Two Angular Dioxygenases Contribute to the Metabolic Versatility of

APPLIEDANDENVIRONMENTALMICROBIOLOGY,June2008,p.3812–38220099-2240/08/$08.00?0doi:10.1128/AEM.00226-08

Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.

Vol.74,No.12

TwoAngularDioxygenasesContributetotheMetabolicVersatilityof

Dibenzofuran-DegradingRhodococcussp.StrainHA01?

HamdyA.H.Aly,1#NguyenB.Huu,1§VictorWray,2HowardJunca,1andDietmarH.Pieper1*

DepartmentofEnvironmentalMicrobiology1andDepartmentofStructuralBiology,2HZI-HelmholtzCentrefor

InfectionResearch,Inhoffenstrasse7,D-38124Braunschweig,Germany

Received25January2008/Accepted19April2008

Rhodococcussp.strainHA01,isolatedthroughitsabilitytoutilizedibenzofuran(DBF)asthesolecarbonandenergysource,wasalsocapable,albeitwithlowactivity,oftransformingdibenzo-p-dioxin(DD).Thisstraincouldalsotransform3-chlorodibenzofuran(3CDBF),mainlybyangularoxygenationattheetherbond-carryingcarbon(theangularposition)andanadjacentcarbonatom,to4-chlorosalicylateastheendproduct.Similarly,2-chlorodibenzofuran(2CDBF)wastransformedto5-chlorosalicylate.However,lateraloxygenationatthe3,4-positionswasalsoobservedandyieldedthenovelproduct2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran.Twogeneclustersencodingenzymesforangularoxygenation(dfdA1A2A3A4anddbfA1A2)wereisolated,andexpressionofbothwasobservedduringgrowthonDBF.HeterologousexpressionrevealedthatbothoxygenasesystemscatalyzeangularoxygenationofDBFandDDbutexhibitedcomplemen-tarysubstratespeci?citywithrespecttoCDBFtransformation.WhileDfdA1A2A3A4oxygenase,withhighsimilaritytoDfdA1A2A3A4oxygenasefromTerrabactersp.strainYK3,transforms3CDBFbyangulardioxy-genationatarateof29%?4%thatofDBF,2CDBFwasnottransformed.Incontrast,DbfA1A2oxygenase,withhighsimilaritytotheDbfA1A2oxygenasefromTerrabactersp.strainDBF63,exhibitedcomplementaryactivitywithangularoxygenaseactivityagainst2CDBFbutnegligibleactivityagainst3CDBF.Thus,Rhodo-coccussp.strainHA01constitutesthe?rstdescribedexampleofabacterialstrainwherecoexpressionoftwoangulardioxygenaseswasobserved.Suchcomplementaryactivityallowsfortheef?cienttransformationofchlorinatedDBFs.

Biarylethers,comprisingdibenzo-p-dioxin(DD),dibenzofu-ran(DBF),diphenylether,andtheirhalogenatedderivatives,arewidespreadenvironmentalpollutants.PolychlorinatedDDsandDBFs,thecontaminatingby-productsformedduringthemanufactureofpesticidesortheincinerationofindustrialanddomesticwastes,cancauseawiderangeofserioushealtheffects(1,4,13).

Microorganismsplayimportantrolesinthedegradationandmineralizationofxenobioticandaromaticcompoundsinnat-uralenvironments.Theiraerobicdegradationisfrequentlyini-tiatedbyRieskenonhemeironoxygenaseswhichcatalyzetheincorporationoftwooxygenatomsintothearomaticringtoformarenecis-diols(15).Thisisthenfollowedbyadehydro-genationreactioncatalyzedbyacis-dihydrodioldehydroge-nasetogivecatecholorsubstitutedcatechols,whichserveassubstratesforoxygenolyticaromaticringcleavage.Rieskenon-hemeironoxygenasesaretypicallycomposedofaterminaloxygenase(iron-sulfurprotein)anddifferentelectrontrans-portproteins(8).Thecatalyticiron-sulfurproteinsarehomo-orheteromultimerswiththe?-subunitcontainingaRieske-

*Correspondingauthor.Mailingaddress:DepartmentofEnviron-mentalMicrobiology,HZI-HelmholtzCentreforInfectionResearch,Inhoffenstrasse7,D-38124Braunschweig,Germany.Phone:(49)53161814200.Fax:(49)53161814499.E-mail:dpi@helmholtz-hzi.de.#Presentaddress:GEBRIGeneticEngineeringandBiotechnologyResearchInstitute,Minu?yaUniversity,SadatCity,Egypt.

