Comparison of pmoA PCR Primer Sets as Tools for Investigating Methanotroph Diversity

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APPLIEDANDENVIRONMENTALMICROBIOLOGY,Sept.2001,p.3802–38090099-2240/01/$04.00 0DOI:10.1128/AEM.67.9.3802–3809.2001

Copyright©2001,AmericanSocietyforMicrobiology.AllRightsReserved.

Vol.67,No.9

ComparisonofpmoAPCRPrimerSetsasToolsforInvestigatingMethanotrophDiversityinThreeDanishSoils

DAVIDG.BOURNE,IANR.MCDONALD,

AND

J.COLINMURRELL*

DepartmentofBiologicalSciences,UniversityofWarwick,CoventryCV47AL,England

Received14February2001/Accepted31May2001

ThreeparticulatemethanemonooxygenasePCRprimersets(A189-A682,A189-A650,andA189-mb661)wereinvestigatedfortheirabilitytoassessmethanotrophdiversityinsoilsfromthreesites,i.e.,heath,oak,andsitka,eachofwhichwascapableofoxidizingatmosphericconcentrationsofmethane.EachPCRprimersetwasusedtoconstructalibrarycontaining50clonesfromeachsoiltype.Theclonesfromeachlibraryweregroupedbyrestrictionfragmentlengthpolymorphism,andrepresentativesfromeachgroupweresequencedandanalyzed.LibrariesconstructedwiththeA189-A682PCRprimersetweredominatedbyamoA-relatedse-quencesornonspeci cPCRproductswithnonsenseopenreadingframes.Theprimersetcouldnotbeusedtoassessmethanotrophdiversityinthesesoils.AnewpmoA-speci cprimer,A650,wasdesignedinthisstudy.TheA189-A650primersetdemonstrateddistinctbiasesbothinclonelibraryanalysisandwhenincorporatedintodenaturinggradientgelelectrophoresisanalysis.TheA189-mb661PCRprimersetdemonstratedthelargestretrievalofmethanotrophdiversityofalloftheprimersets.However,thisprimersetdidnotretrievesequenceslinkedwithnovelhigh-af nitymethaneoxidizersfromthesoillibraries,whichweredetectedusingtheA189-A650primerset.Acombinationofallthreeprimersetsappearstoberequiredtoexaminebothmethanotrophdiversityandthepresenceofnovelmethanemonooxygenasesequences.

Methanotrophsareauniquegroupoforganismswhichcanusemethaneasasolesourceofcarbonandenergy.Theabilityofmethanotrophstooxidizemethaneisduetothepossessionoftheenzymemethanemonooxygenase.Therearetwodistinctformsofthisenzyme,thecytoplasmicsolublemethanemono-oxygenaseandthemembrane-boundparticulatemethanemonooxygenase(pMMO)(reviewedinreferences21and22).OnlythepMMOisfounduniversallyinmethanotrophsandcanthereforebeusedasafunctionalmarkerfortheseorgan-isms.Nogeneticorstructuralhomologyisfoundbetweenthesetwoenzymesystemsdespitetheirsimilarfunctions.How-ever,thepMMOenzymecomplexsharesmanysimilaritieswiththeammoniamonooxygenase(AMO)enzymecomplexfoundinammonia-oxidizingbacteria(15).Thesesimilaritiesincludeahighdegreeofaminoacidsequenceidentity,similarproteincomplexstructures,andbroadlysimilarsubstrateandinhibitionpro les,whileeachplayacrucialroleincellmetab-olism(6,11,29).Methanotrophsandammonia-oxidizingbac-teriacanoxidizebothmethaneandammonia;however,theycanobtainenergyonlyfromtheoxidationofmethaneandammonium,respectively(3).

Oligonucleotideprimers(A189fandA682r)havebeende-signedtoamplifyinternalfragmentsofthegenesencodingthepMMOandAMOenzymecomplexes(11).Thesequencein-formationobtainedfromthesesgenesencodingpMMO(pmoA)andAMO(amoA)hasbeenusedasphylogeneticmarkersforidenti cationofmethanotrophsandammoniaox-idizers(12,19).Thephylogenyofthesefunctionalgenescloselyre ectsthe16SrRNAphylogenyoftheorganismsfromwhichthegenesequenceswereretrieved.Therefore,retrieval

*Correspondingauthor.Mailingaddress:DepartmentofBiologicalSciences,UniversityofWarwick,CoventryCV47AL,England.Phone:441203523553.Fax:441203523568.E-mail:cmurrell@bio.warwick.ac.uk.

