FEMSMicrobiologyLetters,365,2018,fny069 doi:10.1093/femsle/fny069 AdvanceAccessPublicationDate:22March2018 ResearchLetter RESEARCH LETTER –FoodMicrobiology Community-led comparative genomic and phenotypic Pseudomonas analysis of the aquaculture pathogen baetica a390T sequenced by Ion semiconductor and Nanopore technologies Ainsley Beaton1,†, Ce´dric Lood2,3,†, Edward Cunningham-Oakes4,†, Alison MacFadyen5,†, Alex J. Mullins4,†, Walid El Bestawy1, Joa˜o Botelho6, Sylvie Chevalier7, Shannon Coleman8,‡, Chloe Dalzell1, Stephen K. Dolan9, Alberto Faccenda1, Maarten G. K. Ghequire2, Steven Higgins10, Alexander Kutschera11, Jordan Murray1, Martha Redway1, Talal Salih1, Ana C. da Silva12,#, Brian A. Smith13, Nathan Smits3, Ryan Thomson1, Stuart Woodcock14, Martin Welch9,§, Pierre Cornelis7, Rob Lavigne3, Vera van Noort2 and Nicholas P. Tucker1,∗,¶ 1StrathclydeInstituteofPharmacyandBiomedicalScience,UniversityofStrathclyde,161CathedralStreet, Glasgow,G40RE,UK,2CentreofMicrobialandPlantGenetics,KULeuven,KasteelparkArenberg20,bus2460, LeuvenB-3001,Belgium,3LaboratoryofGeneTechnology,KULeuven,KasteelparkArenberg20,bus2460, LeuvenB-3001,Belgium,4CardiffSchoolofBiosciences,CardiffUniversity,SirMartinEvansBuilding,Park Place,CardiffCF103AX,UK,5Royal(Dick)SchoolofVeterinaryStudies,UniversityofEdinburgh,EasterBush Campus,MidlothianEH259RG,Scotland,UK,6UCIBIO/REQUIMTE,Laborato´riodeMicrobiologia,Faculdadede Farma´cia,UniversidadedoPorto,RuadeJorgeViterboFerreirano.228Porto4050-313,Portugal,7Laboratoire MicrobiologieSignauxetMicroenvironnement(LMSM),Universite´ deRouen,55,rueStGermain,Evreux27000, France,8LowerMallResearchStation,UniversityofBritishColumbia,2259LowerMall,Vancouver,BCV6T1Z4, Canada,9DepartmentofBiochemistry,UniversityofCambridge,HopkinsBuilding,TennisCourtRoad, CambridgeCB21QW,UK,10DepartmentofPlantandMicrobialBiology,UniversityofZu¨rich,Zu¨rich8008, Switzerland,11DepartmentofPhytopathology,CenterofLifeandFoodSciences,TechnicalUniversityof Munich,WeihenstephanD-85354,Germany,12CentreforBiomolecularSciences,SchoolofLifeSciences, UniversityofNottingham,NottinghamNG72RD,UK,13SchoolofPlantSciences,TheUniversityofArizona, P.O.Box210036,ForbesBuilding,303Tucson,Arizona85721-0036,USAand14DepartmentofBiological Chemistry,JohnInnesCentre,ColneyLane,NorwichNR47UH,UK ∗Correspondingauthor:StrathclydeInstituteofPharmacyandBiomedicalScience,UniversityofStrathclyde,161CathedralStreet,GlasgowG40RE,UK. Tel:0141-548-2861;E-mail:[email protected] Received:5January2018;Accepted:21March2018 (cid:3)C FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the orig- inalworkisproperlycited. 1 Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 2 FEMSMicrobiologyLetters,2018,Vol.365,No.9 †TheseauthorscontributedequallytothesequencingortointensivewholegenomedataanalysisofP.baetica.ABgeneratedsemiconductorshortreads whilstCLproducedNanoporelongreads.ECOandAMperformedthephylogeneticanalysiswhilstAMacperformedwholegenomeBLASTanalysisand editing. Onesentencesummary:Here,wepresentthehigh-qualitydraftgenomeofPseudomonasbaetica,abacterialpathogenthathasbeendemonstratedto causediseaseinfishcultivatedinaquaculturefacilities. Editor:RobertJackson ‡ShannonColeman,http://orcid.org/0000-0001-9495-6772 #AnaC.daSilva,http://orcid.org/0000-0002-2967-0986 §MartinWelch,http://orcid.org/0000-0003-3646-1733 ¶NicholasP.Tucker,https://orcid.org/0000-0002-6331-3704 ABSTRACT Pseudomonasbaeticastraina390Tisthetypestrainofthisrecentlydescribedspeciesandherewepresentitshigh-contiguity