RESEARCHARTICLE Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6 BetinaCeciliaAgaras1,2*,Andre´sIriarte3,ClaudioFabia´nValverde1,2 1 LaboratoriodeBioqu´ımica,Microbiolog´ıaeInteraccionesBiolo´gicasenelSuelo,DepartamentodeCiencia yTecnolog´ıa,UniversidadNacionaldeQuilmes,Bernal,BuenosAires,Argentina,2 ConsejoNacionalde a1111111111 InvestigacionesCient´ıficasyTe´cnicas(CONICET),BuenosAires,Argentina,3 LaboratoriodeBiolog´ıa a1111111111 Computacional,DepartamentodeDesarrolloBiotecnolo´gico,InstitutodeHigiene,FacultaddeMedicina, a1111111111 UniversidaddelaRepu´blica,Montevideo,Uruguay a1111111111 a1111111111 *[email protected],[email protected] Abstract OPENACCESS Plant-growthpromotionhasbeenlinkedtothePseudomonasgenussincethebeginningof Citation:AgarasBC,IriarteA,ValverdeCF(2018) thisresearchfield.Inthiswork,weminedthegenomeofanArgentineanisolateofthe Genomicinsightsintothebroadantifungalactivity, recentlydescribedspeciesP.donghuensis.StrainSVBP6,isolatedfrombulksoilofanagri- plant-probioticproperties,andtheirregulation,in culturalplot,showedabroadantifungalactivityandseveralotherplant-probioticactivities. PseudomonasdonghuensisstrainSVBP6.PLoS ONE13(3):e0194088.https://doi.org/10.1371/ Asthisspecieshasbeenrecentlydescribed,anditseemslikesomeplant-growthpromoting journal.pone.0194088 (PGP)traitsdonotbelongtotheclassicalpseudomonadstoolbox,wedecidetoexplorethe Editor:Marie-JoelleVirolle,UniversiteParis-Sud, SVBP6genomeviaanbioinformaticapproach.Genomeinspectionconfirmedourprevious FRANCE invitroresultsaboutgenesinvolvedinseveralprobioticactivities.Othergenetictraitspossi- Received:November23,2017 blyinvolvedinsurvivalofSVBP6inhighlycompetitiveenvironments,suchasrhizospheres, werefound.Tn5mutagenesisrevealedthattheantifungalactivityagainstthesoilpathogen Accepted:February24,2018 MacrophominaphaseolinawasdependentonafunctionalgacSgene,fromtheregulatory Published:March14,2018 cascadeGac-Rsm,butitwasnotduetovolatilecompounds.Altogether,ourgenomicanaly- Copyright:©2018Agarasetal.Thisisanopen sesandinvitrotestsallowedthephylogeneticassignmentandprovidedthefirstinsights accessarticledistributedunderthetermsofthe intoprobioticpropertiesofthefirstP.donghuensisisolatefromtheAmericas. CreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginal authorandsourcearecredited. DataAvailabilityStatement:Allrelevantdataare withinthepaperanditsSupportingInformation Introduction files. Funding:Thisworkwassupportedbygrants: AmongallsoilbacterialgenerahavingarepresentativedescribedasaPlant-GrowthPromot- PUNQEXPTE1411/15(UniversidadNacionalde ingMicrobe(PGPM),thegenusPseudomonascompriseawidevarietyofprobioticspecies, Quilmes,Argentina);PIP2012N˚ withdistinctmechanismsofactionthatstrengthenplanthealth,eitherdirectlyorindirectly 11220110100031(ConsejoNacionalde [1,2].Severalpseudomonadshavedemonstratedproductionofdifferentkindsofsecondary InvestigacionesCient´ıficasyTe´cnicas,Argentina); metabolitesinvolvedinantagonismtopathogens,phytostimulationornutrientsupply,andan PIP2015N˚11220150100388(ConsejoNacional abilitytodegradecomplexorganiccompounds,notonlybeingablehencetocontributeto deInvestigacionesCient´ıficasyTe´cnicas, Argentina);PRCT17ActaN˚1459/17(Comisio´nde plantgrowth,butalsotobioremediationofsoils[2–6].Asmembersoftheγ-Proteobacteria PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 1/32 GenomeminingofP.donghuensisSVBP6 InvestigacionesCient´ıficas,MinisteriodeCiencia, subphylum,whichrangefrom1%to34%oftheabundanceoftotalsoilbacterialcommunity Tecnolog´ıaeInnovacio´ndelaProvinciadeBuenos ofdifferentenvironments[7],Pseudomonasspp.arekeymembersofthesoilmicrobiome. Aires,Argentina).Thefundershadnoroleinstudy Consideredascopiotrophs,becausetheyarespeciallypresentinareaswhereresourceavail- design,datacollectionandanalysis,decisionto abilityishighandcarbonsourcesaresimple[8],theirremarkablenutritionalversatilityallows publish,orpreparationofthemanuscript.They themtoexploitdiverserhizosphereenvironments,whereeachplantexudatesavarietyof contributedwiththemoneytocarryoutthe