In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

19 Pages·2017·4.89 MB·English

RESEARCHARTICLE
In vivo and in vitro protein imaging in
thermophilic archaea by exploiting a novel
protein tag
ValeriaVisone1,WenyuanHan2,GiuseppePerugino1,GiovannidelMonaco1,
QunxinShe2,MosèRossi1,AnnaValenti1*,MariaCiaramella1*
1 InstituteofBiosciencesandBioresources,NationalResearchCouncilofItaly,Napoli,Italy,2 Department
ofBiology,UniversityofCopenhagen,Copenhagen,Denmark
a1111111111
a1111111111 *[email protected](MC);[email protected](AV)
a1111111111
a1111111111
a1111111111 Abstract
Proteinimaging,allowingawidevarietyofbiologicalstudiesbothinvitroandinvivo,isof
greatimportanceinmodernbiology.Proteinandpeptidetagsfusedtoproteinsofinterest
providetheopportunitytoelucidateproteinlocationandfunctions,detectprotein-protein
OPENACCESS
interactions,andmeasureproteinactivityandkineticsinlivingcells.Whereasseveraltags
Citation:VisoneV,HanW,PeruginoG,del
aresuitableforproteinimaginginmesophilicorganisms,theapplicationofthisapproachto
MonacoG,SheQ,RossiM,etal.(2017)Invivo
andinvitroproteinimaginginthermophilic microorganismslivingathightemperaturehaslaggedbehind.Archaeaprovideanexcellent
archaeabyexploitinganovelproteintag.PLoS anduniquemodelforunderstandingbasiccellbiologymechanisms.Here,wepresentthe
ONE12(10):e0185791.https://doi.org/10.1371/
developmentofatoolkitforproteinimaginginthehyperthermophilicarchaeonSulfolobus
journal.pone.0185791
islandicus.Thesystemreliesonathermostableproteintag(H5)constructedbyengineering
Editor:GiovanniMaga,IstitutodiGenetica
thealkylguanine-DNA-alkyl-transferaseproteinofSulfolobussolfataricus,whichcanbe
Molecolare,ITALY
covalentlylabeledusingawiderangeofsmallmolecules.Asasuitablehost,wecon-
Received:June16,2017
structed,byCRISPR-basedgenome-editingtechnology,aS.islandicusmutantstrain
Accepted:September19,2017 deletedforthealkylguanine-DNA-alkyl-transferasegene(Δogt).Introductionofaplasmid-
Published:October3,2017 borneH5geneinthisstrainledtoproductionofafunctionalH5protein,whichwassuccess-
fullylabeledwithappropriatefluorescentmoleculesandvisualizedincellextractsaswellas
Copyright:©2017Visoneetal.Thisisanopen
accessarticledistributedunderthetermsofthe inΔogtlivecells.H5wasfusedtoreversegyrase,apeculiarthermophile-specificDNAtopo-
CreativeCommonsAttributionLicense,which isomeraseendowedwithpositivesupercoilingactivity,andallowedvisualizationofthe
permitsunrestricteduse,distribution,and
enzymeinlivingcells.Tothebestofourknowledge,thisisthefirstreportofinvivoimaging
reproductioninanymedium,providedtheoriginal
ofanyproteinofathermophilicarchaeon,fillinganimportantgapinavailabletoolsforcell
authorandsourcearecredited.
biologystudiesintheseorganisms.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Funding:Thisworkwassupportedbya)Italian
MinistryofEducationandResearch,Firb-Futuroin
Ricerca,NematictoAV;b)ResearchFund
Denmark,DFF-0602-02196toQS.Thefundershad
noroleinstudydesign,datacollectionand
Introduction
analysis,decisiontopublish,orpreparationofthe
manuscript. Detectionandanalysisofproteinsintheircellularcontextandundertheirphysiologicalcondi-
tionsiscrucialforunderstandingtheirfunction.Overthelastfewdecades,greatprogresshas
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. beenobtainedtowardthistaskbyexploitingfluorescentproteintags,suchastheGreen
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 1/19
Proteinimaginginthermophilicarchaea
FluorescentProtein(GFP)anditsderivatives,andtheseproteinscanbefusedtoproteinsof
interestenablingawidevarietyofbiologicalstudies[1,2,3].