draftgenome.Tocelebratethe16thInternationalConferenceonPseudomonas,thegenomeofP.baeticastraina390Twas sequencedusingauniquecombinationofIonTorrentsemiconductorandOxfordNanoporemethodsaspartofa collaborativecommunity-ledproject.Theuseofhigh-qualityIonTorrentsequenceswithlongNanoporereadsgaverapid, high-contiguityand-quality,16-contiggenomesequence.WholegenomephylogeneticanalysisplacesP.baeticawithinthe P.koreensiscladeoftheP.fluorescensgroup.ComparisonofthemaingenomicfeaturesofP.baeticawithavarietyofother Pseudomonasspp.suggeststhatitisahighlyadaptableorganism,typicalofthegenus.Thisstrainwasoriginallyisolated fromtheliverofadiseasedwedgesolefish,andgenotypicandphenotypicanalysesshowthatitistoleranttoosmotic stressandtooxytetracycline. Keywords:Pseudomonas;aquaculture;wholegenomesequencing;OxfordNanoporeMinIon;IonTorrent;Pseudomonasbaetica INTRODUCTION ThisanalysisrevealedthatP.baeticaisamemberoftheP.fluo- InSeptember2017,thebiannualconferenceonthebiologyof rescensgroupandisparticularlycloselyrelatedtoP.koreensis.Al- Pseudomonas was held in Liverpool, UK. All aspects of Pseu- thoughinfectedwedgesoleshowednoobviousvisualsignsofP. domonas biology were reported at the meeting from clinical baeticainfection,intraperitonealinjectionandimmersioninfec- toenvironmentalmicrobiology.ThevastmajorityofthePseu- tionassaysyielded100%and10%mortalityrates,respectively domonasliteraturefocusesonjustafeworganisms,mostnotably (Lo´pezetal.2012).Subsequentinfectionassayswithavariety Pseudomonasaeruginosa,P.fluorescens,P.putidaandP.syringae,al- ofotherfishspeciesdemonstratedthatP.baeticacausedhigher thoughthegenusisfarmorediversethanthis.Inordertocon- mortalityinwedgesolethanotherspeciesincludingseabass tributetoaddressingthisimbalance,thegenomeofP.baetica, andseabream,andthatthereisatemperature-dependentef- arecentlydescribedmemberofthegenus,wassequencedfor fectonvirulence(Lo´pezetal.2017).Inordertorapidlyidentify thePseudomonas2017genomicsforum.Theresultinggenome P.baeticacontaminationinanaquaculturesetting,Lo´pezetal. sequencehasbeenanalysedbymembersofthePseudomonas (2017)havedevelopedimmunologicalandmoleculardetection 2017communityfromacrosstheglobeusingSlackasacollabo- assays. rationtool(Perkel2016). AlthoughP.baeticawasoriginallydescribedfromanaquacul- Pseudomonasbaeticastraina390isoneoffiveGram-negative tureinfectionscenario,anumberofrecentstudieshaveidenti- organismsthatwereisolatedfromafishdiseaseoutbreakinan fiedthisspeciesandcloserelativessuchasthenewlydescribed aquaculturefacilityinHuelva,Spain,in2006(Lo´pezetal.2012). P. helmanticensis and P. granadensis in diverse environments in Theinfectedfishwerewedgesole(Dicologlossacuneata),aflatfish therhizosphereincludingbeanrootsinIran,andforestsoilsin thatiswidelyconsumedinAndaluc´ıaandpartsofFrancewhere Spain(Ram´ırez-Bahenaetal.2014;Pascualetal.2015;Keshavarz- there is increasing interest in sustainable aquaculture of this Tohidetal.2017).ThissuggeststhatP.baeticaismorelikelyto species.Aquaculturenotonlyprovidesasustainablesourceof beanopportunisticpathogenoffishasopposedtoagenuine protein,butitisalsoaneconomicallyimportantindustryaround marinepathogen.Wehypothesisedthatthedraftgenomese- theworld.Europeancommissiondatarevealsthat1.25million quenceofP.baeticawouldrevealthegeneticbasisforthetraits tonnesoffoodareproducedbyaquacultureintheEUeveryyear requiredtocausediseaseinwedgesole.Theaimofthisstudy