PCR Primer Design for 16S rRNAs for Experimental ... - Frontiers

16S rRNA amplicons obtained from the environmental metagenome using ... These primers can cover the majority of bacterial 16S rRNA genes and ... ThisarticleispartoftheResearchTopic UsingGenomics,MetagenomicsandOther"Omics"toAssessValuableMicrobialEcosystemServicesandNovelBiotechnologicalApplications Viewall 53 Articles Articles DianaE.Marco ConsejoNacionaldeInvestigacionesCientíficasyTécnicas(CONICET),Argentina TrevorC.Charles UniversityofWaterloo,Canada KeiKitahara HokkaidoUniversity,Japan Theeditorandreviewers'affiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction MaterialsandMethods ResultsandDiscussion AuthorContributions ConflictofInterestStatement Acknowledgments SupplementaryMaterial References SuggestaResearchTopic> DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) Supplementary Material Exportcitation EndNote ReferenceManager SimpleTEXTfile BibTex totalviews ViewArticleImpact SuggestaResearchTopic> SHAREON OpenSupplementalData ORIGINALRESEARCHarticle Front.Bioeng.Biotechnol.,28February2017 |https://doi.org/10.3389/fbioe.2017.00014 PCRPrimerDesignfor16SrRNAsforExperimentalHorizontalGeneTransferTestinEscherichiacoli KentaroMiyazaki1,2*, MitsuharuSato1,2and MiyukiTsukuda1,2 1DepartmentofLifeScienceandBiotechnology,BioproductionResearchInstitute,NationalInstituteofAdvancedIndustrialScienceandTechnology(AIST),Tsukuba,Ibaraki,Japan 2DepartmentofComputationalBiologyandMedicalSciences,GraduateSchoolofFrontierSciences,TheUniversityofTokyo,Kashiwa,Chiba,Japan WerecentlydemonstratedthattheEscherichiacoliribosomeisrobustenoughtoaccommodateforeign16SrRNAsfromdiversegamma-andbetaproteobacteriabacteria(Kitaharaetal.,2012).Therein,weusedthecommonuniversalprimersBac8fandUN1541rtoobtainanearlyfull-lengthgene.However,wenoticedthattheseprimersoverlapvariablesitesat19[A/C]and1527[U/C]inBac8fandUN1541r,respectively,andthus,theampliconcouldcontainmutations.Thisisproblematic,particularlyfortheformersite,becausethe19thnucleotidepairswiththe916thnucleotide,whichisapartofthe“centralpseudoknot”andiscriticalforfunction.Therefore,wemutationallyinvestigatedtheroleofthebasepairusingseveral16SrRNAsfromgamma-andbetaproteobacteria.Wefoundthatboththenativebasepairs(gammaproteobacterial19A–916Uandbetaproteobacterial19C–916G)andthenon-native19A–916Gpairretainedfunction,whereasthenon-native19C–916Uwasdefective16SrRNAs.Wenextdesignedanewprimerset,Bac1fandUN1542r,sothattheydonotoverlapthepotentialmismatchsites.16SrRNAampliconsobtainedfromtheenvironmentalmetagenomeusingthenewprimersetweredominatedbyproteobacterialspecies(~85%).Subsequentfunctionalscreeningidentifiedvarious16SrRNAsfromproteobacteria,allofwhichcontainednative19A–916Uor19C–916Gbasepairs.Theprimersdevelopedinthisstudyarethusadvantageousforfunctionalcharacterizationofforeign16SrRNAinE.coliwithnoartifacts. Introduction