Flipping chromosomes in deep-sea archaea

27 Pages·2017·9.88 MB·English

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]
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Abstract
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Oneofthemajormechanismsdrivingtheevolutionofallorganismsisgenomicrearrange-
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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.
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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].
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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
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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
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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)
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genes;T.sp.4557GQS_t10759geneisorthologoustotheT.nautilitRNALeugene(BD01_0018)whichcorrespondstothe
chromosomalattachmentsiteofplasmidpTN3.Inversion“2”(PanelB)occurredbetweentRNALeu(GQS_t10807)and
tRNAGly(GQS_t10803)genes.
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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
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Fig2.Dimerformation.Supercoiled(SC)plasmidspUC18andpJO322carryingtheLeu2-88fragment(S5A
Fig)wereincubatedwithIntpTN3inastandardreaction(seeMaterialsandmethods)andcomparedwith
linearizedpJO322byagarosegelelectrophoresis.TheintegrasehasnoeffectonpUC18withtheexception
oftheproductionofafaintlinearspecies(indicatedbyanarrow).Theintegraseincreasesconsiderablythe
formationofplasmidpJO322dimersandtoalowerextentthatofmultimers.Noincreaseintheformationof
opencircular(OC)formwasobserved.
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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.
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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).
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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).
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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.
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Thermococcusnautiliundergoesrapidgenomicrearrangementunder
laboratoryconditions
Thelarge-scalegenomicinversionsobservedbetweenT.sp.4557andT.onnurineusdisplay
minorgeneorderrearrangementsneartherecombinationendpointsindicatingthatthese
eventsarenotrecentandmighthaveundergoneremodeling(Fig1C).Inordertoidentify
morerecentrearrangements,weinvestigatedwhetherlarge-scalegenomicinversionscould
occurspontaneouslyunderlaboratoryconditions.T.nautilicarryingitsnaturalplasmids
wassub-culturedintwoindependentexperimentsfor60and66generations(thereforetermed
T.nautili60Gand66G)inrichliquidmediumwithintermittentstorageat4˚Candthe
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