A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Genetics

and

Molecular

Microbiology

A

protein

expression

system

for

tandem

affinity

purification

in

Xanthomonas

citri

subsp.

citri

Giordanni

C.

Dantas

_,

_Paula

_M.M.

_Martins

_,

_Daniela

_A.B.

_Martins

_,

_Eleni

_Gomes

_,

Henrique

Ferreira

a_{Depto.deBioquimicaeMicrobiologia,InstitutodeBiociências,UniversidadeEstadualPaulista,RioClaro,SP,Brazil} b_{Depto.deBioquímicaeTecnologiaQuímica,InstitutodeQuímica,UniversidadeEstadualPaulista,Araraquara,SP,Brazil} c_{Depto.deBiologia,UniversidadeEstadualPaulista,SãoJosedoRioPreto,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7May2015 Accepted23October2015 Availableonline2March2016 AssociateEditor:SolangeInes Mussatto Keywords: Citruscanker Expressionvectors TAP-tag

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Citruscanker,causedbytheGram-negativebacteriumXanthomonascitrisubsp.citri(Xac),is oneofthemostdevastatingdiseasestoaffectcitruscrops.Thereisnotreatmentforcitrus canker;effectivecontrolagainstthespreadofXacisusuallyachievedbytheelimination ofaffectedplantsalongwiththatofasymptomaticneighbors.Anindepth understand-ingofthepathogenisthekeystoneforunderstandingofthedisease;tothiseffectweare committedtothedevelopmentofstrategiestoeasethestudyofXac.Genome sequenc-ingandannotationofXacrevealedthat∼37%ofthegenomeiscomposedofhypothetical ORFs.TostartasystematiccharacterizationofnovelfactorsencodedbyXac,weconstructed integrative-vectorsforproteinexpressionspecifictothisbacterium.Thevectorsallowfor theproductionofTAP-taggedproteinsinXacundertheregulationofthexylosepromoter.In thisstudy,weshowthataTAP-expressionvector,integratedintotheamylocusofXac,does notcompromiseitsvirulence.Furthermore,ourresultsalsodemonstratethatthe polypep-tideTAPcanbeoverproducedinXacandpurifiedfromthesolublephaseofcellextracts.Our resultssubstantiatetheuseofourvectorsforproteinexpressioninXacthuscontributing anoveltoolforthecharacterizationofproteinsandproteincomplexesgeneratedbythis bacteriuminvivo.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:henrique.ferreira@linacre.oxon.org(H.Ferreira).

1 _{Theseauthorscontributedequallytothiswork.}

http://dx.doi.org/10.1016/j.bjm.2016.01.026

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Citruscanker,oneofthemostimportantdiseasesaffecting cit-ruscropsworldwide,iscausedbyaGram-negativebacterium,

Xanthomonas citri subsp. citri (Xac). The complete genome sequenceofXac,whichwasreportedmorethanadecadeago,1

hassignificantlycontributedtoimprovingourunderstanding ofthemolecularprocessesofthisplantpathogen.2–5 _Ofthe

4313XacORFsthathavebeenannotated,nearly63%exhibit homologytogenesofknownfunctionwhereas∼37% corre-spondto hypotheticalORFs thatcould potentially code for novelpolypeptides.1_{SuchadistributionpatternofORFsisa}

commonfeatureinmicrobialgenomesanditishypothesized thatasignificantamountofinformationregardingadaptation andpathogenicitymaybeencodedwithinsuchhypothetical proteins.Thebiggestchallengeincharacterizingthese hypo-theticalORFsisthatthephenotypesgeneratedbythemare usuallyunknown.