Thebacterialribosomeconsistsof3rRNAmoleculesand54proteinsandplaysacrucialroleintranslatingmRNA-encodedinformationintoproteins.Becauseofthestructuralcomplexityoftheribosome(Schuwirthetal.,2005),itisbelievedthateachribosomalcomponentcoevolvestomaintainfunction(Jainetal.,1999).Inparticular,becausethe16Sand23SrRNAsformthestructuralcoreoftheribosome(Schuwirthetal.,2005),theyarebelievedtobeleastlikelytoexperiencehorizontalgenetransferbetweenspecies(Jainetal.,1999).Onthebasisofthespecies-specificnatureofrRNAandtheiromnipresenceinallbacteria,therRNAgenes,especiallythosefor16SrRNA,havelongbeenusedasan“ultimatechronometer”(Woese,1987)forphylogeneticclassificationofbacterialspecies(Laneetal.,1985;Woese,1987). Despitetheapparentspecies-specificnatureof16SrRNAs,werecentlyfoundthattheEscherichiacoliribosomeisabletoaccommodateforeign16SrRNA(Kitaharaetal.,2012).Namely,usingE.coliΔ7,anullmutantoftherrn(ribosomalRNA)operon,asahoststrain,wehaveshownthatvarious16SrRNAgenes,includingthosefromadifferentphylogeneticclass(i.e.,betaproteobacteria),wereabletocomplementgrowth.Thelowestidentityoffunctional16SrRNAgenetothatofE.coliwasaslowas80%,implyingthathundredsofsimultaneousnucleotidechangesarepermittedinthemaintenanceofribosomefunction.ThebasisforthishighmutabilityistheconservationoftheRNAsecondarystructures,whichisconsistentwithapreviousfindingthat16SrRNAistypicallyrecognizedbyribosomalproteinsviasaltbridgesbetweenphosphateoxygenatomsoftheRNAbackbone,butnucleotidebasesarenotstrictlydiscriminated(Brodersenetal.,2002).Furthermore,insertion/deletionisallowedinsomeRNAhelices(e.g.,h6,10,and17)thatarenotinvolvedinproteinbinding.Understandingthesequenceandstructuralvariationsof16SrRNAthatareaccommodatedintheE.coliribosomeshouldbehelpfulforourunderstandingoftheevolutionofrRNAandthesequence–structure–functionrelationshipsoftheribosome. Inourpreviousstudy,toPCRamplifyforeign16SrRNAgenes,weusedBac8f(A)orBac8f(C)[themostcommonlyused“Bac8f”(Edenetal.,1991)]fortheforwardprimerandUN1541r(U)orUN1541r(C)forthereverseprimer(Figure1;oligonucleotidesequencessummarizedinTable1)(Kitaharaetal.,2012),whichallowedamplificationofanearlyfull-lengthgene.Theseprimerscancoverthemajorityofbacterial16SrRNAgenesandthusarecommonlyusedforphylogeneticand/orcommunityanalysis(Laneetal.,1985;Weisburgetal.,1991;Amannetal.,1995).However,wenoticedthattheampliconsobtainedusingtheprimersetcontainedmutationsatcertainfrequencies,whichcouldaffectthefunctionalityofinvivo-reconstitutedmutantribosomes.InE.coli16SrRNA,nucleotides17–19pairwithnucleotides916–918toformashorthelix(h2)(Figure2).Thehelixisinvolvedintheformationofthe“centralpseudoknot,”whosestructureishighlyconservedinbothprokaryotesandeukaryotes.Thisuniquestructureisessentialfortranslationalinitiationandishighlysusceptibletopointmutations(Brinketal.,1993;DammelandNoller,1993;Pootetal.,1998).Despitethisstructuralconservation,however,the19thnucleotidevariesdependingonthespecies,19Aor19C,whichpairswith916Uor916G,respectively(Figure2).Thus,ifBac8f(A)orBac8f(C)isusedasaprimer,thereisapossibilityofgeneratingamismatchbetweenthe19thand916thnucleotides.Similarly,the1,527thpositionisalsovariable(CorU)andcangenerateamismatchintheamplicons(Figure1B),althoughthissitemaynotbeinvolvedinfunction.Thus,inourspecificsystemforfunctionalinvestigationof16SrRNAs,itisessentialtodevelopanewprimersettoavoidtheintroductionofartificialmutations.Inaddition,weneedtotaketheRNAprocessingissueintoconsideration.ForproperprocessingofaprecursortranscriptintomaturerRNAs(16S,23S,and5SrRNAs),theprocessingsites(i.e.,RNasecleavagesites)needtobesimilartotheE.colisequences(GutgsellandJain,2012). FIGURE1 