土壤微生物菌群多样性研究_2001

2023-11-14 来源：易榕旅网

A0099-2240/01/$04.00PPLIEDANDENVIRONMENTALMICROBIOLOGY,Apr.2001,p.1874–1884Vol.67,No.4

forMicrobiology.AllRightsReserved.

CombinedStudytheUseBacterialof16SRibosomalDNAand16SBiphenyl-PollutedCommunityofrRNATo

Soil

Polychlorinated

BALBINARAMONROSSELLO-MORA,NOGALES,1,2*EDWARD3†RUDOLFR.B.MOORE,1AMANN,3ENRIQUELLOBET-BROSSA,3ANDKENNETHN.TIMMIS1,2DivisionGroup,Max-Planck-InstitutofMicrobiology,GBF-Nationalfu¨rMarineResearchCentreforBiotechnology,Braunschweig,1andMolecularEcologyUniversityMikrobiologie,ofEssex,Colchester,Bremen,3UnitedGermany,KingdomandDepartment2ofBiologicalSciences,

Received18September2000/Accepted9January2001

DNAThecorrespondence(rDNA)bacterial(onediversityassessedfromclonelibrariespreparedfromrRNA(twolibraries)andribosomaltheofthelibrary)communityfrompolychlorinatedcompositionfoundbiphenylinthetwo(PCB)-pollutedtypesoflibrarysoilwashasobserved.beenanalyzed.NearlyAgoodofsimilaritytheclonedtotalsequenceslibraries.higherclonedthansequenceinthe97%[98%typesrDNAlibrarywereidenticaltosequencesintherRNAlibraries.Morethan29%60%offoranalyzedBurkholderiaweregroupedandPseudomonasinphylogenetic-typeclones])groups(arepresentedclonegroupinwithbothsequencetypesoftype(s),lackwereSomeobservedofthoseinonlyphylogeneticoneofthegroups,typesofmostlyrepresentedbyasingle(orpair)ofclonedsequencebacterialofclonesrepresentativeoftheActinobacterialibrary.intherDNAAnimportantlibrary.differenceThePCB-pollutedbetweenthesoillibrariesexhibitedwasahightheafﬁliateddiversitybytothebeta-subclasswhichincludedoftherepresentativesProteobacteria,ofandtwotonovelthegenuslineages.BurkholderiaTheapparentabundanceofbacteriaconcentrationsﬂuorescencePCR.wasinsituassessedhybridizationbydilutionanalysis.oftheRNAThepossibleinﬂuenceonapparentinparticular,diversityofwaslowconﬁrmedtemplateconcentrationsAlthoughlibraries,thisstudy.

andthereforeweredifferencesobserved,inthecompositionofthetemplatetworRNApriorlibrariestoampliﬁcationobtainedbyfromreversehightranscription-andlowRNAtheirdetectionthemainwascomponentsnotcompromisedofthebacterialbythelowercommunityconcentrationswererepresentedoftemplateinusedbothinInvestigationsofmicrobialcompositionanddiversityinnat-reﬂectpredominantlythediversityofthemetabolicallyactiveuralandanthropogenicallyimpactedorcreatedhabitatsismembersofthecommunity.Severalreportsontheanalysisofimportantinthecharacterizationofsuchhabitats,sincemi-bacterialcommunitiesusing16SrRNAhavebeenpublishedcrobesarekeyplayersinmanyenvironmentalprocesses.Overthelastfewyears,cultivation-independentmethodologies,par-(7,20,22,36,37).However,itisnotcurrentlyknownwhetherticularlythesequenceanalysisofcloned16SribosomalRNArRNAandrDNAlibrarieswillbesigniﬁcantlydifferent,sincegenes(16SrDNA),haveproventobepowerfultoolsforin-itisnotknownwhichproportionofmicrobialcommunityisvestigatingthemicrobialdiversityofenvironmentalsamplesquiescent.AcomparisonofresultsobtainedfromrRNAand(10).AtleastasimportantisthespeciﬁcidentiﬁcationoftherDNAlibrarieshasbeenattemptedbyMiskinetal.(20)inametabolicallyactivemicroorganisms,sincetheseareresponsi-studyofananoxicsedimentsample.Theseauthorsobservedableforthemicrobiallydrivenenvironmentalprocesses.Forfewidenticalsequencesinthetwotypesoflibraryandcon-example,knowledgeoftheactivemicroorganismsinpollutedcludedthatthelibrariesdidnothaveadegreeofcoverageofhabitatsisrelevanttothedevelopmentofoptimalinsitubiore-thediversityinthesamplehighenoughtoenablevalidcom-mediationstrategies,aswellascontributingtotheidentiﬁca-parisons.

tionofyet-undescribed(i.e.,notyet-cultured)bacteriawhichWehaveundertakensuchacomparisonwithadegreeofmayplayimportant,albeitunknown,rolesinpollutantdegra-diversitycoveragethatshouldpermitconclusions.Inthedationorothercommunityprocesses.

presentstudywedescribea16SrRNAgeneclonelibrary,Sincemetabolicallyactivecellsusuallycontainhighernum-obtainedbyPCRampliﬁcationfromtotalDNAextractedfrombersofribosomesthanquiescentcells(23),a16SrRNAlibraryapolychlorinatedbiphenyl(PCB)-pollutedsoil,andcompareitgeneratedfromtotalextractedrRNAmaybeconsideredto

withapreviouslydescribed16SrRNAlibraryobtainedbyreversetranscription-PCR(RT-PCR)(22)andanunreportedrRNAlibrarygeneratedfroma1:500dilutionoftheoriginaltemplateRNA.Ahighspeciesdiversitywasfoundinbothcal*Correspondingtypesoflibrary,thoughitwasclearfromrarefactionplotsthat,3SQ,Sciences,author.Mailingaddress:DepartamentofBiologi-eventhoughsome404cloneswereanalyzed,notalloftheE-mail:UnitedKingdom.UniversityPhone:ofEssex,44-1206-872547.WivenhoePark,Fax:Colchester44-1206872592.CO4bacterialdiversityinthathabitathadbeenrevealed.Aconsid-erablepercentageofrDNAcloneswerealsorepresentedintheUniversitat†Presentbnogales@essex.ac.uk.

deaddress:lesIllesAreaBalears,deMicrobiologia,PalmadeMallorca,DepartmentSpain.

deBiologia,rRNAlibrariesand,ingeneral,therewasaqualitativecorre-

1874

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spondenceofclonefrequencyinthetwotypesoflibraries,withrepresentativesofthealphaandbetasubdivisionsofPro-teobacteriaandtheAcidobacteriumphylumdominating.