3802

ofpmoAandamoAgenesequenceinformationprovidesinfor-mationonthediversityoftheseorganismsindifferentenvi-ronments(12,19).

TheA189andA682primershavebeenusedextensivelyinenvironmentalstudiestoprovideamolecularpro leofthemethane-oxidizingcommunity(10,13,20).RecentlyanewreversepmoA-speci cprimer,mb661,usedinconjunctionwiththeA189primer,wasdesignedanddemonstratedspeci citytoamplifypmoAsequenceswhilenotdetectingamoAsequences(4).Thisnewprimerwasusedalongside16SribosomalDNAphylogeneticprobestodetermineinsitupopulationsofmeth-anotrophsinfreshwaterenvironments(4).TheuseofPCRprimerstoamplifytheamoAgenesfromammoniaoxidizershasrecentlybeencriticallyevaluated(24),withtheamoAprimersetofRotthauweetal.(26)beingrecommendedasthemostsuitable.Thesolublemethanemonooxygenasegeneshavealsobeenusedtodetectmethanotrophsintheenviron-ment(2,17,18);however,thisproceduredetectsonlythesubgroupofmethanotrophsthatcontainthisenzyme.

ThepmoAPCRprimersetA189-A682hasbeenadaptedfordenaturinggradientgelelectrophoresis(DGGE)analysisasameanstostudypmoAgenediversity(5,9).Theuseofdegen-erateprimersinDGGEanalysismaycausetheappearanceofmultiplebandsforindividualorganisms,whichincomplexenvironmentsmaycauseconfusionininterpretationofresults.TheA682rprimerhasfourredundancieswithinitssequence,whichissuspectedtocausemultiple-bandingproblemsinDGGEanalysis.ThisstudyattemptedtodesignanewpmoAprimersetcontainingnoredundancieswhichcouldsubse-quentlybeappliedinDGGEanalysisofpmoA.

Aerobicsoils,suchasforestsoils,playanimportantroleintheglobalmethanecyclebyactingasamajorsinkforatmo-sphericmethaneintheatmosphere(1,14,30).Toinvestigatemethanotrophpopulationsinvolvedinmethaneoxidationin

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VOL.67,2001pmoAPCRPRIMERSETSTOSTUDYMETHANOTROPHDIVERSITY3803

forestsoils,pmoAclonelibrarieswereconstructedfromthreeDanishsoils,i.e.,heath,oak,andsitka,andcompared.ThesoilswereobtainedfromasiteatHjelmHede,Denmark.Thesoiltypesaresimilar.Allwereoriginallyheath,butpartwasplantedwithsitkaspruceover60yearsagoandparthasbeengraduallycolonizedbyoakwoodland.Thechangeinvegeta-tionhaseffectedachangeintheatmosphericoxidationpo-tentialsofthesoils.Aplantsuccessionfromheathertooakvegetationincreasedmethaneuptakesixfold,whiletheintro-ductionofsitkasprucedoubledmethaneuptakeratesrelativetothoseofthenativeheathland.Adetailedanalysisofthephysiochemicalpropertiesofthesoilsandtheirrelativemeth-aneuptakepotentialswillbepresentedelsewhere(I.R.Mc-Donaldetal.,unpublisheddata;N.Høeghetal.,unpublisheddata).However,themajoraimsofthisstudyontheseheath-landandforestsoilsweretwofold:toevaluatedifferentpmoAprimersetsastoolsforinvestigatingmethanotrophdiversityandtoinvestigatethemethanotrophdiversityinthesesoils,whichdemonstratenovelatmosphericmethaneoxidationpo-tentials.

MATERIALSANDMETHODS

MicrobialstrainsandtemplateDNA.ThemicroorganismsusedinthisstudywereobtainedfromaculturecollectionofmethanotrophsmaintainedattheUniversityofWarwickandfromtheNationalCollectionofIndustrialandMa-rineBacteria(Aberdeen,UnitedKingdom).Culturesweregrowninnitratemineralsaltsmediumwiththeadditionofexcessmethane(20%[vol/vol]inair)asthesolecarbonsubstrateasdescribedpreviously(32).DNAwasextractedfromculturesusingthemethodsofMarmur(16).

