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Sharedstrategiesforβ-lactamcatabolisminthesoilmicrobiomeTerenceS.Crofts1,2,BinWang1,2,AaronSpivak2,TaraA.Gianoulis3,**,KevinJ.Forsberg2,MollyK.Gibson2,LaurenA.Johnsky4,StaceyM.Broomall4,C.NicoleRosenzweig4,EvanW.Skowronski4,HenryS.Gibbons4,MortenO.A.Sommer5,andGautamDantas1,2,6,7,*1DepartmentofPathologyandImmunology,WashingtonUniversityinStLouisSchoolofMedicine,SaintLouis,MO,USA2TheEdisonFamilyCenterforGenomeSciencesandSystemsBiology,WashingtonUniversityinStLouisSchoolofMedicine,SaintLouis,MO,USA3WyssInstituteforBiologicallyInspiredEngineering,Harvard,Cambridge,MA,USA4USArmyEdgewoodChemicalBiologicalCenter,AberdeenProvingGround,MD,USA5NovoNordiskFoundationCenterforBiosustainability,TechnicalUniversityofDenmark,DK-2800,Lyngby,Denmark6DepartmentofMolecularMicrobiology,WashingtonUniversityinStLouisSchoolofMedicine,SaintLouis,MO,USA7DepartmentofBiomedicalEngineering,WashingtonUniversityinStLouis,SaintLouis,MO,USAAbstractThesoilmicrobiomecanproduce,resist,ordegradeantibioticsandevencatabolizethem.Whileresistancegenesarewidelydistributedinthesoil,thereisadearthofknowledgeconcerningantibioticcatabolism.Herewedescribeapathwayforpenicillincatabolisminfourisolates.Genomicandtranscriptomicsequencingrevealedβ-lactamase,amidase,andphenylaceticacidcatabolonup-regulation.Knockingoutpartofthephenylaceticacidcatabolonoranapparentpenicillinutilizationoperon(put)resultedinlossofpenicillincatabolisminoneisolate.Ahydrolasefromtheputoperonwasfoundtodegradeinvitrobenzylpenicilloicacid,theβ-lactamasepenicillinproduct.Totestthegeneralityofthisstrategy,anE.colistrainwasengineeredtoco-expressaβ-lactamaseandapenicillinamidaseortheputoperon,enablingittogrowusingpenicillinorbenzylpenicilloicacid,respectively.ElucidationofadditionalpathwaysmayallowforUsersmayview,print,copy,anddownloadtextanddata-minethecontentinsuchdocuments,forthepurposesofacademicresearch,subjectalwaystothefullConditionsofuse:*Towhomcorrespondenceshouldbeaddressed:GautamDantas(dantas@wustl.edu).**DeceasedAuthorcontributionsT.S.C.,A.S.,T.A.G.,M.O.A.S.,andG.D.conceivedofexperimentsanddesignofwork.T.S.C.,B.W.,A.S.,andT.A.G.performedinvitro,microbial,andtranscriptomicexperiments.L.A.J.,S.M.B.,C.N.R.,E.W.S.,andH.S.G.sequencedstraingenomes.T.S.C.,A.S.,T.A.G.,K.J.F,andM.K.G.providedanalyses.ArticledraftingwasperformedbyT.S.C.withcriticalrevisionperformedbyT.S.C.,B.W.,A.S.,K.J.F,M.K.G.,M.O.A.S.,andG.D.CompetingfinancialinterestsstatementTheauthorsdeclarethattheyhavenocompetingfinancialinterests.HHSPublicAccessAuthormanuscriptNatChemBiol.Authormanuscript;availableinPMC2018October30.Publishedinfinaleditedformas:NatChemBiol.2018June;14(6):556–564.doi:10.1038/s41589-018-0052-1.AuthorManuscriptAuthorManuscriptAuthorManuscriptAuthorManuscriptbioremediationofantibiotic-contaminatedsoilsanddiscoveryofantibiotic-remodelingenzymeswithindustrialutility.IntroductionThediscoveryofantibioticsandtheirdevelopmentintoanarmamentariumagainstbacterialinfectionshasbeenoneofthegreatpublichealthsuccessstoriesofthelastcentury.However,increasingantibioticresistanceinpathogenicbacteriawithconcomitantdecreasingdevelopmentofnewantibioticsthreatensareturntothedarkagesofthepre-antibioticera1.Bacterialresistancetoantibioticsisancient2andubiquitousintheenvironment3,4.Moreover,anthropogenicantibioticusehasledtoameasurableincreaseincarriageofantibioticresistancegenesintheenvironmentwiththepotentialtospreadtotheclinic5.Theultimatefateofantibioticsintheenvironment,andwhatroleresistanceplaysintheirmineralization,isunknown.Mostantibioticsarenaturalproducts,orderivativesthereof,originallyisolatedfromsoilbacteria6.Giventheirsoilorigin,andthelackofenvironmentalaccumulationoftheseorganiccompounds,itisnaturalthatsomeantibioticsareconsumedbysoilbacteriaascarbonornitrogensources.Thiswasrecognizedsoonafterthemassanthropogenicintroductionofantibioticsbystudiesdemonstratingtheabilityofsoilbacteriatomineralizevariousnaturalantibioticsincludingstreptomycin7,penicillinG(alsoknownasbenzylpenicillin,referredtohereaftersimplyaspenicillin)8,andchloramphenicol9.Intheliterature,utilizationofpenicillinhasmostoftenfocusedonPseudomonasstrains,withconflictingevidenceforwhatpartofthemoleculeisusedasacarbonsource10–12,althoughcatabolismofpenicillinsinotherorganisms,suchasKlebsiellapneumoniae,hasbeenreportedaswell13.Littleisknownaboutthepathwaysandenzymesutilizedduringcatabolism,includingwhetherβ-lactamaseactivityisrequired8,11,14.Morerecently,thelistofantibioticscapableofsustainingbacterialgrowthhasexpandedsubstantially,ashasthecatalogofbacterialspeciescapableofsubsistingonantibiotics14–19.However,controversystillremainsoverthecharacterizationofresistant,butnotmetabolizing,versussubsistentgrowthphenotypes.Todate,nospecificgenesorpathwayshavebeenidentifiedthatenablebacteriatouseantibioticsasasolecarbonsource16,20.Hereweprovideevidenceforapathwayforβ-lactamantibioticcatabolisminwhichamidases,foundtobedistinctfromknownpenicillinamidaseenzymes,linkresistanceenzymestocentralme