andthatover85000peoplearedirectlyemployedintheindus- istoprovidegenomicinsightsintothebiologyofP.baeticaand try.Adisadvantagetoaquaculturesystemsisthattheunusually tousethisgenomeasareflectiononthediverseinterestsofthe highpopulationdensityofthefarmedfishleadstoanelevated Pseudomonascommunity. riskofinfectiousdiseaseoutbreaks.Forthisreason,antibiotics suchasoxytetracycline(OT)areroutinelyusedasgrowthpro- MATERIALSANDMETHODS motersinaquaculturefishfoodpellets(Lealetal.2017).Other membersofthePseudomonadaceaearewellknowntocausedis- Bacterialstrainsandgenomesequenceaccessions easeinaquaculturescenarios,mostnotablyP.anguillisepticaisa pathogenofbothfarmedandwildeels(Johetal.2013). Pseudomonas baetica a390T originally isolated from the liver of Pseudomonasbaeticawasformallydescribedin2012usinga wedgesolefish,D.cuneate,anddescribedbyLopezetal.(2012), combinationof16SrRNAsequencingandphenotypicanalyses, was obtained from DSMZ (DSM no. 26532) and is considered allowingabiochemicalprofiletobegenerated(Lo´pezetal.2012). to be the type strain. Unless stated otherwise, all phenotypic Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 Beatonetal. 3 comparisonswereperformedagainstP.fluorescensPF01,P.putida searchedforprophagesusingPHASTER(Arndtetal.2016),and KT2440 and P. aeruginosa PA14. This Whole Genome Shotgun genomicislandsusingtheIslandViewerandantiSMASH(Bertelli project has been deposited at DDBJ/ENA/GenBank under the etal.2017;Blinetal.2017).Forthelatter,thereferencegenome accession PKLC00000000. The version described in this paper used for the alignment was that of P. koreensis strain D26 as isversionPKLC01000000.Rawsequencingreadswerealsode- it was deemed phylogenetically close to P. baetica. CCT maps positedandcanbefoundwithaccessionnumbersSRR6792524 werecreatedusingCCTversion1.0usingdefaultsettingswith andSRR6792523fortheNanoporeandIonTorrentreads,respec- the exception of the following commands; -t –cct –custom tively. scaleblast=F cctblastthickness=60 backboneThickness=10 tickThickness=15 rulerFontSize=100 featureThickness=150 DNAextraction,librarypreparationandIonTorrent tickLength=30legend=Fdetails=F.Afeaturesfilewasusedfor the‘beigeblocks’,anda‘labelstoshow’filewasusedtolabel PGMSequencing theCCTmap(Grant,ArantesandStothard2012). DNAextractionwascarriedoutusingISOLATEIIgenomicDNA kit(BIOLINE)usingthestandardprotocolforbacterialcells.Frag- Phylogeneticanalysis mentation was carried out using the standard protocol from NEBNextFastDNAFragmentation&LibraryPrepSetforIonTor- TreeswereassembledusingMashv1.1.1(Ondovetal.2016)fast rentandadapter-ligatedDNAwaspreparedusingbarcodeset6. genomeandmetagenomedistanceestimationusingtheMin- SizeselectionwascarriedoutusingtheE-gelibase(Invitrogen), Hashalgorithm,andvisualisedinFigTreev1.4.3(Rambaut2012, andanalysedusingthe2100Bioanalyzer(AgilentGenomics).A also available via GitHub). Reference genomes were obtained fragmentsizeof424bpwascarriedforward.Templateampli- fromNCBI.Averagenucleotideidentity(ANI)wasperformedus- fication and enrichment were carried out using the standard ingPyANI(Guptaetal.2016).DatawereprocessedusingCLIMB IonTorrentprotocolusingtheIonOneTouch2system(Thermo- (Connoretal.2016). Fischer).ThestandardsequencingprotocolforIonTorrentwas usedandDNAlibrarywasloadedontoanIon316chipv2withan Azocaseinassay ISPloadingpercentageof87%. OvernightculturesofP.aeruginosaUCBPP-PA14,P.baeticaa390T, Nanoporesequencing P. fluorescens Pf0-1 and P. putida KT2440 were centrifuged at 12000×gfor5minand100μlofsupernatantwaspipettedinto