describedassaysandtopayforthepublicationfee. organiccompounds[9].Theirhighrhizospherecompetenceiscriticalforthecorrectfunction- ingofmicrobialinoculantsandtheireffectonsoilmicrobiome[10,11]. Competinginterests:Theauthorshavedeclared thatnocompetinginterestsexist. ThephysiologicalandgeneticadaptabilityofPseudomonasspp.facilitatedthewidespread distributionofthisgenusinvariousecosystemsaroundtheworld[12–14].Besides,those genomictraitsallowthesebacteriatobecontinuouslystudiedassourcesofnewmetabolic pathwaysandnovelproducts,sometimesrelatedwithplant-probioticactivities[15–18],which couldbeusefulforthedevelopmentofmoreefficientagriculturalbio-inputs. Genomesequencinghasbecomeausefultoolfordifferentmicrobiologyprojects,notonly tounderstandthegeneticbasisofmetabolicprocesses[19,20],butalsotounravelsomeincon- sistencesintaxonomicaffiliationsthattheclassicalapproachescannotdefine,forinstance,by comparingtheaveragenucleotideidentity(ANI)[21,22].AsPseudomonasspp.genomesare soadaptable,thesetechniqueshaverecentlyallowedthedescriptionofseveralnovelspecies [23–26],andtobetterorganizethisgenus,whichisthelargestwithinGram-negativebacteria [27].Anadditionalbenefitofnextgenerationsequencingisthepossibilitytoaccesstoan increasingamountofgeneticinformation.Thus,genomemininghasbecomeanessentialtool forprocessingsuchamountofdatagenerated,inordertosearchfornewcatalysts,targetsor products,particularlyinmicroorganismswithindustrialapplications,asPGPM[28]. ThereareseveralreportsonthedifficultiestoreproducethepositiveeffectsofPGPMthat hadbeenseenatthelaboratoryonplantgrowth,infieldassays,mostprobablyduetotheinflu- enceofseveraluncontrolledbioticandabioticfactors[29–33].Therefore,thefocushasbeen recentlyputintotheisolationofPGPMfromthesameagriculturalplotsorcroprhizospheres wheretheywouldbeappliedlaterasinoculants.Thisstrategymayhelptoovercomedifficul- tiesinadaptingnon-nativebacterialspeciesinanewenvironment[6,34–37].Withthispur- pose,weisolatedandcharacterizedagroupof19pseudomonadsfromthehumidPampean regionofArgentina,whichwereselectedbytheirinvitroantagonisticactivityagainstdifferent fungalpathogens[6].Oneoftheisolates,namedSVBP6,displayedabroadspectrumoffun- gal-growthinhibitionactivity.Besides,SVBP6couldnotbeclearlyassignedtoanyofthePseu- domonasspeciesalreadydescribedatthatmoment.Therefore,theaimofthisworkwasto obtainandexplorethedraftgenomeofstrainSVBP6,inordertobetterdefineitstaxonomic assignment,tosurveythegeneticdeterminantsofthebroadantifungalactivityshowninvitro, andtodiscoverputativeplant-probiotictraitsthatmaybeinstrumentalforthisstrainasan agriculturalbio-input. Materialsandmethods IsolationofSVBP6,andphysiologicalandbiochemicalcharacterization StrainSVBP6wasisolatedfromthebulktopsoil(0–10cm)ofanagriculturalplotlocatedin Viale,EntreR´ıosprovince,Argentina(31˚52’59,6”S;59˚40’07”W)usingGould’sS1selec- tivemedia[38].Atsamplingtime(February2010),theplotwascultivatedwithsoybeanand hadahistoryofno-tillmanagementundergoodagriculturalpractices(croprotation,nutrient replacement,andminimizedagrochemicaluse)for,atleast,13years[39].IsolateSVBP6was selectedbyitsantagonisticpotentialagainstadiversegroupofphytopathogenicfungithat includedmembersofFusarium,Colletotrichum,Phomopsis,Macrophomina andCercospora genera[6]. PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 2/32 GenomeminingofP.donghuensisSVBP6 Colonymorphologywasobservedunderamagnifyingglass(OlympusSZ61,10×),after sowinga20μl-dropofabacterialsuspensionwithDO =1.0onanutrientagar(NA,Biokar) 600 plateandincubatingfor48hat28˚C.Morphologicalcharacterizationofitscellularstructure wasperformedusingTransmissionElectronMicroscopy(JEM1200EXII,Jeol)attheCentral ServiceofElectronMicroscopy(SCME)fromtheFacultyofVeterinaryoftheNationalUni- versityofLaPlata(BuenosAires,Argentina).Forthisassay,SVBP6wasgrownovernighton nutrientyeastbroth(NYB,Biokar)at28˚Cand200rpm. ClassicalbiochemicalassayswereperformedtocharacterizeSVBP6:Gramstaining(Labora- toriosBritania);catalasedirecttestwithadropof3%H O onacolonyandevaluationofbub- 2 2 bling(O production);oxidasetest(BDBBL™Taxo™,N-discswith6%ofp-aminodimethylaniline 