transposasesodu.tw

ArchMicrobiol(2016)198:53–69DOI10.
1007/s00203-015-1154-8ORIGINALPAPERMosaiccompositionofribAandwspBgenesflankingthevirB8D4operonintheWolbachiasupergroupBstrain,wStrGeraldD.
Baldridge1·YangGraceLi1·BruceA.
Witthuhn2·LeeAnnHiggins2·ToddW.
Markowski2·AbigailS.
Baldridge3·AnnM.
Fallon1Received:27April2015/Revised:9September2015/Accepted:14September2015/Publishedonline:23September2015TheAuthor(s)2015.
ThisarticleispublishedwithopenaccessatSpringerlink.
comvirproteinsareexpressedatsimilar,aboveaverageabun-dancelevels.
InwStr,bothribAandwspBaremosaicsofconservedsequencemotifsfromWolbachiasupergroupA-andB-strains,andwspBisnearlyidenticaltoitshomologfromwCobU4-2,anA-strainfromweevils(Coleoptera).
WedescribeconservedrepeatedsequenceelementsthatmapwithinornearpseudogenelesionsandtransitionsbetweenA-andB-strainmotifs.
ThesestudiescontributetoongoingeffortstoexploreinteractionsbetweenWolbachiaanditshostcellinaninvitrosystem.
KeywordsWolbachia·LC–MS/MS·Proteomics·Mosaicgenes·T4SS·RibA·RibB·WspBIntroductionWolbachiapipientis(Rickettsiales;Alphaproteobacteria)isanobligateintracellularbacteriumthatinfectsfilar-ialnematodesandawiderangeofarthropodsincluding≥60%ofinsectsand≈35%ofisopodcrustaceans,butdoesnotinfectvertebrates(Hilgenboeckeretal.
2008).
WolbachiaisconsideredtobeasinglespeciesclassifiedintocladesbymultilocussequencetypinganddesignatedassupergroupsAtoN(Baldoetal.
2006b;Comandatoreetal.
2013;Loetal.
2007).
TheC-andD-strainsthatinfectfilarialwormshavephylogeniesconcordantwiththoseofnematodehosts,consistentwithstrictverticaltransmissionasobligatemutualists(Comandatoreetal.
2013;Dedeineetal.
2003;LiandCarlow2012;Strubingetal.
2010;Tay-loretal.
2005;Wuetal.
2004).
Althougharthropod-asso-ciatedA-andB-strainsmayprovidesubtlefitnessbenefitstohosts(ZugandHammerstein2014),theyarebestknownasreproductiveparasites,causingphenotypesthatmain-tainorincreaseWolbachiainfectionfrequencies,includingAbstractTheobligateintracellularbacterium,Wol-bachiapipientis(Rickettsiales),isawidespread,verti-callytransmittedendosymbiontoffilarialnematodesandarthropods.
Ininsects,Wolbachiamodifiesreproduction,andinmosquitoes,infectioninterfereswithreplicationofarboviruses,bacteriaandplasmodia.
DevelopmentofWol-bachiaasatooltocontrolpestinsectswillbefacilitatedbyanunderstandingofmoleculareventsthatunderliegeneticexchangebetweenWolbachiastrains.
Here,weusednucle-otidesequence,transcriptionalandproteomicanalysestoevaluateexpressionlevelsandestablishthemosaicnatureofgenesflankingtheT4SSvirB8-D4operonfromwStr,asupergroupB-strainfromaplanthopper(Hemiptera)thatmaintainsarobust,persistentinfectioninanAedesalbop-ictusmosquitocellline.
Basedonproteinabundance,ribA,whichcontainspromoterelementsatthe5′-endoftheoperon,isweaklyexpressed.
The3′-endoftheoperonencodesanintactwspB,whichencodesanoutermembraneproteinandisco-transcribedwiththevirgenes.
WspBandCommunicatedbyMarkusNett.
ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s00203-015-1154-8)containssupplementarymaterial,whichisavailabletoauthorizedusers.
*AnnM.
Fallonfallo002@umn.
edu1DepartmentofEntomology,UniversityofMinnesota,1980FolwellAve.
,St.
Paul,MN55108,USA2DepartmentofBiochemistry,MolecularBiologyandBiophysics,UniversityofMinnesota,Minneapolis,MN55455,USA3FeinbergSchoolofMedicine,NorthwesternUniversity,Chicago,IL60611,USA54ArchMicrobiol(2016)198:53–69feminization,parthenogenesis,andcytoplasmicincompat-ibility(SaridakiandBourtzis2010;Werrenetal.
2008).
Interferencewithhostimmunemechanismsandreplicationofarboviruses,bacteriaandmalarialplasmodia(Kambrisetal.
2009;Panetal.
2012;ZugandHammerstein2014)hasencouragedeffortstoexploitWolbachiaforbiocontrolofarthropodvectorsofvertebratepathogensand/orcroppests(Bourtzis2008;Rioetal.
2004;SinkinsandGould2006;Zabalouetal.
2004).
AnunderstandingofmoleculardifferencesbetweenA-andB-strains,andhowtheyhavebeeninfluencedbyhorizontaltransmissionandgeneticexchange(NewtonandBordenstein2011;Schuleretal.
2013;Werrenetal.
2008;ZugandHammerstein2014)willfacilitatemanipulationofWolbachia.