LongreadsbyNanoporesequencingwereobtainedusingaMin- centrifugetubescontaining400μlofreactionmixture;200μlof ION sequencer from Oxford Nanopore Technology (ONT) with 2%azocaseinin0.5%bicarbonatesodiumbufferand200μlof thegoaltoimprovethegenomeassemblyofP.baetica.Thestrain 0.5%sodiumbicarbonatebuffer.Thereactionmixturewasthen wasgrowninLBmediumandgrownat30◦CtoanOD600of0.850 incubatedat30◦Cfor10,70and140min. anditsgenomicDNAwasisolatedusingtheMoBioDNAeasy Followingincubation,0.5mlof10%trichloroaceticacidwas UltraCleanMicrobialkit.QualityofthegDNAwascheckedus- addedtoterminatethereaction.Tubeswerethenvortexedfor5 ingaNanoDropandanagarosegelforintegrity.ThegDNAwas minfollowedbycentrifugingfor5minat4◦Cand10000×g.Five shearedmechanicallytoanaveragefragmentsizeof10kbpus- hundredmicrolitreofsupernatantwasthenaddedtocuvettes ingaCovarisgTube.Thesequencinglibrarywaspreparedusing containing500μlof0.5MNaOH,andabsorbancewasmeasured the1DligationprotocolfromONTwithnativebarcodingofthe at440nminanUltrospec3100prospectrophotometer.Allsam- sample.TheresultwassequencedonaR9.4flowcellforaperiod pleswererunintriplicate. of8h. Skimmilkassay CombinedGenomeAssemblyofIonPGMand NanoporeReads FivemicrolitreofovernightculturesofP.aeruginosaUCBPP-PA14, P.baeticaa390T,P.fluorescensPf0-1andP.putidaKT2440werein- TheIonPGMreadswerecheckedforqualityusingFastQCand oculatedontoMOPSminimalmediaagarplates,supplemented processedwithBBDukinordertoremovepotentialadaptercon- tamination,sizeexclusion(50>readlength<250),andfortrim- withdehydratedskimmedmilkandincubatedfor72hat30◦C. ming (phred score < 28) (Andrews 2014). Basecalling of the Photographsweretakenat24-htimeintervals.Ironsupplement, NanoporereadswasperformedwiththesoftwareAlbacorev2.1.3 FeCl2,wasnotincludedinthe10×stock,and5g/Lofagarand skimmilkwereaddedinordertoobserveclearhaloformation. fromONT,andthebarcodeswereremovedusingPorechop.Read lengthdistributionandqualityoftheNanoporereadswasas- sessedusingNanoPlot. OTresistanceassay ThegenomewasfirstassembledwithSPAdes,version3.11.1 usingdefaultparameters(Bankevichetal.2012)usingIonPGM Pseudomonasbaeticawastestedforantibioticsensitivitybyagar readsonly,thenwithUnicycler,versionv0.4.3(Wicketal.2017) dilutionaccordingtotheEuropeanCommitteeforAntimicrobial alsousingdefaultparameterswithbothIonPGMandNanopore SusceptibilityTestingguidelines(EuropeanCommitteeforAn- reads.Forthelatter,thegenomewaspolishedwithRacon(Vaser timicrobialSusceptibilityTesting(EUCAST)oftheEuropeanSo- etal.2017)andPilon(Walkeretal.2014).Thequalityofbothas- cietyofClinicalMicrobiologyandInfectiousDiseases(ESCMID) semblieswasassessedwithQUAST(Gurevichetal.2013). 2000).ResistancetoOTwastestedduetoitsfrequentuseasa growthpromoterinaquaculture(Lealetal.2017).Mueller-Hinton agar plates with final concentrations ranging from 0.125 to Genomicislandpredictionandgenomevisualisation 128μgmL−1wereinoculatedwithP.baeticaa390Tsuspensions The Ion PGM and the hybrid assemblies were annotated with dilutedin0.85%NaCltoaturbidityequivalentofa0.5McFarland Prokka (Seemann 2014) using a custom protein database built standard.Plateswereincubatedovernightat30oC.Pseudomonas from the Pseudomonas genus. The annotations were then aeruginosaPAO1wasusedasapositivecontrol. Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 4 FEMSMicrobiologyLetters,2018,Vol.365,No.9 Table 1. Assessment of genome assembly quality for both ap- (Bravo,LozanoandHandelsman2017).ANIwasusedtocompare proachesrevealsavastimprovementintheassemblydespitealow thewholegenomesequenceofP.baeticaagainstthesixavailable nanoporesequencingcoverage(9X). referencegenomesforP.koreensisaswellasstrainIrchel3E19, andconfirmsthatitismostcloselyrelatedtoIrchel3E19and Metric IonPGMonly Hybrid CI12, with an ANI of 98.02% and 89.6% similarity,respectively Assemblylength 6602908 6773804 (Fig.1A)(Arahal2014).WealsousedJSpeciesWSasanindepen- No.ofcontigs(>1000bp) 338 16 dentANImethodandthisanalysisagreedwiththosedescribed N50 36090 973739 above(seesupplementaryANItables)(Richteretal.2016).Finally, N75 19947 745006 BLASTwasusedtocomparethegyrBandrpoDgenessequences Longestcontig 122046 1654292 ofP.baeticaa390TandCI12againstavailablepartialrpoDandgyrB sequencesavailableforP.baetica(Lo´pezetal.2012).Thisshowed a93.6%similarityinrpoDand95.0%similarityingyrBbetween CI12andthewholegenome-sequencedP.baeticaa390T. RESULTS Sequencingresultsandassembly Genomicislands,biosyntheticgeneclustersand prophagesdetectedintheP.baeticagenome TheinitialIonTorrentsequencingproducedover3.5millionbar- codedreadswithameanreadlengthof299bp.SPAdesassembly Genomic islands can be readily detected when long contigu- resultedinfinalcontignumberof338.TheNanoporesequenc- ous regions are assembled. As such, searching for prophages ingrunyieldedaround22000reads,ofwhich7771passedqual- inthehighlyfragmented,ionPGM-onlyassemblydidnotyield ity control during the basecalling process. The average phred anyresults,whereasinspectionofthehybridassemblyshowed quality score of the reads is 11 and their length distribution four intact prophages in the genome of P. baetica, as well as wasaround9kbp(seeFigureS1,SupportingInformation).With five incomplete ones (Table 2a). Two integrative conjugative agenomesizeofabout6.6Mbp,estimatedfromaninitialIon elements were also detected. A combination of IslandViewer PGMassembly,weinferagenomecoverageofabout9×forthe andantiSMASHwerealsousedtopredictgenomicislandsand Nanoporesequencingdata(60Mbp),and80×withIonPGMdata the secondary metabolite biosynthetic potential of P. baetica (540MbpafterQC). (Bertelli et al. 2017; Blin et al. 2017). These results are sum- Thehybridapproachthatcombinesboththedatasetofshort marisedontheCGViewComparisonToolmapinFig.2,anda IonPGMreadswiththelongNanoporereadssignificantlyim- moredetailedsummaryoftheantiSMASHresultsisprovidedin provedthegenomeassemblyoverthatusingIonPGMreadsonly Table2b(Grant,ArantesandStothard2012).Manyofthelocire- giving16contigsover1000 bp(Table1).Quastanalysisofthe ferred to throughout this manuscript are labelled around the combinedassemblygivesanN50valueof973739whichishigher CCT map in Fig. 2 as well as the results of IslandViewer. The than 96% of all draft genomes in The Pseudomonas Genome CCT BLAST analysis confirms the close phylogenetic relation- database. shipbetweenP.baeticaa390andIrchel3E19(Fig.2).Ofthenine TheIonPGM-onlyandthehybridassemblygraphswerealso biosyntheticgeneclusters(BGCs)predictedbyantiSMASH,four inspectedvisuallyusingBandage(Wicketal.2015)inordertoas- areNrps(non-ribosomalpeptidesynthetases)biosyntheticgene sessthefragmentationofthegenomeassembliesandthege- clusters.Clusters3and8hadrelativelylowsimilaritiestothe nomic organisation, including the presence of potential plas- nearestknownclusters;however,cluster6sharedahighsim- mids(seeFiguresS2andS3,SupportingInformation).Aftergene ilaritywithanorfamideBGCwith80%ofgenesinthiscluster annotationwithProkka,plasmid-associatedgeneslikerecombi- sharinghomologywiththeputisolvinbiosurfactantBGC.Puti- nases,transposasesandintegrasesweresearchedforandwere solvin has been implicated in motility (Ca´rcamo-Oyarce et al. notpresentorwerenotindicativeofbeingplasmidassociated. 