Amongtechniquesthathavethepotentialtobeusedfor the characterization of hypothetical proteins we cite: two dimensional (2-D) protein gels allied to mass spectrome-try, DNA microarrays, and next generation sequencing of nucleicacids(NGS).6–15_{Allofthesemethodsallowfor}

high-throughputprocessingofcandidategenes/proteins andthe dataobtainedbyanalyzingpatternsofco-expressionand co-repression(exhibitedbyaparticulargene/polypeptideduring different growth or developmentconditions) in turn helps intheattributionoffunction.Inaddition tothe aforemen-tionedtechniques,invivoproteinlocalizationviafluorescence microscopyhasalsoproventobeapowerfultoolfor annotat-ing functionto unknown proteinfactors.16,17 _However,the

mostpromisingtechniques in this field are those that are capableofdemonstratingadirectcontactbetween hypotheti-calproteinsand(an)othercellularfactor(s).Onesuchmethod istheyeast-two-hybrid (YTH)techniqueanditsvariantthe bacterial-two-hybrid(BTH)system.18,19_{Oneofthefirstmajor}

successesoftheYTHsystemwastheconstructionofa com-plexprotein–proteininteractionmapofthehumanpathogen

Helicobacterpylori.20_{InXacthismethodhasbeenappliedfor}

theidentificationand characterizationofseveralpreviously unknownproteincomponentsofthetypeIIIandIVsecretion systems.2,3_{InspiteoftheobviousvalueandutilityoftheYTH}

technique,themethodologysuffersfromcertainserious dis-advantagessuchasthe needtoconstructagenomic/cDNA expressionlibraryoftheorganismunderstudy.Another tech-nique,aninterestingalternative to the YTH/BTHmethods, istheTandemAffinityPurificationstrategy(TAP-tagging).21,22

TAP-taggingwasdevelopedtoaidinthepurificationofactive macromolecular complexes from yeast; it differs from the two-hybridsystemsintheaspectthatitdoesnotrequirethe construction ofprotein expressionlibraries tobe screened withbaits.ThebasisofTAP-taggingis theexpression ofa fusion-proteincomprisingoftheproteinofinterestfusedto aTAP-tageitherattheC-or theN-terminus.Expressionof thisfusionproteininsideacellularenvironmentwhereinthe expressionoftheproteinunderstudyisindigenouscauses the TAP-tagged proteinto interact with other cellular fac-tors in the same fashion as an untagged protein would. Followingcellpropagation,proteincomplexescomprisingof

TAP-protein fusionand theirassociatedcellularfactorsare tobepurifiedandidentifiedbymassspectrometry.The Tap-taggingtechniquehasbeensuccessfullyadaptedtoavariety oforganismsincludingthericepathogenXanthomonasoryzae

pv.oryzae.23–35

Herein,wedescribeaproteinexpressionsystemfor tan-demaffinitypurificationinXac(TAPexpressionvectors).The vectorsinvolvedinthissystemhavethecapabilitytostably integrate into a specificgenomic region (the amy locus)of Xac withoutaltering pathogenicityorvirulence.Finally, we expressedandpurifiedtheTAP-tag fromXac,corroborating theuseofourexpressionvectorsforproteinstudiesinthis bacterium.

Materials

and

methods

Bacterialstrainsandgrowthconditions

TheX.citrisubsp.citriusedinthestudy36,37_{wasthesequenced}

strain 306.1 _{Escherichia coli strain DH10B (F}− _mcrA

(mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 endA1 ara139 (ara, leu)7697 galU galK -rpsL (StrR_{) nupG) was used for}

cloning.DH10Bwascultivatedat37◦CinLB38_andXacat30◦_C

inNYG(Peptone5g/L,yeastextract3g/Landglycerol20g/L). Fortheamylasetests,solublestarchwasaddedtoNYG-agar atafinalconcentrationof0.2%.Duringthecloningprocedure, ampicillin(20␮g/mL)andkanamycin(10␮g/mL)wereaddedto themediatoallowforselectionofplasmid-carryingcolonies.