AsanalternativetoGFP-basedsystems,otherproteinandpeptidetagshavebeenrecently
introduced[2].Oneofthemostpromisingistheso-calledSNAP-tag,proposedbyK.Johnsson
andcolleagues[4,5].Thisapproachexploitstheuniquepropertiesofaproteininvolvedinrepair
ofalkylationdamageinDNA,theO6-alkylguanine-DNAalkyl-transferase(AGTorOGT).
UponreactionofthisproteinwithO6-benzylguanine(BG),asmallmoleculeactingasirrevers-
ibleinhibitoroftheenzyme,acovalentbondbetweenthebenzylgroupandaspecificcysteine
residueintheproteinactivesiteisformed.IfBGisconjugatedwithasuitablechemicalgroup
(suchasafluorophore,biotin,andsoon),thislatteristransferredtotheproteinmolecule,thus
resultingincovalentlabelingofAGTandAGT-containingchimericproteins(Fig1A)[6].
Fig1.H5labeling.A.SchemeofthereactionleadingtoirreversiblelabelingofH5withafluorescent
derivativeofbenzyl-guanine.B.SDS-PAGEofpurifiedH5labeledwithtwodifferentbenzyl-guanine
derivatives.
https://doi.org/10.1371/journal.pone.0185791.g001
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 2/19
Proteinimaginginthermophilicarchaea
Althoughthisapproachreliesontheadditionofanexternalsubstrate,whichcellsmaynotbe
permeableto,itholdsseveraladvantagesascomparedwithGFPs:itisextremelyspecific,highly
versatile,andoffersthepossibilitytolabelproteinswithvirtuallyunlimitedchemicalgroups[6].
BecausebothGFPandSNAP-tagaremesophilicproteins,invitrotheycanonlyfunction
undermildreactionconditions,andtheiruseforinvivoimaginghasbeenessentiallyrestricted
toorganismslivingintherangeofmesophilictemperatures.AlthoughthermotolerantGFPs
variantshavebeenrecentlyproduced[7,8],theapplicationofproteintagstothermophilicand
hyperthermophilicmicroorganisms(bothbacteriaandarchaea)haslaggedbehind.
WehavepreviouslyobtainedanengineeredversionoftheOGTproteinfromthehyper-
thermophilicandacidophilicarchaeonSulfolobussolfataricus[9,10].Thismodifiedprotein,
namedH5,wasobtainedbymutationoffiveaminoacidresiduesintheproteinhelix-turn-
helixdomain,thusimpairingtheDNAbindingactivity;likethewild-typeOGT,H5canbe
effectivelylabeledwithfluorophoresorotherchemicalgroupsconjugatedwithabenzyl-gua-
nine(Fig1B),andisthussuitableasacandidateproteintagforthermophilicorganisms.
AsatypicalS.solfataricusprotein,H5displayedastrongstabilityunderharshconditions,
includinghightemperature,extremesofpH,ionicstrength,presenceoforganicsolventsand
digestionwithproteases[10].TheH5proteinwasfusedtotheS.solfataricusβ-glycosidase,giv-
ingrisetoachimericproteinwhichwascorrectlyexpressed,folded,functionalandstablein
bothEscherichiacoliandthethermophilicbacteriumThermusthermophilus,andcouldbe
imagedinlivingcellsaswellasincell-freeproteinextracts[10].Thus,H5behavedlikeather-
mostableversionofthecommercialSNAP-tagprotein,whichiswidelyusedforstudiesin
mesophilicorganisms[4,11].
Sofarnoproteintaghasbeenreportedforhyperthermophilicarchaea.Thecellbiologyof
theseorganismsisofparticularinterest,notonlyfortheirpeculiarlifestyle,butalsobecause
theircellmachineriesdevotedtoDNAreplicationandrepair,andgenetranscriptionshare
commonevolutionaryoriginwiththoseofeukaryotes.Therefore,theseorganismsprovide
goodmodelsystemstostudythemostbasicmechanismsofgenome-relatedprocessesinthe
lifebranchofEukaryaandArchaea.