2 monohydrochloride);gelatinliquefactionafter24hofincubationofNYBwith12%ofgelatin; glucosefermentationinHughandLeifson’sOFbasalmedium(pH=7.4);growthonNAplates atdifferenttemperatures(4˚C,25˚C,28˚C,35˚C,37˚Cand45˚C);growthonLuria-Bertani(LB) brothatseveralpHvalues(from3.0to13.0)andNaClconcentrations(from0%to11%);reduc- tionofnitritesandnitratesafter24hofculturegrowthonNYBsupplementedwithKNO or 2 KNO ,respectively,bytheevaluationofnitritepresencewithn-(1-naphtyl)-ethylenediamine 3 dihydrochloride[40]andgasformationinsideaDurhamtube[41].Dependingonthetestper- formed,EscherichiacoliK12orPseudomonasaeruginosaPA01wereemployedaspositiveorneg- ativecontrols.Allincubationsweredoneat28˚C,unlessotherwisespecified. ToanalyzethecarbonsourcesthatSVBP6wasabletometabolize,weassayeditsgrowthin BiologEcoPlatesTM[42],after108hofstaticincubationat30˚C.Antibioticresistancewasana- lyzedbygrowingSVBP6inNYBmediumsupplementedwiththecorrespondingantibioticat theconcentrationscommonlyemployedforthePseudomonasspp.genus:chloramphenicol (20μg/ml),ampicillin(100μg/ml),gentamicin(10μg/ml),kanamycin(50μg/ml)andtetracy- cline(150μg/ml). EvaluationofPGPproperties Wetestedseveralplant-growthpromoting(PGP)activitiesaspreviouslydescribed[6]:exopro- tease,phospholipaseand1-aminocyclopropanecarboxylicacid(ACC)deaminaseactivities; hydrogencyanide(HCN)production;siderophoreandindoleaceticacid(IAA)synthesis; swimmingandswarmingmotilities;inorganicphosphatesolubilizationability;biofilmdevelop- ment;secretionofacylhomoserinelactone-like(AHL)quorumsensingsignalsandthepresence ofantifungal-relatedgenes(phlD,phzF,pltBandprnD).Exoprotease(milkagarplates),phos- pholipase(eggyolkagarplates)andsiderophore(CASagarplates)activitieswererelativizedto thecolonydiameterasfollows:[halodiameter(inmm)—diameterofeachbacterialspot(in mm)]/diameterofeachbacterialspot(inmm).Relativevalueswereexpressedaspercentage. Additionally,wetestedthechitinaseactivitybyafluorometricassaywith4-methylumbelli- ferone-N-acetyl-b-D-glucosaminideassubstrate[43].Briefly,wegrewSVBP6onsynthetic medium(SM)liquidmediumsupplementedwith10%v/vofLBbrothand0.2%w/vofcolloi- dalchitin[44]for72hat200rpmand28˚C.Then,wecollected200μlofsupernatantbytripli- cateandmixeditwith50μlofsubstrateat200μMinMESbuffer0.1M(pH=6.1),tomeasure thefluorescenceduring15minutes.Controlreactionsandcalculationsofactivityweredone aspreviouslydescribed[43].Chitinaseproductionwasexpressedastheenzymeactivity (nmol/min)relativetotheOD valueoftheSMculture. 600 Fordetectionoflipopeptideproduction,wecarriedoutthedrop-collapsetestonParaf- ilm1[19].Thepresenceofbiosurfactantsinadropofcell-freesupernatant(fromabacterial culturegrownonNYBfor16hat28˚Cand200rpm)decreasesthesurfacetensionoftheliq- uidand,therefore,resultsinitscollapse.Methylenebluewasaddedtostainthedropsfor PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 3/32 GenomeminingofP.donghuensisSVBP6 photographicpurposesandhadnoinfluenceontheresults.Besides,aspecificassayforrham- nolipidsdetectionwasperformedinSiegmundWagner(SW)agar[45],evaluatingtheproduc- tionofhalosaroundthecoloniesafterplateincubationfor48hat28˚C.Rhamnolipidsform aninsolublecomplexwiththeCTABpresentintheSWmedium,anditinteractswiththe methylenebluealsopresent.Thus,rhamnolipidsproducersaredetectedbyadarkbluehalo aroundthecolony[46]. Antagonismbytheproductionofvolatileorganiccompounds(VOCs)wasevaluatedbyco- cultivationassaysinpartitionedPetridishes[47].Briefly,aheavystreakofSVBP6wasper- formedinonesideoftheplateontoNA,anda1cm2plugofafreshM.phaseolinaculturewas depositedontotheothersideoftheplatecontainingpotatodextroseagar(PDA).Plateswere sealedwithParafilm1andincubatedfor5daysat28˚Cinthedarkness.Theassaywasper- formedbytriplicateandcontrolswithoutbacteriaorfungalinoculumwereincluded. Antibacterialactivitywasevaluatedintwoassaystoanalyzeifitwasduetoadirectcontact ofcells(co-culture)ortothesecretionofatoxinmolecule(overlaidlayer).Forthefirstassay, NAplatesweresownwith100μlofabacterialsuspensionofthepreywithaOD =1.0.Once 600 platesweredried,a10μldropofaSVBP6bacterialsuspensionwithOD =1.0wasspotted 600 oneachone,andplateswereincubated24hat28˚CfortheoptimalSVBP6growth.Forthe secondassay,a10μldropofasimilarSVBP6bacterialsuspensionwasspottedintheNAplates andincubatedovernightat28˚C.Then,after30minutesofUVexposuretokilltheSVBP6 cells,4mlofsoftNA(0.8%agar)with10%v/vofasaturatedculturesoftheconfrontedbacte- rialstrainwereoverlaidabovethedropsandplateswereincubatedfor24hattheoptimaltem- peratureofthebacterialprey.SVBP6wasconfrontedagainstEscherichiacoliK12,aBacillus subtilisstrainfromthebacterialcollectionoftheMicrobiologyAreaoftheDepartmentofSci- enceandTechnology(UniversidadNacionaldeQuilmes)andPseudomonasfluorescens1008 fromRizobacterArgentinaS.A.NAplateswiththeoverlaidlayersofE.coliorB.subtilisstrains wereincubatedat37˚C,orwithP.fluorescensstrain,at28˚C. Fattyacidsandwhole-cellsproteinprofileanalyses FattyacidanalysiswasperformedbygaschromatographyusingtheMIDISherlock1Micro- bialIdentificationSystemandthestandardprotocol[48].TheRTSBA6methodwasemployed forcomparisonoftheFAMEprofilewiththeavailablelibrary(http://www.midi-inc.com/pdf/ RTSBA_6.21%20.(Environmental%20Aerobes).pdf,asavailableinAugust2017). Whole-cellproteinprofileofSVBP6wasobtainedusingtheUltraflexIIIUV-MALDI-TOF/ TOFmassspectrometerandMALDIBiotyper3.1software(BrukerDaltonics,Bremen,Ger- many)attheCEQUIBIEMInstitute(CONICET,Argentina)inassociationwiththeInstitute ofBiotechnologyandMolecularBiology(IBBM,CONICET,LaPlata,Argentina).Theprepa- rationofthesampleswasperformedaccordingtomanufacturers’recommendationfromasin- glecolonyculturedonLBfor24hat28˚C[49].Bacterialidentificationwasperformedwith MALDIBiotyperOfflineclassificationsoftwareusingscorevaluesproposedbythemanufac- turerasfollows:ascorevaluehigherthan2.0indicatesspeciesidentification;ascorevalue between1.7and1.9indicatesgenusidentification;andascorevaluelowerthan1.7indicates notaxonomicmatching[50].ThePseudomonasspp.databaseintheMALDIBiotypersoftware included168representativespecies(S1Table). GenomicDNApreparationandsequencing ForDNAisolation,wecollected20mgfreshweightofSVBP6cellsfromanovernightculture onNA.AfterprocessingwiththeZRSoilMicrobeDNAMicroPrep™kit(ZymoResearch), weobtainedca.9μgofDNAwithanOD of1.9andagoodintegrityasjudgedby 260/280 PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 4/32 GenomeminingofP.donghuensisSVBP6 electrophoresisina0.8%agarosegel.PurifiedDNA(approx.300ng)wassenttoINDEAR (Rosario,Argentina)topreparethecorrespondinglibrariesandtoobtainadraftgenomevia theIlluminaHiSeq1500sequencingsystem. Genomeassemblyandgeneprediction GenomeassemblywasperformedwiththeA5pipeline[51].Genepredictionandgenome analysisofPGP-relatedtraitsinthedraftgenomeofSVBP6strainwereperformedwiththe RapidAnnotationwithSubsystemsTechnology(RAST)serverdatabase2.0[52],BasicLocal AlignmentSearchTool(BLAST1,[53]),Pseudomonasgenomedatabase(www.pseudomonas. com,[54]),Antibiotics&SecondaryMetaboliteAnalysisShell(antiSMASH)v.3.0.5[55]and theIslandViewer3software[56].SyntenyanalyseswereperformedwiththeSimpleSynteny platform(https://www.dveltri.com/simplesynteny/index.html,[57]). Selectionofgenomes,identificationofputativeorthologousgenes, alignments,andphylogeneticanalysis Alldraftandcompleteassemblies,andproteincodingsequencesofthegenusPseudomonas weredownloadedviaftpfromftp.ncbi.nlm.nih.gov/genomes(April19th,2017).Thisfirst datasetcomprised3545assemblies.Forty-fourhighlyconservedribosomalprotein-coding geneswereusedforgeneticcomparisonandsamplinggenomes(S2Table).All44conserved geneswereidentifiedin3162genomesandthesegenomeswereusedforfurtheranalysis (S3Table).The44groupsofputativeorthologousgeneswereindependentlyalignedusing Muscle[58]withthefastestalgorithm(options:-maxiters1-diags1).Poorlyalignedpositions wereeliminatedusingGblocksoftwarewithdefaultparameters[59],andsubsequently concatenatedforsequencedistanceestimation.Genomesdisplayinga99%ormoresimilarity intheseconservedgeneswereclustered.Onegenomeforeachclusterwasrandomlyselected forsubsequentphylogeneticanalysiswithpreferenceforthosewithanassignedspecies,if