Generalmethods

Basicmolecularbiologyexperimentswereconductedasper prescribedprotocols.38_{ElectrotransformationofXacwas}

per-formed as described previously.39 _{DNA polymerases and}

restrictionandmodifyingenzymeswereobtainedfrom Fer-mentas (Thermo Scientific). The construction of pHF5Ca (GenBank FJ562210) has been previously described in the literature40_{;pHF5Na(GenBankFJ573043)isavariantofpHF5Ca}

in which the tap1479 has been replaced by the tap176121

(Fig. 1A). The tap1761 cassette was isolated by PCR using pBS1761astemplate21_{andtheprimerpairPBS1761F5}_-TGA

GGATCCATG ATAACTTCGTATAGC ATACandPBS1761R 5-TCATTCTAGACTATAGGGCGAATTGGGTACC. Follow-ingPCR,thepurifiedfragmentwasdigestedwithBamHI/XbaI

andligatedtothebackboneofpre-digestedpHF5Ca.For detec-tingthepresenceofpHF5CaintegratedintotheXacgenome diagnosticPCRwasperformed.Theprimerpairusedforthis purposewas:pxyl5-GTACTTACTATATGAAATAAAATG and1479R5-ATCAAGCTTCAGGTTGACTTCCCCG.

Proteinexpression

StrainsofE.coli/pHF5CaandXacamy::pHF5Cawereactivated bytheinoculationof2–3isolatedcoloniesin3mLofLBorNYG, respectively,alongwiththerequiredantibiotics.The inocu-lumswereculturedfor8hat30◦Cand180rpm.Attheendof thedesignatedgrowthperiod,thecultureswerediluted1:50 (E.coli)or1:10(Xac)inErlenmeyerflaskscontaining50mLLB orNYGbrothplusantibiotics.Culturesweregrownfor14hat

30◦Cand180rpm.Attheendofthetimeperiod,strainswere againdiluted1:50(E.coli)or1:10(Xac)inErlenmeyerflasks con-taining50mLLBorNYGbroth(forWesternblotting)or500mL LBorNYG(forproteinpurification).Cultureswereincubated at30◦Cand 180rpmtillanOD600 valueof0.5wasreached.

Forinductionofproteinexpression,xylosewasaddedtothe growthmediumtoafinalconcentrationof0.5%;timeperiod forinductionwas1h(E.coli)and4h(Xac)usingconditions identicaltothoseemployedforculturegrowth.Subsequentto induction,cellswereharvestedbycentrifugationat4000×g

for10minfollowedbywashingwithcellwashbuffer(30mM Tris–HCl,200mMNaCl);cellpelletswerestoredat−80◦_Ctill

use.

Westernblotting

Totalproteinextractswerepreparedbyresuspending20mg of each cell pellet in100␮L ofSDS-sample buffer. Sample processing,SDS-PAGE,andtransfertoPVDFmembranewas conducted using standard methodology.38 _{For detection of}

theimmobilizedTAP-tag,thePVDFmembranewaswashed withPBS-Tween-20(0.2%)containing5%skimmedmilkand incubated withasecondary antibodycoupled toHRP (rab-bit Anti-Horse IgG; SIGMA A6917) in a dilution of 1:3000. The principle underlying this methodology is that Protein A, which ispart ofthe TAP-tag, isknown tobind IgG iso-types directly. Following incubation with the antibody, the

EcoRI xylR Kan EcoRI EcoRI ori xyIR pxyI pHF5Na 6651bp tap1761 amy Xmal Xmal Xbal BamHI ClaI HincII Sal I Xhol EcoO109I Apal PspOMI Acc65I Kpnl ori Ap Kan Ap pHF5Ca