Figure1.Coverageratesandnucleotidecompositionatthe(A)5′-and(B)and3′-endsofthebacterial16SrRNAgene.TheannealingregionsforBac1f,Bac8f,andUN1541rareshownbyarrowswiththesequences.Wobblenucleotidesaremarkedbyasterisks.Colors:blue,A;red,T;green,G;purple,C;black,gap. TABLE1 Table1.PCRprimersusedforamplificationof16SrRNAgenes. FIGURE2 Figure2.CentralpseudoknotstructureinEscherichiacoli16SrRNA.Nucleotidenumbers19and916arelabeledinwhitelettersinablackcircle. Takingthesepointsintoconsideration,wefirstevaluatedtheeffectsofmismatchesbetweenthe19thand916thnucleotides.Inadditiontothenative19A–916Uand19C–916Gpairs,thenon-native19A–916Gpairretainedfunction,whereasthenon-native19C–916Uwasdetrimental.Second,wedesignednewprimers,Bac1fandUN1542r,whichencompassednucleotidepositions1–18forBac1fand1542–1528forUN1542r,sothattheydidnotoverlapthepotentialmismatchsites.TheseprimerswereusedforPCRamplificationof16SrRNAgenesandtheresultantlibrarywasfunctionallyscreened.DNAsequencingofthe16SrRNAgenesinthefunctionalclonesconfirmedtheabsenceofunwantedmismatchesinthefunctionalgenes. MaterialsandMethods Reagents KOD-Plus-NeoandKODFX-NeoDNApolymeraseswerepurchasedfromToyobo(Osaka,Japan).Trimethoprim(Tmp),ampicillin(Amp),kanamycin(Km),andsucrose(Suc)werepurchasedfromWakoPureChemicals(Tokyo,Japan).Zeocin™(Zeo)andtheIn-FusionCloningKitwerepurchasedfromInvitrogen(Carlsbad,CA,USA).LennoxLBmedium[1%(w/v)tryptone,0.5%(w/v)yeastextract,0.5%(w/v)NaCl]waspurchasedfromMerck(Tokyo,Japan).TheExtrapSoilDNAKitPlusver.2waspurchasedfromJ-Bio21(Tsukuba,Japan).FastDNAKitwaspurchasedfromBIO101(LaJolla,CA,USA).Oligonucleotideprimers(Table1)werepurchasedfromSigma(Hokkaido,Japan). BacterialStrainsandCultureConditions ThefollowingbacterialstrainswerepurchasedfromtheBiologicalResourceCenter(NBRC),NationalInstituteofTechnologyandEvaluation,Japan:Serratiaficaria(NBRC102596),Caldimonasmanganoxidans(NBRC16448),Hydrogenophagaflava(NBRC102514),Hydrogenophilusthermoluteolus(NBRC14978),Oxalicibacteriumhorti(NBRC13594),Oligellaurethralis(NBRC14589),andRalstoniapickettii(NBRC102503).Burkholderiasacchariwasalaboratorystock.CompetentE.coliJM109cellswerepurchasedfromRBCBioscience(Taipei,Taiwan).Antibioticswereaddedwhennecessaryatthefollowingconcentrations:Tmp,10μg/ml;Amp,100μg/ml;Km,25μg/ml;andZeo,50μg/ml.Agar(1.5%[w/v])wasaddedtosolidifythemedium.Sucwasaddedat5%(w/v)forcounterselectionpurposeswhennecessary. GenomicDNAPurification,PCRAmplificationof16SrRNAGenes,andLibraryConstruction GenomicDNAofbacterialisolateswaspurifiedusingtheFastDNAKit.TheNitrosomonaseuropaeawasagiftfromDr.NaohiroNoda(AIST,Japan).EnvironmentalmetagenomicDNA(soils,fermentedproducts,andseawater)waspurifiedusingtheExtrapSoilDNAKitPlusver.2.The16SrRNAgeneswereamplifiedbyPCRusingasetofprimers,Bac1f,Bac8f(A),orBac8f(C)andUN1542r.Thereactionmixturecontained100ngoftemplateDNA(bacterialgenomeorenvironmentalmetagenome),1×PCRbuffer,0.4mMeachofdNTPs,0.25μMeachofprimersand1UofKODFX-NeoDNApolymeraseinatotalvolumeof50μl.Themixturewasheatedat94°Cfor2minandsubjectedto30roundsofthermalcyclingat98°Cfor10s,48°Cfor30sforBac1for57°Cfor30sforBac8f(A)andBac8f(C),and68°Cfor1.5minandfinalincubationat68°Cfor5min.Theampliconwasseparatedbyagarosegel(0.8%[w/v])electrophoresis;asinglebandwasexcisedfromthegel,purifiedanddissolvedin30μlofwater. Anexpressionvectorforthe16SrRNAgenewasmodifiedfrompRB103