Wolbachia'sinteractionwithhostcellslikelyinvolvesthetypeIVsecretionsystem(T4SS),amacromolecularcomplexthattransportsDNA,nucleoproteinsand"effec-tor"proteinsacrossthemicrobialcellenvelopeintothehostcell,wheretheymediateintracellularinteractions(Alvarez-MartinezandChristie2009;Zechneretal.
2012).
HomologsofallgenesexceptvirB5ofAgrobacteriumtumefaciensT4SShavebeenidentifiedinWolbachiaandothermembersoftheRickettsiales(Gillespieetal.
2009,2010),includingAnaplasma,Ehrlichia,Neorickettsia,OrientiaandRickettsia.
AmongsequencedWolbachiagenomes,T4SSgenesareorganizedintwooperons:virB3-B6containingvirB3,virB4andfourvirB6paralogsandvirB8-D4containingvirB8,virB9,virB10,virB11,virD4and,insomegenomes,thewspBparalogofthewspAmajorsurfaceantigen(Pichonetal.
2009;Rancesetal.
2008).
InthesupergroupB-strainwPipfromCulexpipiensmosqui-toes,wspBisdisruptedbyatransposonandispresumablyinactive(Sanogoetal2007).
T4SSeffectorproteinsthatmanipulatehostcellshavebeenidentifiedfromAnaplasmaandEhrlichia(Liuetal.
2012;Lockwoodetal.
2011;Niuetal.
2010),andWolbachiaexpressbothviroperonsinovariesofarthropodhosts,whereinT4SSeffectorsaresuspectedtoplayaroleincytoplasmicincompatibilityandotherreproductivedistortions(Masuietal.
2000;Rancesetal.
2008;Wuetal.
2004).
AlthoughWspAandWspBarelikelycomponentsoftheWolbachiaoutermembrane,theirfunctionsremainunknown.
InthecaseofwBm,WspBisexcreted/secretedintofilarialhostcells(Bennuruetal.
2009)andco-localizeswiththeBm1_46455hostproteinintissuesthatincludeembryonicnuclei(Melnikowetal.
2011).
WspBisthereforeitselfacandidateT4SSeffectorthatmayplayaroleinreproductivemanipulationofthehost.
TheWolbachiastrainwStrinsupergroupBcausesstrongcytoplasmicincompatibilityintheplanthopper,Laodelphaxstriatellus(Nodaetal.
2001a),andinaddi-tionmaintainsarobust,persistentinfectioninaclonalAedesalbopictusmosquitocellline,C/wStr1(Fallonetal.
2013;Nodaetal.
2002).
BecauseinvitrostudieswithwStrprovideadvantagesofscaleandeaseofmanipulationforexploringmechanismsthatmayfacilitatetransformationandgeneticmanipulationofWolbachia,wehaveunder-takenproteomics-basedstudiesthatprovidestrongsupportforexpressionofT4SSmachineryincellculture.
Here,wereportthesequenceofthevirB8-D4operon,includingflankinggenesribA,upstreamofvirB8,andwspBdown-streamofvirD4.
WeshowthatwspBisintact,describepro-teinstructurepredictedfromthededucedWspBsequence,andverifyco-transcriptionofwspBwithupstreamvirgenes.
RelativeabundancelevelsofWspBandtheVirB8-D4proteinsinwStrarewellaboveaverage,whileRibAisamongtheleastabundantofMS-detectedproteins.
InwStr,ribAandwspBaremosaicsofsequencemotifsthataredif-ferentiallyconservedinsupergroupA-(WOL-A)andB-(WOL-B)strains,andtheycontainconserved8-bprepeatelementsthatmaybeassociatedwithgeneticexchange.
Finally,wediscussimplicationsforfunctionalintegrationoftheWolbachiaT4SSwithWspBandwiththeribofla-vinbiosynthesispathwayenzymesGTPcyclohydrolaseII(RibA)anddihydroxybutanonephosphatesynthase(RibB).
MaterialsandmethodsCultivationofcellsAedesalbopictusC7-10andC/wStr1cellsweremaintainedinEagle'sminimalmediumsupplementedwith5%fetalbovineserumat28–30°Cina5%CO2atmosphere(Fal-lonetal.
2013;Shihetal.
1998).
Cellswereharvesteddur-ingexponentialgrowth,underconditionsfavoringmaximalrecoveryofWolbachia(Baldridgeetal.
2014).
Polymerasechainreaction,cloningandDNAsequencingThepolymerasechainreaction(PCR)wasusedtoamplifywStrgenesfromDNAextractspreparedfromWolbachiaenrichedbyfractionationofC/wStr1cellsonsucroseden-sitygradientsandrecoveredfromtheinterfacebetween50and60%sucrose(Baldridgeetal.
2014).
TemplateDNAwasusedtoobtain21PCRproductsusingapanelof31primers(TableS1),GoTaqDNApolymerase(Promega,Madison,WI),andaTechneTC-312cycler(Staffordshire,UK).
Cycleparameterswere:1cycleat94°Cfor2min,35cyclesat94°Cfor35s,53°Cfor35s,72°Cfor1min,followedby1cycleat72°Cfor5min.
Extensiontimewasincreasedto2minforproducts≥1000bp.
PCRproductswereclonedinthepCR4-TOPOvectorwiththeTOPO-TACloningKitforSequencing(LifeTechnologies,GrandIsland,NY),andtwoormorecloneseachweresequenced55ArchMicrobiol(2016)198:53–69attheUniversityofMinnesotaBioMedicalGenomicsCenter.
