstillanactivefieldofinvestigation.Itisknownthattheinitialresectioningstepafter double-strandbreakinvolvestheRad50–Mre11–HerA–NurAcomplextogenerate3’single- strandsubstrates[36,37].TheRecAparalogRadAanditsaccessoryfunctionsassociatewith thisssDNAtoconstitutethepresynapticfilament,whichwillscanandpairwithhomologous sequences[38].InthearchaeonThermococcuskodakarensis,homologousrecombinationhas beendetectedexperimentallybetweenstretchesofidenticalDNAsequencesequaltoorgreater than500bp[39]. Toourknowledge,adirectoverlapbetweensite-specificandhomologousrecombination processeshasnotbeendescribedsofar.Inthepresentwork,wereportthediscoveryand PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 3/27 Thermococcalesgenomeevolution characterizationofanewintegrasefromthehyperthermophilicarchaeonThermococcusnautili [40,41]capableofcatalyzingbothsite-specificrecombinationandlowsequencespecificity recombinationreactionsmimickinghomologousrecombination.Thewidedistributionofthis particularY-recombinaseamongtheThermococcusgenusprovidesavalidrationaleforthe observedgenomicrearrangementsintheseArchaea. Results DotplotcomparisonsidentifysyntenybreakpointsinThermococcus chromosomes Wecomparedthechromosomesofthe13completelysequencedThermococcusspeciesavail- abletodatebydotplotanalysisandobservedhighlevelsofgenomescramblingasshowninFig 1A.Strikingly,comparisonofT.onnurineusandT.sp.4557chromosomesbythisapproach revealedonlytwolargeinversionsof139/143Kband102/74Kbrespectively(Fig1B&1C). Thisrelativelysmallnumberofinversionsfacilitatedtheinvestigationofthesyntenybreak- pointsborderingbothinversions.UsingtheSyntTaxwebtool[42],acompositerepresentation wasobtainedasshowninFig1C.Geneorderisconservedimmediatelyupstreamanddown- streamofeachinversionborderandwasusedtoidentifythesyntenybreakpoints.Foreach inversion,thebreakpointsarelocatedwithintRNAgenepairs,transcribedinopposite orientations.Interestingly,T.nautiliplasmidpTN3integratesinthetRNALeugene BD01_0018[41,43](S2Fig)andthisgenedisplaysover97%sequenceidentitywithtRNALeu (GQS_t10759),whichbordersalargechromosomalinversionbetweenT.onnurineusandT. sp.4557(Fig1B).TheconcordancebetweenthechromosomalattachmentsiteofthepTN3 integrase(IntpTN3)andtherecombinationtargetsborderingeachinversion(inoppositeorien- tations)ledustodefineaworkingmodeltoexplaintheformationofgenomicinversions observedintheThermococcusgenus.Wehypothesizethatthefrequentgenomicinversions observedintheevolutionoftheThermococcalesorderarearesultofenzymaticactivityofthe integraseencodedbyhorizontallymobileelements,suchaspTN3. IntpTN3isabonafidetyrosinerecombinase TheintegraseofpTN3sharessignificantsequencesimilaritywithcanonicalY-recombinases anditspredictedactivesitecanbedefinedasR..K..AxxR..Ywhichonlyslightlydivergesfrom theconsensus(S3AFig).Inaddition,IntpTN3displaysahighdegreeofconservationwithtwo biochemicallycharacterizedhyperthermophilicY-recombinases,thearchaealIntSSV1[44]and IntSSV2[30](S3BFig).Thus,itseemedworthwhiletocomparetheenzymaticactivitiesof IntpTN3tothoseofotherenzymesofthesamefamilysuchasphageλintegraseandSaccharo- mycescerevisiae2μplasmidFLPproteinandtovalidatethemagainstthecanonicalY-recombi- nasemodel. IntpTN3isanactivesite-specifictyrosinerecombinase