(KitaharaandSuzuki,2009;Kitaharaetal.,2012)bydeletingthegenesfortRNA,23SrRNA,and5SrRNA,replacingtheantibioticselectionmarkerfromZeotoTmpandreplicationoriginfrompSC101top15A.TheresultantplasmidwasnamedpMS205aTp1(mapillustratedinFigureS1AinSupplementaryMaterial).Theentirevector(withoutthe16SrRNAgene)wasPCR-amplifiedusingtheprimersetBac1R,Bac8r(A),orBac8r(C)andUN1542f.ThePCRmixturecontained1×PCRbuffer,0.2mMeachofdNTPs,1.5mMMgSO4,0.25μMeachofprimers,10ngofpMS205aTp1,and1UofKOD-Neo-DNApolymeraseinatotalvolumeof50μl.Themixturewasheatedat94°Cfor2minandsubjectedto25cyclesat94°Cfor10s,60°Cfor30s,and68°Cfor2.5min,followedbyafinalincubationat68°Cfor5min.TheproductsweretreatedwithDpnI(10U,37°C,6h),gel-purifiedanddissolvedin30μlofwater. The16SrRNAgene(ca.,200ng)andthelinearizedpMS205aTp1(ca.,200ng)fragmentswerecombinedandligatedusingtheIn-FusionCloningKitinatotalvolumeof10μl.Afterincubationat50°Cfor1h,thereactionproducts(2μl)wereintroducedintocompetentE.coliJM109cells(100μl)andgrownonLB/Tmpagarplatesat37°Covernight.Someofthecolonieswererandomlypickedandusedforsequencedeterminationforphylogeneticanalysis.Restofthecolonieswerecombinedandplasmidswereextractedtoyieldalibrary. FunctionalScreeningof16SrRNAGenes EscherichiacoliMY201rna−isaderivativeofE.coliΔ7rna−(KitaharaandSuzuki,2009),whichcontainsthegrowthrescueplasmidpMY201(modifiedfrompRB101bysubstitutingthepSC101oritop15Aori,FigureS1BinSupplementaryMaterial)andpML103Δ(expressionplasmidfor23SrRNA,5SrRNA,andtRNAs,createdbydeletingthe16SrRNAgenefrompRB103,mapillustratedinFigureS1CinSupplementaryMaterial).CompetentMY201cellsweretransformedwithapMS205aTp1libraryandgrownonLB/Km/Zeo/Tmpagarplatesat37°Cforovernight.Colonieswerecollected,mixedin1mlofLBbroth,vigorouslyvortexed,appropriatelydiluted,andspreadoverLB/Km/Zeo/Tmp/Sucagarplates.Someofthecolonieswererandomlypickedandusedforsequencedeterminationforphylogeneticanalysis.Restofthecoloniesgrownontheplateswerecollectedandusedforfurtherstudies. GrowthAssay MutantE.colistrainsweregrownin1mlofLB/Km/Zeo/Tmp/Sucbrothina96deep-wellplate.Theplatewasincubatedat37°Cwithvigorousagitation(1,200rpm)inanMBR-024microplateshaker(Taitec,Saitama,Japan).After14h,1μloftheculturewastransferredtoafreshLB/Km/Zeo/Tmp/Sucbroth(1ml)in96-wellplateandgrownatvarioustemperatures(30,37,or42°C)withvigorousagitation(1,200rpm).After14h,200μloftheculturewastransferredtoa96-wellplateandOD600wasmeasured. DNASequencingandBLASTSearch DNAsequencingwascarriedoutusingtheSangermethodwithanAppliedBiosystems(FosterCity,CA,USA)automaticDNAsequencer(ABIPRISM3130xlGeneticAnalyzer)andanAppliedBiosystemsBigDye(ver.3.1)kit.Blastsearch(Altschuletal.,1990)wascarriedoutusingtheNCBInucleotidedatabase“16SrRNAsequences(BacteriaandArchaea)”withtheprogramselectionoptimizedfor“Highlysimilarsequences(megablast).” DatasetandSequenceAlignmentof16SrRNAGenes All16SrRNAgenesequences(plus50additionalnucleotidesatthe5′and3′ends)wereretrievedfromthegenomicsequencesintheNCBIdatabase(asofAugust2014)(Table2).Atotalof9,624geneswereidentifiedin2,476genomesof23phyla.MultiplesequencealignmentofthesegeneswasperformedusingtheMAFFTv7program(KatohandStandley,2013). TABLE2 Table2.Listof16SrRNAgenesretrievedfromtheNCBIdatabase.a NucleotideSequenceAccessionNumbers