MATERIALSANDMETHODS

TotalDNAandRNAextraction.Thesampleusedfortotalnucleicacid(DNAandRNA)extractionwastakenfromtheupperfewcentimetersofthesurfaceofasoilinanareanearWittenberg,Germany,wherehighconcentrationsofPCBweredetected(22),weighed,andfrozenatϪ70°Cuntilprocessing.Totalnucleicacidswereextractedfromthesoilusingaprotocoldescribedpreviously(22).Theextractednucleicacidswerepelletedandwashedwith70%ethanol,dried,andresuspendedin300␮lofdeionizedwater.Analiquotofthesamplewasdigestedwith30UofRNase-freeDNaseI(RocheDiagnostics,GmbH,Mannheim,Germany)at37°Cfor2hin10mMsodiumacetate–0.5mMMgSORNAandtotalDNAwerepuriﬁedusingMicroconmicroconcentra-4(pH5.0).Bothtotaltors100(MilliporeGmbH,Eschborn,Germany),accordingtothemanufactur-er’sinstructions.AliquotsofpuriﬁedandnonpuriﬁedtotalRNAandtotalDNAwereanalyzedbyelectrophoresisona1%(wt/vol)agarosegelandstainingwithethidiumbromide.

RT-PCRampliﬁcationof16SrRNA,PCRampliﬁcationof16SrRNAgenes,andcloningoftheampliﬁcationproducts.Theregionofthe16SrRNAbetweennucleotidepositions27and518(Escherichiacoli16SrRNAgenesequencenumbering),correspondingtoapproximatelyone-thirdoftheentire16SrRNA,wastargettedforreversetranscription-PCR(RT-PCR)ampliﬁcationfromtheextractedtemplateRNA.RT-PCRanalyseswereperformedwithca.230ngand460pg(dilution,1:500)ofthetotalRNA,usingrTthDNApolymerase(AppliedBiosystems,Weiterstadt,Germany)asdescribedpreviously(22).Nearlytheentire16SrRNAgene,betweenpositions27and1492(E.coli16SrRNAgenesequencenumbering),wasampliﬁedbyPCR.PCRmixturescontained10mMTris-HCl(pH8.3),50mMKCl,1.5mMMgCl2,200␮Mconcentrationsofdeoxynucleosidetriphosphates,0.5␮Mconcentrationsofprimers,approximately80ngofDNA,and2.5UofAmpliTaqDNApolymerase(AppliedBiosystems).PCRswereperformedinaGeneAmp9600thermocycler(AppliedBiosystems)withthefollowingconditions:aninitialdenaturationstepat94°Cfor2min,followedby30cyclesof1minat94°C,1minat55°C,and2minat72°C,andaﬁnalextensionstepof10minat72°C.

ThePCRandRT-PCRswerecarriedoutintriplicate,andtheresultingproductswerepooledbeforegelpuriﬁcationandcloning.Thecloningprocedurehasbeendetailedpreviously(22).Threeclonelibrariesweregenerated:onewiththePCRampliﬁcationproductsfromtotalDNAandtwowiththeRT-PCRampliﬁcationproductsfromundilutedand1:500-dilutedRNA,respectively.SequencingofclonedRT-PCRproducts.ThenucleotidesequencesoftheclonedproductsweredeterminedfromplasmidDNApreparations(obtainedusingQiawell8orQiaSpinplasmidextractionkits[QiagenGmbH,Hilden,Germany])usingtheABIPRISMdRhodamineandBigDyeTerminatorCycleSequencingkitsandABI373andABI377Sequencers(AppliedBiosystems)accordingtothemanufacturer’sinstructions.VectorprimersT3andT7wereusedforthesequencingreactions.

Assignmentofclonedsequencestoestablishedphylogeneticdivisions.Cloned16SrRNAsequenceswerecomparedinitiallywithreferencesequencescon-tainedintheEMBLNucleotideSequenceDatabase(2)usingtheFASTApro-gram(25)andsubsequentlyalignedwith16SrRNAreferencesequencesintheARBpackage(http://www.mikro.biologie.tu-muenchen.de)(32).Ambiguouspo-sitionswereexcludedfromsimilaritycalculations.Evolutionarydistances,de-rivedfromsequence-pairdissimilaritiesusingtheJukesandCantoralgorithm(12),werecalculatedusingtheDNADISTprogramfromthePhylogenyInfer-encePackage(PHYLIP)includedintheARBpackage.ForthecalculationofthedendrogramshowninFig.2,clonedsequenceswerealignedwith16SrRNAsequencesrepresentativeofthemainbacterialdivisions.Dendrogramswerecalculatedusingneighborjoining;theleast-squaresalgorithmofFitch-Margo-liashoftheFITCHprogram;parsimony(DNAPARS),andmaximum-likelihood(DNA_ML)algorithmsofthePHYLIPpackageincludedintheARBsoftware.Hypervariableregionsinthe16SrRNAmoleculewereexcludedfromthecal-culationasdescribedelsewhere(14).Brancheswhosephylogeneticpositioninthedendrogramchangeddependingonthemethodofanalysisusedwerecol-lapsedbacktothepreviousconsistentnodebyintroducingmultifurcations.Rarefactionanalysesanddiversityindexes.RarefactioncalculationsweredoneusingthesoftwareAnalyticRarefaction(version1.2;StratigraphyLabora-tory,UniversityofGeorgia[www.uga.edu/ϳstrata/Software.html]).Shannondi-versityindex(H)andequitability(J)valueswerecalculatedaspreviouslyde-scribed(3).