Oneofthemostfascinatingandmysteriousproteinsofhyperthermophilicorganismsis
reversegyrase(RG).ItisauniqueDNAtopoisomerasethatintroducespositivesupercoilsinto
DNAmolecules(forreviews,see[12,13,14])andisexclusivelyfoundinorganismsliving
above60˚C[15,16],thussuggestingthattheenzymeplaysaroleinadaptationtohightemper-
atures.Accordingly,positivesupercoilingispredictedtoprotectDNAfromdenaturationat
thegrowthtemperaturesofhyperthermophiles.AnumberofstudiessupportaroleforRGin
DNAprotection,repairandresponsetoDNAdamage:itactsasaDNArenaturase,promoting
annealingofcomplementarysingle-strandedDNAcircles[17];bindstosinglestrandedbreaks
onDNAandpreventsDNAthermaldenaturationattheDNAbreaks[18];isdegradedafter
treatmentofS.solfataricuscellswithalkylatingagents,inconcomitancewithdegradationof
genomicDNA[19];interactswithandinhibitsthetranslesionDNApolymerasePolYandthe
single-strandDNAbindingprotein,SSB[20,21];isrecruitedtoDNAafterultravioletirradia-
tion[22].Inaddition,RGisabletoresolveinvitroHollidayjunctionsfollowingbothATP-
dependentandATP-independentmechanisms[23,24].
Despitebiochemicalandstructuraldata,thefunctionofthisenzymeisstillunderdebate.
Geneticstudiesindifferentarchaealspeciesgavecontradictoryresults:aRGknockoutstrain
ofThermococcuskodakaraensiswasviable,butshowedslowergrowthathighertemperatures,
ascomparedwiththewildtype[25].Incontrast,deletionoftheRGgeneinPyrococcusfuriosus
waslethalattemperatureshigherthan95˚C[26];finally,thetwoRGencodinggenesofthe
crenarchaeonSulfolobusislandicuswerebothessentialforgrowthatanytemperature[27].
Thus,whereasRGislikelyinvolvedinthermotoleranceandfundamentalDNA-relatedprocess
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 3/19
Proteinimaginginthermophilicarchaea
athightemperature,itsabsenceseemstohavedifferentdegreesofseverityindifferentspecies.
Thereasonsforthesediscrepanciesarecurrentlyunknownandmanygapsarestillpresentin
ourunderstandingofthefunctionofthispeculiartopoisomerase.
Here,wepresentthedevelopmentofathermostableproteintagsuitableforproteinvisuali-
zationinthehyperthermophiliccrenarcheaonS.islandicus,oneofthemostusefularchaeal
modelsforgeneticstudies[28].Tothisaim,weconstructed,byaCRISPR-basedgenome-edit-
ingmethod[29],aS.islandicusmutantstraindeletedfortheogtgene(Δogt).Thisstrainwas
transformedwithanautonomouslyreplicatingplasmidexpressingtheH5protein.Theprotein
wassuccessfullyexpressedandcouldbeefficientlylabeledusingspecificfluorescentsubstrates
inS.islandicuslivingcells.Inordertodemonstratetheutilityofthistag,wefusedH5tothe
TopR1RGfromS.solfataricus.Thechimericproteinwascorrectlyfoldedwhenexpressedin
E.coliandretainedboththealkyl-transferaseandpositivesupercoilingactivities;moreover,
thefusionproteinwassuccessfullyexpressedandfunctionalinS.islandicusΔogtcellstrans-
formedwithasuitableexpressionvector.Thepotentialapplicationsofthistechnologyforcell
biologystudiesinthermophilicorganismswillbediscussed.
Resultsanddiscussion
ConstructionofaS.islandicusogt-KOstrain
Inaproof-of-conceptwork,wehavepreviouslyshownthattheH5proteincanbesuccessfully
usedasaproteintaginthethermophilicbacteriumT.Thermophilus[10];thisorganismwasa
convenientmodeltodemonstratetheusefulnessofourtagbecauseitisgeneticallytractable
andlacksendogenousDNAalkyl-transferaseactivity[30].