available.Thisresultingseconddatasetcomprise140selectedgenomesplustheonefromPseu- domonassp.SVBP6reportedhere(SampledGenomesinS3Table).Sixty-sevenputativeortho- logousgeneswereidentifiedamongthese141genomes.TheOrthoMCLmethod[60]was implementedintheGet_homologoussoftwareandusedforhomologousidentification[61]. Blastsearcheswereperformedwithaminimalidentityvalueof30%andminimalquerycover- ageof75%.OrthologousproteinsequenceswerealignedusingClustalOv1.2.0[62].Again, poorlyalignedpositionswereeliminatedusingGblockwithdefaultparameters[59]andthen concatenatedforphylogeneticanalysis.Aphylogenetictreewasinferredusinganapproxi- matelymaximumlikelihoodmethodwithanaminoacidLG+GmodelusinginFastTree2.1 [63].TheShimodaira-Hasegawa(SH-like)testwasusedtoevaluatebranchsupport.Thephy- logeneticpositionofSVBP6wasestablishedandallgenomesfromthemostinclusiveand robustmonophyleticgroupwereselected.Genomesfromthismonophyleticgroupwereused totrackbackallthecloselyrelatedgenomestoSVBP6strainavailableintheoriginalfirstdata- set.Asaresult,122closelyrelatedgenomesfromthefirstdatasetwereretrievedandusedfora furtherphylogeneticanalysis(CloselyrelatedgenomesinS3Table).Aspreviouslydescribed, putativeorthologousgenesamongthe122genomesplusSVBP6strainwereidentifiedusing Get_homologuespackage.Sixhundredandseventy-sixclustersoforthologousgeneswere foundandsubsequentlyalignedandtrimmedusingClustalOv1.2.0andGblock,respectively. Blockswerefinallyconcatenated,andaphylogenetictreewasbuiltbymeansofFastTree2.1, asaforementioned.ThewholeapproachisdescribedasaworkflowinS1Fig.AVenndiagram wasconstructedwithRsoftwarev.3.4.0[64],employingtheVennDiagrampackageandallthe ORFs(PEGs)foundineveryP.donghuensisgenomeasdatabase. PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 5/32 GenomeminingofP.donghuensisSVBP6 Averagenucleotideidentityscore(ANI) PairwiseTwo-wayANIscorewascomputedamongallcloselyrelatedgenomesofSVBP6 usingtheani.rbscriptdevelopedbyLuisM.Rodriguez-Randavailableatenveomics.blogspot. com.ANIscoreisaresultfromawholegenomecomparison.ANIindexisusedtodelineate speciesfromgenomessequencedata[65].Therefore,iftwogenomesdisplayanANIvalueof 95%orhigher,bothstrainsarebelievedtobelongtothesamespecies.Phylogeneticanalysis wasusedtosupportANIresults. Tn5mutagenesisandscreeningofmutantcloneswithreducedantifungal activity ToobtainaclonelibraryofrandomTn5mutantsofSVBP6,weperformedatriparentalconju- gationwithSVBP6astheacceptorstrain,EscherichiacoliCC118λpirwiththepBAMD1-4 plasmidasthedonorstrainandE.coliHB101withthepRK600plasmidasthehelperstrain,as previouslydescribed[66].Briefly,from5mlofNYBovernightculturesthatwereincubatedat 37˚C(forE.colistrains)and35˚CforSVBP6strain(toimprovethePseudomonasabilityto acceptheterologousDNA,[67]),wecombinedequalvolumesofthethreebacterialcultures, centrifugedtoobtaincellularpellets,andmixedtheminasingle1.5mltube.Weresuspended themwith50μloffreshNYBmediumandweplateditontheborderofaNAplate.Theincu- bationwasperformedat37˚Cfor5h.Cellswerecollectedwith1mloffreshNYBandappro- priatedilutionswereplatedontoM9minimalmediumagarplates[49]with0.2%ofcitrateas thesolecarbonsource,supplementedwith100μg/mlofstreptomycin[47].Weobtained approximately2500clonesthatwereconservedin384-wellplatesat-80˚Cin20%glycerol. Forselectionofputativeclonesthathadlosttheirantagonisticpotential,weperformedco-cul- tivationassaysbystreakingindividualclonesontoagarplatespreviouslyoverlaidwithasus- pensionofM.phaseolina131.2010conidia.Afterincubationfor48hat28˚C,wesearchedfor clonesthatdidnotproduceahalooffungalgrowthinhibition.ToidentifytheTn5insertion siteineachselectedclone,wecarriedoutanarbitrarynestedPCRamplificationwiththemeth- odologypreviouslydescribed[66],followedbypartialsequencingofthecorrespondingampli- consatMacrogenInc.