2015)andourpreliminaryexperimentshavedemonstratedthat Thehighcontinuityassemblyprovidedbythecombinatorialap- P.baeticaiscapableofbothswimmingandswarming(seeFig- proachofIonTorrentandNanoporetechnologiesallowedfora ureS4,SupportingInformation).Sinceputisolvinbiosynthesisin comprehensiveanalysisofthegenome. certainstrainsofP.putidaisknowntobeaquorumsensing(QS)- regulatedphenotype,wesearchedforhomologuesofknownQS PhylogeneticplacementandANIofP.baeticaa390T systemsintheP.baeticagenome(Dubernetal.2008).Nocon- vincinghomologuesoftheLasRorRhlRregulatedsystemsfrom We have produced a phylogeny to place the sequenced P. P.aeruginosa,orthePpuI,PpuR,PpuA,RsaLsystemfromP.putida baetica a390T genome against reference genomes from 19 werefound. speciesclades,comprisingtheentiregenusPseudomonas(Fig.1B) (Gomilaetal.2015).ThiswasdoneusingMash,apipelinethat Bacteriocins,tailocinsandmodularbacteriocinsofP. convertsagenomeintoasketch(orgroupsofk-mers).Sketches baetica arethencomparedtoproduceaJaccardindex,whichisgener- atedbaseduponsharedk-mersbetweengenomes(Ondovetal. Pseudomonads are capable of producing a variety of 2016). Our phylogeny places P. baetica amongst the P. koreensis antagonism-mediating peptides and proteins. A subset of subclade,aspreviouslyshowninphylogeniesconstructedusing thesemoleculesarebacteriocins,andassistintheelimination partial23SrRNAP.koreensisgenesequences.AP.koreensisphy- of phylogenetically related competitors (Ghequire and de logenyconstructedinthesamemannerplacesP.baeticaclos- Mot 2014). Historically, these compounds have been mainly esttoPseudomonassp.Irchel3E19,astrainisolatedfromapond studied in P. aeruginosa, there termed pyocins (Michel-Briand inZurich,Switzerland,suggestingthatthisisolateisinfacta and Baysse 2002), but they have been equally detected and strainofP.baetica(Butaite˙ etal.2017).Thenextmostcloselyre- characterizedinanumberofotherPseudomonasspecies.Evolu- latedorganismisP.koreensisstrainCI12,whichwasrecentlyco- tionarilyrelatedtobacteriophagetails,tailocinsarefunctional isolated with Bacillus cereus from a soybean plant rhizosphere standaloneunits—ratherthandefectiveprophages—lackingan Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 Beatonetal. 5 Figure1.PlacementofnovelbaeticagenomeinthegenusPseudomonasbaseduponANI,andsharedk-mersA)ANIheatmapgeneratedthePython3modulepyani. Thesequencedbaeticagenome,Pseudomonassp.Irchel3E19andreferencegenomesfromthecloselyrelatedkoreensissubcladeweresubjecttoANIanalysis.B)A Mash-basedtreegeneratedfromreferencegenomes(n=86)from19speciesclades,comprisingtheentiregenusPseudomonas.Thistreewasgeneratedbasedupon theJaccardindex,calculatedfromsharedk-mers.CellvibriojaponicumUeda107wasusedasoutgroup. Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 6 FEMSMicrobiologyLetters,2018,Vol.365,No.9 Table2a.Listofprophageregionsdetectedinthehybridassembly. Contigno. Length Completeness Score No.ofproteins Regionposition Mostcommonphage GC% 1 6.7Kbp Questionable 70 6 681684–688458 PHAGEStx2c1717NC011357(3) 53.77% 2 6.5Kbp Incomplete 40 10 512098–518615 PHAGEEnterophi92NC023693(2) 60.11% 2 10.2Kbp Incomplete 30 8 742363–752628 PHAGEClostrphiCT453ANC028991(3) 56.63% 2 5.5Kbp Incomplete 60 6 925750–931322 PHAGEStx2c1717NC011357(3) 54.15% 3 40.7Kbp Intact 150 48 383269–424017 PHAGEVibrioVP882NC009016(12) 58.74% 3 57.2Kbp Intact 150 49 430072–487323 PHAGEVibriovBVpaMMARNC019722(8) 58.69% 4 35.1Kbp Intact 150 42 357957–393153 PHAGEPseudophiCTXNC003278(18) 57.32% 4 50.1Kbp Intact 150 70 564492–614686 PHAGEEnteroAryaNC031048(10) 56.98% 5 21.3Kbp Incomplete 40 11 26364–47679 PHAGEPseudovBPsyMKIL1NC030934(6) 49.79% 5 8.7Kbp Incomplete 50 13 540790–549505 PHAGEBacillBMBtpLANC028748(1) 57.37% accompanying phage head (Ghequire and de Mot 2015). Pseu- and2(cid:5)arepossibleduetosequencehomologyofputativeLpxD domonas phage tail-like bacteriocins are typically recruited to and the presents of possible HtrB1 and HtrB2 homologs (Lam trpE-trpG and mutS-cinA intergenic regions (Ghequire and de etal.2011;Hittleetal.2015).Ingeneral,itislikelythatP.baetica Mot2015).InspectionofthecorrespondingregionsinP.baetica possessesalipidAstructuresimilartoknownstructuresfrom revealsthepresenceoftwoadjacentR-typegeneclustersatthe P.aeruginosawhenpossiblepost-synthesismodificationsarenot mutS-cinAlocus.Phylogeneticassessmentandgenesyntenyis takenintoaccount(Knireletal.2006). indicative of an Rp3-Rp4 organisation, and a similar tailocin Furthergenomeanalysisresultedintheidentificationofa configuration was recently described in P. chlororaphis 30-84 geneclusterhomologoustothecoreoligosaccharidegeneclus- (Dorosky et al. 2017). An interesting observation is that four terwhichwasdescribedandstudiedinP.aeruginosa.Directcom- putative tail fibres are likely present in the Rp4 cassette of parisonoftheclusterrevealedthatP.baeticalackshomologues P. baetica, whereas usually only one or two of these can be oftheP.aeruginosagenesPA4998(putativekinase)andPA5008 detectedintailocins. (heptosekinase),whichpossiblysuggestsalessphosphorylated ReferredtoasS-typepyocinsinP.aeruginosa,modularbac- coreoligosaccharide(Lametal.2011).ReciprocalBLASTsearch- teriocinsrepresentaheterogeneousgroupofpolymorphictox- ing of different Pseudomonas proteomes resulted in sequence ins (Zhang et al. 2012; Jamet and Nassif 2015), comprising a identities between the wide range of 14% and 98%. The high- receptor-bindingdomain,asegmentenablingmembranetrans- estoverallsequencesimilaritywasobservedtoP.fluorescensPf0- ferandatoxindomainatthecarboxy-terminus(Ghequireand 1coreoligosaccharidebiosynthesisproteins,rangingfrom85% de Mot 2014). To protect from self-inhibition, bacteriocin pro- to98%whichisconsistentwiththephylogeny.Ingeneral,the ducerstrainsco-expressanimmunitygene,typicallyencoded clusterstructureandpresenceofputativeglycosyltransferases adjacent to the bacteriocin killer gene. Pseudomonas nuclease (WapR, WapH, WaaG, WaaC, WaaF) indicates the presence of bacteriocins(DNase,tRNase,rRNase)areconsistentlyequipped aninnercoreoligosaccharidestructuresimilartoP.aeruginosa withacentralpyocinSdomain(Pfam06958),andsuggestedto (Knireletal.2006). playapivotalroleintranslocationofthetoxintothecytoplasm (GhequireanddeMot2014;Sharpetal.2017).Searchingforthis ExtracellularproteaseandeffectorsecretionbyP. domainrevealedanarrayofputativemodularbacteriocinsinP. baetica baetica,sevenofwhichhostanHNH-DNaseandthreeanrRNase domain.Inonebacteriocin,aputative(non-HNH-type)DNase Extracellular protease secretion is an important virulence de- homologoustothekillerdomainfoundincarocinD—butdis- terminant of P. aeruginosa. In order to determine if P. baetica tinctfromthenon-HNH-typeinpyocinS3—wasidentified. has a