A

Fig.1–ExpressionvectorsforTandemAffinitypurificationinXac.(A)SchematicrepresentationofpHF5CaandpHF5Na:the proteinexpressionvectorsforTAP-tagginginXac.EachvectorcarriesadifferentTAP-codingDNA,tap1479andtap1761 respectively,whichproduceTAPfusionstoeithertheC-orN-terminalendsofproteinsunderstudy.Bothvectorsarebased onthepCR2.1-TOPObackbonewhichcarriesapUCreplicationoriginandDNAcassettesforampicillin(Ap)andkanamycin (kan)resistance.(B)DNAsequenceofthexylosepromoterregioncommontopHF5CaandpHF5Nashowingthelocationof theribosomebinding-site(RBS)andthestartcodon(ATG).(C)TheDNAsequencesofthe3-endsoftap1479andtap1761. Underneatheachsequenceistheschematicviewoftheproteinfusionthatcanbeproducedusingthetag.Notethatthe organizationofthemodulesthatcomposetheTAP-tags(CBP,TEV,andProtA)differwithrespecttoTAP1479andTAP1761 (seetext);fortheC-terminalfusions,theTAP1479hasProtAlocatedattheextremeC-terminusofthepolypeptide,while ProtAisatthebeginningoftheproteinintheN-terminalfusions.UniquerestrictionsitesforDNAcloningareshownin black;non-uniquesitesingray.CBP,calmodulinbinding-protein;TEV,TobaccoEtchVirusProteaserecognitionsequence; ProtA,ProteinAfromStaphylococcusaureus;andEK,enterokinaserecognitionssequence(DDDDK,presentonlyinTAP1761); pxyl,xylosepromoter;xylR,thexyloserepressorgene;amy,an800bpfragmentofthealpha-amylasegenefromXac.

B

C

N

C

C

N

C-terminal TAP-tag

N-terminal TAP-tag

HincII Xhol ApaI KpnI

xbaI

Fig.1–(Continuación)

westernblotwasdevelopedbyimmersingthemembranein ECLdetectionreagents(AmershamECLWesternBlotting Sys-temkit-GE).Thechemiluminescenceproducedwascaptured onX-rayfilm(Hyperfilm,GE)forfurtheranalysis.

Proteinpurification

Cellpelletsobtainedfrom500mLculturesweredissolvedin pre-chilled10mLTST(50mMTris–HClpH8.0,150mMNaCl, and0.05% Tween20) containingaone-fourthofatablet of EDTA-free protease inhibitors (Roche 1.873.580). Cells were lysedbysonicationusing aVibra-CellVCX 130(Sonics)(10 pulses of 10s each with intervals of 1min) and the soni-catedsampleswerethencentrifugedat48,000×gfor30min at4◦C.Batch binding ofproteins tothe affinity resin was donebyincubatingtheclarifiedsuspensionswith500␮Lof IgG-Sepharose (previously equilibrated in TST;GE 17-0969-01) on an ice bath with gentle agitation through a rotary shaker(120rpm).Aftera2hincubation,resinwastransferred to a 10mL disposable column (Poly-prep BioRad 731-1550) andwashed3–4timeswith20column-volumesofTSTusing

gravity flow. Samples of 100␮L were collected throughout theprocessforSDS-PAGEanalysis.TheTAP-tagwasreleased from the resin byacid elutionusing 0.5M acetic acid (pH 3.4;adjustedwith10Mammoniumacetate).Eluatefractions of 1mL each were precipitated using TCA (3% final) and centrifugedfor10minusingamicrocentrifugeatmaximum speed;proteinpelletswerewashedoncewithabsoluteethanol andthen dissolvedin100␮LofSDSsamplebufferfor SDS-PAGE.

Pathogenicitytests

The plant host used for this study was the sweet orange cultivar‘Bahia’(CitrussinensisL.Osbeck).Orangetreeswere cultivated undergreen-house conditions.Cells tobetested were cultivated in the appropriate medium until OD600

reached∼0.6(∼108_{CFU/mL).Subsequenttogrowth,cell}

sus-pensions ordilutions wereused toinoculateleaveson the abaxialsurfacewiththehelpofhypodermicsyringes(1mL) fittedwithneedles.Symptomswereobservedinthecourseof threeweeksfromthedayofinoculation.