ReversetranscriptasepolymerasechainreactionTotalRNAwaspurifiedfromA.
albopictusC7-10andC/wStr1cellsusingthePureLinkRNAMiniKit(LifeTechnologies)andtreatedwithDNaseI(RNase-free;LifeTechnologies)followedbyheatinactivation,assuggestedbythemanufacturer.
RT-PCRwasexecutedwithprimersvirD4F1764–1784andwspBR152–172(TableS1)usingtheRNAPCRCoreKit(LifeTechnologies)assuggestedbythemanufacturerwiththeexceptionthatsynthesizedcDNAwastreatedwithDNase-inactivatedRNaseAbeforethefinalPCRreaction.
ThePCRreactionincluded1cycleat95°Cfor4min,35cyclesat95°Cfor35s,56°Cfor40s,72°Cfor40s,followedby1cycleat72°Cfor3min.
Reactionproductswereelectrophoresedon1%agarosegels,cloned,andsequencedasabove.
SequencealignmentsandproteinstructurepredictionDNAandproteinsequencealignmentswereexecutedwiththeClustalOmegaprogram(Sieversetal.
2011).
Align-mentswereeditedbyvisualinspectionandmodifiedinMicrosoftWord.
WspBproteinstructurepredictionswereobtainedusingtoolsavailableatwww.
predictprotein.
org,includingthePROFtmbprogram(Delletal.
2010)forpre-dictionofbacterialtransmembranebetabarrels(Bigelowetal.
2004)andper-residuepredictionofup-strand,down-strand,periplasmicloopandouterlooppositionsofresidues.
ThePROFisisprogram(OfranandRost2006)wasusedtopredictWspBaminoacidresiduesthatarepotentiallyinvolvedinprotein–proteininteractions.
TreeswereproducedusingPAUP*version4(Swofford2002).
AminoacidswerealignedwithClustalW,usingpairwisealignmentparametersof25/0.
5andmultiplealignmentparametersof10/0.
2forgapopeningandgapextension,respectively.
TheproteinweightmatrixwassettoGonnet.
ThealignmentwassavedasanexusfileandloadedintoPAUP*,andthetreeswerecreatedusingaheuristicsearchwiththecriterionsettoparsimony.
Bootstrap50%major-ity-ruleconsensustreesarebasedon1000replicates,withwBm(WOL-D)astheoutgroup.
Massspectrometry,peptidedetection,proteinidentificationandstatisticalanalysisMassspectrometrydata,generatedusingLC–MS/MSonLTQandOrbitrapVelosmassspectrometersasfourdatasets,weredescribedpreviously(Baldridgeetal.
2014).
TheMSsearchdatabasewasmodifiedtoincludededucedORFsfromwStrsequencedatadescribedherein.
AlltestsofassociationwereperformedwithSASversion9.
3(Cary,NC;http://www.
sas.
com/en_us/home.
html/).
ResultsStructureofthewStrvirB4D8operonTherobust,persistentinfectionofA.
albopictusmos-quitocellline,C/wStr1withBwStr(inthetextbelow,straindesignationsaredenotedbysuperscripts),isolatedfromtheplanthopperL.
striatellus,providesaninvitromodeltoidentifyproteinsthatmodulatethehost–microbeTable1MS-detectedpeptidesfromwStrproteinsencodedbyribA,ribBandthevirB8-D4operonaProteinmassinkilodaltons.
bNumberof95%confidenceuniquepeptides;(1)designatesoriginalsearch[7];(2)designatesarefinedsearchinwhichthedatabaseincludedpeptidesbasedonthepresentwStrnucleotidesequencedata;(T)combinedtotalpeptidesfrombothsearches.
cPercentproteinsequencecoveragerepresentedbydetectedpeptides.
dMeannumberofpeptidesfromfourindependentMSdatasets.
eStudentizedresidualbasedonthemodifiedunivariablemodeloftherefinedsearch(TableS3,col-umnR);SRvalue0indicatesaverageabundanceprotein,0–1aboveaverage,1–2abundantand>2highlyabundant.
Valuesbelow0indicatelowerthanaverageabundance.
fA94%confidencepeptideindicatedinFig.
1Adidnotmeetthethresholdforproteomeinclusionintheoriginalsearch.
ForVirB10,oneoriginallydetectedpeptidewasabsentfromtherefinedsearchProteinakDabPep(1)bPep(2)bPep(T)cCov.
dRALeSRRibA4122260.
52.
30RibB2471212897.
01.
20VirB82691010585.
00.
59VirB93110810456.
20.
84VirB1054141618538.
80.
94VirB1137121414427.
00.
82VirD477121414266.
20.
45WspB312f11507.
21.
0856ArchMicrobiol(2016)198:53–69interaction.
ApotentialrolefortheT4SSissupportedbystrongrepresentationofpeptidesfromVirB8,VirB9,VirB10,VirB11,VirD4(Table1)andassociatedproteinsintheBwStrproteome(Baldridgeetal.
2014).
Despiteitsemergenceasausefulstrainthatgrowswellinvitro,theBwStrgenomeisnotyetavailable.
InWolbachiastrainsforwhichgenomeannotationisavailable,geneorderwithinthevirB8-D4operonisconserved.
Basedontranscriptionalanalysesintherelatedgenera,AnaplasmaandEhrlichia(Pichonetal.
2009),thepromoterlikelymapswithinthe3′-endofribAextendingintotheintergenicspacer(Fig.
1a,blackhorizontalarrowatleft)andisfollowedbyfivecon-secutivevirgenes(Fig.
1b).
InBwPipfromCulexpipiensmosquitoes,wspBisdisruptedbyinsertionofanIS256elementthatencodesatransposaseontheoppositestrand(Fig.