InordertocharacterizetheactivitiesofIntpTN3,itwasnecessarytoover-produceandpurify theenzyme(S4Fig)andtoconstructDNAsubstratescarryingappropriateattachmentsites (asdeterminedbysequentialdeletions(S5Fig).Anintegrasevariant(IntpTN3Y428A)inwhich thecatalytictyrosineissubstitutedwithanalaninewasconstructed,purifiedandtested(S6 Fig).WeusedtheseproteinsandDNAcomponentsinaseriesofinvitroandinvivoexperi- ments,detailedbelow,toascertainthepropertiesofIntpTN3. IntpTN3catalyzesattP-attBintegration. Inordertomeasuretheactivityofpurified IntpTN3,weinitiallydevelopedasimpletestinwhichintegrase-catalyzedintegrationofone PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 4/27 Thermococcalesgenomeevolution Fig1.Genomicdotplotsandsyntenyanalysis.Genomicdotplots(A)betweenT.kodakarensisandT.nautiliand(B) betweenT.onnurineusandT.sp.4557.Allgenomesarecenteredontheirputativepredictedoriginofreplication[33].C. ThetwosyntenybreaksinthegenomicalignmentbetweenT.onnurineusandT.sp.4557(PanelB)werefurtheranalyzed. Geneorderconservationandrecombinationendpointsofthetwomajorinversionswereidentifiedusingcompositeimages generatedbytheSyntTaxwebtool.Inversion“1”occurredbetweentRNALeu(GQS_t10759)andtRNAThr(GQS_t10745) PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 5/27 Thermococcalesgenomeevolution genes;T.sp.4557GQS_t10759geneisorthologoustotheT.nautilitRNALeugene(BD01_0018)whichcorrespondstothe chromosomalattachmentsiteofplasmidpTN3.Inversion“2”(PanelB)occurredbetweentRNALeu(GQS_t10807)and tRNAGly(GQS_t10803)genes. https://doi.org/10.1371/journal.pgen.1006847.g001 plasmid-encodedattBsiteinanidenticalsiteonasecondplasmidresultsinformationofa plasmid-plasmiddimer(S1AFig),whichcanbedetectedbygelelectrophoresis.Inaccordance withouridentificationoftRNALeuasapotentialattBsite,wegeneratedasupercoiledDNA templatecarryingaquasi-full-lengthT.nautilitRNALeugene,Leu2-88(seebelow).We observedtheformationofdimericDNAmoleculesonlywithDNAtemplatescarryingattB tRNALeu,andonlyinthepresenceofIntpTN3(Fig2).Thus,theIntpTN3isabletocatalyzethe site-specificrecombinationofoneattsitewithanother. IntpTN3catalyzesattL-attRexcision. ThecapacityofIntpTN3tocatalyzetheinversereac- tioni.e.theexcisionofaDNAsegmentlocatedbetweenattLandattRsiteswastestedusing thetemplatepMC479,whichcarriesaLeu2-88siteandaminimalLeu2-44siteinthesameori- entation,separatedbya762bpsegment.InthepresenceofIntpTN3,therestrictiondigestion patternrevealedthepresenceoftwobandsof2358and849bp,consistentwiththeexcisionofa circularDNAspeciesbetweentwoattBsites(Fig3).Therecombinationreactionalsogener- atedanadditionalbandof4056bp,explainablebytheintegrationofthe849bpcircularproduct intotheinitialpMC478template.ThisdemonstratesthatIntpTN3isabletoefficientlycatalyze bothDNAintegrationandexcisionreactions. IntpTN3canre-activaterelatedintegratedmobileelements. ThespeciesT.kodakarensis carriesinitsgenomethestablyintegratedelementTKV4[45],whichiscloselyrelatedtopTN3 ofT.nautili.AsshownforpTN3(S2Fig),theintegrationofTKV4intotheT.kodakarensis genomehasdisruptedthegeneencodingIntTKV4,renderingTKV4incapableofspontaneous chromosomalexcision.ConsideringthatIntpTN3andIntTKV4displayextensivesequencesimi- larity(S3Fig)andpromoteintegrationinorthologoustRNALeugenes[45],weinvestigatedthe