Results

TheTAPexpressionvectorsforXac

Inthisstudy,thetwoTAPexpressionvectorsusedforXac, pHF5Ca and pHF5Na, carrythe xylose promoter (pxyl),the xylose repressor (xylR), and the TAP encoding sequences

tap1479ortap1761(Fig.1A).Thesecomponentsareseparated byashortstretchofDNAcontainingaRibosomeBindingSite (RBS)whosesequenceisaconsensusforbothBacillussubtilis

aswellasE.coli41_{(Fig.1B).Thexylosepromoterusedhasbeen}

previouslyevaluatedbyourgroupinXacandfoundto func-tionsatisfactorily.17,40 _{ThetwoTAPsequences,tap1479and}

tap1761,encodefortagsusedinTandemAffinityPurification ofproteincomplexesandarecomposedofthreecomponents (Fig.1C):(a)TwodomainsofProteinA(ProtA;Staphylococcus aureus) that is known tohave a strong affinity forIgG; (b) Apeptide that binds calmodulin (CBP); (c)A shortpeptide stretchcontainingtherecognitionsequencefortheprotease TEV (Tobacco Etch Virus) separating the above mentioned components.21,22_{TheexpressionvectorspHF5CaandpHF5Na}

differonlyintheTAP-tagsthattheyencode;pHF5Cacarries thetap1479andisusedfortheexpressionofproteinfusions containingthepeptideTAP1479attheC-terminiwhereasthe pHF5Nacarriesthetap1761andisusedtoproducefusionsof TAP1761totheN-terminiofproteinsofinterest.Additionally, boththevectorsusedareintegrativeastheycarrythe amy106-914fragmentofXacwhichdrivestheirintegrationintotheamy

locusofthebacteriumviaasinglecrossoverevent.Upon inte-gration,theamygeneofXacisdisruptedandthebacterium becomesincapableofdegradingstarch.

XacmutantsharboringtheTAPexpressionvectors colonizecitrus

In a previous study, we demonstrated that Xac mutant strainscarryingtheGFPexpressionvectorspPM2aorpPM7g integrated into the amy locus were capable of inducing disease symptoms in citrus plants.17 _{Herein, in}

continua-tion of the previous study, we wanted to evaluate if the presence of the TAP coding DNA altered or affected the virulence/pathogenicity associated with Xac. To test this hypothesis, pHF5Cawas electroporated into Xac;and kanR

transformants (candidates potentially carrying the vector pHF5Ca)were selectedfor furthercharacterization. To cer-tifythatthe TAPexpressionvector hadintegratedintoamy

locus,asetofputativemutantsweretestedfortheirability todegradestarchonatestplate.17_{Theresultsclearly}

demon-stratedthatthecandidatemutantswere deficientinstarch degradationhenceprovingthatpHF5Cawasstablyintegrated intotheamylocusofXacsuchthatthesemutantswereunable toproduce alpha-amylase(datanotshown). Alongside the starchdegradationtest,wealsocarriedoutdiagnosticPCRs onaselectionofsixkanR_{mutantssoastocertifythe}

pres-enceofpHF5Ca(Fig.2).ThediagnosticPCRsdetectedaDNA fragmentof∼640bpcorrespondingtothetap1479inallthe mutantsanalyzed(comparelanes2–7withthepositive con-trolinlane8whereinpHF5CawasusedasDNAtemplate).No

kb

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Fig.2–ColonyPCRtodetectthepresenceofpHF5Ca integratedintothechromosomalDNAofXac.Subsequent toelectrotransformationofXacwiththeexpressionvector pHF5Ca,sixkanR_{mutantswereselectedandsubjectedto}

colonyPCRusingtheprimersPBS1479F/Rspecificforthe amplificationofthetap1479(approximately640bp).The PCRampliconswerevisualizedaftermigrationthrougha 0.7%agarosegel.Lane:1,WTXac(negativecontrol);2–7, thesixselectedmutants;8,PCRusingpHF5CaastheDNA template(positivecontrol);and9,DNAmolecularweight marker(1kbDNAladder,NEB).

bandsweredetectedinthenegativecontrol(lane1,wildtype Xac).