Wesoughttoextendtheapplicationofoursystemtohyperthermophilicarchaea,forwhich
noproteintagisavailable.Althoughtheseorganismsareprokaryotes,theyuseregulatoryele-
mentsandmechanismsdistinctfrombacteria.Geneticmanipulationhasrecentlybecamepos-
sibleonlyforalimitednumberofhyperthermophilicarchaealspecies,thusweneededto
identifyanorganismsuitableforourpurpose,i.e.,transformableandselectable,lacking
endogenousDNAalkyl-transferaseactivityandpermeabletotheH5substrate.Wechoose
S.islandicus,forwhichvectors,transformationandgeneknockoutsystemsareavailable.Pre-
liminarytestsshowedthatS.islandicuscellsarepermeabletotheH5substrate(datanot
shown;seebelow).However,theS.islandicusgenomecontainsanORF(SiRe_0281)poten-
tiallycodingforanOGTortholog,whichshares99%similaritywiththeS.solfataricusogtgene
(datanotshown).Sincetheactivityofthisproteinmightinterferewithourassay,aprerequi-
sitefortheapplicationoftheH5tagwastheconstructionofaS.islandicusmutantstrainin
whichtheogtgenewasdeletedbyusingtheCRISPR-basedgenome-editingmethodwhichhas
recentlybeendevelopedforthisspecies[29].Theknockoutplasmid(pKO-ogt)wascon-
structedasdescribedinMaterialsandMethods.TheplasmidcontainsadonorDNAfragment
carryinganin-framedeletionintheogtgeneandanartificialmini-CRISPRarraywithaspacer
matchingaprotospacerintheogtgenesequence,andimportantly,theprotospacerisabsent
fromtheogtdeletionallele(Fig2A).UpontransformationoftheplasmidintoS.islandicus
E233S1,twogeneticeventswouldoccurinthetransformants:(a)recombinationbetweenthe
donorDNAandthewild-typeogtgenelocuswouldyieldthedesiredogtmutantalleleonthe
chromosome,and(b)crRNAgeneratedfromtheexpressionoftheplasmid-bornemini-
CRISPRarraywouldformribonucleoproteincomplexeswithCasproteinsencodedbythe
endogenousCRISPR-Cassystemandguidethemtomediateself-targetingonthechromo-
somescontainingthewild-typeogtgene(Fig2A).Sinceogtdeletionmutantslacktheproto-
spacer,theyarenottargetedbytheCRISPRactivity,whereasthewild-typechromosomeis
targetedforDNAdegradation.Therefore,coloniesofpKO-ogttransformantsgrownon
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 4/19
Proteinimaginginthermophilicarchaea
Fig2.ConstructionandanalysisofaS.islandicusΔogtstrain.A.SchemeoftheconstructionoftheS.
islandicusΔpyrEFogt-KOderivativestrain(Δogt).B.PCRtestofnineΔogtstrainsandΔpyrEFasacontrol.
Theprimers(T-UPandT-DW)areindicatedin(A).C.Westernblotanalysisoftotalcellextracts(400μg/lane)
preparedfromculturesofΔpyrEF(lane1)andΔogt(lane2)strains.Thefilterwasprobedwiththeantibody
againsttheS.solfataricusOGTprotein[9];asacontrolforproteinloading,thesamefilterwasstrippedand
probedagainsttheS.solfataricusTopR1protein[21].
https://doi.org/10.1371/journal.pone.0185791.g002
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 5/19
Proteinimaginginthermophilicarchaea
Table1. OligonucleotidesusedfortheconstructionoftheS.islandicusΔogtstrain.
Name Sequence
KOSiRe_0281spF 5’-aagCTTGGCTATATAACTGTTGCTAAGGACGATAAGGGATTTA-3’
KOSiRe_0281spR 5’-agcTAAATCCCTTATCGTCCTTAGCAACAGTTATATAGCCAAG-3’
KOSiRe_0281Lf 5’-ctttgcatgcCCGCGTTGCAAGAATCGGGC-3’
KOSiRe_0281Lr 5’-CCACATCATTCCCCATACACTAGCACAAGTATTAAT-3’
KOSiRe_0281Rf 5’-GCTAGTGTATGGGGAATGATGTGGAAAAATTTAACAG-3’
KOSiRe_0281Rr 5’-gtttctcgagCCATCCCTTGTTTCTCTACG-3’
KOSiRe_0281T-UP 5’-CTAAGACAGTGGAAGTTTGGC-3’
KOSiRe_0281T-DW 5’-CCACGTCTTGGTTGTCCAGTC-3’
https://doi.org/10.1371/journal.pone.0185791.t001
uracil-freenutrientplatesshouldbethedesignedogtdeletionstrain.Indeed,characterization
ofninecoloniesbyPCRamplificationoftheogtdeletionallelefromthecandidatemutants
usingtheT-UPandT-DWprimerset(Table1)andagarosegelelectrophoresisoftheresulting
PCRproductsrevealedthatallninecoloniesonlycontainedtheogtdeletionallele(Fig2B).