§Presentaddress:DepartmentofEnvironmentalBiotechnology,InstituteofBiotechnology,VietnameseAcademyofScienceandTech-nology,18-HoangQuocViet,CauGiay,Hanoi,Vietnam.?

Publishedaheadofprinton25April2008.

3812

type[2Fe-2S]cluster,amononuclearnonhemeironoxygenactivationcenter,andasubstrate-bindingsite(8)thatisre-sponsibleforsubstratespeci?city(http://wendang.chazidian.comparisonoftheaminoacidsequencesofthe?-subunitshasrevealedthattheyformafamilyofdiversebutevolutionarilyrelatedsequences.Distinctmajorlineageshavebeenidenti?ed(15),andalthoughnoneoftheenzymesarecompletelyspeci?c,abroadcorrela-tionbetweenthegroupingintoluene/biphenyl,naphthalene,benzoate,orphthalatesubfamiliesandtheirnativesubstrateshasbeenobserved.

Naphthalene-orbiphenyl-degradingPseudomonasorSphin-gomonasstrainstransformDBF,DD,andchlorinatedderiva-tivesintodead-endproducts(9,30,31).Thesesubstratesareattackedatthelateral1,2-and2,3-positionstogivedihydro-diols,whicharesubsequentlydehydrogenatedtodihydroxycompoundsandinsomecasesundergoringcleavageinamannerthatisanalogoustobiphenylornaphthalenetransfor-mation.Whilesuchlateraldioxygenationisappropriatetoinitiatedegradationofbiphenylandnaphthalene,variousau-thorshaveshownthatitisinappropriateforthedegradationofbiarylethers(5,45).Itwasthusarguedthatcleavageoftheetherbridgeiscriticalforthedegradationofbiarylethers(12),andanalysisofSphingomonaswittichiiRW1indicatedthatDBFdegradationproceedsviainitialdioxygenationattheetherbond-carryingcarbon(theangularposition)andanad-jacentcarbonatom(angulardioxygenation),resultingintheformationofahighlyunstablehemiacetal,whichaftersponta-neouscleavageandrearomatizationgivesriseto2,2?,3-trihy-droxybiphenyl(THB)(7).THBissubjecttometacleavageandsubsequenthydrolysistogivesalicylate,analogoustothetrans-

VOL.74,2008DIBENZOFURAN-DEGRADINGRHODOCOCCUSSP.STRAINHA01

TABLE1.Bacterialstrainsandplasmidsusedinthisstudy

3813

BacterialstrainorplasmidRelevantcharacteristic(s)Sourceorreference

Bacterialstrains

SphingomonaswittichiiRW1Rhodococcussp.strainATCC12674

Rhodococcussp.strainHA01E.coliBL21(DE3)(pT7-5RW)E.coliJM109Plasmids

pGEM-TEasypUC119pRSG43pDFDEpDFDRpDBFA12apDBFA12

ab

Dibenzofuran-utilizingbacterium

Bacteriumincapableofutilizingdibenzofuran

Dibenzofuran-utilizingbacterium

pT7-5with1.4-kbPstI/SalIinsertcarryingdbfBgeneofS.wittichiiRW1recA1endA1gyrA96thi-1hsdR17(rK?mK?)e14?(mcrA)supE44relA1?(lac-proAB)/F??traD36proAB?lacIqlacZ?M15?Cloningvector

AprlacZ;pMB9replicon

E.coli-Rhodococcusshuttlevector;RhodococcusrhodochrouscrypticplasmidpRC4integratedintotheA?IIIsiteofpHSG299

Apr;pUC119with4.4-kbEcoRI/PstIinsertcarryingdfdA1A2A3A4genesofstrainHA01

Kmr;pRSG43with4.4-kbEcoRI/PstIinsertcarryingdfdA1A2A3A4genesofstrainHA01

Apr;pUC119with3-kbHindIII/EcoRIinsertcarryingdbfA1A2genesofstrainHA01

Kmr;pRSG43with3-kbXbaI/EcoRIinsertcarryingdbfA1A2genesofstrainHA01

DSMZaDSMZbThisstudy18

Stratagene

Promega

TakaraShuzoCo.MasahiroTakeoThisstudyThisstudyThisstudyThisstudy

DSMZ,DeutscheSammlungvonMikoorganismenundZellkulturen,Braunschweig,Germany.ThisstrainwasDSMZ6014intheDSMZculturecollection.ThisstrainwasDSMZ43287intheDSMZculturecollection.

formationof2,3-dihydroxybiphenyl(DHB)tobenzoatebybi-phenyl-degradingbacteria.Angulardioxygenationwasalsoshowntobetheinitialbasicstepinthedegradationofotherbiarylethercompounds(11).