SamplecollectionandDNAextraction.CoresoilsamplesweretakenfromthreesiteslocatedatHjelmHedeinNorthernJutland,Denmark.Thesamplingsiteswere(i)nativeheathland(heath),(ii)establishedoak(oak),and(iii)sitkaspruce(sitka).Coresoilsamples(15-cmdiameter,35-cmdepth)wereobtainedfromeachofthethreevegetationsitesusingthemethodofHalletal.(7).Theoakandsitkacoreswereextrudedfromthesampletubeandsectionedinto5-cmsectionsbeforebeingplacedinairtightcollectionbags.Theheathcorewassectionedinto5-cmsectionsdownto15cmandtheninto2-cmsections(15to27cm).Thesoilsamplesweresieved(4-mmmesh)toremovestoneandrootsandtohomogenizethesoil.TotalDNAwasextractedfrom2gofeachcoresectionusingthemethodsofMcDonaldetal.(17).High-molecular-massDNAwasexcisedfroma1%(wt/vol)agarosegelandpuri ed(GenecleanIIkit;Bio101)toremovehumiccompoundswhichinterferedwithPCRampli cation.Thismethodyieldedconsistentlyhigh-qualityDNA,whichcouldbeeasilydigestedwithrestrictionendonucleasesandwassuitableasatemplateinPCRampli -cationexperiments.Inthisstudy,onlyDNAextractedfromthecoresectionsdemonstratingthehighestmethaneoxidationpotentialswasused(heath,21to23cm;oak,5to10cm;sitka,20to25cm)(Høeghetal.,unpublisheddata)DesignofanewpmoA-speci cprimer,A650.ThepmoAandamoAsequencesofmethanotrophsandnitri erspresentlyavailablefromtheGenBankdatabasewerealignedandthenscannedforconservedregionswithinthepmoAgenewhichcouldprovideasuitableprimertargetsite.Fromthisanalysis,areverseprimer,A650r(5 ACGTCCTTACCGAAGGT3 ),wasdesigned.NouniqueregionatthestartofthepmoAsequencecouldbeidenti edasbeingsuitableforanewforwardprimer.TheA650primerwasusedinconjunctionwiththeA189fprimertoamplifya478-bpinternalsectionofthepmoAgene.Theprimerwastestedagainstarangeofmethanotrophsandnitri ers,includingMethylococcuscapsulatus(Bath),Methylococcuscapsulatus(strainM),Methylomicrobiumagile(A30),Methylobacterwhittenburyi,Methylocaldumtepidum(LK6),Methylomonasmethanica(S1),Methylomicrobiumalbum(BG8),Methylosinustrichosporium(OB3b),Methylocystisparvus(OBBP),Methylosphaerahansonii,Nitrosomonaseuropaea(NCIMB11850),Nitrosospirasp.(Np22),Nitrosococcusoceanus(NCIMB11848),Nitrosomonaseutropha,andNitrosospiramultiformis(NCIMB11849).

PCRampli cation.PCRampli cationreactionswereperformedin50- l(totalvolume)reactionmixturesin0.5-mlMicrofugetubesusingaDNAthermalcyclerwithahotlid(Touchdownmodel;Hybaid,Teddington,Middlesex,UnitedKingdom).AllPCRampli cationsofthepmoAgeneusedtheA189fprimerincombinationwitheithertheA682,A650,ormb661primer.Individualreagents

