Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation

32 Pages·2017·13.61 MB·English

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
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InvestigacionesCient´ıficasyTe´cnicas(CONICET),BuenosAires,Argentina,3 LaboratoriodeBiolog´ıa
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Computacional,DepartamentodeDesarrolloBiotecnolo´gico,InstitutodeHigiene,FacultaddeMedicina,
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UniversidaddelaRepu´blica,Montevideo,Uruguay
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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].
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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
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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
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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.
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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
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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
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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.
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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.
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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%).
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