Insituhybridization.ThesamesoilsamplesfromthePCB-pollutedsoilusedfortheelaborationofthelibrarieswereﬁxedat4°Cfor16hin4%paraformal-dehyde–phosphate-bufferedsaline(PBS),composedof0.13MNaCl,7mMNa2HPOin4,and3mMNaHPBSthreetimesand2POstored4(pH7.2).Afterﬁxation,thesampleswerewashedinethanol-PBS(1:1[vol/vol])atϪ20°C.Thesoilslurrywasvortexedfor1minanddilutedinPBS.Hybridizationswerecarriedouton0.22␮m-pore-sizepolycarbonateﬁlters(Millipore)afterﬁltrationofthedilutedsoilslurries.OligonucleotideprobesweresynthesizedwithCy3ﬂuorochromeatthe5Јend(InteractivaBiotechnologieGmbH,Ulm,Germany).TheprobesusedwereEUB338forthedomainBacteria(1),ALF1bforthealphasubclassofProteobacteria,BET42aforthebetasubclassofProteobacteria(usedwithcompetitor),GAM42aforthegammasubclassofProteobacteria(usedwithcompetitor)(19),PLA886forplanctomycetes(usedwithcompetitor)(21),HGC69aforActinobacteria(formerlygram-positivebacteriawithahighGϩCcontent)(28),SUBU1237forBurkholderiaandSuterellaspp.(31),andtheantisenseprobeNON338(1).Hybridizationsandmicroscopycountsofhybrid-izedandDAPI(4Ј,6-diamidino-2-phenylindole)-stainedcellswereperformedaspreviouslydescribed(29),exceptthatanadditionalprehybridizationstepusing1%(forEUB338)or2%(fortheotherprobes)ofblockingreagent(Roche)wasintroducedinordertoreduceunspeciﬁcbindingoftheprobestosoilparticles.TheslideswereexaminedwithanAxiophotIImicroscope(Zeiss,Jena,Germa-ny).

Nucleotidesequencedata.Thesequencedataofthecloned16SrRNAob-tainedbyRT-PCRwithundilutedRNAtemplatewasdepositedintheEMBLdatabaseundertheaccessionnumbersAJ233467toAJ233589.Thenewse-quencedatareportedinthisstudyhavebeendepositedunderaccessionnumbersAJ292571toAJ292689forthesequencedatacorrespondingtocloned16SrDNAandAJ292771toAJ292925forcloned16SrRNAobtainedwiththedilutedRNAtemplate.

RESULTS

Comparisonofthecompositionofthe16SrDNAlibraryandthe16SrRNAlibraries.ThebacterialdiversityinanacidicPCB-pollutedsoilnearWittenberg(Germany)wasanalyzedbyampliﬁcationof16SrDNAfromtotalDNAextractedfromthesoilandcomparedwiththediversityobservedintwoclonelibrariesgeneratedfromextractedRNA,whichshouldbemorerepresentativeofthemetabolicallyactivebacteriainthesoil.The16SrDNAclonedsequencesdeterminedweredesig-natedwithanumberprecededbytheletters“W”(forWitten-berg)and“D”(fromDNA)todifferentiatethemfromtheclonedsequencesWR(fromRNA).

Thepredominantbacterialdivisionspresentinthe16SrDNAlibrarywerealsothemostnumerousinbothlibrariesobtainedfrom16SrRNA,i.e.,clonedsequencetypesafﬁliatedtothealpha,beta,andgammasubdivisionsoftheProteobac-teria(30)andtotheHolophaga-Acidobacteriumphylum(16).The5Ј-partialsequencesof34clonesfromthe16SrDNAclonelibrary(28.6%ofthetotalnumberof16SrDNAclonesanalyzed)wereidenticaltothoseofclonesfromthe16SrRNAlibrariesandbelongedtothefourpredominantdivisionsmen-tionedabove:21,6,and4clonedsequencesclusteredwithinthebeta,alpha,andgammasubdivisionsoftheProteobacteria,respectively,andthreeclonedsequenceswithintheHol-ophaga-Acidobacteriumphylum.

Inordertosimplifythecomparisonofthesequencesob-tainedintheanalysisofthethreelibrariesfromWittenberg,clonedsequencetypeswithϾ97%similaritywereconsideredtoconstituteaphylogeneticgroup,exceptforthecollectionofclonedsequencetypesrelatedtoPseudomonasandBurkhold-eriaspp.,forwhichahighersimilaritythreshold(ca.98%)wasset.

Thenumberofphylogeneticgroups,clonedsequencesandclonedsequencetypesforthedifferentbacterialdivisionsare

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TABLE1.Numberobservedofphylogeneticineitherthe16Sgroups,rDNAclones,library,andtheclonedtwosequence16SrRNAtypeslibraries,fromorPCB-pollutedbothtypesofsoillibraryforeachsimultaneously

ofthebacterialdivisions

No.ofphylogeneticgroupsaobservedinPCB-pollutedsoil16SrDNAand16SrRNAlibraries

Bacterialdivision

Total

DNAlibrary

RNAlibraries

DNAplusRNA

libraries

DNAlibraryonly

RNAlibrariesonly

Proteobacteria

AlphaBetasubdivision4625(128/99)201840181411(88/61)67(6/6)26Holophaga-AcidobacteriumGammasubdivisionsubdivision10(124/81)23(42/26)1(8/8)7(34/32)ActinobacteriaPlanctomycetales

6(68/58)1161995(105/62)4(1/1)124(11/11)(6/6)Candidate5(9/8)067(35/19)0

(5/5)Plastids

divisionOP104(9/8)240

(41/34)16(22/19)Low3(7/7)1410

(1/1)3(9/8)Cytophaga-Flavobacterium-BacteroidesGϩCgrampositives

2(3/3)211(3/3)23(5/4)Notafﬁliated(WPS-1andWPS-2)1(2/2)(1/1)020(2/2)0(2/2)1(5/5)2(1/1)6(11/10)031040

1(2/2)0

2(2/2)1(2/2)3(1/1)(7/6)Total

131(404/303)

108

40(276/183)

23(25/25)

68(103/95)

athePhylogeneticnumberofclones/clonedgroup:aclonesequencegroupwithtypes.

sequencesimilarityhigherthan97%(98%forBurkholderiaandPseudomonas-typeclones).ValuesinparenthesesindicateshowninTable1.Atotalof29.3%ofthephylogeneticgroups,sequencetypesafﬁliatedwiththreebacterialdivisionsnotob-whichincludedmostofthesequencetypesobserved(183servedinthe16SrDNAlibrarywereobservedexclusivelyinclonedsequencetypes,representing60.4%ofthetotalse-therRNAlibraries,namely,clonedsequencetypesclusteringquencetypes;Fig.1),werefoundinbothtypesoflibraries.withintheActinobacteria(constitutingadiversesetrelatedtoTable2showstheafﬁliationofrDNAclonedsequencetypesthe16SrRNAsequencesofgenerasuchasGordonia,Curto-representativeofeachofthephylogeneticgroupsobservedinbacterium,Geodermatophilus,andTerrabacterandthesoilrDNAandrRNAlibraries.TheidentiﬁcationoftheclonesclonedsequencetypeTM146,amemberofthegroupITMpresentinbothtypesoflibrariesagreedwiththeresultsde-clones[27]),thelow-GϩC-contentgrampositives(Clostridi-rivedfrompreviousanalysesonthemicrobialcommunityfromum-likesequencetypes),andtheCytophaga-Flavobacterium-WittenbergPCB-contaminatedsoil(22),i.e.,(i)adiversesetofBacteroidesphylum,withsequencetypesrelatedto16SrRNAclonedsequencetypesafﬁliatedwiththealphasubclassofsequencesofSphingobacterium.