andtheirconcentrationsoramountswereasfollows:1 PCRbuffer,1.5mMMgCl2,0.05%W-1(suppliedwiththeTaqDNApolymerase),20 gofbovineserumalbumin(BoehringerMannheim),200 molofeachdeoxynucleosidetriphosphate,20pmolofeachprimer,1 loftemplateDNA(approximately5to50ng),and5UofTaqpolymerase(LifeTechnologies).Eachprimersetusedthesamethermalpro le.Taqpolymerasewasaddedaftertheinitialdenaturationstepof96°Cfor5min,followedby30cyclesof94°Cfor1min,56°Cfor1min,and72°Cfor1min.A nalextensionperiodof5minat72°Cwasincluded(11).Constructionofclonebanksandrestrictionfragmentlengthpolymorphism(RFLP)analysis.ThesizeandpurityofeachPCRproductwerecheckedon1%(wt/vol)agarosegels(27),andtheproductswerethenligatedintothepCR2.1vectorsuppliedwiththeTAcloningkit(Invitrogen,SanDiego,Calif.)accordingtothemanufacturer’sinstructions.Individualcoloniescontaininginsertsweresuspendedin3mlofnutrientbrothcontainingampicillin(50 g/ml)andgrownovernightat37°C.Small-scalepreparationsofplasmidswereperformedusingthemethodsofSaundersandBurke(28).PlasmidsweredigestedwiththerestrictionenzymecombinationsofEcoRI-RsaIandEcoRI-PvuII-HincII.Di-gestswereresolvedon2%(wt/vol)agarosegelsandgroupedmanually,basedontherestrictionpatternobtained.

DNAsequencingandanalysis.Small-scalepreparationsofclonesfromlibrar-iesweredonebythemethodofSaundersandBurke(28),andDNAfordirectsequencingofDGGEbandswaspreparedbypuri cationofPCRproductsusingaWizardPCRpuri cationkit(Promega,Southampton,UnitedKingdom).DNAsequencingreactionswerecarriedoutbycyclesequencingwiththeDyeTermi-nationkitofPEAppliedBiosystems(Warrington,Cheshire,UnitedKingdom).PhylogeneticanalysesoftheDNAanddeducedaminoacidsequenceswerecarriedoutusingtheARBprogram(http://www.mikro.biologie.tu-muenchen.de).SequencesweremanuallyalignedwiththepmoAandamoAsequencesobtainedfromtheGenBankdatabase.Regionsofsequenceambiguityandin-completedatawereexcludedfromtheanalyses.Resultsweredepictedasaconsensustree,combiningtheresultsofevolutionarydistance(Dayhoffpercent-ageofacceptablepointmutationsmodel),maximum-parsimony,andmaximum-likelihoodanalysesofthedatasets.Multifurcationsindicatepointswherethebranchingorderwasnotsupportedbyallthreemethods.

DGGEanalysisofthepmoAgene.AGCclamp(23)wasattachedtothe5 endofthepmoA-speci cA650primer.PCRampli cationwasperformedwiththeGC-A650primerandtheA189primer.AtouchdownPCRprogramwasopti-mizedandconsistedofaninitialdenaturationstepof5minat94°C,followedby20touchdowncycles(65to55°C)and10furthercyclesat55°Cfor1min,followedby72°Cfor1minanda nalextensionof72°Cfor5min.PCRampli cationwiththeGC-A189–A682primersetwasperformedasoutlinedbyHenckeletal.(9).PCRproductswereanalyzedasdescribedabove.

PCRproductswereseparatedusingaDcodesystem(Bio-Rad,Munich,Ger-many)on1-mm-thickpolyacrylamidegels(7.5%[wt/vol]acrylamide-bisacrylam-ide[37.5:1])(Bio-Rad)preparedwithandelectrophoresedin0.5 TAE(0.02MTrisbase,0.01Msodiumaceticacid,0.5mMEDTA,pH7.4)at60°Candaconstantvoltage.PCRproductsampli edwiththeA189–A650-GCprimersetwererunonagradientof55to65%(65%correspondsto7.5%[wt/vol]acryl-amide,4.55Murea,and26%[vol/vol]deionizedformamide)ataconstantvoltageof150Vfor6h.PCRproductsampli edwiththeGC-A189–A682primerswererunaccordingtotheproceduresofHenckeletal.(9).Gelswerestainedwith1:50,000(vol/vol)SYBR-Gold(MolecularProbes)for30minbeforebeingphotographed.DistinctDNAbandswereexcisedandsuspendedin100 lofwaterovernighttoeluteDNA.Thebandswerereampli edandrunagainontheDGGEsystemtoensurepurityandcorrectmobilitywithinthegels.DirectsequencingoftheDNAbandswasperformedasdescribedabove,andtheanalysisofderivedsequenceswasalsoperformedasdescribedabove.