capacityofIntpTN3toexciseTKV4invitro.ExcisionandcircularizationofaDNAmoleculeis detectablebyPCRamplificationusingsuitablyorientedprimers(Fig4A).TreatmentofT. kodakarensisgenomicDNAwithpurifiedIntpTN3resultedinproductsconsistentwithTKV4 circularization(Fig4B),demonstratingthatIntpTN3couldexcise,andhencere-activatethis dormantmobileelement.Inlightofthisinvitroactivity,weendeavoredtotestthisTKV4res- urrectionreactioninvivo.ThisexperimentinvolvedtheconstructionofspecializedT.koda- karensisexpressionvectorspRC524andpRC526expressingwildtypeIntpTN3andmutant IntpTN3Y428Arespectively(Fig4C)(seeMaterialandmethods).Surprisingly,bothIntpTN3 andtheactivesitemutantIntpTN3Y428AwereabletoreviveTKV4invivo(Fig4D).Notonly doesthisresultdemonstratetheabilityofpTN3toexcise,andthereforere-activateintegrated mobileelements,italsostronglysuggeststhattheactivityofmutatedIntpTN3Y428Acouldbe complementedbythetruncatedIntTKV4encodedbytheintegratedelement,whereasbothvari- antsareinactiveontheirown.Asimilarphenomenonofcomplementationhasbeenreported betweenaDNA-bindingimpairedmutantandacatalytictyrosineresiduemutantofIntSSV1 [44]. IntpTN3catalyzesDNAinversionbetweenattsites. TheabilityofIntpTN3tocatalyzethe inversionofDNAsequencesiskeyinourmodeloflarge-scaleintegrase-mediatedchromo- somalrearrangementsintheThermococcusgenus.TotesttheIntpTN3invertaseactivity,we constructedaplasmid(pMC478)withtwoattachmentsitesininvertedorientation:thefull- lengthtRNALeugeneandtheminimalLeu2-44.Therestrictiondigestionpatternshowedthe presenceoftwonewbandscorrespondingtotheinversionoftheDNAsegmentbetweenthe PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 6/27 Thermococcalesgenomeevolution Fig2.Dimerformation.Supercoiled(SC)plasmidspUC18andpJO322carryingtheLeu2-88fragment(S5A Fig)wereincubatedwithIntpTN3inastandardreaction(seeMaterialsandmethods)andcomparedwith linearizedpJO322byagarosegelelectrophoresis.TheintegrasehasnoeffectonpUC18withtheexception oftheproductionofafaintlinearspecies(indicatedbyanarrow).Theintegraseincreasesconsiderablythe formationofplasmidpJO322dimersandtoalowerextentthatofmultimers.Noincreaseintheformationof opencircular(OC)formwasobserved. https://doi.org/10.1371/journal.pgen.1006847.g002 PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 7/27 Thermococcalesgenomeevolution Fig3.Intptn3excisionandintegration.PlasmidpMC479carriestwocopiesoftRNALeuclonedindirect orientationandseparatedbya762bpspacerfragment(seeMaterialandmethods).Thedirectrepeatsconsist oftheminimaltRNALeu2–44andthelongertRNALeu2–88,bothproficientindimerizationreactions.Plasmid pMC479wasincubatedwithIntpTN3inastandardreaction(seeMaterialsandmethods).TheNdeIrestriction enzymegeneratestwofragmentsof3207and1366bprespectivelyinpMC479.UponincubationwithIntpTN3, NdeIdigestiongeneratesadditionalfragmentsof2358bpcorrespondingtorecombinedpMC479*and849bp correspondingtothecircularizedspacerandrecombinedattsite.Bothconstitutetheproductsoftheexcision reaction.Alarger4056bpfragmentisgeneratedaswellandcorrespondstotherecombinationproduct generatedbyintegrationofthe3207and849bpspecies.Therelativeintensityofthebandsiscompatiblewith anexpectedequilibriumreaction. https://doi.org/10.1371/journal.pgen.1006847.g003 attBsitesonlywhenDNAwastreatedwiththeintegrase(Fig5).Thisresultindicatesthat,like