1a,atright;Sanogoetal.
2007).
BecauseVirB8-D4proteinswerehighlysimilartohomologsfromBwPip(Baldridgeetal.
2014),weevaluatedwspBinBwStranditspotentialexpressionasavirB8-D4operonmember,asisthecaseinAwMelandAwRifromDrosophilaspp.
andAwAtab3fromthewaspAsobaratabida(Rancesetal.
2008;Wuetal.
2004).
Intheoriginalproteomicanalysis,threeWspBpeptides(Fig.
1a,tallblackandgrayarrowsrepresent95and94%confidencepeptides,respectively)mappedproximalanddistaltothetransposoninsertioninBwPip,whiletheabsenceofpeptidescorrespondingtothetransposonsuggestedthatwspBisintactinBwStr.
NucleotideanddeducedaminoacidsequencecomparisonsToexaminethevirB4-D4operoninBwStr,wesequencedoverlappingPCRproductsfrom20primerpairs(TableS1)spanning9.
1kbbeginning43bpdownstreamofthe5′-endofribAinotherWolbachiastrainsandendingwithintopAencodedimmediatelydownstreamoftheoperonontheoppositestrand(Fig.
1b,c).
WiththenotableexceptionoftheBwPiptransposon,thenucleotidesequencealignedmost**wMelwPippromoterB8BB10DD4wspBtopAtransposaseribAribB10kb5kbBwVitBwPipwVulCwMelwBmwRiB9B110CDWOL-BBBAADRT-PCRAFig.
1SchematicmapoftheWolbachiaT4SSvirB8-D4operonandcloningstrategyfortheribAtotopAsequencefromBwStr.
aLeftexpandedviewoftheBwStrribAORFdepictedasanarrowshowingthedirectionoftranscription.
Blackhorizontalarrowindicatesaputa-tivepromoterthatextendsintoanintergenicspacer(blackrectangle).
BlackarrowheadsindicatepositionsofMS-detecteduniquepeptides(95%confidence).
Gradientshadingfromwhitetoblackdesignates5′-sequenceidentityresemblingWOL-Atransitioningto3′-sequencemorecloselyresemblingWOL-B-strains.
aRightexpandedviewoftheinterruptedwspBhomologinBwPip.
Blackellipsesindicateposi-tionsofIS256invertedrepeatelementsflankinga1.
2-kbinsertionencodingaMULEdomainsuperfamilytransposase(gi|190571636;pfam10551)ontheoppositestrand(indicatedbythedirectionoftheopenarrow);flankinggrayshadingindicateswspB.
Tallverticalblackandgrayarrowheadsindicatepositionsofuniquepeptides(95and94%confidence,respectively)identifiedintheoriginalMSdatasearch.
Smallgrayarrowsindicate95%confidencepeptidesmatchedinarefineddataset(includingtheBwStrsequencedescribedhere)thatareconservedinWOL-B-strains,andopenarrowheadswithstarsindicatepeptidesuniquetoBwStr.
bSchematicdepictionoftheWol-bachiavirB8-D4operonandflankinggeneswitharrowsdesignat-ingthedirectionoftranscription.
Virgenesaredesignatedinwhitefontonablackbackground;blacksquaresindicateintergenicspac-ers.
GradientshadingindicatesmosaicstructureofanintactwspBinBwStr.
cFilledlinesabovethe10-kbscalemarkerrepresentclonedPCRamplificationproducts(seeTableS1forprimers)thatweresequencedandassembledintotheBwStrribBandribA–topAconsen-sussequence.
ThedoubleslashsymbolsatleftindicatethatribBisnotcontiguouswithdownstreamgenes.
TheopenboxindicatestheRT-PCRamplificationproductfromFig.
2.
dBLASTnalignmentofthe9133-bpBwStrribA–topAsequencetocorrespondingsequencesinBwVitBBwPip,BwVulC,AwRi,AwMelandDwBmgenomes.
Darkfilledlinesindicatesequenceidentity>70%;lightlinesindicatelowsequenceidentity,andtheopenspaceinBwPiprepresentsanalign-mentgap57ArchMicrobiol(2016)198:53–69closelytohomologoussequencesfromBwVitBandBwPip.
Inaddition,wenotedvariabilityinan~0.
3-kbregionofvirB10inBwStrthatwasconservedinBwVitB,BwPipandAwRi,butnotinBwVulC,AwMelandDwBm(Fig.
1d;seeTableS2forGenBankAccessions).
PairwisesequencecomparisonsofthevirB8-D4operonfromBwStrtohomologsfromWolbachiasupergroupA,B,C,DandFstrains(Table2)confirmthatvirB10,withnucleotideidentitiesrangingfrom74–99%,istheleastconservedofthefivevirgenes,andwenotethatKlassonetal.
(2009)attributeddiver-genceofvirB10inAwMelandAwRitogeneticexchangewithaWOL-B-strain.
Collectivelyandasindividuals,thevirgenesfromBwStrhavethehighestnucleotideidentities(~99%)withBwVitBandBwPip.
IdentitieswithfiveA-strainsarelower(range87–91%),loweryet(range80–89%)withtheF-strain,FwCleandfalltoarangeof74–88%withthreenematode-associatedstrains,DwBm,CwOoandCwOv.
Atthe5′-endoftheoperon,ribAwasdistinct,withapproximatelyequiva-lentnucleotideidentitywithhomologsfromA-andB-strains(range91–94%),whilethepartialsequenceoftopAdownstreamoftheoperonhadaconservationpatternsimilartothatofthevirgenes.