(Seoul,Korea). RNAextractionandpurification,andNorthernblotanalysis RNApreparationfromSVBP6cellswascarriedoutessentiallyasdescribedpreviouslyforP. protegensCHA0[68],withminormodifications.Briefly,200–500μlofcellculturewascentri- fuged,andcellswereresuspendedin500μlofTKMbuffer(10mMTris-HCl,10mMKCl,5 mMMgCl ,pH7.5).Washedcellsweremixedwith75μloflysissolution(320mMsodium 2 acetateatpH4.6,8%SDS,16mMEDTA).Lysedcellsweremixedfor5minwith575μlof water-saturatedphenolat65˚C.Aftercentrifugation,thesupernatantwasextractedoncewith phenol-chloroformandprecipitatedwith3volumesofethanol.TheresultingRNApelletwas dissolvedindiethylpyrocarbonate-treatedH Oandkeptat-80˚C.RNAconcentrationwas 2 determinedat260nm.PurityandintegrityofRNApreparationswereassessedbydenaturing agaroseelectrophoresisandethidiumbromidestaining.Northernblotanalyseswereper- formedasreportedelsewhere[69].ThreemicrogramsoftotalRNAfromeachsamplewasini- tiallyelectrophoresedfor45minataconstantcurrent(15mA)inpolyacrylamidegels(8.3M urea,8%[wt/vol]acrylamide,0.2%[wt/vol]bisacrylamidein1×Tris-borate-EDTA[TBE] buffer),withthelow-rangeRNAladder(ThermoScientific,USA)servingasamolecular weightmarker.Themarkerlanewascutandstainedseparatelywithethidiumbromideand theimageregisteredwithaUVtransilluminator.Theremaininggelwaselectroblottedat150 PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 6/32 GenomeminingofP.donghuensisSVBP6 mAfor30minontoaHybond-Nmembranein1×TBEbuffer.Afterwashingthemembrane twicewith2×SSCsolution(30mMsodiumcitrate,0.3MNaCl),theRNAwascross-linkedto themembranebyexposuretoUVlightfor5min.Themembraneswerethenblockedwith prehybridizationbuffer(50%[wt/vol]formamide,5×SSC,50mMphosphatebuffer[pH7.0], 2%[wt/vol]blockingreagent,0.1%[wt/vol]N-laurylsarcosine,7%[wt/vol]sodiumdodecyl sulfate[SDS])for1hat50˚Cinahybridizationovenandthenincubatedovernightat50˚C withthehybridizationbuffercontainingthespecificanti-rsmYdigoxigenin-labeleddouble- strandedDNA(dsDNA)probe(previouslygeneratedbyamplificationofthersmYgenomic locusofP.protegensCHA0)[69].Thehybridizedmembraneswerewashedunderstandard stringentconditions,incubatedwithanalkalinephosphatase-coupledanti-digoxigeninanti- bodysolution,washedwiththesamebuffer,andcoveredwiththeLumiphoschemilumines- centreagent(Lumigen,USA)inthedarkatroomtemperaturefor5min.Themembranes wereexposedfor60mintophotographicfilmsandthenfurtherdeveloped. Resultsanddiscussion BiochemicalandmorphologicalfeaturessupporttheassignationofSVBP6 tothePseudomonasgenus SVBP6isaGram-negative,rod-shaped(between1.3–3.2μminlengthand0.5–0.9μmin width)andpolar-flagellatedbacterium(Fig1A)oftheorderPseudomonadales,belongingto theGammaproteobacteriaclass.Itisoxidase,catalaseandgelatinasepositive,andnonsporulat- ing.SVBP6cangrowbetween4˚Cand35˚Cinrichmedia,likeNA,eventhoughtheoptimal growthtemperatureis28˚C,anditbelongstother-strategist(copiotrophs)groupofmicroor- ganisms[8].After48honnutrientagar(NA)plates,SVBP6coloniesarewhiteopaque,rough (non-mucoid)andhaveirregularborders(Fig1B).Besides,thisstraincangrowinapHrange from6.0to10.0andinasalinityrangefrom0%to5%NaClinNYBmedium.SVBP6can reducenitratebutnotnitrite,soitcouldnotparticipateinthesoildenitrificationprocess.Its respirationisstrictlyaerobicanditdoesnotproduceanyfluorescentpigment,neitherpyocya- ninorpyoverdine,inKing’sAorBorGould’sS1media[70].Finally,SVBP6strainisnaturally resistanttochloramphenicol(Cm,20μg/ml),ampicillin(Amp,100μg/ml)andgentamicin (Gm,10μg/ml)inbothliquidNYBandsolidNAmedia.Fivecopiesofbeta-lactamasepro- teins,auniquecopyofanaminoglycoside6’-N-acetyltransferase,whichcouldberesponsible fortheGmresistance[71],andtheeffluxoperonTtgABC,whichgiveCmresistanceinP. putidaKT2440[72],werefoundintheSVBP6genome.Nevertheless,thesegeneswerenot associatedtomobilegeneticelementsandusuallyfoundinenvironmentalisolates[73–75]. Fig1.MorphologicalfeaturesofSVBP6strain.SVBP6wasisolatedfromabulksoilsample(0–10cmdepth)ofan agriculturalplotlocatedinViale,EntreRiosprovince,Argentine(31˚52’59,6”S;59˚40’07”W).Morphological features,i.e.bacillarstructureandpolarflagella,ofP.donghuensisSVBP6seenwithTEM(A).Colonymorphologyona NAplateafter48hofgrowthat28˚C(B). https://doi.org/10.1371/journal.pone.0194088.g001 PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 7/32 GenomeminingofP.donghuensisSVBP6 Besides,knowledgeaboutnaturalantibiotics’resistanceallowedustocorrectlyselectthemuta- genesistoolsforthemolecularbiologystudiesontheSVBP6metabolism,asweconfirmedwe couldemployplasmidsmarkedwithtetracyclineandkanamycinresistancegenesintheTn5 approach. Withregardstocarbonsourceutilization,SVBP6wasabletorespirethefollowingsub- strates:pyruvicacidmethylester,Tween40,Tween80,glycogen,D-xylose,D-mannitol,N- acetyl-D-glucosamine,D-galactonicacidγ-lactone,4-hydroxybenzoicacid,itaconicacid,D- malicacid,L-arginine,L-asparagine,L-serine,L-threonine,glycyl-L-glutamicacidandputres- cine.GrowthonL-threonineandD-mannitolcandifferentiateP.donghuesisspeciesfromthe recentlydescribeP.wadenswilerensissp.nov.