Proteobacteria,withahighproportionofclonedsequencetypesNewbacteriallineages.Atotalof10clonedsequencetypesrelatedto16SrRNAsequencesofSphingomonasandacidi-retrievedfromWittenbergsoilwerenotafﬁliatedwithanyphilicgenerawithinthissubdivision,(ii)alargenumberofdescribedbacterialdivisionsandareproposedhereasrepre-clonedsequencetypesrelatedtoBurkholderiaandVariovorax-sentativesoftwonewbacteriallineages,whichwedesignatedrelated16SrRNAsequencesinthebetasubclassofthePro-WPS-1for“Wittenbergpollutedsoil”(nineclonedsequenceteobacteria,(iii)aprevalenceofclonedsequencetypesafﬁli-types)andWPS-2(onesequencetype).ThedendrograminatedwithNevskiaramosawithinthegammasubclassoftheFig.2showsthephylogeneticpositionsamongtheBacteriaforProteobacteria,(iv)ahighdiversitywithintheclonedsequenceWPS-1andWPS-2.

typesafﬁliatedwiththeHolophaga-Acidobacteriumphylum,TheWPS-1sequencetypesformedadiversecollectionofand(v)thepresenceofclonedsequencetypesrelatedto16SrelatedsequenceswithsimilaritiesbetweenthemrangingfromrRNAsequencesofIsosphaeraspp.withinthePlanctomy-82.7to99.8%andincludedclonedsequencetypesfromthecetales.

rDNAlibraryanalyzed(twosequencetypes)andbothrRNAAninterestingsetofclonedsequencetypesrepresentedinlibrariesanalyzed(sevensequencetypes).ComparisonoftherRNAandrDNAlibrariesappearedtobedistantlyrelatedtoalmost-complete16SrDNAsequenceoftheclonesfromtheclonesretrievedfromatrichlorobenzene-transformingconsor-rDNAlibrarywithsequencesavailableindatabasessuggeststiumwhichwerereportedtobemembersofthecandidatethatWPS-1mightbeadeeplybranchinglineage,distantlydivisionOP10(34).NotethatfouroftheseclonedsequencerelatedtothePlanctomycetales(similaritiesofϽ76%tothetypeshad,onthebasisofpartialsequences,beenpreviouslyclosestrelatives).ThephylogeneticrelationshipbetweenassignedasmembersoftheclassActinobacteriasincetheyWPS-1andthePlanctomycetaleswassupportedbyallthetree-appearedtoberelatedtoAcidimicrobiumferrooxidans(22).ingmethodsemployedintheanalysis.

About52and18%ofthephylogeneticgroupswerepresentTheWPS-2lineagewasrepresentedbyasingleclonedse-exclusivelyintherRNAlibrariesorintherDNAlibrary,re-quencetype,WD272,whichwasonlyobservedinthe16Sspectively(Fig.1),althoughthemajorityofthesewererepre-rDNAlibrary.Analysisofthisclonedsequencewiththepro-sentedbyasingleortwoclonedsequencetypes(afewcon-gramCHIMERA_CHECK(version2.7)(18)andcarefultainedmore[uptoﬁve]).Mostofthephylogeneticgroupscheckingofitsbasepairing(withtheaidoftheARBpackage)uniquetotherDNAlibrarywerecloselyrelatedtogroupsruledoutthepossibilitythatthissequencewasachimericfoundinbothrRNAandrDNAlibraries.However,cloneproduct.ThesequencesimilarityofcloneWD272tocloned

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groups.FIG.Holophaga-Acidobacterium(B)1.ComparisonClonedsequenceofthetypes.representationALPHA,ofBETA,differentandbacterialGAMMA,divisionsthealpha,inthebeta,16SrDNAandgammalibrary,subdivisions16SrRNAlibraries,orboth.(A)Phylogeneticlow-GϩC-contentgrampositives;;ACTINOB,CFB,ActinobacteriaCytophaga-Flavobacterium-Bacteroides;PLAN,Planctomycetales;NA,;OP10,notcandidateafﬁliated(lineagesdivisionOP10;oftheWPS-1PLAST,ProteobacteriaandWPS-2).

plastids;;HOL-ACID,LOWGϩC,sequencetypesintheWPS-1lineagerangedbetween63.2andthecyanobacteria,itappearedtobranchoutsidethecyanobac-67.8%.Thephylogeneticpositionofthisclonedsequencetypeteriallineageinthemaximumparsimonytree.InthetreecouldnotbedeterminedconsistentlybythedifferenttreeingcalculatedusingFITCH,WPS-2branchedfromtheradiationmethodsusedinthepresentanalysis.Threetreeingmethodstothedeinococci.ByusingFASTAsearches,theclosest16S(neighborjoining,parsimony,andmaximumlikelihood)indi-rDNAsequencestothatofWD272werethesequencesofcatedtheproximityofWPS-2tothecyanobacteria.WhileforclonesSJA-5,SJA-22,andWCHB1-84retrievedfromchlori-theﬁrsttwomethodsWPS-2branchedfromtheradiationtonatedhydrocarbon-degradingcommunities(5,34).

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TABLE2.Identiﬁcationthe16SofrDNArepresentativeandthe16SclonedrRNAsequenceclonelibrariestypespresentfrominPCB-pollutedphylogeneticsoil

groupsfoundinboth

Bacterialdivision

PhylogeneticNo.group

no.ofofsequences/sequence

Representative

types

clone

Closestrelativeindatabase(EMBLaccessionno.)