InordertoverifyifXacamy::pHF5Cawascapableofcausing diseasesymptomsinasusceptiblehost,twoindependently selectedmutantswereinoculatedintoleavesofsweetorange (Fig.3A–C).Sevendayspost-inoculation, the bordersofthe areainoculatedwithXacclearlyexhibitedthetypical water-soaking symptom associated with the disease (Fig. 3D–F). These areas developedbrownishcanker-like lesionsduring thecourseofthenextthreeweeks(datanotshown).Apoint to noteis thatthe mutantstrains displayedthe same dis-tinct lesion pattern asthe wild type.In contrast, the area inoculatedwithNYG-medium(negativecontrol)showedonly a mild chlorosis. In summary,the resultsobtained in this study clearly demonstrated that the ability to cause dis-ease remained unaffected followingintegration ofthe Xac

amy::pHF5Camutants.

TAP-tagexpression

TheproductionofaTAP-tagcanbedetectedinWestern blot-tingassays byexploitingthe abilityofProtA tobind tothe IgGantibody.42_{AstrainofE.colicarryingpHF5Ca(multicopy)}

vectorandtwoXacamy::pHF5Camutantstrains(harboringa singlecopyoftheexpressioncassetteintegratedintotheamy

locusofXac)werecultivatedandinducedwithxylose.Total protein extractswere separated bySDS-PAGE (Fig. 4A) and transferredontoaPVDFmembranesoastofacilitate detec-tionofTAP1479usingantibodyreactions.Thebandsofthesize expectedfortheTAP1479(∼21kDa)weredetectedforboththe mutantstrainsofXacaswellasforE.colitransformedwith pHF5Ca(Fig.4B,lanes1–2,and5–8).Theresultsalsoshowed

A

B

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D

E

F

1 and 2=Xac am

y ::pHF5Ca

1

Xac

Wt

NYG

2

Fig.3–TheintegrationoftheTAPexpressionsystemintothechromosomeofXacdoesnotalteritspathogenicity.Two independentlyselectedmutantsofXacamy::pHF5Ca(1and2)wereinoculatedintoleavesofsweetorangeBahiaalongside thewildtypestrain(Xacwt)andasampleofNYG-medium(negativecontrol).Amapoftheinoculationisshownonthe right-handsideofthefigure.Picturesweretakenimmediatelyafterinoculation(A–C)andalso7dayspost-inoculation(D–F). Thetestwasconductedintriplicate(A–C).

thattheproductionofTAP1479wasresponsivetoinductionby xylose(lanes1and2,5and6,7and8).Forthenegative con-trol,wildtypeXac,nosignalcorrespondingtothebandsize ofTAP1479wasdetected(lanes3and4).Itwasobservedthat

E.coliproducedmoreofthetagthantheXacmutants,aresult thatisconsistentwiththemulticopyconditionofpHF5Cain

E.coli.Theresultsobtainedclearlyshowthattheexpression systemisfunctionalinbothbacteriaandalsothatanintact andactiveProtAmoietywasproducedinXac.

PurificationoftheTAP-tagfromXac

The TAP-tag detected in the previous section appeared to bearesilientpolypeptide capableofretaining theproperty of IgG-binding even after treatment with SDS and boiling asis mandatoryforsample preparationin SDS-PAGE elec-trophoresis.However,resultsobtainedbywesternblottingdo notguaranteefunction,as thepositive outcomecan easily beexplainedasabyproductofresidualIgGbindingactivity ofProtA.Additionally,fortheproteincomplexesproducedin Xactobeuseful,itisessentialtocheckiftheTAP1479could beproducedinlargeramountsandinasolubleform.Inorder toevaluatethis,anattemptwasmadetopurifyTAP1479by affinityseparation.AnE.colistraincarryingpHF5CaandaXac