Todate,alargenumberofbacterialstrainscapableofde-gradingbiarylethershavebeenisolatedandcharacterized.Phylogenetically,almostallofthesebelongtothephylaProteobacteriaandActinobacteria(predominantlyJanibacterstrains)(23,37,60),andinallstrainssofaranalyzed,degra-dationofDBFisinitiatedbyangulardioxygenation.Interest-ingly,likethe?-subunitofdioxindioxygenasefromS.wittichiiRW1,theDbfA1?-subunitofDBFdioxygenasesfromTerra-bactersp.strainDBF63(28)andPaenibacillussp.strainYK5(24),aswellastheDfdA1DBFdioxygenasefromTerrabactersp.strainYK3(22)andcarbazole1,9a-dioxygenasefromPseudomonasresinovoransstrainCA10(42),allrepresentnovelbranchesinthephylogenyof?-subunitsofRieskenon-hemeironoxygenases.

Incontrasttotheimmenseamountofinformationavailablefortransformationofpolychlorinatedbiphenylcongenersbybiphenyldioxygenases,informationonthesubstraterangeofangulardioxygenasesregardingthetransformationofchlori-natedsubstratesremainsscarce.S.wittichiiRW1iscapableoftransformingseveralmono-anddichlorinatedDDsandDBFs(55),whereashighlychlorinatedcongenersareratherrecalci-trant.Currently,allstrainsharboringangulardioxygenasessharethecapabilityoftransforming2-chlorodibenzofuran(2CDBF)and2-chlorodibenzo-p-dioxin(14,16,55),althoughdifferencesareobservedbothintheregioselectivityofattackandsubstratespeci?city.Additionally,Sphingomonassp.strainHH69,S.wittichiiRW1,andSphingomonassp.strainRW16arecapableoftransforming3-chlorodibenzofuran(3CDBF)(19,55,58);however,suchatransformationbyangulardioxy-genasesofgram-positivebacteriahasyettobedescribed.Inthisstudy,weidentifytwoangulardioxygenasesinRhodococ-

cussp.strainHA01andcharacterizetheirregioselectivityofattackonchlorinatedDBFsandsubstratespeci?city.

MATERIALSANDMETHODS

Bacterialstrainsandisolatesandcultureconditions.EscherichiacolicellswereculturedinLBmediumat37°Ccontainingtheappropriateselectionmark-ers,andRhodococcussp.strainATCC12674wasculturedinGYMStreptomycesmedium(4gglucose,4gyeastextract,and10gmaltextractperliter)at30°C.Rhodococcussp.strainHA01wasculturedat30°Cinmineralmediumsupple-mentedwiththeappropriatecarbonsourcesatconcentrationsof2mM.DBFwasaddedfroma100mM?lter-sterilizedstocksolutionindimethylsulfoxide(DMSO),andincubationwasperformedinsealed,screw-cappedErlenmeyer?askstoavoidevaporation.Growthwasmonitoredbymeasuringturbidity(A600).

DBF-degradingbacteriawereisolatedfromsoilsamplescollectedinareassurroundingchemical-,insecticide-,andpesticide-producingfactoriesinKafrElZiat,Egypt.Soil(1g)wasincubatedina1-literErlenmeyer?askcontaining100mlofmineralmedium(10)withDBF(2mM)asthesolecarbonandenergysource.Following1monthofcultivationat30°C,10%oftheculturewastrans-ferredtofreshmediumandculturedforafurthermonth.DilutionsoftheculturewerespreadontominimalmediumagarplatessupplementedwithDBFascrystalsinthelidoftheagarplatesandincubatedfor7daysafterwhichcoloniesweresprayedwith?lter-sterilizedDHB(10mM).Yellowcoloniesduetoextra-diolcleavageofDHBwerepuri?edonminimalmediumagarplateswithDBFasthesolecarbonsource.Onepredominantcolonymorphotype,whichgrewrap-idlyat30°Candexhibitedaslightlyredcolor,wasselectedforfurtherstudies.Bacteria,vectors,media,andcultureconditions.MostchemicalsusedinthisstudywereobtainedfromSigmaandAldrichandwereofthehighestgradeavailable.DHBwasobtainedfromWakoChemicalsGmbH.THBand2,2?,3-trihydroxybiphenylether(THBE)wereavailablefromprevioussyntheses(2).3CDBFwaskindlyprovidedbyStefanSchmidt,BiocenterKleinFlottbek,Uni-versityofHamburg.2CDBFwasobtainedfromtheSigma-Aldrichlibraryofrarechemicals,andDDwasfromPromochemGmbH.RestrictionenzymesandreagentsformolecularexperimentswerepurchasedfromNewEnglandBiolabs,BoehringerMannheim,Promega,Qiagen,Q-Biogene,UnitedStatesBiochemi-cals,andSigma.BacterialstrainsandplasmidsusedinthisstudyarelistedinTable1.