Length(nt)a%Identity

Alpha-Proteobacteria

A-1A-26/5Sphingomonas95.32A-37/4WD208Phenylobacteriumasaccharolytica1,41097.08A-45/3WD225MagnetospirillumimmobileIFO10564(Y09639)1,40493.75A-74/4WD229WD2103Azospirillumsp.strainMSM-4(Y18216)

(Y17390)1,40990.13A-8Rhodopilasp.strainASP-1(X92464)1,40892.58A-910/62/2WD238Acidosphaeraglobiformis1,42894.70A-1016/9WD248SphingomonasrubrifaciensDSM161(D86513)1,41496.17A-115/4WD249SphingomonassubarticaKF1HS-AP3(X94102)(D86512)1,41198.58A-124/4WD252WD267AﬁpiapruniIFO15498(Y09637)1,40995.46A-13Gluconacetobactergenosp.13strain1,41192.43A-1511/52/1WD271BradyrhizobiumliquefaciensG8991(U87784)

1,41399.01A-164/4WD2756/6WD295WD297Gluconacetobactersp.1,410CaulobactersaccharistrainPe4LMG1382(X75617)IF2-6(AF159437)(AF127412)1,41394.5597.23Beta-Proteobacteria

A-19B-2GluconacetobactervibriodesATCC11764(AJ227755)1,40896.47B-311/36/4WD2108Xylophilus1,41798.14B-510/4WD291VariovoraxampelinussacchariATCCIF2-6(AF127412)1,45599.18B-138/5WD2115Rubrivivaxsp.strainWFF5233914(AF078758)1,45796.35B-146/6WD2102Burkholderiagelatinosus(AB003627)1,451B-158/8WD221Burkholderiasp.(D16213)

1,45373997.29B-165/4WD202Burkholderiasp.strain1,45698.97B-175/5WD263WD206Burkholderiasp.strainLB400(U86373)strainN3P2NF100(U37344)(AB025790)1,45597.2597.80B-18Burkholderiasp.strainNF100(AB025790)B-1938/175/3WD2373/3WD227Burkholderiasp.Burkholderiasp.strainN2P5(U37342)1,45574898.801,45198.28Gamma-Proteobacteria

B-21G-16/4WD268Burkholderiasp.strain1,45597.80G-22/1WD21163/2WD259WD260PseudomonasglatheistrainDha-54(AJ011508)ATCCN2P529195(U37342)

Ectothiorhodospirasp.strainPsK(AF105389)(AB021374)1,45997.251,46299.3287.41G-3(AF084511)

sp.strainBogoriaRedG-413/4WD280Nevskia1,450G-5

15/102/2WD284WD2124NevskiaramosaEctothiorhodospiraramosa(AJ001343)(AJ001343)

1,45096.141,46396.6986.74Holophaga-AcidobacteriumH-2(AF084511)

sp.strainBogoriaRedH-47/57/7WD207Acidobacterium1,424H-56/4WD247Clonecapsulatum(D26171)1,42295.37Holophaga-Acidobacterium

H-65/3WD217CloneUA1(AF200696)1,42696.06H-75/3WD228CloneUA3(AF200699)1,39892.50H-104/4WD243CloneTRB82(AF047646)1,40297.14H-117/7WD261CloneTRB82(AF047646)1,46196.36Planctomycetales

P-43/3WD277CloneDA052(Y07646)1,40093.29CandidateO-12/2WD2112IsosphaeraTRB821,44495.14Notafﬁliateddivision(WS-I)

OP10N-3

2/2

WD294WD2101

Clonesp.(AF047646)

strainSchlesner640PlanctomyceteSJA-22(AJ009456)

(X81959)strain394(AJ231192)

1,43992.451,395

86.1775.41

ant,nucleotides.

Rarefactionanalysisanddiversityindexes.Theclonedse-inthelibraryobtainedwithdilutedRNAwaslowerthaninthequencesrepresentedinthedifferentlibrariesfromWittenbergonegeneratedfromundilutedRNA,asexpected.Shannonweresubjectedtorarefactionanalysis.Twosetsofdatawerediversityindices(calculatedforthethreelibrariesfromclonedrareﬁed:onecomprisingtheclonedsequencetypesandan-sequencetypes)werehigherandverysimilarforthelibrariesothercomprisingthephylogeneticgroupsestablished.DespitegeneratedfromRNA(Hϭ4.71and4.72forthelibraryob-thefactthatonly362of404clonedsequences(65%)inthetainedfromundilutedanddilutedRNAs,respectively),asop-threelibrarieswereunique,therarefactionanalysissuggestsposedtothelibraryobtainedfromDNA(Hϭ4.41).Equita-thatthenumberofclonesscreenedisinsufﬁcienttocircum-bilitywashigherforthelibraryobtainedfromundilutedRNAscribethebacterialdiversityinthePCB-pollutedsoil(Fig.3A).(Jϭ0.97),slightlylowerforthelibraryobtainedfromdilutedThedatafromtheclonedsequencetypesineachoneoftheRNA(Jϭ0.94)and,ﬁnally,lowerforthelibrarygeneratedthreelibrariesgeneratedinthisstudywerealsorareﬁed.Ac-fromDNA(Jϭ0.92).Theseresultsagreewiththerarefactioncordingtotherarefactionplots,theexpectednumberofse-plotsandshowahigherdiversityofthelibrariesgeneratedquencetypesinthe16SrDNAlibrarywaslowerthanthatforfromRNA.

the16SrRNAlibraries,suggestingalowerdiversitywithintheEffectoftemplatedilutionpriortoRT-PCRonthecompo-16SrDNAlibrary(Fig.3B).Thisresultisconsistentwiththesitionofthe16SrRNAlibraries.OnegeneralconcerninthehigherpercentageofredundantclonedsequencesobservedinanalysisofbacterialdiversitybyPCRampliﬁcationof16Sthe16SrDNAlibrary.TheexpectednumberofsequencetypesrRNAgenes,particularlywithsamplesfromsoilenvironments,

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tionaryFIG.2.DendrogramofthenovelbacteriallineagesWPS-1andWPS-2andsequencesrepresentativeofcompletedistancesweresequencesandfromthephylogeneticthe16SrDNArelationshipscloneswerewereincludedcalculatedintheusingdistance,parsimony,andmaximum-likelihooddifferentbacterialmethods.divisions.Onlynearly-Evolu-Thearchaealcollapsedsequencesbacktothewereprevioususedconsistentasoutgroup.

node.Thehypervariablecalculation.regionsintheBranches16SrRNAwhosemoleculeposition(14)variedwerewithexcludedthetreeingfromthemethodcalculation.usedisthefrequentnecessitytodilutetheextractedDNAtoover-0.97inthelibraryobtainedwithundilutedRNA).Eighty-onecomeinhibitionofthePCRandtheeffectthatdilutionmightcloneswithidenticalsequenceswereobtainedfrombothli-haveonthecompositionsofderivedclonelibraries.Thiseffectbraries.

