71Urbanparksoilmicrobiomesarearichreservoi

UrbanparksoilmicrobiomesarearichreservoirofnaturalproductbiosyntheticdiversityZacharyCharlop-Powersa,ClaraC.Pregitzerb,ChristopheLemetrea,MelindaA.Terneia,JeffreyManikoa,BradleyM.Hovera,PaulaY.Callea,KristaL.McGuirec,d,JeanneGarbarinoe,HelenM.Forgioneb,SarahCharlop-Powersb,andSeanF.Bradya,1aLaboratoryofGeneticallyEncodedSmallMolecules,TheRockefellerUniversity,NewYork,NY10065;bNaturalAreasConservancy,NewYork,NY10029;cDepartmentofBiology,BarnardCollege,ColumbiaUniversity,NewYork,NY10027;dDepartmentofEcology,EvolutionandEnvironmentalBiology,ColumbiaUniversity,NewYork,NY10027;andeScienceOutreachProgram,TheRockefellerUniversity,NewYork,NY10065EditedbyJerroldMeinwald,CornellUniversity,Ithaca,NY,andapprovedOctober20,2016(receivedforreviewSeptember17,2016)Numeroustherapeuticallyrelevantsmallmoleculeshavebeeniden-tifiedfromthescreeningofnaturalproducts(NPs)producedbyenvironmentalbacteria.Thesediscoveryeffortshaveprincipallyfocusedonculturingbacteriafromnaturalenvironmentsrichinbiodiversity.WesoughttoassessthebiosyntheticcapacityofurbansoilenvironmentsusingaphylogeneticanalysisofconservedNPbiosyntheticgenesamplifieddirectlyfromDNAisolatedfromNewYorkCityparksoils.Bysequencinggenesinvolvedinthebiosynthesisofnonribosomalpeptidesandpolyketides,wefoundthaturbanparksoilmicrobiomesarebothrichinbiosyntheticdiversityanddistinctfromnonurbansamplesintheirbiosyntheticgenecomposition.AcomparisonofsequencesderivedfromNewYorkCityparkstogenesinvolvedinthebiosynthesisofbiomedicallyimportantNPsproducedbybacteriaoriginallycollectedfromnaturalenvironmentsaroundtheworldsuggeststhatbacteriaproducingthesesamefamiliesofclinicallyimportantantibiotics,antifungals,andanticanceragentsareactuallypresentinthesoilsofNewYorkCity.TheidentificationofnewbacterialNPsoftencentersonthesystematicexplorationofbacteriapresentinnaturalenvironments.Here,wefindthatthesoilmicrobiomesfoundinlargecitieslikelyholdsimilarpromiseasrichunexploredsourcesofclinicallyrelevantNPs.naturalproducts|metagenomics|biosynthesisBacterialnaturalproducts(NPs)havearichhistoryofservingastheinspirationforthedevelopmentofdiversesmall-moleculetherapeutics(1).Thediscoveryofnewbioactivebacterialmetabo-litesconventionallybeginswiththeculturingofbacteriafromtheenvironmentandthesubsequentexaminationofthemoleculestheyproduceinpureculture.ThesearchfornewbacteriatointroduceintoNPdiscoverypipelineshasledtoaglobalsearchfornaturalecosystemsfromwhichdiversebacteriacanbecultured(2,3).Insomeinstances,moleculesisolatedfrombacteriaoriginallyobtainedinremoteenvironmentshavesubsequentlybeenfoundtobepro-ducedbyorganismsacquiredclosertohome.Forexample,themacrolactinsareaseriesofantiviralcompoundsoriginallyisolatedfrommarinemicroorganisms(4)butlaterfoundtobeencodedbycommonroot-associatedBacillusspecies(5).WewonderedwhetherurbanparksoilsmightrepresentrichreservoirsofNPbiosyntheticdiversity.Here,weusedtargetedmetagenomesequencingtoexplorethebiosyntheticdiversityoftheurbanparksoilmicro-biomesofNewYorkCity(NYC).Inthesestudies,phylogeneticanalysisofindividualnext-generationsequencingreads,derivedfromtheconservedbiosynthesisgenesamplifiedfromenviron-mentalDNA(eDNA),wasusedtopredictthegeneclusterfamiliespresentinanenvironment.OuranalysisrevealedthepresenceofrichNPbiosyntheticdiversitythroughoutNYCparksoils.AcomparisonofeDNAsequencestogeneclustersthatencodebiomedicallyimportantNPfamiliesrevealedthatparksoilslikelycontainbacteriathatencodecongenersofmanybiomedicallyimportantNPs,includingclinicallyusedantibiotics,antifungals,andanticanceragents.Althoughpreviouseffortstoidentifymetaboliteshavefocusedontheglobal-scaleculturingofbacteriafromnaturalenvironments,NPscapableofimprovinghumanhealthmayliehiddenmuchclosertohome,intheurbanparksoilmicrobiomesofourlargecities.ResultsandDiscussionDespitesupportingalmost9millionpeopleatoneofthehighestpopulationdensitiesintheUnitedStates(6),thediverseeco-systemsfoundinNYCparkssupportalargecollectionoffloraandfauna,including2,000speciesofplantsand350speciesofbirds(7,8).MicrobialdiversitysurveysindicatethatNYCisalsohometodiversebacterialandfungalcommunities(9,10).Althoughbacteriaproduceatremendousdiversityofsmallmolecules,nonribosomalpeptide(NRP)andpolyketide(PK)biosynthesisisresponsibleforproducingmanyofthemostbio-medicallyimportantNPscharacterizedfrombacteria(1,11).Bothbiosyntheticsystemsfollowthesameparadigmwhereinmoleculesaregeneratedinaniterativebuildingprocess,usingenzymesthatarecomposedofconserveddomainsorganizedintomodules(Fig.1).Aprototypicalmodulecontainsthreedomains:oneforselectingbuildingblocks,oneforconnectingbuildingblocks,andoneforcarryingthegrowingNP.Weusednonribosomalpeptidesynthetase(NRPS)adenylation(AD)andpolyketidesyn-thase(PKS)ketosynthase(KS)domain-specificdegeneratepri-mers(12,13)toamplifybiosyntheticdomainsfromeDNAisolatedSignificanceBacterialnaturalproducts(NPs)haveservedasinspirationformanytherapeutics.ThehuntfornewbioactiveNPshasledtoaglobalsearchfornaturalecosystemsfromwhichbacteriacanbecultured.Here,weusedNP-focusedmetagenomesequenc-ingtoexplorebiosyntheti