Transformationofsubstrates.Toquantifygrowthrateandsubstrateuptake,Rhodococcussp.strainHA01wasgrownasdescribedabove.Harvestedcellswereinoculated(A600of0.1)into10-mlglasstubescontaining2mlmineralmediumandDBFasthesolecarbonsource.ToestimatethenumberofCFU,

3814ALYETAL.aliquotswereseriallydilutedontosolidLBmediumandincubatedfor48h.Tomeasuresubstratedepletion,1mlofculturewassupplementedwith8mlmeth-anolforextractionofresidualDBFfromcellwallsandtoachievecompletedissolution.Sampleswerecentrifuged,andthesupernatantwasanalyzedbyreverse-phasehigh-performanceliquidchromatography(HPLC).Noninoculatedtubesandtubeswithoutsubstrate(withDMSOonly)servedascontrols.

Toquantifythetransformationratesandidentifythemetabolites,Rhodococ-cussp.strainHA01wasgrowninminimalmediumwithDBF(2mM)orfructose(2mM)asthesolecarbonsourcesasdescribedabove,whereasRhodococcussp.strainATCC12674derivativesweregrowninGYMStreptomycesmediumsupplementedwithkanamycin(100?gml?1).E.coliJM109derivativeswereculturedinLBmediumsupplementedwiththeappropriateantibiotic.Ifneces-sary,isopropyl-thio-?-D-galactopyranoside(IPTG)(0.5mM)wasaddedwhenculturesreachedanA600of0.6.Cellswereharvestedduringlateexponentialgrowthbycentrifugation,washedtwicewith50mMphosphatebuffer(pH7.4),andresuspendedtoanA600of5to20.CellswereharvestedasdescribedaboveforRhodococcussp.strainHA01whentheculturereachedanA600of1.

HarvestedcellswereincubatedwithDBF,DD,2CDBF,or3CDBFsuppliedataconcentrationof0.2to2mMinaliquotsof1mlin10-mlreagenttubesclosedwithTe?on-coatedscrewcapsandincubatedat30°Conanoverheadshaker.InthecaseoftransformationbyE.coliJM109(pDBF12),transformationmixturesweresupplementedwithglucose(10mM).Atappropriatetimeinter-vals,tubeswereanalyzedforresidualsubstrateasdescribedabove.Forquanti-?cationoftransformationproducts,cell-freesupernatantsweredirectlyanalyzedbyHPLC.Rateswerecalculatedbyquantifyingsubstratedepletionorproductaccumulation,incaseauthenticstandardswereavailable.Productformationratefrom2CDBFbyE.coliJM109(pDBF12)wascalculatedassumingthattheprod-uctexhibitedabsorptioncharacteristicssimilartothoseofTHB.

Speci?cactivitiesareexpressedasmicromolesofsubstratedepletedorprod-uctformedperminuteandgramofcellweight(dryweight)(U/g[dryweight]).Dryweightwasdeterminedforexponentiallygrowingcells,anditwascalculatedthataturbidityofA600of1correspondsto0.208?0.010g/literofRhodococcussp.strainHA01,0.214?0.011g/literofRhodococcussp.strainATCC12674,and0.295?0.015g(dryweight)perliterofE.coli.

Toinactivateextradioldioxygenasesandthusaccumulateandidentifyringcleavageintermediatesduringtransformationofbiarylethers,restingcellsofRhodococcussp.strainHA01wereincubatedwiththerespectivesubstratesasdescribedaboveinthepresenceof3-chlorocatechol(0.1mM)(56).

Tocharacterizetheproductformedaftertransformationof3CDBFbyrestingcells,thereactionmixturewassupplementedwithanaliquotofcellextractofE.coliBL21(DE3)(pT7-5RW)overexpressingDbfB,a2,2?,3-trihydroxybiphenyldioxygenasefromS.wittichiiRW1(50mU/ml[activitydeterminedwithDHB])(18).