Insomecom-parisons,virB8,virB11,virD4andtopAaminoacididentitiesexceednucleotideidentities.
AlthoughribBisnotphysicallyadjacenttothevirB8-D4operoninanno-tatedWolbachiagenomes,ribBfromBwStrismostsim-ilartohomologsfromBwNo(97%nucleotideidentity)andAwMel(90%),butwasexceptionalbecauseidenti-tieswiththreeotherinsect-associatedA-andB-strains(~80%)werelowerthanwithF-,C-andD-strains(range85–87%).
Consistentwithearlierproteomicdata(Baldridgeetal.
2014),inallcomparisonsthatdiscriminatebetweenA-andB-strains,BwStrresem-bledWOL-B,whilevariabilityinribAandwspBflank-ingthevirB8-D4genesexceededthatofthevirgenesthemselves.
Table2PairwisenucleotideandaminoacidcomparisonsWolbachiastrainsfromsupergroupsA,B,C,DandFareindicatedbysuperscripts,withpercentagesofnucleotide(N)andaminoacid(AA)sequenceidentitiestoBwStr.
Dashesindicatesequencesnotavailable,andxxindicatespseudogenes;GenBankAccessionnumbersaregiveninTableS2aPartialgeneandproteinsequences:ribA1040bp,ribB592bp;topA825bp.
Hostassociations:wPip,Culexpipiens—mosquito;wVitB,Nasoniavitripennis—wasp;wTai,Teleogryllustaiwanensis—cricket;wVulC,Armadillidiumvulgare—isopod;wMel,wRi,wAna,wNo,Drosophilaspp.
—fruitfly;wKue,Ephestiakuehniella—moth;wAtab3Asobaratabida—wasp;wBm,wOoandwOvfromfilarialnematodesBrugiamalayi,OnchocercaochengiandO.
volvulus,respectively.
Inthecomparison,valuesof97%orgreaterareshowninitalicsGeneBwPiPBwVitBBwNoBwTaiBwVulCAwMelAwRiNAANAANAANAANAANAANAAribAa9489948993889490939293919289virB89910099100999999100949488868887virB99999999898979797949391899189virB109999999890869896887487748885virB119999979996989799909389958995virD49999999999999999949789928993wspB56xx98968568––––85708570topAa99100991009999––––88878786ribBa8180––9796––––90917978GeneAwAnaAwKueAwAtab3FwCleDwBmCwOoCwOvNAANAANAANAANAANAANAAribAa91889391––8481838082748275virB88887888688888583858183818482virB99189918991898484848482768176virB108884877487738071847076647464virB118995899589958995889486898789virD48993899388928792878786918894wspB83688570857072xx736172497149topAa8685––––8892868884838488ribBa8088––––8787868785xx85xx58ArchMicrobiol(2016)198:53–69ExpressionandrelativeabundancesoftheBwStrvirB4D8proteinsTorefineanearlieroriginalproteomicanalysis(Baldridgeetal.
2014),weincorporatedthePCR-amplifiedBwStrsequencesdescribedheretothedatabaseforpeptideiden-tification[Table1,seecolumnlabeledPep(2)].
Statisti-calanalysisindicatedthatinaunivariablemodel,proteinmolecularweightwasweakly(r2=0.
2221)butsignifi-cantly(p1.
0)foranabundantproteinandroughlyequivalenttoSRval-ues(range1–1.
17)ofhousekeepingproteinssuchasisoci-tratedehydrogenase,ftsZ,ATPsynthaseF0F1αsubunit,andribosomalproteinsS2,S9,L3,L7/L12andL14(TableS3).
Incomparison,WspAwithanSRof2.
17(TableS3,entry63)rankedashighlyabundant,andthemostabundantproteinintheproteomewastheGroELchaperone(entry586),withanSRof3.
66.
ReversetranscriptasePCRconfirmscotranscriptionofwspBwithvirgenesSimilarSRvaluesforWspB,relativetoVirB8-D4,wereconsistentwithevidencethatwspBisco-transcribedwithvirB8-D4inAwMel,AwRiandAwAtab3(Rancesetal.
2008;Wuetal.
2004).
WeusedRT-PCRwithRNAtem-plateverifiedbyPCRtobefreeofDNAcontamination(Fig.
2b,lanes2and3)toamplifya528-bpproductthatwasproducedinreactionscontainingRNAfromC/wStr1cells(Fig.
2a,lane4),butnotinnegativecontrolreac-tions(lanes1and2)orthosewithRNAfromC7-10cells(lane3).
ItssequencematchedtheexpectedBwStrgenomicsequence(Fig.
1c,RT-PCRboxatright),confirmingthatinBwStr,wspBisamemberofthevirB8-D4operon.
InBwStr,ribAisamosaicofconservedWOLAandWOLBsequencemotifsTheribAnucleotidesequencehasbeenshowntocontainregulatoryelementsforexpressionoftheT4SSoperoninAnaplasmaandEhrlichia(Ohashietal.
2002;Pichonetal.
2009).
IncontrasttohighesthomologiesofBwStrvirB8-D4genestoWOL-B-strains,ribAsequenceidentitiesshowedlittledifferencebetweenWOL-Aand-Bhomologs(Table2),butthetwoMS-detectedpeptidescorrespondedtoAwMelandBwPiphomologs,respectively(Fig.
1a).
Alignmentofaminoacidsfrom10RibAhomologs(Fig.