Nucleotidesequenceaccessionnumbers.TheenvironmentalclonesequenceshavebeendepositedintheGenBankdatabaseunderaccessionnumbersAF368354toAF368374.

RESULTS

Clonelibraryconstruction.ThreedifferentpmoA-speci cprimerssets(A189-A682,A189-A650,andA189-mb661)wereusedtoconstructclonelibrariesfromthreesoilsamples(heath,oak,andsitka).PCRampli cationproductsofthepredictedsizewereobtainedfromeachofthesoilsusingthethreeprimersets.Librariesofapproximately50cloneswereconstructedforeachsoilandfromeachpmoAprimerset(ninelibrariesintotal).ThelibrariesweresubjectedtoRFLPanal-

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3804BOURNEETAL.APPL.ENVIRON.MICROBIOL.

TABLE1.RepresentativeproportionsofclonesineachOTUgroupforeachclonelibrary

Resultsawithprimerset

Soil

Clonetype

A189-A682

%oflibrary

Clonetype

A189-A650

%oflibrary

Clonetype

A189-mb661

%oflibrary

Heath

A1A2

NomatchNosequence5922910

B2F4982

B1C1C2D1E1F1F3

NosequenceB4C1C4D2F2B1B5F1

464264282824522121068923

Oak

A1B1

MiscellaneousNomatchNosequenceA1

MiscellaneousNomatch

44344513661915

B1G1

Nosequence8794

Sitka

B1B3G2G382297

Nosequence,OTUgroupsforwhichnorepresentativesweresequenced;nomatch,OTUgroupsforwhichsequenceinformationwasnotvalidorforwhichnodiscernibleopenreadingframecouldbeidenti ed;miscellaneous,OTUgroupscontainingindividualcloneswhichhavenotbeencharacterizedbysequencing.

a

ysisandgroupedbasedontheirrepresentativeRFLPpatterns.Thisuseofmethanotroph-speci cprimersandoperationaltaxonomicunits(OTUs)hasbeenshowntobeeffectiveinscreeningenvironmentalclonelibrariesandprovidinganindi-cationofmethanotrophdiversity(4,12).Inthisstudy,randomsequencingwithinclassi edOTUgroupsalwaysdemonstratedidenticalclonesequences,suggestingthattheclonegroupingsbasedonrestrictionanalysiswererobust.

PhylogeneticanalysisofsequencesofrepresentativeclonesfromeachOTUgroupofeachlibrarywasperformed.AllpmoAsequencesobtainedwereaf liatedcloselywithestab-lishedpmoAgroupingsandweresubsequentlyassignedtobroadphylogeneticgroups,designatedAthroughG.Table1providesanoverviewoftheproportionofOTU-groupedclonesfoundineachlibrary,whileFig.1indicatesthephylo-geneticaf liationsoftheseclonesequences.ThegroupAsequences(A1andA2)wererelatedtotheamoAgenesofNitrosomonas.GroupBsequences(B1,B2,B3,B4,andB5)wererelatedtothepmoAgenesofthegenusMethylococcus.GroupCsequences(C1,C2,C3,andC4)includerelativesofthepmoAgenesfromunculturedbacteria(presumedmeth-anotrophs).GroupDsequences(D1andD2)areaf liatedwiththepmoAgenesofMethylomonas.GroupE(E1)includessequencesalsoaf liatedwithpmoAsequencesfromunculturedbacteria(presumedmethanotrophs).GroupF(F1,F2,F3,andF4)containssequencesrelatedtothepmoAgenesofthetypeIImethanotrophsMethylosinusandMethylocystis.Finally,groupGsequences(G1,G2,andG3)wererelatedtotherecentlydescribedenvironmentalpmoAcloneRA14.