Preparationofcellextracts.Cellextractswerepreparedaspreviouslyde-scribed(39).ProteinconcentrationsweredeterminedbytheBradfordmethod(6).Supernatant?uidsweresubjectedtosodiumdodecylsulfate-12.5%poly-acrylamidegelelectrophoresis(35),andgelswerestainedin1%(wt/vol)Coo-massiebrilliantblueG-250(47).Dilutionsweredesignedsothattheamountofproteinloadedrangedfrom1to30?g.PageRulerunstainedproteinladder(Fermentas)wasusedtoevaluatethemolecularweightsofthedenaturedproteinsubunits.

Analyticalmethods.DepletionofbiaryletherswasmonitoredbyHPLCanal-ysisaspreviouslydescribed(59)usinganaqueoussolventsystem(?owrate,1ml/min)containing0.01%(vol/vol)H3PO4(87%)and80%(vol/vol)methanol.Productaccumulationwasquanti?edusing60%(vol/vol)methanol.HPLC-massspectrometry(HPLC-MS)wasperformedaspreviouslydescribed(59).

Theone-dimensional1Hnuclearmagneticresonance(NMR)spectrawererecordedaspreviouslydescribed(59).Forrespectivetransformationexperi-ments,thesubstrateswereaddedfroma100mMstocksolutionindeuteratedDMSOinordertoavoidinterferencefromprotonatedDMSO-derivedsignals.DNAisolation.GenomicDNAofRhodococcussp.wasextractedwiththeFastDNAspinkitforsoil(Q-Biogene).DNAfromrecombinantE.colistrainswasobtainedbydissolvingcoloniesin50?lwaterfollowedbyboilingfor10min(27).SupernatantobtainedaftercentrifugationwasusedastemplateforPCRs.PCRampli?cationof16SrRNAgenes.Thephylogeneticrelationshipoftheisolatewasderivedusingthenearlycompletesequenceofthe16SrRNAgenes(correspondingtopositions15to1480intheEscherichiacolinumberingsystem)determinedusingprimersandconditionsdescribedbyLane(36).

PCRampli?cationofRieskenonhemeironoxygenaseencodinggenes.PartofthedfdA1geneofRhodococcussp.strainHA01wasampli?edwithRieskeFandRieskeRprimers(28).A2.5-kbfragmentwasobtainedbyPCRwiththeDfdDOF1forwardprimer(5?-AGGTCTGCCGCGCCGACTGG-3?)designedbasedontheobtainedsequenceandtheDfdDOR1reverseprimer(5?-GAYA

APPL.ENVIRON.MICROBIOL.

GMGGBGGKCGYTSRTASGG-3?)designedbasedonconservedaminoacidsequencesofferredoxinreductasesofstrainRW1,Rhodococcussp.strainM5,RhodococcuserythropolisTA421,andRhodococcusgloberulusP6.Speci?cfor-wardandreverseprimers(DfdDOF2[5?-TCCACGACAGCTCGGTCCTGC-3?]andDfdDOR2[5?-CCGGTGCGCAAGTTGAACTTA-3?],respectively)an-nealingatthebordersoftheobtainedsequenceweredesignedinordertobetterresolvetheinnersequence,andadditionalprimersweresuccessivelydesignedtoobtainthecompletesequenceofthefragmentonbothstrands.Ampli?cationofthecompletedfdA1A2A3A4geneclusterofRhodococcussp.strainHA01(4.4kb)wasachievedusingprimersDfdF546(5?-GGGGGGATATTTGGCCTCACCC-3?)andDfdR4900(5?-TGCGGGTGGACCGGGCGACG-3?).Severaloli-gonucleotidesweredesignedtoperformtheprimerwalkingandsequenceas-semblage.

TolocalizedbfAgenes,primersDxn1F(5?-TGYASNTAYCAYGGVTGG-3?)andDxn2R(5?-TGYASNTAYCAYGGVTGG-3?)weredesignedtodetectandamplifya700-bpfragmentencodingthe?-subunitofthedioxindioxygenaseofS.wittichiiRW1andweremodi?edwithdegeneratepositionstointegratethecodonusageoftherelativelyscarceaminoacidsequencestretchesthissequencehasincommonwithitsclosestrelativesRhodococcussp.strainM5bpdC1,RhodococcuserythropolisTA421bphA,andRhodococcusgloberulusP6bphA.Ampli?cationandsequencingprimersweredesignedbasedontheangulardi-oxygenaseencodinggeneregionfromTerrabactersp.strainDBF63.