3;WOL-AandWOL-B-strainsareidentifiedatleftinredandblue,respectively)suggestedthatBwStrRibAisatwo-partmosaic,eachcontainingaproteinfunctionaldomain.
Theaminoterminal150residuesinBwStrRibA(Fig.
3)includeashortdihydroxybutanonephosphatesynthasedomainandthefirstdetectedpeptide(residues94–104).
ThisportionofBwStrRibAmatchedsequencesfromthefourA-strainsandasingleB-strain,BwVulC,at29of36variableaminoacids(showninred),whileonlythree(4,39and168inblue)matchedtheotherthreeB-strainsandfour(ingreen)wereunique.
Incontrast,theC-terminal151–347residues,encompassingthesecondpeptide(residues250–258)withinaGTPcyclohydrolasedomain,includedasingleaminoaciduniquetoBwStr,while23(inblue)uni-formlymatchedB-strainsexceptBwVulC,whichcontinuedtoresembletheA-strainsuntilresidue239.
AmongthefourA-strains,theBwRihomologismostsimilarthroughoutthealignmenttotheB-strains,butwithinresidues129–150immediatelyprecedingthecyclohydrolasedomain,itcloselymatchedBwTai,BwPipandBwVitB,whileBwStrABFig.
2ReversetranscriptasePCR(RT-PCR)analysisshowsco-transcriptionofwspBwithvirD4.
aLanes1and2RT-PCRnegativecontrolswithnoRNAorwithnoreversetranscriptase,respectively.
Lanes3and4RT-PCRofRNAfromuninfectedC7-10andinfectedC/wStr1cells,respectively,withvirD4forwardandwspBreverseprimers.
Lane5RT-PCRpositivecontrolwithC/wStr1RNAandWolbachiaprimersS12F/S7R,whichamplifyportionsofaribosomalproteinoperondescribedpreviously(Fallon2008).
bLane1PCRnegativecontrolwithnoTaqenzyme.
Lanes2and3negativecontrollackingRT,withRNAfromuninfectedC7-10andinfectedC/wStr1cells,respectively59ArchMicrobiol(2016)198:53–69Fig.
3AminoacidsequencealignmentofRibAhomologsfromBwStrandWolbachiasupergroupsA(red),B(blue)andD(black)respectively.
Asterisksbelowthealignmentindicateuniversallyconservedresidues.
Uniqueresiduesareingreenfont.
ResiduesconservedinBwStrandamajorityofB-strainsareindarkblue,boldfont,whilethoseindarkred,boldfontareconservedwithamajorityofA-strains.
Residuesconservedintwotofourstrainsareinlightblue,orangeororangeboldfont.
Residueshighlightedingraycorrespondto95%confidencepeptidesdetectedbyLC–MS/MS.
Thedihydroxybutanonephosphatesynthase(RibB)andGTPcyclohydrolaseIIdomains(RibA)areindicatedabovethealignmentwithingreaterthanlessthansymbols.
BoldunderlinedresiduesinAwMelandBwStrindicateconservedactivesiteaminoacids,includ-ingcriticalcysteineresidues.
Doubleunderlinedresiduesindicateaminoacidsinvolvedinthedimerizationinterface.
SeeTables2andS2forhostassociationsandGenBankAccessions.
ThePCR-amplifiedBwStrsequencedoesnotencodetheN-terminalaminoacids;position1correspondstothe15thaminoacid1>DHBPsynthasedomainRibAGTPcyclohydrolaseII180wKueNNHEMRNWCEKNDVIALDTSFINNFQENQDVYEVCKTSLFLKQTQEVNIISYRTESGGREwMelNNHEMRNWCEKNDVIALDTSFINNFQENQDVYEVCKTSLFLKQTQEVNIISYRTESGGREwHaNNHEMRNWCEKNDVIALDTSFINNFQENQDVYEVCKTSLFLKQTQEVNIISYRTESGGREwRiNKYEMRNWCEENDIIALDTLLVNDFQQNQSVYEVCKTSLFLKQTQEVDIISYRTESGGREwVulCNNHEMQNWCEKNDVIALDTSFINNFQENQDVYEVCKTSLFLKQTQEVDIISYRTESGGREwStrNNHEMRNWCEKNDVIALDKSFINNFQENQDVYEVCKTSLFLKQTQEVDIISYRTKSGGREwTaiNKHEMRNWCEENDIIALNTLLVNDFQRNHSVYEVCKTSLFLKQTQEVDIISYRTKSGGREwPipNKHEMRNWCEENDIIALNTLLVNDFQQNHSVYEVCKTSLFLKQTQEVDIISYRTKSGGREwVitBNKHEMRNWCEENDIIALNTLLVNDFQQNHSVYEVCKTSLFLKQTQEVDIISYRTKSGGREwBmDEYEMRGWCEKSDVIALDVLFINNFQQNQDIYEVCKTPLFLKQTQKVNIISYRTCNGRKE181>RibAGTPcyclohydrolaseIIdomainRibAGTPcyclohydrolaseIIdomain300wKueDGRGIGLTNKLRAYSMQRGHNLDTVDANRILGFEDDERSFAVAAKMLKKLNINKIQLLTNwMelDGRGIGLTNKLRAYSMQRGHNLDTVDANRILGFEDDERSFAVAAKMLKKLNINKIQLLTNwHaDGRGIGLTNKLRAYSMQREHNLDTVDANRILGFEDDERSFVVAAKMLKKLNINKIQLLTNwRiDGRGIGLTNKLRAYSVQREHNLDTVDANRILGFEDDERSFVVAAKMLKKLNINKIQLLTNwVulCDGRGIGLANKLRAYSMQRRHNLDTVDANRVLGFEDDERSFAVAVEILKKLDIKKIQLLTNwStrDGRGIGLTNKLRAYSMQRKYNLDTVDANRVLGFEDDERSFAVAAKILKKLNINKIQLLTNwTaiDGRGIGLTNKLRAYSMQRKYNLDTVDANRVLGFEDDERSFAVAAKILKKLNINKIQLLTNwPipDGRGIGLTNKLRAYSMQRKYNLDTVDANRVLGFEDDERSFAVAAKILKKLNINKIQLLTNwVitBDGRGIGLTNKLRAYSMQRKYNLDTVDANRVLGFEDDERSFAVAAKILKKLNINKIQLLKNwBmDGRGIGLTNKLRAYDMQRKYNLDTVDANRILGFEDDERSFAVAAEMLKKLGIKKIQLLTN201>RibAGTPcyclohydrolaseIIdomain98%nucleotideidentityFig.