Thenucleotidesequencesfor16SrRNAgenehavebeendepositedinGenBank/EMBL/DDBJundertheaccessionnumbersLC213146–LC213207,LC213207–LC213252,andLC213253–LC213296. ResultsandDiscussion PrimerDesign Nucleotidecompositionaroundthe5′-and3′-endregionsofallbacterial16SrRNAgenes(Table2)isshowninFigure1.Forthe5′-end,thesequencesurroundingthe19thnucleotide,particularlyfromthe8thto27th,ishighlyconserved(Figure1A),whichcorrespondstotheBac8fprimer-bindingsite.TheBac8fprimercovers97%ofbacterial16SrRNAsequences(Figure1A),confirmingtheappropriatenessoftheprimerforphylogenetic/communityanalysis(Laneetal.,1985;Amannetal.,1995).However,duetothepresenceofapotentialmismatchsiteatthe19thnucleotideposition,thisisnotappropriateforourspecificpurpose(i.e.,functionalanalysis),andthus,wedesignedanewprimerBac1f,whichencompassesthe1stto18thnucleotides.Althoughtheverybeginningofthesequence(fromfirsttoseventhnucleotides)ishighlyvariableamongallbacteria(Figure1A),theregioniscriticalforRNAprocessing(GutgsellandJain,2012),sowestrictlyfollowedtheE.colisequenceforthissite. WenextcheckedthecoveragerateoftheBac1fprimerforeachphylum(FigureS2inSupplementaryMaterial).Asdescribedabove,the5′endofthe16SrRNAsequencevariesamongbacteria(Figure1A).Nevertheless,Bac1fshowedrelativelyhighspecificitytosomebacterial16SrRNAsthatincludedBacteroidetes–Chlorobi(FigureS2BinSupplementaryMaterial),Chlamydiae-Verrucomicrobia(FigureS2CinSupplementaryMaterial),andProteobacteria(FigureS2GinSupplementaryMaterial).Inourpreviousstudy(Kitaharaetal.,2012),nofunctional16SrRNAswereobtainedfromphylaotherthanproteobacteria.Thus,inpractice,althoughtheBac1fprimerhassomebiastospecificphylogeneticgroups,thisbiasisadvantageoustoenrichinglibrarieswithapotentiallyfunctionalfractionandtoreducingbackground. FigureS3inSupplementaryMaterialsummarizesthecoveragerateoftheBac1fprimerforeachclassofproteobacteria.Overall,thereisaslightpreferenceforalpha-,beta-,andgamma-classesofproteobacteria,andthedelta-epsilonclasshasalargernumberofpotentialmismatches. EffectsofNon-NaturalBasePairingbetweenthe19thand916thNucleotidesonRibosomalActivity Toinvestigatehownon-naturalbasepairingbetweenthe19thand916thnucleotidesaffectsribosomalactivity,weused16SrRNAgenesfromthefollowingbacteria:gammaproteobacterialE.coli(Eco)andS.ficaria(Sfi)andbetaproteobacterialB.sacchari(Bsa),C.manganoxidans(Cma),H.flava(Hfl),H.thermoluteolus(Hth),N.europaea(Neu),O.horti(Oho),O.urethralis(Our),andR.pickettii(Rpi). PCRamplificationwascarriedoutusingthreetypesofforwardprimers:Bac1f,Bac8f(A),orBac8f(C);UN1542rwasusedasacommonreverseprimer.AspecificallyamplifiedfragmentwasthenclonedbackintopMS205aTp1.Afterconfirmingthesequenceoftheentire16SrRNAgene,theresultantplasmidwastransferredintoE.coliMY201rna−(Δ7strain).AftersacB-basedcounterselectiontoeliminaterescueplasmidsexpressingE.coli16SrRNA,alltheclonesweresuccessfullyobtainedat37°C,implyingthatmutationinthe19th–916thbasepairismoreorlesspermissiveunderthiscondition. Wenextexaminedthegrowthpropertiesofeachmutantatvarioustemperatures(30°C,37°C,and42°C).Figure3AillustratesOD600aftergrowthfor14h.Ingeneral,forthenativebasepairs(19A–916Uforgammaproteobacterialand19C–916Gforbetaproteobacterial16SrRNAs)allcloneshadhigherOD600at37°Cthanat30°C.Fortheclonescarryingnon-nativepairs(Figure3B,19C–916Uforgammaproteobacterialand19A–916Gforbetaproteobacterial16SrRNAs),nogrowthperturbationwasobservedforbetaproteobacterialclonesandtheyappearedtogainabroadenedtemperatureoptimum;finalOD600shiftedupwardat30°C.Incontrast,gammaproteobacterialclones(EcoandSfi)showedgreatlyreducedOD600valuesatalltemperatures.Virtuallynogrowthwasobservedat30°C,indicatingtheappearanceofacold-sensitive(orheat-tolerant)phenotype. FIGURE3 Figure3.Growthprofile.