ThePCRproductswerecleanedusingtheQiaquickPCRcleaningkit(Qiagen)andclonedinthepGEM-Tsystem(Promega),andtheligationprod-uctsweretransformedinE.coliJM109competentcells(Promega).

DNAsequencingandhomologyresearch.Puri?edPCRproductswerese-quencedusinganABIPRISMBigDyeterminatorv1.1readyreactioncyclesequencingkit(AppliedBiosystems)andanABIPRISM3100geneticanalyzer(AppliedBiosystems).PrimersusedforsequencingreactionswerethesameasthoseusedintheoriginalPCR,exceptinthecaseofinsertsinthepGEM-Teasyvector(Promega),whichwereampli?edandsequencedwithvector-speci?cM13forwardandM13reverseprimers(47).RawsequencechromatogramsfrombothstrandswereassembledwithSequenchersoftwareversion4.0.5(GeneCodesCorporation).AssembledcontigswereusedforDNAandproteinsimilaritysearchesonGenBankdatabasesperformedwithBlastNandBlastPprogramsoftheNationalCenterforBiotechnologyInformationwebsite.16SrRNAgenenucleotidesequenceswerealignedusingClustalWimplementedinMEGAsoft-wareversion3.1(33).Nucleotidesequencesofaromaticdioxygenasesweretranslatedandalignedwiththeprogramfunctionsavailableinthesamesoftwarepackage.Phylogenetictreeswereconstructedusingtheneighbor-joiningalgo-rithm.DistancesweregeneratedusingtheKimuramatrix,andtreestabilitywassupportedthroughbootstrapanalysis(100replicates).Bootstrappedconsensusofneighbor-joiningtreeswasvisualizedwithMEGAsoftware.

CloningandtransformationinE.coliandRhodococcussp.ThecompletegeneclustercomprisingthedfdA1A2A3A4geneswasampli?edfromDNAfromRhodococcussp.strainHA01usingtheprimersDfdPstF(5?-CACGACGACTGCAGCGGTGTGAT-3?)andDfdEcoR(5?-CTACTCTTCGAATTCCTGCGGCATG-3?),whichincludearti?cialPstIandEcoRIrestrictionsites(shownunder-lined)andwhichannealed240bpupstreamand137bpdownstream,respectively,ofthegenecluster.A3-kbDNAfragmentcontainingthegeneregioncomprisingthedbfA1A2genesfromRhodococcussp.strainHA01wasampli?edusingtheforwardprimerDbfHindF(5?TGATACC-3?)andreverseprimerDbfEcoR(5?-GTACCGGGAATTCTCCACCAGT-3?),whichincludesarti?cialHindIIIandEcoRIrestrictionsites(shownunderlined)forcloninginpUC119.ForcloninginpRSG43,ampli?cationwasperformedwithforwardprimerDbfXbaF(5?ATACC-3?)insteadofDbfHindFtointroducethenecessaryXbaIrestrictionsite(shownunderlined).Aftercon?rmationofthesequences,thefragmentswereclonedintotherespectiverestrictionsitesofpUC119,givingpDFDE(containingthedfdA1A2A3A4genes)andpDF32(containingthedbfA1A2genes),orclonedintorespectiverestrictionsitesofpRSG43,givingpDFDR(containingthedfdA1A2A3A4genes)andpDBFA12(containingthedbfA1A2genes).VectorswereintroducedintoE.coliJM109competentcells(Promega)orelectrocom-petentcellsofRhodococcussp.strainATCC12674.

ExtractionofmRNA,cDNAsynthesis,andRT-PCR.Forgeneexpressionstudies,Rhodococcussp.strainHA01wasgrownonDBF(2mM)asthesolecarbonsource.Toassessconstitutiveexpression,thestrainwasgrowninparallelonfructose(2mM).Cultureswereharvestedduringexponentialgrowthbycentrifugation.TotalRNAwasisolatedfrom3mlofDBF-orfructose-growncells.InthecaseofDBF-growncells,residualcrystalsofDBFwereremovedby?ltration(5951/2?lterpaper;Schleicher&Schuell)beforeharvesting.Har-vestedcellswereresuspendedin100?lwaterandimmediatelyprocessedaspreviouslydescribed(59).TheRNAwassubsequentlypuri?edusingtheRNeasy