theS.cerevisiaeFLPrecombinase,IntpTN3iscapableofefficientlyperformingallthreecanoni- calreactionscharacteristicofsite-specificY-recombinases:integration,excisionandinversion. Norecombinationproductscouldbeobservedininversionreactionsperformedwiththeinac- tivatedintegrasevariantIntpTN3Y428A(S6Fig). SyntenyanalysisoftheinversionendpointsobservedbetweenT.sp.4557andT.onnurineus indicatesthatrecombinationmayhaveoccurredbetweendifferenttRNAgenes,namely betweentRNALeu(GQS_t10759)andtRNAThr(GQS_t10745)aswellasbetweentRNALeu (GQS_t10807)andtRNAGly(GQS_t10803).Interestingly,inversiontemplatescombining tRNALeuandtRNAThrfailedtoproducerecombinationproducts(Fig5). PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 8/27 Thermococcalesgenomeevolution Fig4.TKV4excisioninvitroandinvivo.APCRamplificationassaywasdesignedtoassertartificial IntpTN3-mediatedTKV4circularization(PanelA).Theassaywasfirstperformedinvitroonfoursamplesof purifiedT.kodakarensisgenomicDNAincubatedwithwildtypeIntpTN3orinactiveIntpTN3Y428Amutated enzymeinastandardreactionanalyzedbyagarosegelelectrophoresis(seeMaterialsandmethods).Only reactionsusingwild-typeenzymegenerateda1710bpbandoftheexpectedexcisionsize(PanelB).The sameTKV4excisionreactionwastestedinvivobytransformingT.kodakarensisKUW1withshuttleplasmids pRC524(expressingwildtypeintegrase)andpRC526(expressingmutatedIntpTN3Y428A)orwiththevector alone(PanelC).TotalDNAwasextractedfromthetransformantsandamplifiedasdescribedabove.Inthisin vivoexperiment,bothenzymeswereTKV4excision-proficient(PanelD). https://doi.org/10.1371/journal.pgen.1006847.g004 PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 9/27 Thermococcalesgenomeevolution Fig5.Intptn3inversion.PlasmidpMC477carriestwocopiesoftRNALeuclonedininvertedorientationand separatedbya892bpspacerfragment(seeMaterialandmethods).Theinvertedrepeatsconsistofthe minimaltRNALeu2–44andthelongertRNALeu2–88,bothproficientindimerizationreactions.Plasmid pMC473carriestRNALeu2–44andtRNAThrGQS_t10745,ininvertedorientationaswell.Bothplasmidswere incubatedwithIntpTN3inastandardreaction(seeMaterialsandmethods).TheNdeIrestrictionenzyme generatesineachcasetwofragmentsof2796and1777bp.UponincubationwithIntpTN3,NdeIdigestionof pMC477generatesadditionalfragmentsof2358and2215bpcorrespondingtotherecombinantpMC477*.As fortheintegration/excisionreactions,therelativeintensityofthebandsiscompatiblewithanexpected equilibriumreaction.WecouldnotdetectanyinversionbetweentRNALeuandtRNAThrinplasmidpMC473. https://doi.org/10.1371/journal.pgen.1006847.g005 Thermococcusnautiliundergoesrapidgenomicrearrangementunder laboratoryconditions Thelarge-scalegenomicinversionsobservedbetweenT.sp.4557andT.onnurineusdisplay minorgeneorderrearrangementsneartherecombinationendpointsindicatingthatthese eventsarenotrecentandmighthaveundergoneremodeling(Fig1C).Inordertoidentify morerecentrearrangements,weinvestigatedwhetherlarge-scalegenomicinversionscould occurspontaneouslyunderlaboratoryconditions.T.nautilicarryingitsnaturalplasmids wassub-culturedintwoindependentexperimentsfor60and66generations(thereforetermed T.nautili60Gand66G)inrichliquidmediumwithintermittentstorageat4˚Candthe PLOSGenetics|https://doi.org/10.1371/journal.pgen.1006847 June19,2017 10/27