amy::pHF5Camutantwerecultivatedandinducedwithxylose. FollowingexpressionoftheTAP-tag,cellswerelysedby son-ication and clarifiedcell extractswere subjected toaffinity chromatography usingIgG-immobilizedresin.TAP1479 was successfullypurifiedfromthesolublephaseofbothextracts (Fig.5;seearrows).AsobservedintheWesternblotting exper-iments,E.coliproducedarelativelyhigheramountofthetag(a tagconcentrationof∼1.4mg/mLwasestimatedforthe sam-pleinlane8)ascomparedtotheyieldfromXac(∼0.2mg/mL, lane7).However,theamountoftagproducedbyXac,though less,shouldsufficeformassspectrometryidentification.The contaminantbandsabovetheTAP1479representdenatured humanIgGwhichwasstrippedoutfrom thematrixbythe acidelutiontogetherwithotherbacterialproteinsthatmight beinteractingwiththeresin.Theseresultscorroboratethe useoftheexpressionvectorspHF5CaandpHF5Naas integra-tion/proteinexpressionsystemsforTAP-tagginginXac.

Discussion

Inthepresentworkwecharacterizedanovelprotein expres-sionsystemintendedforhigh-throughputanalysisofprotein complexes inXac. These vectors are integrativeand allow

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Fig.4–Westernblottinganalysistodetectexpressionof TAP1479byXacamy::pHF5Camutants.Thefunctionalityof theTAPexpressionsystemwasverifiedbytheabilityofan E.coliDH10Bstrain,withpHF5CaandXackanR_mutants,

carryingthevectorintegratedintothechromosome,to producetheTAP1479(approximately21kDa)(detailsofthe inductionprocedurehavebeendescribedin“Materialsand methods”section).(A)Coomassieblue-stained10% SDS-PAGEshowingtheseparationofproteinsfromcell extractsofE.coliandXac;lanes:1,E.coli/pHF5Ca;2, E.coli/pHF5Ca+0.5%xylose;3,Xac(WT);4,XacWT+0.5% xylose;5,Xacamy::pHF5Camutant1;6,Xacamy::pHF5Ca mutant1+0.5%xylose;7,Xacamy::pHF5Camutant2,8; Xacamy::pHF5Camutant2+0.5%xylose;and9,protein molecularweightmarker.(B)X-rayfilmdisplayingbands detectedbytheWesternblottinganalysis.Proteinsina replicaofthegelshowninAweretransferredtoaPVDF membraneandexposedtoasecondaryantibodycoupledto HRP.Lanesarethesameasin(A).Thepositionofthe TAP1479ismarkedwithablackarrowontherighthand sideofthefilm.

fortheproductionoffusionproteinstaggedwiththeTAP-tag eitherattheC-orattheN-terminalends.21,22_{Integrationof}

theexpressionvectorsintothechromosomeofthebacterium mayoccursite-specificallyintotheamylocus(asshownhere) or into the ORF as demonstrated previously in a prelimi-narycharacterizationofpHF5Ca.40Integrationmediatedbya

E. coli/pHF5Ca

Xac amy::pHF5Ca

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Fig.5–PurificationofTAP1479fromE.coli/pHF5CaandXac amy::pHF5Ca.E.coliDH10BcarryingpHF5CaandakanR mutantofXacharboringpHF5Caintegratedintoits chromosomewereinducedfortheproductionofthe TAP1479.Followingexpression,cellswerelysedand proteinextractssubjectedtoaffinitychromatography throughIgG-sepharose.(A)E.coli/pHF5Ca;and(B)Xac amy::pHF5Ca,Coomassieblue-stained10%SDS-PAGE showingtheseparationofproteinsfromthefractions collectthroughouttheprocess.(A)lane:1,clarifiedlysate;2, flowthrough;3–6,washes(20column-volumeseach);7, proteinmolecularweightmarker;8–14,elutionfractions. (B)Lane:1,clarifiedlysate;2,flowthrough;3–5,washes;6, proteinmolecularweightmarker;7–14,elutionfractions. ThepositionoftheTAP1479ismarkedwithblackarrowsin bothgels.