VOL.74,2008DIBENZOFURAN-DEGRADINGRHODOCOCCUSSP.STRAINHA013815

FIG.1.Dendrogramshowingtherelationshipofthenearlycomplete16SrRNAgenesequenceofRhodococcussp.strainHA01withthosesequencesreportedforseveralRhodococcussp.typestrains.Thetreewasconstructedusingtheneighbor-joiningmethod.Thereliabilityoftheinferredtreeswastestedbybootstrapanalysisusing100resamplings.Bootstrapvaluesbeyond50%aredenotedatthebranchingpoints.Thescalebarindicates0.01nucleotidesubstitutionperposition.GenBank/EMBL/DDBJaccessionnumbersusedforphylogeneticanalysisareshownafterthebacterialstrainname.

kit(Qiagen),and2?loftheelutedRNA(60?l)wasseparatedin1%agarosegelsandstainedwithethidiumbromide.TheratiobetweenthetotalRNAcontentofDBFandfructose-growncellswasquanti?edbyImageQuantsoftware(MolecularDynamics)as0.7:1.0.cDNAwassynthesizedfrom1?loftotalRNAusinga?rst-strandcDNAsynthesiskitforreversetranscriptasePCR(RT-PCR)(Roche,Mannheim,Germany).Thereversetranscriptionreactionmixtureswereseriallydiluted(3.2-fold)withnuclease-freewater(Qiagen),and1?lofeachdilutionwassubjectedtoampli?cationbyPCRusingtheprimersetDfdRNAF(5?-CAACGTGTTCCCCAACTTCT-3?)andDfdRNAR(5?-GGTCGATTACCTCGGTCGTA-3?)toamplifyadfdAgenefragmentandprimersetDbfRNAF(5?-TCTACCGCAAGGAATTGGAC-3?)andDbfRNAR(5?-ATCCCGACGTCGTTCTGATA-3?)toamplifyadbfAgenefragment.Ampli?cationproductswereseparatedon1%agarosegelsandstainedwithethidiumbromide.Productbandswerepuri?edfromagarosegelsusingaQIAquickPCRpuri?cationkit(Qiagen)andsequencedtoverifytheiridentity.

Nucleotidesequenceaccessionnumbers.ThenucleotidesequencesreportedinthisstudyareavailableunderGenBankaccessionnumbersEU622789toEU622791.

RESULTS

CharacterizationofstrainHA01.Analysisofthenearlycomplete16SrRNAgenesequence(correspondingtoposi-tions15to1494accordingtotheE.colinumberingsystem)revealedthatthenovelDBF-degradingisolatedesignated

HA01belongstothegenusRhodococcus(Fig.1)andisthemostsimilartothetypestrainsofRhodococcusrhodochrous,Rhodococcuspyridinivorans,andRhodococcussp.strainT104,whichhaspreviouslybeenreportedtogrowonbiphenylaswellasonterpenoidsasthesolecarbonandenergysource(21).GrowthofRhodococcussp.strainHA01onDBFandtrans-formationofsubstrateanalogues.Rhodococcussp.strainHA01canutilizeDBFasthesolecarbonandenergysourcewithadoublingtimeof3h,whileneitherDD,2CDBF,nor3CDBFcouldbeusedasacarbonsource.CellspregrownonDBFandhavingaturnoverrateof20.5?U/g(dryweight)wereabletoremoveDD,2CDBF,and3CDBFatratesof1.8?0.1,2.3?0.1,and5.8?1.0U/g(dryweight),respec-tively.DepletionofDBFbyfructose-growncellswasnegligible(?0.4U/g[dryweight]),indicatingthattheenzymesinvolvedinDBFdegradationbystrainHA01areinducible.

TheremovalofbothDBFandDDoccurredwithoutsignif-icantaccumulationofintermediates.Inthepresenceof3-chlo-rocatechol(0.1mM),whichisknowntobeastronginhibitorofextradioldioxygenases(3,54),theintermediateaccumulationofupto20%ofappliedDBF(1mM)asTHBandupto

40%

3816ALYETAL.APPL.ENVIRON.MICROBIOL.

FIG.2.The1HNMRspectrumofthemetabolitemixtureformedfrom2CDBFindicatesthepresenceoftwomajormetabolitesinapproxi-matelyequalamounts.Onemetaboliteisidenticalto5-chlorosalicylate(??).TheNMRspectrumofthesecondmetabolite(?)indicatesitsidentityto2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran.Thestructuresofthemetabolitesareshownatthetopofthe?gure.Thesignalsatthebottomofthe?gurearemarkedwithnumbersandlettersaccordingtotheassignmentsoftherespectiveprotons.

ofappliedDDasTHBEindicatedthatthedegradationisinitiatedbyangulardioxygenation.However,3-chlorocatechol-mediatedinactivationwasnotquantitative,andtrihydroxybi-phenylmetaboliteswereslowlytransformedfurther.