S2)isconsistentwithexchangeofanapparentlyintactgenebetweenmembersofdistinctWolbachiasupergroupsbyamechanismthatrequiresfurtherinvestigation.
Inten-siveanalysisofthewspAparalogdemonstratesthatintra-genicrecombinationbreakpointsareconcentratedincon-servedregionsoutsideoftheHVRs(Baldoetal.
2005,2010).
CAARTARYrepeatsarenotpresentinwspA,andinwspB,theyoccuronlywithinanddirectlyadjacenttoHVR2atpositionsthatcorrespondtopseudogenelesionsinAwCobU4-2andinBwPip(duetoatranspositioneventinBwPip;Sanogoetal.
2007).
Furthermore,Pichonetal.
(2009)suggestedthattranspositioneventsmayexplainabsenceofwspBinthevirB8-D4operonsofmanyWOL-B-strains.
Inapracticalsense,CAARTARYrepeatsatwspBpseudogenelesionsandWOL-A/Bsequencemotiftransitions(Figs.
S1,S2,S3)suggesttheirinvolvementingeneticexchange.
BecausetransformationofWolbachiahasnotyetbeenachieved,engineeringofCAARTARYrepeatsintovectorsusedsuccessfullytointroduceselecta-blemarkersintoothermembersoftheRickettsiales(seeBeareetal.
2011)meritsinvestigation.
PotentialfunctionsofWspBAlthoughbacterialoutermembraneproteinsareimpor-tantmediatorsofinteractionswithhostcellsandspecificfunction(s)ofbothWspAandWspBremaintobeidenti-fied,theymayhaveuniquefunctionsasporinproteinsinWolbachia,whichlackcellwalls.
ThevirB8-D4operonsofWolbachiaanditssistergenera,AnaplasmaandEhrlichia,aresimilarlyorganized(Gillespieetal.
2010;Hotoppetal.
2006)with3′-terminalgenesencodingmajorsurfacepro-teinsthat,analogoustowspB,areco-transcribedwiththevirgenes(Ohashietal.
2002).
InA.
marginale,afamilyofmsp2pseudogenesundergo"combinatorialgenecon-version"attheexpressionsite(Braytonetal.
2002)andMSP2variantschangeduringgrowthindifferenthostcelltypes,whichlikelyreflectsaresponsetohostimmunitymechanisms(Chávezetal.
2012).
Similarly,Baldoetal.
(2010)proposedthatchangesinWspAHVRregionsplayaroleinhostadaptationandinnateimmunityinteractions,consistentwithvariationinthehigher-orderstructureoftheproteinindifferenthosts(UdayandPuttaraju2012).
HVRsequencechangesinthewspBparalogmayreflectasimilardynamic.
AdditionalevidenceindicatesthatMSP2proteinsareglycosylated(Sarkaretal.
2008),whichisnowanestablishedprocessinpost-translationalmodifica-tioninbacteria(Delletal.
2010;NothaftandSzymanski2010),andwenotethatWspBcontainspotentialglycosyla-tionsites.
AlthoughaninactivatedpseudogeneorabsenceofwspBinvirB8-D4operonsofsomeWolbachiastrainsindicatesthatitisnotabsolutelyrequiredforsurvival,asecretomeanalysisofBrugiamalayishowedthatWspBfromDwBmisexcreted/secretedintofilarialhostcells(Bennuruetal.
2009).
Furthermore,itco-localizeswiththeBm1_46455hostproteinintissuesthatincludeembryonicnuclei(Melnikowetal.
2011).
WspBisthereforeitselfacandidateT4SSeffectorthatmayplayaroleinreproduc-tivemanipulationofthehost.
MosaicisminwspBanditshighrateofevolution(Comandatoreetal.
2013)maythusreflectgeneticchangesthatoptimizeadaptationtoparticu-larhostcellssuchasthoseinreproductivetissuesandfacil-itateexploitationofnewarthropodnichesbyWolbachia.
GeneticplasticityofribAinthevirB8D4operonAsidefromwspBatthe3′-endoftheT4SSvirB8-D4operon,ribAexhibitsgeneticplasticityatits5′-end.
InbothBwStrandBwVulC,ribAisatwo-partmosaicofN-termi-nalWOL-AandC-terminalWOL-Bmotifs.
Incontrast,theinternalvirB8-D4geneshavetypicalB-strainidenti-ties,andinsomestraincomparisons,aminoacididentitiesslightlyexceednucleotideidentities,whichPichonetal.
