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AnalysisofGeneTranscriptsinaCrudeOil-DegradingMarineMicrobialCommunitySouichiroKATO1andKazuyaWATANABE1;2;y1HashimotoLightEnergyConversionProject,ERATO,JST,Hongo,Bunkyo-ku,Tokyo113-8656,Japan2ResearchCenterforAdvancedScienceandTechnology,TheUniversityofTokyo,Komaba,Meguro-ku,Tokyo153-8904,JapanReceivedJanuary30,2009;AcceptedApril7,2009;OnlinePublication,July7,2009[doi:10.1271/bbb.90072]Weestablishedaprocedureforanalyzinggenetran-scriptsinamicrobialcommunityofunknowngenomicbackground.Analysisofacrudeoil-degradingmarinemicrobialcommunitydetectedtheexpressionofgenesrelatedtothebiodegradationoffattyacidsandthebiosynthesisofglycolipidsprobablyinvolvedintheemulsificationofcrudeoil.Keywords:bioremediation;petroleum;communitytranscriptomeDetectionandmonitoringofgenetranscriptshavebecomepowerfultoolstoinvestigatethestructureandfunctionofmicrobialcommunities.Recently,severalstudieshaveemployedmeta-transcriptomicapproachestoinvestigatenaturalmicrobialcommunities.1–3)Inthesestudies,totalRNAsextractedfrommicrobialcommunitieswereusedtosynthesizecDNAforse-quenceanalysis.Althoughthesestudiesprovideabasisforanalyzinggenetranscripts(rRNAandmRNA)inmicrobialcommunities,theyincludedsomeintrinsictechnicallimitations,asfollows:(i)itisdifficulttoprepareRNAinsufficientquantityandqualityfromanaturalmicrobialcommunity,(ii)mRNAcomposesonlyasmallfraction(usually5%4))ofthetotalRNAextractedfromamicrobialcommunity,and(iii)theprimersusedforRTareinefficient(e.g.,randomprimers)and/orcausebias(e.g.,Shine-Dalgarnose-quenceprimers).Alloftheselimitationsshouldbeobviatedforsuccessfulcommunitygene-transcriptanal-ysis.Inthepresentstudy,weattemptedtoobviatethetechnicallimitationsevidentinpreviousstudiesandestablishedaprocedureforcommunitygene-transcriptanalysis.Itincludesthefollowingsteps;(i)polyadeny-lation(poly[A]-tailing)ofmicrobialRNA,(ii)rRNAsubtractionbyhybridizationtorRNAprobes,(iii)amplificationofthepoly(A)-tailedmRNAbyinvitrotranscription,and(iv)RTusinganoligod(T)primertosynthesizecDNA.Byincorporatingpoly(A)-tailingandsubsequentlyusinganoligod(T)primerincDNAsynthesis,weimprovedtheefficiencyofRTwithoutintroducingprimer-associatedbiasestowardspecificsequences.Thein-vitroamplificationstepprovedusefulforobtainingasufficientamountofRNAfromasmallamountofenvironmentalRNA.Furthermore,subtrac-tivehybridizationremovedalargeportionoftherRNA,whichfacilitatedsubsequentmRNA-orientedanalysis.Usingthisprocedure,genetranscriptsinacrudeoil-degradingmarinemicrobialcommunitywasanalyzedincomparisonwiththoseinapurecultureofamarinehydrocarbon-degradingbacterium,Alcanivoraxborku-mensisSK-2,5)knowntobeimportantinmarinespilled-oilbioremediation.6)A.borkumensisSK-2wascultivatedasdescribedpreviously5)inthepresenceofeitherpyruvate(SK2-Pyr)orcrudeoil(SK2-Oil)asthesolecarbonsource.RNAwasextractedfromthecellsatthelogarithmicgrowthphaseusingthefollowingprocedure:Cellswereharvestedbyfiltrationthrougha0.22-mmpore-sizemembranefilter.TotalRNAwasextractedusingaTRIZOLreagent(Invitrogen,Tokyo)accordingtothemanufacturer’sinstructions,withthefollowingmodifi-cation:Thechloroformvolumeusedforthecell-lysisstepwasincreasedby5timesinordertodissolveresidualoilcomponents.AftercrudeRNAextraction,polysaccharideswereremovedusingalithiumchloride-precipitationsolution(Ambion,Tokyo).TotalRNAwaspurifiedusinganRNeasyMinikit(Qiagen,Tokyo),includingon-columnDNaseIdigestionusingRNase-freeDNase.TheRNAwasquantifiedusingaspectro-photometer,anditsqualitywasevaluatedusinganAgilent2100Bioanalyzer,RNA6000Picoreagents,andRNAPicoChips(AgilentTechnologies,Tokyo).ToconstructacDNA-clonelibrary,the30endofthepurifiedRNAwaspolyadenylatedusingaPoly(A)tailingkit(Ambion).BacterialrRNAwasselectivelyremovedfromthepoly(A)-tailedRNAsamplebythecapture-hybridizationmethodusingaMICROBExpressbacterialmRNApurificationkit(Ambion).ThelinearRNAamplificationmethod(invitrotranscription)wasappliedtotherRNA-removedsampleusingaSMARTmRNAamplificationkit(Clontech,Tokyo).WealsousedthePCR-selectcDNAsubtractionkit(Clontech)andExTaqPolymerase(Takara)fordoublestrandcDNAsynthesisusinganoligod(T)primer,restriction-enzyme(RsaI)digestion,adaptorligation,andnestedPCRusingprimerswithcomplementsequencestotheadaptors.ThePCRproductswereligatedintopGEM-TEasyvector(Promega,Tokyo)andclonedintoEsche-richiacoliJM109competentcells.SequencesoftheclonedPCRproductsweredeterminedbytheDragonGenomicsCenter(TakaraBio,Ohtsu,Japan)usingtheSP6primertargetingavectorfrankingsequence.Data-yTowhomcorrespondenceshouldbeaddressed.Fax:[phone];E-mail:[e-mail].,73(7),1665–1668,2009NotebasesearcheswithcDNAsequenceswereperformedbyBLASTNandBLASTX.7)ThetopBLASThits,withane-value0:001,wereusedtoassigncDNAreads.SequencesofcDNAderivedfrommRNAwerefurtheranalyzedbyNCBIConservedDomainsSearch(),andwerecategorizedaccordingtotheCOGdatabase.8)Ninety-eightand111cDNAcloneswereanalyzedfortheSK2-PyrandSK2-Oillibraries,respectively(Table1).TheaveragelengthsofthesequencedcDNAcloneswere239(SK2-Pyr)and254(SK2-Oil