hasbeenexploredwithsedimentsamplesinwhichthePCRsTable3showsthenumberofphylogeneticgroups,clones,werecarriedoutwithverylowtemplateDNAconcentrations,andclonedsequencetypeswhichwereobservedintherRNAintheorderofpicograms,andclonelibrarieswerescreenedbylibraries.Nearlytwo-thirdsoftheclonedsequencetypeswererestrictionfragmentlengthpolymorphismanalysis(4).OntheobservedinbothrRNAlibrariesandwerepreciselytheclonedotherhand,althoughtheabundanceofacertain16SrRNA(orsequencetypeswithinthepredominantphylogeneticgroupsrDNA)sequencetypeinaclonelibrarycannotbedirectlyobservedinbothlibraries,includingclonedsequencetypescorrelatedwiththeabundanceintheenvironmentofthebac-similartothe16SrRNAsequenceofspeciesof(i)Sphingomo-teriumrepresentedbythatsequence,thepresenceofclonednas,Caulobacter,Bradyrhizobium,Phenylobacterium,Magneto-sequencetypesinlibrariesobtainedwithhighlydilutedtem-spirillum,Acidosphaera,andGluconoacetobacter,withintheal-platemightbeconsideredtobeindicativeoftheirpredomi-phasubclassoftheProteobacteria,(ii)Burkholderia,Xylophilusnanceinthatparticularsample.Wehavestudiedtheeffectofampelinus,andRubrivivaxgelatinosusinthebetasubclass,(iii)RNAtemplatedilutionconcentrations(innanograms)mostNevskiaramosawithinthegammasubclassoftheProteobacte-frequentlyobtainedwithsoilsamplesbycomparingthecom-ria,and(iv)Acidobacteriumcapsulatumandtheclonedse-positionsoftwolibrariesobtainedbyRT-PCRampliﬁcationofquencesUA1andUA3(26),TRB82(6),andHRS-56(24)in16SrRNAfromtotalRNA(undilutedandata1:500dilution)theHolophaga-Acidobacteriumphylum.

andcloningoftheresultingproducts.Asexpected,ahigherCertainclonedsequencetypes,almostexclusivelyrepre-percentageofsequenceredundancycharacterizedtheclonedsentedbyasinglesequencetype,wereonlyfoundinthelibrarysequencesobtainedfromdilutedRNA(27%sequenceredun-preparedfromdilutedRNA,includingclonesequencetypesdancyandJϭ0.94comparedwith17%redundancyandJϭafﬁliatedwiththelow-GϩC-contentgram-positivebacteria,

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theFIG.analyzedtotal3.sequenceRarefaction(DNA,s;dataanalysis.undilutedfromWittenberg(A)ExpectedRNA,F;1:500-dilutedPCB-pollutednumberofRNA,soil.phylogeneticE(B)).

Expectedgroupsnumber(clusters)ofcloned(F)andsequenceclonedtypessequenceforeachtypesone(E)ofaftertheclonerarefactionlibrariesofplastids,andtheCytophaga-Flavobacterium-Bacteroides.Dilu-AnalysisofthecommunitycompositionbyFISH.InordertotionofthetemplateRNAalsohadaneffectontheabundanceconﬁrmthehighabundanceofbacteriarelatedtothebetaofcertainsequencetypesintheresultinglibraries.Forexam-subclassoftheProteobacteria,andBurkholderiaspp.inpartic-ple,sequencetypesrelatedtothe16SrDNAsequenceofular,inthebacterialcommunityoftheWittenbergPCB-pol-Nevskiaramosawererepresentedby5clonesinthelibrarylutedsoil,ﬂuorescenceinsituhybridization(FISH)analysisfromundilutedRNAandby18clonesinthelibraryfromwithspeciﬁcoligonucleotideprobeswascarriedout.ThesamedilutedRNA.soilsampleemployedfortheconstructionoftheclonelibraries

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TABLE3.Numberobservedofphylogeneticineithergroups,thelibraryclones,andclonedsequencetypesfromandgeneratedPCB-pollutedsoilforeachofthebacterialdivisions

inbothfromtypesundilutedoflibrariesRNA,simultaneously

theonegeneratedfromdilutedRNA,

aBacterialdivision

No.ofphylogeneticgroupsobservedin16SrRNAlibrariesobtainedwithundilutedanddilutedRNATotal

BothrRNAlibraries

UndilutedRNAonly

1:500-dilutedRNAonly

Proteobacteria

AlphaBetasubdivision4018(97/78)1310(67/50)178(19/18)100

(11/10)Holophaga-AcidobacteriumGammasubdivisionsubdivisionActinobacteria199(74/57)(33/22)3(59/44)3(15/13)3Planctomycetales

6(48/42)8(25/15)8(4/4)Candidate4(9/8)1(37/31)3(3/3)2(8/8)Plastids

divisionOP104(7/6)2(2/2)3(3/3)1(4/3)Low1(6/6)0(5/4)1(3/3)1(1/1)Cytophagales

GϩCgrampositives2(1/1)03(1/1)1(2/2)Notafﬁliated(WPS-1)1(2/2)00(4/4)4(1/1)(8/7)0

01(5/4)02(1/1)01(2/2)3(1/1)(3/3)Total

108(285/230)

38(200/150)

38(48/45)

32(37/35)

athePhylogeneticnumberofclones/clonedgroup:aclonesequencegroupwithtypes.