Furthermore,DNAsequencingofthePCRproductsrevealedthatthedesignedmutationwas
obtained,whichretainedashorttruncatedpeptideof6aminoacidsintheogtdeletionallele.
Thestrain,hereaftercalledΔogt,wasviableat75˚CandtheabsenceoftheOGTproteinwas
confirmedbywesternblotoftotalcellextracts,whichshowedabandreactingwiththeanti-
bodydirectedagainsttheS.solfataricusOGTprotein[9]intheΔpyrEFstrain,butnotinΔogt
(Fig2C).
HeterologousexpressionofSsOGT-H5inS.islandicusandinvivo
labeling
Havingobtainedasuitablehost,thenextstepwastointroducetheH5proteininS.islandicus
cells.Tothisaim,weconstructedanexpressionvectorsuitabletotransformtheΔogtstrain
basedonuracilselection.StrainscarryingtheΔpyrEFmutationareunabletoproduceuracil
thatisnecessaryforthegrowthinminimalliquidmedium(SCVmedium);transformantscan
beselectedforbycomplementation,providedthatafunctionalpyrEFcassetteispresentinthe
plasmid.Complementation-basedstrategiesareaconvenientchoiceforSulfolobusspecies,
alsobecauseofthelimitedsuccessintheuseofantibioticsasselectivemarkersinhyperther-
mophilicArchaea[31].Toconstructtheexpressionvector,theSsOgt-H5genewasclonedin
thepSeSDplasmid(Fig3A)[32],whichcarriesasyntheticarabinose-induciblepromoterthat
confershighlevelsofproteinexpression.TheresultantrecombinantplasmidwascalledpSH5.
ThepSH5plasmidwasintroducedintheΔogtstrainbyelectroporation,andtransformants
wereselectedbygrowthinaliquidselectivemedium(SCV,seeMaterialsandmethods)at
75˚C;after5days,transformedculturesreached*0.7/0.8OD .ThepresenceoftheH5pro-
600
teinintransformantswasconfirmedbywesternblot(Fig3B).TheH5proteinwasexpressed
fromtheplasmid-bornegeneatabout0.2ngμg-1oftotalproteinextract,whichwascompara-
bletotheleveloftheendogenousOGTproteinintheparentalΔpyrEFstrain(Fig3C).
WenextwantedtotestwhethertheH5proteinwasfunctionalintransformants.Tothis
aim,weappliedafluorescentassaywepreviouslydeveloped,whichutilizesafluorescentderiv-
ativeoftheBGsubstrate(BG-FL)[9,10].BG-FLwasincubatedwithcellfreeextractsprepared
fromculturesoftheΔogtstraintransformedwitheitherpSH5orthepSeSDemptyvector.
Afterincubationat70˚Cfor30min.,samplesweredenaturedandloadedonSDS-PAGEfor
fluorescenceimaginganalysis.Afluorescentbandoftheexpectedmolecularweightwas
observedinextractsfromcellstransformedwithpSH5,butnotpSeSD,thusconfirmingthat
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 6/19
Proteinimaginginthermophilicarchaea
Fig3.HeterologousexpressionofH5intheS.islandicusΔogtstrain.A.ThepSeSDplasmidusedto
transformS.islandicus[32].TheH5andH5-TopR1geneswereinsertedintheNdeI-SalIandNdeI-NheI
restrictionsites,respectively.B.Westernblotanalysisofcellextracts(400μg/lane)preparedfromculturesof
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 7/19
Proteinimaginginthermophilicarchaea
theΔogtstraintransformedwitheitherpSH5(lane1)orthepSeSDemptyvector(lane2).Thesamefilterwas
strippedandprobedwithantibodiesagainsttheindicatedproteins.C.Westernblotanalysisusingtheanti-
OGTantibody[9].Lane1:purifiedH5protein(400ng);lane2:S.islandicusΔpyrEFcellextracts(400μg);
lane3:ΔogttransformedwithpSH5cellextracts(300μg).Thearrowsindicatethemigrationofendogenous
S.islandicusOGT(Si-OGT)andheterologousH5.