(A)Nativebasepairsbetweennucleotides19and916[19C–916GforbetaproteobacterialBurkholderiasacchari(Bsa),Caldimonasmanganoxidans(Cma),Hydrogenophagaflava(Hfl),Hydrogenophilusthermoluteolus(Hth),Nitrosomonaseuropaea(Neu),Oxalicibacteriumhorti(Oho),Oligellaurethralis(Our),andRalstoniapickettii(Rpi)and19A–916UforgammaproteobacterialEscherichiacoli(Eco)andSerratiaficaria(Sfi)]and(B)non-nativebasepairsbetweennucleotides19and916(19A–916GforbetaproteobacterialBsa,Cma,Hfl,Hth,Neu,Oho,Our,andRpiand19C–916UforgammaproteobacterialEcoandSfi).yaxisrepresentsOD600aftercultivationatvarioustemperatures(30°C,openbars;37°C,shadedbars;42°C,solidbars)for14h. Pootetal.(1998)haveanalyzedtheroleofh2throughmutagenesis.UsingE.coli16SrRNAasatemplate,theyintroducedapointmutationtoalterthenative19A–916Ubasepairto19A–916G,19C–916G,and19C–916U.Theyusedthemutantribosomeinaninvitrotranslationalassay(at42°C)andfoundthattheformertwomutantsretainednearlyfullactivity(>80%),whereas19C–916Uhadmuchreduced(30%)activity.Althoughtheassaysystemsaredifferent,thegeneralconclusionofboththeirstudyandoursisthat19C–916Uisdefective. MetagenomicScreeningforFunctional16SrRNAGenesinE.coli Wenextusedenvironmentalmetagenomesasasourcefor16SrRNAgenes.Forallprimersets,specificamplificationwasobtained(FigureS4inSupplementaryMaterial).Toinvestigatesequencediversity,theclonedgeneswerephylogeneticallycharacterized.AsshowninFigure4,whenBac1fwasusedasaforwardprimer,thegenewasdominatedbyproteobacterial16SrRNAs[~84%(32/38)],butwasmuchlessforthoseobtainedusingtheBac8f(A)andBac8f(C)primers[~56%(25/45)and~73%(35/48),respectively]. FIGURE4 Figure4.Phylogeneticanalysisof16SrRNAgenesclonedinpMS205aTp1(beforefunctionalselection)amplicons.Theforwardprimersusedwere(A)Bac1f,(B)Bac8f(A),and(C)Bac8f(C).“All”istheclassificationbasedonphylumand“Proteobacteria”istheclasscompositionintheproteobacterialphylum. Thegeneswerethensubjectedtofunctionalscreening.Functional16SrRNAgeneswerecollected,andtheirmicrobialoriginsandthebasepairpatternsbetweenthe19thand916thnucleotideswereinvestigated.AsshowninTable3,whenBac1fwasused,85%(52/61)offunctional16SrRNAswerefromgammaproteobacteriaandtherestwerefrombetaproteobacteria.PhylogenetictreeofthesesequencesisillustratedinFigureS5AinSupplementaryMaterial.Basepairpatternswere78%19A–916U(48/61)and22%(13/61)19C–916G.Itisnoteworthythatnoartificiallyshuffledbasepairs(A–GandC–U)wereobserved,implyingthatthenewlydesignedprimersdidnotintroducenon-nativebasepairsandareadequateforfunctionalstudies.Therearesomemismatchesbetweenproteobacterial16SrRNAandBac1fsequences(FigureS3inSupplementaryMaterial),butinpractice,wesucceededinretrievingvariousfunctionalgenes,suggestingthatthemismatchesatthe5′-endcanaffectannealingefficiency,butstillremaineffectiveforamplification.Thelackofanyalphaproteobacterial16SrRNAsinourfunctional16SrRNAcollectionmaybeduetofunctionalincompatibilityinE.coli. TABLE3 