[24,26].UnlikeHYSstrain,SVBP6canrespire itaconicacidafter108hofincubation[24].ThegroupofcompoundsthatSVBP6canrespire containsrepresentativesfromalltestedguilds:polymers,carbohydrates,carboxylicoracetic acids,aminesandaminoacids.Therefore,thisstrainseemstohavethepotentialtoinhabitthe rhizosphereofmultipleplants,asthosetypesofcompoundsaregenerallyexudedbyroots fromdifferentspecies[76–79]. MIDIandMALDI-TOFanalysesdidnotallowidentifyingSVBP6atthe Pseudomonasspecieslevel Inourpreviousstudies,phylogeneticanalysesplacedisolateSVBP6withintheP.putida complex.Partialsequencingof16SrDNA,oprFandrpoBgenespositionedSVBP6closeto isolatesP.vranovensisT-16andP.alkylphenolicaKL28,butwithsimilaritypercentageslower than96%[6].Maldi-TOFBiotyperanalysisgaveIDscorevaluesranging1.75–1.84(n=4) withtheclosestmatchbeingP.graminisDSM11363T(amemberoftheP.luteagroup)[80]; however,suchi.d.scoresonlycorrespondtoidentificationatthelevelofgenus.Besides, MIDIanalysisdidnotfindanymatchbetweenthefattyacidprofileofSVBP6andthose fromtheSherlocklibraries.Therefore,wecomparedtheSVBP6profilewiththosereported forP.donghuensisHYS,P.putidaATCC12633,P.vranovensisDSM16006[24]andP.alkyl- phenolicaKL28[25],andwefoundthetypicalfattyacidsassociatedwiththePseudomonas genuslikeC 3-OH,C andC 2-OH[13],aswellasseveraluniquefattyacidspecies 10:0 12:0 12:0 foreachstrain(S4Table).Particularly,SVBP6straincontainsauniquefattyacid,C13:1Δ12, thatwasnotpreviouslydescribedforpseudomonads,butonlyforVibrionaceae andClostrid- iumspecies[81,82]. Genomesequencinginformationandgeneannotation Inparallelwiththebiochemicalcharacterization,wedecidedtosequencethegenomeof SVBP6strainwiththeaimsof:1)gettingadditionalsupportforthetaxonomicpositioningof thisisolateatthespecieslevel;2)decipheringthemoleculardeterminantsofthestrongand broadspectrumantifungaltraitofthisbacterium.Withtheprotocolpreviouslydescribed,we sequenced93%ofthegenomebasedonthedatafromthetypestrainP.donghuensisHYS[24]. TheaverageGCcontentwas62.4%matchingvaluesdescribedforpseudomonads(58%-69%, [13]).Atotalof7,136,473readswereassembleddenovointo40scaffolds,withameanscaffold sizeof142534bases(N scaffoldlengthof288994bases).MinimumInformationaboutthe 50 GenomeSequence[83]ofstrainSVBP6issummarizedinTable1.WeconfirmedthatSVBP6 doesnotcontainanyplasmid. RASTanalysispredicted5253genesintotal:5179proteinencodinggenes(PEGs)and74 RNAs,ofwhich65aretRNAs.54.0%ofPEGs(2757)werearrangedinto527subsystems,and atotalof1125(21.72%)wereassignedashypotheticalproteins.RASTanalysisestimateda numberof90missinggenes,i.e.undercalledPEGsintheremaininggapsbetweenfeatures. PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 8/32 GenomeminingofP.donghuensisSVBP6 Table1. ClassificationandminimuminformationaboutthegenomesequenceofP.donghuensisstrainSVBP6. MIGSID Property Term Evidencecode1[84] Generalfeatures Classification Domain:Bacteria TAS Phylum:Proteobacteria TAS Class:Gammaproteobacteria TAS Order:Pseudomonadales TAS Family:Pseudomonadaceae TAS Genus:Pseudomonas IDA Species:Pseudomonasdonghuensis IDA strain:SVBP6 TAS Gramstain negative IDA Cellshape Rod IDA Motility Motile IDA Sporulation None IDA Temperaturerange Mesophilic(4–35˚C) IDA Optimumtemperature 28˚C IDA pHrange 6–10 IDA Carbonsource Heterotrophic IDA MIGS-22 Oxygenrequirement Aerobic IDA MIGS-14 Pathogenicity Unknown NAS MIGS-6 Habitat Soil TAS MIGS-5 Samplecollection February2010 TAS MIGS-15 Bioticrelationship free-living/rhizospheric NAS MIGS-4 Geographiclocation Viale,EntreR´ıosprovince