sequencesimilarityhigherthan97%(98%forBurkholderiaandPseudomonas-typeclones).ValuesinparenthesesindicatewasusedfortheFISHexperiments.Highbackgroundﬂuores-reﬂectthemetabolicallyactivebacteria.DespitetheinherentcencewasobservedwithWittenbergsoilsamplesduetonon-methodologicaldifferencesinthegenerationofthePCRandspeciﬁcbindingoftheﬂuorescentlylabeledprobes.Theprob-lemwaspartiallysolvedbyintroducingaprehybridizationRT-PCRampliﬁcationproductsusedforcloning,agoodcor-treatmentwithblockingreagent(seeMaterialsandMethodsrespondencewasobserved,withbothtypesoflibrariesexhib-fordetails)priortotheadditionoftheﬂuorescentlylabeleditingsimilarbacterialcommunitycompositions,intermsoftheprobe,whichresultedinlowerbackgroundlevels,thoughthismajorconstituents.AconsiderablenumberofidenticalclonedmadethecountingofDAPI-stainedcellsmoredifﬁcult.Totalsequenceswereobservedinalllibraries.Phylogeneticgroupscellcounts,determinedbyDAPIstainingofthesoilsample,representedinbothtypesoflibrariesalsocontainedthema-were1.3ϫ109cellsgofsoilϪ1,valueswhichwereinaccor-jorityoftheclonedsequencetypesobserved.Theresultscon-dancewithwhathasbeenreportedpreviouslyforWittenbergﬁrmedtheabundanceofsequencetypesrelatedtothebetasoil(35).About66%oftheDAPI-stainedcellshybridizedwithsubclassoftheProteobacteria,andthepredominanceofse-theEUB338probeforBacteriaandmostexhibitedstrongquencetypesrelatedtoBurkholderia,Variovoraxparadoxus,ﬂuorescentsignals(Fig.4).TheabundanceofmembersoftheXylophilusampelinus,Nevskiaramosa,Sphingomonassp.,alphasubclassoftheProteobacteriawascomparabletothatofmembersoftheRhodopilaglobiformisphylogeneticbranch,thebetasubclass,with4.2and5.3%ofthetotalDAPI-stainedandAcidobacteriumcapsulatum.Theseresultssupportthepo-cellshybridizingwithprobesALF1bandBET42a,respectively.tentialfunctionalimportanceofthesebacterialgroupsinPCB-Some3.4%oftheDAPI-stainedcellshybridizedwiththepollutedWittenbergsoil.TherewerealsosomedifferencesinprobeSUBU1237forBurkholderiaandSuterellaspp.(31),thecompositionoftherDNAandrRNAlibraries.Forexam-whichrepresentedalmost64%ofthebacteriabelongingtotheple,somephylogeneticgroupswerefoundonlyinoneorthebetasubclassoftheProteobacteriadetected(Fig.4).TheseotheroftherDNAorrRNAclonelibraries(althoughtheyproportionsareconsistentwiththoseobtainedfromthecloneweremostlyrepresentedbyoneortwoclonedsequencetypes),libraries,whichindicatedthatbacteriaafﬁliatedtothegenusandclonedsequencetypesafﬁliatedwiththeActinobacteria,BurkholderiaareabundantandmostprobablymetabolicallyCFB,andlow-GϩC-contentgrampositiveswereobservedonlyactiveinthissoil.ThePLA886probehybridizedwithsphericalintherRNAlibraries.

cells,whosemorphologyisthatofIsosphaerapallida(9),theInastudycarriedoutwithfreshwatersedimentsamples,closestcultivatedrelativeoftheplanctomyceteclonedse-Miskinetal.(20)wereunabletocomparetheresultsofthequencetypesfoundinWittenbergsoillibraries.

rDNAandrRNAlibrariesbecauseofthelownumberofiden-ticalsequencesobservedandthelowdegreeofcoverageoftheDISCUSSION

diversitypresentinthesedimentthatwasreﬂectedintheclonelibraries.Although,asshownbytherarefactionplots,thedi-Aknowledgeofthediversityofmicrobialcommunitiesin-versityinWittenbergsoilwasconsiderable,thecoveragevalueshabitingpollutedenvironmentsisusefulsinceitprovidescluesforWittenbergPCB-pollutedsoillibrarieswerehigh(33.6%aboutthetypeofbacteriaabletoadapttoandtoexploitsuchforrDNAand29%forrRNA,calculatedasdescribedprevi-habitats.Inthisstudy,thebacterialcommunityinasoilhighlyously[20])incontrasttovaluesof4and6%inthefreshwaterpollutedwithPCBshasbeenanalyzedbycloningandsequenc-sediment(20).Thismightbeduetothehighabundanceofing16SrDNAampliﬁedfromtotalDNAextractedfromthecertainsequencetypesinWittenbergclonelibraries(suchassoil,andtheresultsobtainedhavebeencomparedwiththoseBurkholderia)andtothehighernumberofclonesanalyzedinoftwo16SrRNAclonelibraries,whichareassumedtobetterourstudy.

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microscopicFIG.4.FISHofaPCB-pollutedsoilsamplefromWittenberg.(A)HybridizationwithprobeBurkholderiaﬁeldandforSuterellaDAPIspp.staining.(C)and(CofandstainingD)IdenticalwithDAPImicroscopic(D).Bar,ﬁelds10␮showingm(appliesresultstoallofpanels).

hybridizationEUB338,speciﬁcwithprobeforSUBU1237Bacteria.(B)speciﬁcIdenticalforItisimportanttopointoutthattheresultspresentedhereonlyonceortwiceinthelibraries.Thisaspectemphasizesoncearemerelyindicativeandnotdeﬁnitive.Determinationofthemorethefrequentlydiscussedissueofhowrepresentativearesigniﬁcanceoftheobservedsimilaritiesanddifferencesbe-clonelibrariesofthehighbacterialdiversityinsoilsandhowtweentherDNAandtherRNAlibrariesfromthisPCB-pol-manyclonesshouldbescreenedinordertoobtainarepresen-lutedsoilwillrequiremore-comprehensivestudiesinvolvingtativepictureofthecompositionofthebacterialsoilcommu-statisticalanalysis.SomeofthedifferencesobservedinrDNAnity.Second,therewasaslightdifferenceinthespeciﬁcityofandrRNAlibrariesmightbeexplainedwithregardtobiologythereverseprimersusedforthePCRandtheRT-PCR(prim-and/ormethodology,however.Thepresenceofsinglese-er1492Rhasanarrowerspeciﬁcitythanprimer518R[14]).quencetypesexclusivelyintherDNAlibrarycould,theoreti-Therationalebehindtheuseofprimer518RfortheRT-PCRscally,beindicativeofquiescentbacteriapresentinthesample.insteadofprimer1492RwastominimizebiascausedbyearlyThecontrarysituation,namely,thepresenceofrareclonedterminationoftheRTatmodiﬁedbasesinsome16SrRNAsequencetypesexclusivelyinthe16SrRNAlibrary,isunex-molecules(apotentialbiasforRT-PCRampliﬁcationfrompectedsincethecells(activeornot)containbothRNAandrRNA),aswasshownbyWelleretal.(36,37).Finally,athirdDNA.Severalmethodologicalfactorsmighthavecontributedfactormightbedifferentstartingconcentrationsofthediffer-tothesedifferencesbetweentherDNAandrRNAlibraries.enttemplatesintheampliﬁcationreactions(sincethiswillFirst,cloneselectionfromacomplexcollectionisarandomdependontheabundancesintheenvironmentofeachoftheevent,whichmayresultintheselectionofararecloneanddifferentbacterialpopulations,thenumberofrRNAgenes,distortcomparisonsofdifferentclonelibraries.Therefore,andtheribosomalcontentpercell),whichwouldresultinbiascomparisonsshouldnotbebasedonrareclonesthatappearintheproportionofdifferentPCRamplicons(33).