Accumulationofintermediateswasobservedduringthetransformationofboth2CDBFand3CDBF.HPLCanalysisrevealedtheaccumulationofonemajorintermediateconcom-itantwiththedepletionof3CDBF,whichshowedaretentionbehaviorandanabsorptionspectrumidenticalwithanauthen-ticstandardof4-chlorosalicylate.Theidentityofthemetabo-litewasfurthercon?rmedbyHPLC-MSanalysis(negativeionizationmode),whichshowedametabolitewithamolecularionatm/z171and173(ratio3:1).Transformationto4-chlo-rosalicylatewasnearlystoichiometric(95%?5%ofapplied3CDBFaccumulatedas4-chlorosalicylate).However,thecul-turesupernatantduring3-CDBFdepletionwasyellow,whichisindicativeoftheaccumulationofanextradiolringcleavageproduct.Theyellowcolorwasnotdepletedduringfurtherincubation,indicatingthatthiscompoundmightbeadead-endmetabolite,probablyformedbylateraldioxygenationof3CDBFfollowedbydehydrogenationandringcleavage.Spec-trophotometricanalysisoftheculturesupernatantaftercom-pleteremovalof2mM3CDBFshowedthattheyellowproductexhibitedanabsorptionmaximumat455nm(A455of?1)similartothereportedabsorptionmaximumofthemetacleav-ageproductof1,2-dihydroxydibenzofuran(464to470nm[49,50]).

HPLCanalysisofthesupernatantsduringthetransforma-tionof2CDBFrevealedtheaccumulationoftwomajorme-tabolites.Oneofthemetaboliteswasidenti?edas5-chlorosa-licylatebasedontheidenticalretentionbehaviorandUVspectrumcomparedwithanauthenticstandard.Moreover,HPLC-MSanalysiscon?rmedtheidentityofonemetaboliteas5-chlorosalicylate.Quanti?cationwithanauthenticstandardrevealedthat40%?5%oftheapplied2CDBFwastrans-formedinto5-chlorosalicylate.HPLC-MSanalysisindicatedthatthesecondmetabolitehasanegativemolecularionatm/z235/237correspondingtoaparentcompound,C12H9ClO3,thatiseitherachlorosubstitutedtrihydroxybiphenyloradihy-drodiolof2CDBF.

Thestructuresofbothmetaboliteswereelucidatedusinginsitu1HNMRspectroscopicanalysisofafreshlypreparedproductmixture(Fig.2).Thisanalysisshowedthepresenceoftwomajormetabolites,oneofwhichwas5-chlorosalicylate.Thethreearomaticprotonsofthiscompoundshowedchemicalshifts,?,at6.96(H-3),7.45(H-4),and7.83(H-6)ppm,respec-tively,withcharacteristiccouplingconstantsofJ(H-3/H-4)?9.0HzandJ(H-4/H-6)?2.2Hz.

Thesecondmetabolitepossessed?vearomaticorvinylicprotons,exhibitingchemicalshiftsat??7.75(H-A),7.42(H-B),7.45(H-C),7.67(H-D),and6.99(H-E)ppm,respec-tively.AlthoughthesignalsofH-4of5-chlorosalicylateandofH-Cofthesecondmetabolitecouldnotbeseparated,theintegralclearlyshowedtwodistinctprotonsat7.45ppm.Thepresenceof?vearomaticorvinylicprotonsexcludesthechlo-rosubstitutedtrihydroxybiphenyls(either2,2?3-trihydroxy-5-chlorobiphenylor2,2?3-trihydroxy-5-chlorobiphenylcanbeformedafterangulardioxygenation),whichharborsixaro-maticprotonsandindicatesthepresenceofachlorosubsti-tutedDBFdihydrodiol.Asthemetabolitecontainedatleastfouraromaticprotonsshowingvicinalcouplings(J?8to10Hz)withatleastoneotherproton,dioxygenolyticattackhasoccurredinthechlorosubstitutedring.Thus,theobservedNMRdataarecompatibleonlywiththestructureoftheme-taboliteas2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran,withasingletvinylicprotonresonatingat??6.99ppm(H-E).ProtonsFandG(seethechemicalstructuresinFig.2),areexpectedtoresonateat??http://wendang.chazidian.comparisonoftheintegralsoftheresonancelinesshowedthatindifferentpreparations,theratioinwhichtheseproductshavebeenformed(5-chlorosalicylateto2-chloro-3,4-dihydro-3,4-dihydroxydibenzofuran)was

al-