(2009)attributetostrongselectionagainstnon-synony-mouscodonsubstitutions.
AmongtheinternalvirB8-D4genes,however,Klassonetal.
(2009)suggestthatinAwRi,anespeciallyvariableregioninvirB10islikelyderivedfromgeneticexchangewithaB-strain.
WenoteherethatribAfromAwRicloselyresemblesB-strainhomologswithinavariableregionthatimmediatelyprecedestheGTPcyclohydrolasedomain,whereitshomologinBwStrtran-sitionsfromWOL-AtoWOL-Bsequencemotifs(Fig.
S1,positions387–450).
IncontrasttoDwBm,inwhichribAandvirB8areco-transcribedandbindcommontranscriptionfactors(LiandCarlow2012),relativeabundancelevelssuggestthatinBwStr,ribAistranscribedindependentlyofthevirB8-D4operon.
SomeWOL-B-strains,suchasBwVulC,lackwspBatthe3′-terminusofthevirB8-D4operon,whileourdataconfirmthatinBwStr,wspBisco-transcribedwiththevirgenes,consistentwithsimilarrelativeabundancesofWspBandthefiveVirproteins.
Inaggregate,theseobser-vationssuggestthatWOL-DandWOL-A-/B-strainsmaydifferinhowRibAandWspBexpressioninterfaceswith66ArchMicrobiol(2016)198:53–69T4SS-mediatedtransportofeffectorsinfilarialwormsandarthropodhosts(Felixetal.
2008;Masuietal.
2000;Rancesetal.
2008;Wuetal.
2004),anditwillbeofinteresttoexplorewhethersuchdifferencesrelatetoriboflavinpro-visioning.
Infilarialnematodes(LiandCarlow2012;Stru-bingetal.
2010;Wuetal.
2009)andbedbugs(Hosokawaetal.
2010),evidencesuggeststhatWolbachiaprovisionshostwithriboflavin,theprecursorofflavincofactorsthatareessentialformanycellularredoxreactions.
Incontrast,riboflavindepletionreducesBwStrabundanceinC/wStr1cells,suggestingthatBwStrutilizeshostriboflavinanddoesnotaugmentriboflavinlevelsinmosquitohostcells(Fallonetal.
2014).
PotentialfunctionsofRibAandRibBIninitialcommitmentstepsinriboflavinbiosynthesis,enzymaticactivitiesencodedbytheribAandribBfunc-tionaldomainsuseGTPandribulose-5-phosphateassub-stratestocatalyzeriboflavinbiosynthesis,consuming25moleculesofATPpermoleculeofriboflavin(Bacheretal.
2000).
WenotethatinWolbachiagenomes,ribAistheannotatedhomologofribBAinEscherichiacoli(Brutineletal.
2013)andencodesadihydroxybutanonephosphatesynthasedomainwithputativeRibBfunctionneartheN-terminus,upstreamofaGTPcyclohydrolaseIIdomainwithconserveddimerizationandactivesiteresidues(RibAfunction).
AsinE.
coli,WolbachiagenomesalsoencoderibB,butatadistinctchromosomallocus,suggestingthatribAandribBarenotcoordinatelyexpressed.
InSinorhizo-biummeliloti(Rhizobiales;Alphaproteobacteria),knock-outmutationsofribBAdecreasedflavinsecretionbutdidnotcauseriboflavinauxotrophyorblockestablishmentofsymbiosis,suggestingthatRibBAmayhaveanundefinedroleinmoleculartransport(Yurgeletal.
2014).
AsisthecasewithBwStr,RibBisatleastthreefoldmoreabundantthanRibAinthebacteriumAcidithiobacillusferrooxidans(Knegtetal.
2008).
Inyeast,RibBhasthiol-dependentalternativeredoxstates(McDonaghetal.
2011),partiallylocalizestothemitochondrialperiplasm,andhasanunex-plainedfunctioninoxidativerespirationthatisindependentofriboflavinbiosynthesis(Jinetal.
2003).
Theseobserva-tionsraisethepossibilitythatinWolbachia,RibAandRibBmayhavefunctionsotherthanriboflavinbiosynthesisthatintegratewithpathwaysinvolvedincellularoxidativestate,suchasironmetabolism.
Intracellularbacteriaarechal-lengedbyhost-imposedoxidativestressandironstarvation(reviewedbyBenjaminetal.
2010)andriboflavinbiosyn-thesisisassociatedwithironacquisitioninbacteriasuchasHelicobacterpylori(Worstetal.
1998)andCampylobacterjejuni(Crossleyetal.
2007).
Wolbachiainterfereswithironmetabolismandsequestrationininsects(Brownlieetal.
2009;Kremeretal.
2009)andinfluencesiron-dependenthostprocessessuchashememetabolism,oxidativestress,apoptosisandautophagy(Gilletal.
2014).
Wenotethattheperiplasmiciron-bindingcomponentofamembranetrans-porterisanabundantproteininBwStr(TableS3,entry778andBaldridgeetal.
2014).
AcknowledgmentsThisworkwassupportedbyGrantAI081322fromtheNationalInstitutesofHealthandbytheUniversityofMin-nesotaAgriculturalExperimentStation,St.
Paul,MN.
CompliancewithethicalstandardsConflictofinterestTheauthorshavenoconflictsofinteresttodeclare.
OpenAccessThisarticleisdistributedunderthetermsoftheCrea-tiveCommonsAttribution4.
0InternationalLicense(http://creativecom-mons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
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