crossover betweenORFs(ligatedinthevectorand its chro-mosomalcopy)putstheexpressionoftheTAP-taggedpeptide underthecontrolofthenativepromoterofthechromosomal ORF.Hence,useofpHF5Caisrecommendedsoastohavea C-terminalTAP-taggedprotein.Theintegrationintotheamy

locusisthepreferablesiteofintegrationasiteliminatesthe possibilityofperturbationsofchromosomalregions contain-ing the ORFs under investigation.Genome integrationalso guarantees thatthe expressioncassettewillbepropagated asasinglecopypercell,allowingforabettercontrolof pro-teinexpressionunderthexylosepromoter.43,44_{Finally,inthis}

study,wehaveshownthattheintroductionofaTAPcoding DNA intoXac had nodetectable effectonvirulence,which emphasizesandunderlinestheutilityoftheTAP-expression vectorsforproteininteractionstudiesinthisplantpathogen.

Resultobtainedfromthis studyprovedthat theTAP-tag couldbestablyexpressedandpurifiedfromthesolublephase ofXaccellextracts.Currently,weareintheprocessof test-inganumberofTAP-proteinfusions(hypothetical,previously characterized and control factors) in our model organism inanattempttoconductfunctional assignmentsusingthe TAP-taggingstrategy.ApartfromitsuseinTAP-tagging exper-iments,theexpressionvectorspHF5CaandpHF5Nacanalso beappliedforproteinexpressionandpurificationfrom Xan-thomonas spp. thus eliminating the need for heterologous expressioninE.coli.Anexampleofthisapplicationisthe strat-egyforexpressionandpurificationofTAP1479outlinedabove. Another feasible application would be in genetic comple-mentationtestsinvivo.Insuchexperiments,theTAPcoding DNAmayberemovedduringthecloningstepssuchthatthe polypeptideproducedinXacwillnotcarryanytag.

Althoughextensively explored in eukaryotes,the utility oftandemaffinitypurificationforprotein–proteininteraction analysisinprokaryotesislimited.24,26,45–49 _{TAP-taggingwas}

originallyusedinE.coliasatoolforexploringthe biologi-calrolesofcomponentsofproteincomplexesinvolvingACP andothercellularfactors,aswellasforthecharacterization ofYbdB(EntH),athioesteraseproducedinE.coliunderiron starvation.26,45,47,48_{Abroaderhigh-throughputprotein}

inter-actionstudyusingTAP-taghasbeenreportedinE.coliinwhich 857proteinsweretagged24_{;morethansixhundredproteins}

weresuccessfullypurified;ofwhich,530werefoundtobea partofprotein–proteincomplexes.Proteininteractantswere subsequentlyidentifiedbymassspectrometryandthe resul-tantdatawerecompiledtobuildanetworkofprotein–protein associations coveringa vast variety ofbiological activities. Additionally,thenetworkraisedthepossibilityofannotating uncharacterizedfactorswiththeirdesignatedfunction.More recently,TAP-tagginghasbeenusedtolabelfactorsinX.oryzae

pv.oryzae34,35_{allowingforthequantitativeanalysesof}

pro-teinexpressionandsecretion.TAP-labelingwasalsousedby ourgroup,inapreviousstudy,todetecttheexpressionofthe chromosomesegregationproteinParBinXac.40_{Inconclusion,}

theresultsoutlinedabovehavedemonstratedthestabilityof theTAPmoietyandtheexpressionvectorsdescribedherein havethepotentialtodevelopintovaluabletoolsforexploring proteinnetworksinXacespeciallywherefactorsofunknown functionmaybeparticipants.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

GCD and PMMM received scholarships from Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2013/19806-5and2006/59494-9)andCAPES/Brazil.Thiswork was funded by FAPESP grant numbers 2004/09173-6, and 2013/50367-8.

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