TAS MIGS-4.1 Latitude S31˚52’ IDA MIGS-4.2 Longitude W59˚41’ IDA MIGS-4.3 Depth 0–10cmlayer TAS MIGS-4.4 Altitude 80mabovesealevel TAS MIGS-23.1 Isolation Bulksoilfromanagriculturalplot TAS Projectinformation MIGS-31 Finishingquality High-qualitydraft MIGS-31.2 Foldcoverage 125× MIGS-28 Librariesused Illuminapaired-endlibrary(Nextera1XT) MIGS-29 Sequencingplatforms Sanger/Illumina1.9(Illumina1500HiSeq) MIGS-30 Assemblers A5pipeline(Phred) MIGS-32 Genecallingmethod RASTv.2.0 BioProjectID PRJNA407750 GenBankaccessionnumber NWCB00000000 GenBankDateofRelease December1st,2017 Projectrelevance Biocontrol,Plantgrowthpromotion MIGS-13 Sourcematerialidentifier SVBP6 1Evidencecodes—IDAInferredfromDirectAssay,TASTraceableAuthorStatement(i.e.,adirectreportexistsintheliterature),NASNon-traceableAuthorStatement (i.e.,notdirectlyobservedfortheliving,isolatedsamplebutbasedonagenerallyacceptedpropertyforthespeciesoranecdotalevidence).Theseevidencecodesare fromtheGeneOntologyproject. https://doi.org/10.1371/journal.pone.0194088.t001 GenomepropertiesandstatisticsaresummarizedinTable2.Geneswithsignalpeptideswere detectedwiththeSignalP4.1Server[85],andtheCRISPRrepeats,withtheCRISPRreconig- tiontoolv1.0[86].DetailsofclustersoforthologsgroupsareshowninS5Table. PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 9/32 GenomeminingofP.donghuensisSVBP6 Table2. Genomestatistics. Attribute Value Percentage Genomesize(bp) 5,701,342 100 DNAcoding(bp) 5,005,841 87.8 DNAG+C(bp) 3,557,637 62.4 DNAscaffolds 40 - Totalgenes 5253 100 Proteinencodinggenes(PEGs) 5179 98.6 RNAgenes 74 1.4 Genesininternalclusters 118 2.2 Geneswithfunctionprediction 4054 78.3 GenesassignedtoCOGs 4865 92.6 GeneswithPfamdomainsa 4450 85.9 Geneswithsignalpeptides 512 9.9 Geneswithtransmembranehelicesb 1170 22.6 CRISPRrepeats 0 0 aDetectedwithPfamv.31.0(http://pfam.xfam.org) bDetectedwithTMHMMServerv.2.0(http://www.cbs.dtu.dk/services/TMHMM/) https://doi.org/10.1371/journal.pone.0194088.t002 PhylogeneticidentificationwithANIanalysisshowedthatstrainSVBP6 belongstotherecentlydescribedspeciesPseudomonasdonghuensis Onehundredandfortygenomeswereselectedfromafirstdatasetasrepresentativesofthe geneticdiversityofthegenusPseudomonas(seeMethods).Thephylogeneticpositionof SVBP6wasestablishedamongthesegenomes(S2Fig).Despiteminordifferences,theobtained phylogenetictreewasingoodagreementwithpreviousonesreportedforthegenus[27].Pseu- domonassp.SVBP6wasclearlypositionedwithintheP.putidacomplex.Onehundredand twenty-twocloselyrelatedgenomesofSVBP6wereselectedandincludedinasecondphyloge- neticanalysis.SVBP6belongstoabasallineagewithintheP.putidacomplex(Fig2A),which includesatleast12differentspecies,accordingtotheestimatedANIscore(S6Table).SVBP6 wascloselyrelatedtoPseudomonassp.482andP.donghuensisHYS(Fig2AandS6Table).The siderophoreproducingisolateHYS,whichservedtodefinethetypespeciesP.donghuensis, wasisolatedfromthewaterofEastLakeofWuhan,China[24],whereasthebactericidalstrain Pseudomonassp.P482wasisolatedfromtherhizosphereofagarden-cultivatedtomatoin Gdynia,Poland[87].ANIscoresupportsthephylogeneticresultandsuggeststhatthesethree strainsbelongtothesamespecies,asSVBP6showedsimilarityvaluesof99.60%±0.70and 99.52%±0.99withPseudomonassp.P482andP.donghuensisHYS,respectively[24,88].This resultwassupportedbyaMulti-LocusPhylogeneticAnalysis(MLPA)performedwith concatenated16srDNA,gyrB,rpoBandrpoDsequencesobtainedfromdifferentgenome sequencingprojectsofreferencestrains[21,27].WhenwecomparedORFsfromthethreeP. donghuensisrepresentative’sgenomesbyahomologousclusteranalysis(minimum50%iden- tity,75%coverage),wefoundthatSVBP6sharesahighernumberofORFswithstrainP482 thanwithstrainHYS(Fig2B).Fromthe284ORFsfoundonlyinP.donghuensisSVBP6(5.6% ofthetotal),wecoulddetectseveralmobilegeneticelements(9.9%),membranecomponents (8.2%),putativesecretedelements(3.4%),genesinvolvedinregulationand/ormetabolism (23.7%)andanimportantnumberofhypotheticalproteins(54.7%). PLOSONE|https://doi.org/10.1371/journal.pone.0194088 March14,2018 10/32
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