CompleteanditegratedpryenedegradationinMycobacteri

JOURNALOFBACTERIOLOGY,Jan.2007,p.464–472Vol.189,No.20021-9193/07/$08.00
0doi:10.1128/JB.01310-06CompleteandIntegratedPyreneDegradationPathwayinMycobacteriumvanbaaleniiPYR-1BasedonSystemsBiology
†Seong-JaeKim,1‡OhgewKweon,1‡RichardC.Jones,2‡JamesP.Freeman,3RickyD.Edmondson,2andCarlE.Cerniglia1*DivisionofMicrobiology,1DivisionofSystemsToxicology,2andDivisionofBiochemicalToxicology,3NationalCenterforToxicologicalResearch,U.S.FoodandDrugAdministration,Jefferson,Arkansas72079Received17August2006/Accepted23October2006MycobacteriumvanbaaleniiPYR-1wasthefirstbacteriumisolatedbyvirtueofitsabilitytometabolizethehigh-molecular-weightpolycyclicaromatichydrocarbon(PAH)pyrene.Weusedmetabolic,genomic,andproteomicapproachesinthisinvestigationtoconstructacompleteandintegratedpyrenedegradationpathwayforM.vanbaaleniiPYR-1.Genomesequenceanalysesidentifiedgenesinvolvedinthepyrenedegradationpathwaythatwehaveproposedforthisbacterium.Toidentifyproteinsinvolvedinthedegradation,weconductedaproteomeanalysisofcellsexposedtopyreneusingone-dimensionalgelelectrophoresisincom-binationwithliquidchromatography-tandemmassspectrometry.DatabasesearchingperformedwiththeM.vanbaaleniiPYR-1genomeresultedinidentificationof1,028proteinswithaproteinfalsediscoveryrateof1%.Basedonbothgenomicandproteomicdata,weidentified27enzymesnecessaryforconstructingacompletepathwayforpyrenedegradation.Ouranalysesindicatethatthisbacteriumdegradespyrenetocentralintermediatesthrougho-phthalateandthe
-ketoadipatepathway.Proteomicanalysisalsorevealedthat18enzymesinthepathwaywereupregulatedmorethantwofold,asindicatedbypeptidecountingwhentheorganismwasgrownwithpyrene;threecopiesoftheterminalsubunitsofring-hydroxylatingoxygenase(NidAB2,MvanDraft_0817/0818,andPhtAaAb),dihydrodioldehydrogenase(MvanDraft_0815),andringcleavagedioxygenase(MvanDraft_3242)weredetectedonlyinpyrene-growncells.TheresultspresentedhereprovideacomprehensivepictureofpyrenemetabolisminM.vanbaaleniiPYR-1andausefulframeworkforunderstandingcellularprocessesinvolvedinPAHdegradation.Polycyclicaromatichydrocarbons(PAHs)areproducedbyincompletecombustionoffossilfuels,wasteincineration,andindustrialprocessesandoccurnaturallyduetoforestandprai-riefires(8,62).Pyrene,aPAHcontainingfourfusedbenzenerings,iscommonlyfoundasapollutantofair,water,andsoil(20).Althoughthereisnoevidencethatpyreneiscarcinogenictohumans,nonhumanprimates,orlaboratoryanimals,whentestedonmouseskinsimultaneouslywithbenzo[a]pyrene,itenhancedthecarcinogeniceffectsofbenzo[a]pyrene(20).PyreneistoxictotheaquaticmicroinvertebrateGammaruspulexandcanbetransformedintoquinonemetabolitesthataremutagenicandtoxictoorganismsintheenvironment(3,5,51).Microbialdegradationstudieshaveshownthatitismuchmoredifficulttoremovehigh-molecular-weight(HMW)PAHswithfourormorefusedaromaticringsthanitistoremovelow-molecular-weightPAHs(24)sinceHMWPAHsarether-modynamicallystableandhydrophobicandtheyareadsorbedtosolidparticles(59).Pyrenehasbeenusedasamodelcom-poundtostudybiodegradationofHMWPAHssinceitisstructurallysimilartoseveralcarcinogenicPAHs(24).Al-thoughanumberofbacterialisolateshavebeenreportedtogrowonormineralizepyrene,themajorityoftheseisolatesarenocardioformactinomycetes,suchasmembersofthegeneraMycobacterium(4,12,19,26,40,48,56,60)andRhodococcus(6,61).MycobacteriumvanbaaleniiPYR-1(29,33)wasoriginallyiso-latedfromoil-contaminatedsedimentbasedonitsabilitytode-gradepyrene(18).Thisbacteriumwasthefirstisolatereportedtomineralizepyrenebymono-anddioxygenasereactions(19).PYR-1canalsodegradeortransformbiphenyl,naphthalene,anthracene,fluoranthene,1-nitropyrene,phenanthrene,benzo-[a]pyrene,benz[a]anthracene,and7,12-dimethylbenz[a]anthra-cene(16,17,27,28,35,43–47).Becauseofitsmetabolicversa-tility,thisbacteriumhasbeenthoughttobeapotentialcandidateforbioremediationofPAH-contaminatedareas(41).PyrenedegradationpathwayshavebeenproposedforM.van-baaleniiPYR-1(19,30,35,57,58)(Fig.1).Basedonidentifica-tionofmetabolitesandon18O2incorporationexperiments,thisbacteriumcanoxidizepyrenebytwopathways.First,itcanoxi-dizepyreneviainitialdioxygenationattheC-1andC-2positionstoformO-methylatedderivatives(Fig.1)ofpyrene-1,2-diol(36),asadetoxificationstep.However,thepredominantsecondpath-wayisdioxygenationinitiatedattheC-4andC-5positions(K-region)(19)togivecis-4,5-dihydroxy-4,5-dihydropyrene(pyrenecis-4,5-dihydrodiol).Rearomatizationofthedihydrodiolandsub-sequentringcleavagedioxygenationleadtotheformationof4,5-dicarboxyphenanthrene,whichisfurtherdecarboxylatedto4-phenanthroate.Thesubsequentintermediate,cis-3,4-dihy-droxyphenanthrene-4-carboxylate,isproducedbyaseconddioxygenationreaction.Rearomatizationthenforms3,4-dihy-droxyphenanthrene,whichisfurthermetabolizedto1-hydroxy-2-*Correspondingauthor.Mailingaddress:DivisionofMicrobiology,NCTR/USFDA,3900NCTRRoad,Jefferson,AR72079.Phone:[phone].Fax:[phone].E-mail:[e-mail].†Supplementalmaterialforthisarticlemaybefoundat‡S.-J.K.,O.K.,andR.C.J.contributedequallytothiswork.
Publishedaheadofprinton3November2006.464naphthoate.The