A189-A682clonelibraries.TheclonelibrariesconstructedwiththeA189-A682primersetresultedinlimitedsuccessfulretrievalofmethanotrophpmoAsequences.TheheathandsitkalibrariesweredominatedbytheclonesequenceA1(59and66%ofclonelibraries,respectively),whichdemonstratedhighidentity( 98%)totheamoAgeneofNitrosomonaseu-ropaea.Only4%ofcloneswereaf liatedwiththissequenceintheoaklibrary.Thelibrariesconsistedofahighproportionofclonetypeswhichcontainednoopenreadingframesofsignif-icantlengthandwhichfailedtoshowhomologytootherse-quencesafterdatabaseanalysis(heath,29%;oak,45%;andsitka,15%).Theprimersappearedtoretrievealargenumberofhybridnonsenseampli edbandsofthecorrectinsertsize,althoughthesequenceswerenotidenti edaschimeric.Asmallnumberofclonesinthelibrariesalsofailedtoprovideanysequencedata,asindicatedinTable1(heath,10%;oak,13%).

Theonlyclonesrecoveredfromthelibrariesthatwereaf- liatedwithmethanotrophicsequenceswerefoundintheoaklibrary.Theretrievedsequence(B1)showedhighsequenceidentity( 99%)withthepmoAgeneofMethylococcuscapsu-latus.However,thisclonetypeconstitutedonly4%oftheclonelibrary(Table1).Atotalof34%ofcloneswithintheoaklibrary,whenanalyzedbyRFLP,werefoundtobesingleclonesrepresentingdifferentOTUgroups.Sequenceinforma-tionwasnotrecoveredfromtheseindividualOTUpatterns.FromtheseresultsitcanbeconcludedthattheprimersetA189-A682wasinadequatetoassessmethanotrophdiversityinthesesoils,andthereforetheuseofotherprimersetswasinvestigated.

DesignofprimerA650.AllavailablepmoAsequenceswerealigned,andanewpmoA-speci creverseprimer,A650,wasdesignedforuseinbothclonelibraryconstructionandDGGEanalysis.TofacilitateDGGEanalysis,theA650primerwasdesignedwithoutredundancies.Designingaprimerspeci ctoallpmoAsequenceswasproblematic;therefore,mismatcheswithsomepmoAsequencesoccur.AselectionofpmoAandamoAsequencesshowingbothmatchingandmismatchingre-gionscomparedtoallthreereverseprimersusedinthisstudyisshowninTable2.TheA650primerdidnotmatchanyammoniamonooxygenaseamoAgenesequences.

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VOL.67,2001pmoAPCRPRIMERSETSTOSTUDYMETHANOTROPHDIVERSITY3805

encodedFIG.1.Bar,sequences10inferredbyPhylogeneticpmoAanalysisofthederivedaminoacidsequencessubstitutionsandamoAgenesretrievedfromtheDanishsoils.groupedUnculturedintoandestablishedtheirrelationshipsper100phylogeneticwithaminofamiliesknownacidpositions.RetrievedA,pmoAB,C,D,sequencesE,areusedastheoutgroup.

methanotrophpmoAsequencesRA21andMR1F,andwereG.Theprimerwastestedforspeci cityviaPCRagainsttargetandnontargetorganismsatmoderatestringency(seeMaterialsandMethods).Allmethanotrophstested(Methylococcuscap-sulatus[Bath],Methylococcuscapsulatus[strainM],Methylomi-crobiumagile[A30],Methylobacterwhittenburyi,Methylosphaerahansonii,Methylocaldumtepidum[LK6],Methylomonasmeth-anica[S1],Methylomicrobiumalbum[BG8],Methylosinustri-chosporium[OB3b],andMethylocystisparvus[OBBP])gavepositiveampli cationofpmoAproducts,withtheexceptionsofMethylomicrobiumalbum(BG8)andMethylosphaerahansonii.Table2showsa1-bpmismatchwiththeprimerandMethylo-microbiumalbum(BG8).Despite1-bpmismatchesforMethy-lomonasmethanicaandMethylocaldumtepidum,positiveam-pli cationwiththisprimerwasobservedunderthePCRconditionsused.ThepmoAgenesequencesfromtheorgan-ismsMethylomicrobiumagile(A30),Methylobacterwhittenburyi,andMethylosphaerahansoniihavenotbeendeterminedand

arethereforenotpresentedinTable2.NoPCRproductswereproducedwithDNAfromammonia-oxidizingnitri ers(Nitro-somonaseuropaea[NCIMB11850],Nitrosospirasp.[Np22],Nitrosococcusoceanus[NCIMB11848],Nitrosomonaseutro-pha,andNitrosospiramultiformis[NCIMB11849]).