https://doi.org/10.1371/journal.pone.0185791.g003
theH5proteiniscorrectlyexpressedandproficientforfluorescentlabelingintheheterologous
host(Fig4A).
InordertotestthepermeabilityofS.islandicuscellstotheBG-FLsubstrate,intactΔogt
cellstransformedwitheitherpSH5orpSeSDwereincubatedinthepresenceofBG-FLat70˚C
fordifferenttimes;afterreaction,cellswerewashed,denaturedandloadedonSDS-PAGEfor
fluorescenceimaginganalysis.AsshowninFig4B,theresultswereverysimilartothose
obtainedwithcellsextracts(compareFig4Aand4B),withastrongfluorescentbandseenin
ΔogtcellstransformedwithpSH5,butnottheemptyplasmid.ThisresultsuggeststhatS.islan-
dicuscellsarepermeabletotheBG-FLsubstrate.
Thisresultwasconfirmedbyfluorescencemicroscopy(Fig5).IntactΔogtcellstransformed
withtheemptypSeSDincubatedwithBG-FLandwashedasdescribedintheMaterialand
Methodssection,showedlittlenofluorescentsignals;incontrast,strongfluorescentsignals
appearedincellstransformedwiththepSH5plasmid,indicatingthatS.islandicuscellsareper-
meabletotheBG-FLsubstrateandthattheobservedlabelingisspecificfortheH5protein.
Quantitativeanalysisshowedthatmorethan80%ofcellstransformedwithpSH5,butonly
about5%ofthosetransformedwithpSeSDshowedfluorescentsignals,suggestingthatthe
plasmidisstablyretainedduringgrowthandtheH5proteiniscorrectlyexpressed,foldedand
activeinthevastmajorityoftransformedcells.TheseresultsshowedthatourthermostableH5
tagissuitableforproteinlocalizationandanalysisinthishyperthermophilicarchaeon.
ApplicationoftheH5tagtodetectionofRGinS.islandicus
InordertovalidatethesuitabilityofH5asathermostabletagforproteinimaging,wesought
tovisualizeRGinS.islandicuscells.Tothisaim,theS.solfataricustopR1genewasfuseddown-
streamtoandinframewiththeH5gene,obtainingthechimericH5-topR1gene.Tobesure
thatthepresenceoftheH5tagattheN-terminalendoftheenzymedoesnotaffectRGactivity,
wefirstsoughttotestthefunctionalityofthefusionprotein.Tothisaim,thechimericgene
wasclonedinthepQEvectorandintroducedinE.coliABLECstrain.Thefusionproteinwas
successfullyexpressedinE.coli,asshownbywesternblotanalysis(Fig6A).Theabilityofthe
fusionproteintointroducepositivesupercoilsintoDNAmoleculeswasassayedintotalpro-
teinextractsaspreviouslyreported[20,33].AsshowedinFig6B,proteinextractscontaining
thefusionproteinexhibitpositivesupercoilingactivity,indicatingthatthepresenceoftheH5
tagdoesnotimpairtheenzymeactivity.
Purificationofthefusionproteinbyaffinitychromatographythroughanickelcolumnwas
performed,exploitingthepresenceofaHisx6-tagattheN-terminusandthethermostabilityof
bothRGandH5toperformathermoprecipitationstep(datanotshown).Totestwhetherthe
fusionproteinmightbelabeledbythefluorescentsubstrate,weappliedthesamealkyl-trans-
feraseassaydescribedabove[9].SDS-PAGEshowedthepresenceofafluorescentbandofthe
expectedmolecularweight(Fig6C),indicatingthattheH5portionisfunctionalinthefusion
protein.AspreviouslyshownforH5[10],thechimericproteincouldbelabeledatboth25and
70˚C,although,asexpected,moreefficientlyatthelattertemperature(Fig6C).