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Despitetheseuncertainitiesincomparingthelibraries,thecombineduseofrDNAandrRNAtoanalyzethebacterialcommunityinthisPCB-pollutedsoilhasresultedinanex-pandedviewofthebacterialdiversityinthissoil.TheresultsfromtherRNAlibrariesallowedustoidentifybacteriawhichwerepresumablymetabolicallyactiveandthereforeresponsi-bleforthefunctionalityofthecommunityinthispollutedsoil.Ontheotherhand,resultsfromtherDNAlibraryhaveallowedustoidentifyothermembersofthecommunity,aswellasenablingmoreprecisephylogeneticassignmentoftheclonedsequences,whichinthecaseofrRNAclonesislimitedbytheshortlengthoftheampliﬁedproduct(17).

DataobtainedfromthePCB-pollutedWittenbergsoilconﬁrmedthepresenceofsequencetypesafﬁliatedwiththeproposedcandidatedivisionOP10(11),whichappeartoberelatedtosequencetypesretrievedfromatrichlorobenzene-transformingconsortium(34).SincethesesequencetypeswereobservedinbothrRNAandrDNAlibraries,thereisstrongevidenceforthemetabolicactivityofthebacteriarepresentedbythesesequencesinthePCB-pollutedsoil.However,becausenoculturedrepresentativesofcandidatedivisionOP10havebeendescribed,themetaboliccapabilitiesofbacteriawithinthisdivisionremainunknown.Ourresultsalsoshowthepres-enceoftwoputativenewbacteriallineagesinWittenbergsoil,oneofwhich,WPS-1,appearedtobephylogeneticallyrelatedtothePlanctomycetalesandwasrepresentedbyseveralclonedsequencetypesinbothrRNAandrDNAlibraries.Thephylo-geneticpositionofthesecondnovelbacteriallineage,WPS-2,remainsunclear(theanalysishadtobebasedinauniqueclonedsequencesincenootherrepresentativesofthisphylo-geneticgroupwerefoundintheclonelibraries).

ThemainfeatureofthebacterialcommunityinthishighlyPCB-pollutedsoil,comparedwithothersoilcommunities,wastheabundanceofalow-diversitysetofsequencetypesafﬁliatedtothebetasubclassoftheProteobacteria,mostlyrelatedtothegenusBurkholderia.FISHanalysisindicatedabundancesofapproximately4.3ϫ107cellshybridizingwiththeprobeSUBU1237pergofsoil,andastrongﬂuorescentsignalwasobservedforthecellshybridizingwiththisprobe(asshowninFig.4).FISHresultsconﬁrmedtheabundanceandthuspre-sumablythehighactivityofbacteriaofthegenusBurkholderiainthePCB-pollutedsoil.AlsoconsistentwiththisconclusionisthehighnumberofBurkholderiaisolatesobtainedfromthisWittenbergsitesamples(W.R.Abrahametal.,unpublisheddata).Althoughrepresentativesofthisgenusarefrequentlyfoundintherhizosphere,theyarenotusuallyabundantinbulksoil(8,13).TheonlypublicationreportingabundantBurkhold-eria-related(andJanthinobacterium-related)sequencesinsoilisoneofaclonelibraryobtainedfromanacidicthermalsoilinNewZealand(15).Inthatcase,theauthorsspeculatedonthepossibilitythattheseclonedsequencesmightrepresenteitherinactiveornonviablecells,sincethesebacteriaareconsideredtobemesophilicandcharacteristicofneutralpHenviron-ments.Thedetectionhereofabundant,presumablymetabol-icallyactive,Burkholderia-typecellsinalow-pHPCB-pollutedsoilatteststotheirabilitytoﬂourishinacidicsoils.AhighabundanceofBurkholderiahasalsobeenreportedinabacte-rialcommunitydegradingaromatichydrocarbonsinatrickle-bedbioreactor(31).

Finally,comparisonofthetworRNAlibrariesobtained

fromundilutedtemplateand1:500-dilutedtemplaterevealeddiscrepanciespreviouslyobserved(4)ofclonedsequencetypesappearingonlyinthelibraryobtainedfromdilutedRNA.ThesedifferencesmaybeattributedtoPCRkineticsbias,whichresultsintheearlyinhibitionofampliﬁcationofabun-danttemplates,whileless-abundanttemplatescontinuetobeampliﬁed(33).Theeffectsoftemplatedilutionseemedtobecomplex,affectingboththeoccurrenceandtheabundanceofsequencetypes,andbiasedaccordingtosequencetypes(i.e.,sequencetypesafﬁliatedwiththegammasubclassofthePro-teobacteriaandwiththeHolophaga-Acidobacteriumphylumwerecharacteristicallymorenumerousinthelibraryfromdi-lutedRNA).However,ascomparisonoftherDNAandrRNAlibrariesrevealed,bothrRNAlibrariesreﬂectedthemajorphylogeneticgroupsandsequencetypesrepresentingthebac-terialcommunityinthePCB-pollutedsoil.Therefore,dilutionofthetemplatepriortoampliﬁcationbyPCR,whichisfre-quentlynecessaryafternucleicacidextractionfromsoilenvi-ronments,doesnotseemtocompromiseassessmentofthebacterialdiversityinthesample(atleastfornotverylowtemplateconcentrations).Ontheotherhand,theabundanceinbothrRNAlibrariesofrelated16SrRNAsequencegroupsisconsistentwiththepredominanceofcertainbacterialpop-ulationsinthispollutedsoil,bacteriarelatedtoSphingomonas,Burkholderia,Nevskia,andAcidobacteriumspp.,amongothers.

ACKNOWLEDGMENTS

EducationThisworkwork,andwasResearchsupported(projectbyagrantno.0319433C).fromtheGermanDuringMinistryforSpanishB.N.supportMinistrywastheforrecipientEducationofandapostdoctoralpartoftheCulture.fellowshipfromthesequencingWethankoftheAnnetteFondsderKru¨gerChemischenandCarstenIndustrie.K.N.T.acknowledgestheStro¨fruitfulexpertisediscussionswork.B.N.andacknowledgesFrank-OliverA.GloM.mplfortheirexcellent¨Osbornformanylate-hourHollandAnalyticRarefactionatinthetheUniversityuseoftheARBpackage.cknerWeforalsokindlythanksharingStevehisM.1.2.

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