Table3.Sequenceanalysisoffunctional16SrRNAgenesinEscherichiacoliΔ7retrievedfromthemetagenome. WhenBac8f(A)wasusedasaforwardprimer,87%(39/45)offunctional16SrRNAswereofgammaproteobacteriaandtherest(13%;6/45)wereofbetaproteobacteria.PhylogenetictreeofthesesequencesisillustratedinFigureS5BinSupplementaryMaterial.Whenthebasepairpatternswereinvestigated,non-canonicalbasepairswerefrequentlyobserved.Approximatelyhalf(21/45)ofthesequencescontainedthe19A–916Gbasepair,whichmayhaveresultedfromthemis-annealingoftheprimertotemplate16SrRNAgenescontaining19Cand916G.Becausethisartificialbasepairispermissive(orevenencouraged)undernormalgrowthconditions(Figure3B),itisreasonabletofindthesespecies;simplePCRconditioningmaynoteasilyremovethesemis-annealedproducts. WhenBac8f(C)wasusedasaforwardprimer,66%(29/44)offunctional16SrRNAswereofbetaproteobacteria,30%(13/44)wereofgammaproteobacteria,and5%(2/44)wereofdeltaproteobacteria.PhylogenetictreeofthesesequencesisillustratedinFigureS5CinSupplementaryMaterial.Inthislibrary,non-naturalbasepairpatternswererarelyobserved.Mostwereofthe19C–916Gbasepairandthenon-native19C–916Ubasepairwasfoundinoneclone(closestrelativewasgammaproteobacterialRahnellaaquatilis,NR_074921).Thislow-leveloccurrenceofthe19C–916Ubasepairagreedwiththedetrimental(yetstillnon-lethal)effectofthepaironcellgrowth(Figure3B). Inconclusion,wedevelopedanewprimerset,Bac1fandUN1542r,forthefunctionalstudyof16SrRNAsinE.coli.Theeffectiveutilizationoftheprimerswasdemonstratedbyretrievalofarangeoffunctional16SrRNAsfromtheproteobacteriallineage,allofwhichcontainedanativebasepairbetweenthe19thand916thnucleotides. AuthorContributions KM,MS,andMTdesignedthestudy,conductedthedataanalysis,andwrotethemanuscript. ConflictofInterestStatement Theauthorsdeclarethattheresearchwasconductedintheabsenceofanycommercialorfinancialrelationshipsthatcouldbeconstruedasapotentialconflictofinterest. Acknowledgments TheauthorsthankDr.NaohiroNoda(AIST,Japan)forthegenomicDNAofNitrosomonaseuropaea.ThisworkwassupportedbyaJSPSGrant-in-AidforScientificResearch(B)(Grant26292048toKM),Grant-in-AidforScientificResearchonInnovativeAreas(Grant26670219toKM),Grant-in-AidforChallengingExploratoryResearch(Grant15H01072toKM),andGrant-in-AidforJSPSFellows26-7760(toMT). SupplementaryMaterial TheSupplementaryMaterialforthisarticlecanbefoundonlineathttps://www.frontiersin.org/article/10.3389/fbioe.2017.00014/full#supplementary-material. 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GoogleScholar Keywords:bacterialphylogeny,16SrRNA,ribosome,horizontalgenetransfer,molecularclock,functionalcomplementation,metagenome,centralpseudoknot Citation:MiyazakiK,SatoMandTsukudaM(2017)PCRPrimerDesignfor16SrRNAsforExperimentalHorizontalGeneTransferTestinEscherichiacoli.Front.Bioeng.Biotechnol.5:14.doi:10.3389/fbioe.2017.00014 Received:19December2016;Accepted:09February2017;Published:28February2017 Editedby: DianaElizabethMarco,NationalScientificCouncil(CONICET),Argentina Reviewedby: KeiKitahara,HokkaidoUniversity,JapanTrevorCarlosCharles,UniversityofWaterloo,Canada Copyright:©2017Miyazaki,SatoandTsukuda.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)orlicensorarecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:KentaroMiyazaki,[email protected] COMMENTARY ORIGINALARTICLE Peoplealsolookedat SuggestaResearchTopic>