InordertovisualizeRGinS.islandicus,cells,theH5-TopR1codingsequencewasclonedin
thepSeSDplasmidandtheresultantvector,calledpSH5-TopR1,wasusedtotransformtheS.
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 8/19
Proteinimaginginthermophilicarchaea
Fig4.ActivityoftheH5proteininS.islandicus.A.Cellfreeextracts(200μg)preparedfromculturesoftheΔogtstraintransformed
withpSH5(lane1)orthepSeSDemptyvector(lane2);lane3contains100ngoftheH5proteinpurifiedfromE.coli.Sampleswere
incubatedwiththeBG-FLsubstrate(2.5μM)for30min.at70˚CandloadedonSDS-PAGE;thegelwasexposedforfluorescence
imaginganalysis,blottedandstainedwithCoomassieblue.Finally,thefilterwasprobedwiththeanti-OGTantibody(bottompanel).B.
PermeabilityofS.islandicustotheBG-FLsubstrate.WholetransformedcellswereincubatedinSCVmediuminthepresenceof3.0μM
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 9/19
Proteinimaginginthermophilicarchaea
ofBG-FL,at70˚Cfortimesasindicated.Lane1contains100ngofpurifiedH5protein;lanes2and3containΔogtcellstransformedwith
pSH5;lanes4and5containΔogtcellstransformedwithpSeSD.Thegelwasexposedforfluorescenceimaginganalysis,blottedand
stainedwithCoomassieblue.Finally,thefilterwasprobedwiththeanti-OGTantibody(bottompanel).
https://doi.org/10.1371/journal.pone.0185791.g004
islandicusΔogtmutant.Transformantswereabletogrowat75˚Cshowingnoapparentgrowth
defect(datanotshown).ThepresenceoftheH5-TopR1proteinintransformantswascon-
firmedbywesternblot(Fig7A):anantibodydirectedagainstS.solfataricusTopR1recognized
onebandintheΔogtstraintransformedwithpSeSD,correspondingtoendogenousS.islandi-
cusprotein(whichshares91%sequenceidentitywithTopR1).Inthesamestraintransformed
withpSH5-TopR1theantibodyrecognizedtwobands,correspondingtoendogenousRGand
H5-TopR1,respectively;assumingthattheaffinityoftheantibodyforthetwoproteinsissimi-
lar,weestimatedthatthefusionisexpressedatapproximatelythesamelevelsastheendoge-
nousRG.Interestingly,theanti-OGTantibodyrevealedthepresenceofsmallerbandsin
extractsofcellstransformedwithpSH5-TopR1(Fig7A,right).Sincetheseshorterfragments
werenotevidencedbytheanti-TopR1antibody,weconcludethatthesefragmentscontainH5
andN-terminalportionsofTopR1,andcouldbeduetoeitherdegradationorprematureter-
minationofthechimericprotein(seealsobelow).Degradationand/orprematuretermination
hasbeenreportedforbothendogenousandheterologouslyexpressedS.solfataricusRG
[19,34].
TheH5-TopR1proteinwasvisualizedinvivobyfluorescencemicroscopyanalysisofintact
S.islandicuscells(Fig7B).AsshownaboveforcellsexpressingH5alone,weobservedspecific
fluorescentsignalsincellstransformedwithpSH5-TopR1,thusshowingthattheH5-TopR1
proteinisproficientforlabelinginlivingcells.Quantitativeanalysisshowedthatabout70%of
Fig5.Fluorescencemicroscopy.S.islandicusΔogtcellstransformedwiththeemptyvector(top)orwith
thepSH5plasmid(bottom)wereincubatedwithBG-FL(3μM)andthenanalysedatfluorescencemicroscopy.
Imagesshowbrightfield(BHF),AlexaFluor488(green)andmergedimages.
https://doi.org/10.1371/journal.pone.0185791.g005
PLOSONE|https://doi.org/10.1371/journal.pone.0185791 October3,2017 10/19