A189-A650clonelibraries.ClonelibrariesconstructedwiththeA189-A650primersetweredominatedbypmoAsequencesB1andB2,showinghighidentity( 98%)toMethylococcuscapsulatus(Bath)pmoAsequences(heath,98%;oak,87%;andsitka,82%ofthelibraries).Limitedmethanotrophdiversitywasdetectedwithinthesoilswiththisprimerset.Forexample,onlytwoOTUgroupswereidenti edintheheathandoaklibraries.

Theoakandsitkalibrariescontainedagroupofclones(G1,G2,andG3)closelyaf liatedwiththepmoAcloneRA14,presumablyfromanunknownorganism.Thesetypesofse-quenceshavebeenfoundinarangeofsoilsthatoxidizeatmo-sphericmethane(10,12).Withintheoaklibrarythesequencescontributed9%ofclones(G1),whilewithinthesitkalibrarytheyrepresented16%ofclones(G2andG3).ThisprimersetalsoretrievedapmoAsequencefromtheheathlibraryaf li-atedwiththepmoAgenefromthetypeIImethanotrophMethylocystissp.strainM(clonetypeF4, 98%sequenceidentity).

Thespeci cityoftheA189-A650primersetforsubgroupsofmethanotrophswascon rmedbytheretrievalofonlymeth-anotrophpmoAsequences.NoamoAsequencesfromammo-nia-oxidizingbacteriawereretrieved.Also,onlytwoambigu-oussequences,wheresequenceinformationcouldnotbeobtained,werefoundintheoaklibrary.However,duetothetargetingoftheprimerstosubgroupsofmethanotrophpmoAsequences,apronouncedbiaswasobservedwithinthelibrariesinrecoveringMethylococcuscapsulatuspmoAsequences.NopmoAsequencesfromothertypeIorganismswereidenti ed,notablynoMethylomonas-orMethylomicrobium-associatedpmoAsequences.ThealignmentofsequencesinTable2dem-onstratesthattheA650primerhasoneortwomismatchestotheseorganisms.

mb661clonelibraries.CostelloandLidstrom(4)designedanewreversepmoAprimer(mb661)tospeci callyamplifypmoAsequencesandnotamoAsequences.TheA189-mb661primersetwasfoundtobemoreusefulforstudyingmeth-anotrophdiversityinfreshwaterenvironments.

Whenusedinthisstudy,clonelibrariesconstructedwiththeA189-mb661primersetcontainedpmoAsequencesaf liatedwithbothtypeIandtypeIImethanotrophpmoAsequences(Table1andFig.1).Methylococcuscapsulatus-af liatedpmoAsequences( 98%sequenceidentity)againrepresentedalargeproportionofcloneswithinthelibraries(heath,clonetypeB1 46%,oak,clonetypeB4 24%;andsitka,clonetypesB1andB5 77%ofthelibraries).Thelargestrepresentativeofclonesintheoaklibrary(52%)wasclonetypeC1,whichisrelatedtothepmoAsequenceofanunculturedproteobacte-riumaf liatedwithMethylomicrobium,atypeImethanotroph.Clonesshowingsimilarhighidentitiestothisunculturedor-ganismwerefoundintheheathlibrary(C1andC2),althoughtogethertheyconstitutedonly6%oftheclonesrecovered.OthertypeI-relatedpmoAsequencesfoundinthelibrariesincludedMethylomonas-af liatedpmoAsequencesD1(6%ofheathlibrary)andD2(12%ofoaklibrary).However,

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gra.g....te......ht......fo..ttttecn......eu......qes....g.eh......to......trefers..ttehc....ta....msi..t.mg..g.ni....woh....ss....esa...gbe....hT.....st....od....yb....det....acia.tadni....er....asgniria)0)p58d84n18a41

s111s12egcBnAAinrRReMBiuIMaphhqC21sppeN2Cioos(pmrrttAaNN(a

roooenshofnnaamapiuanphhaoraodrerttttrseceeeuu ie.etmmzponissdddassediieexnauacrrrooicnsuupooettamscmdillioooouunsssstoccoooooennUUmrrrrttttliiiicmNNNNuANa

TypeIIpmoAsequencesHigh-af nitypmoAsequences