similar phenotype, azocasein and skimmed milk pro- TwootherpredictedP.baeticabacteriocinswithhomologyto teaseassayswereperformedwithP.aeruginosaUCBPP-PA14,P. atoxinmoduleofacontact-dependentinhibitioncassette(type baetica, P. fluorescens Pf01, and P. putida KT2440. As expected, IV)inBurkholderiapseudomalleiwerealsodetected.Nobacteri- P.aeruginosaUCBPP-PA14secretedprodigiousamountsofpro- ocinsactingattheperiplasmiclevel(colicinM-like,Pfam14859; tease(seeFigures5and6,SupportingInformation).Pseudomonas pesticins,Pfam16754;orporeformers(ColIa/ColN),Pfam01024) baeticaa390TsecretedsignificantlylessproteasethanP.aerugi- couldberetrieved. nosabutmarginallymorethantheotherstrains.Thisphenotype wasmostevidentonMOPSminimalmilkagarafter72h(see FigureS6b,SupportingInformation). LipidAandcoreoligosaccharidebiosynthesisgene AmajorvirulencedeterminantinGram-negativepathogens clusters istheabilitytoinjecteffectorproteinsintohostcellsusingatype Putative homologues of the confirmed lipid A biosynthesis IIIsecretionsystem(T3SS)(Hauser2009).BLASTanalysisusing (Raetzpathway)enzymesinP.aeruginosaPAO1couldbeiden- theP.aeruginosaPscT3SSproteinsrevealedaT3SStobepresent tifiedinthesequencedgenome.ComparisontodifferentPseu- intheP.baeticagenome,providingapossibleexplanationforthe domonasproteomesrevealedsequenceidentitiesoftheputative virulenceofthisstrainashypothesisedabove. proteinsrangingfrom59%to97%.Thosehigh-sequenceidenti- tiesandthehydrocarbonrulermotiveinputativeLpxA(UDP-N- OTresistanceinP.baetica acetylglucosamineacyltransferase)hinttowardsalipidAwitha C10acylchainatposition3and3(cid:5)(Smithetal.2015).Besides,C12 Followingovernightincubation,antibioticplateswereexamined secondaryacylchainsorC12primaryacylchainsatposition2 for bacterial growth, and the minimum inhibitory concentra- Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 Beatonetal. 7 Figure2.OverviewofthegenomicarchitectureofP.baeticaa390T.CGViewComparisonToolwasusedtoplotthepercentagesequenceidentityofcloselyrelatedstrains. Theouterlabelsandbeigeblocksrefertolocithatarementionedthroughoutthepaper.TheP.baeticaa390Tgenomeissplitintotheforwardandreversestrandsand thecodingsequencesarerepresentedbybluearrows.Thenext6ringsrepresentthepercentageBLASTidentityofthegenomesofthefollowingcloselyrelatedstrains; fromtheoutsideringtoinsidetheorderisP.spIrchel3E19,P.koreensisCI12,P.koreensisD26,P.fluorescensSBW25,P.putidaKT2440,P.aeruginosaUCBPP-PA14.Thepercentage identityisindicatedbythecolourofthe6BLASTringsasindicatedbythekeyonthebottomlefthandsideofthefigure.Movinginwards,thenexttworingsindicate thechangesin%GCcontentandtheGCskewrespectively.Finally,thecentraldiagramindicatesthegenomicislandspredictedbytheIslandViewerpackagewhere Red=predictedbymultiplemethods,blue=IslandPath-DIMOB,orange=SIGI-HMMandgreen=IslandPick. Table2b.SecondarymetabolitebiosyntheticclusterspredictedbyantiSMASH. Cluster Type Clustersize(bp) Mostsimilarknownbiosyntheticgenecluster Percentagesimilarity 1 Unknown 43395 Mangotoxin 71 2 Arylpolyene 43613 APEVf 40 3 Nrps 63804 Nataxazole 11 4 Bacteriocin/Ripp 10887 – – 5 Transatpks-Nrps 94211 Sorangicin 13 6 Nrps 77485 Orfamide/Putisolvin 70 7 Terpene-Nrps 73119 Pyoverdine 16 8 Nrps 52998 Pyoverdine 22 9 Bacteriocin/Ripp 10845 – – Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 8 FEMSMicrobiologyLetters,2018,Vol.365,No.9 tion(MIC)ofOTforP.aeruginosaPAO1wasdeterminedas1μg OrthologsofMexAB-OprMandMexEF-OprN mL−1,aquarterthatofP.baeticaa390T.OTisaproteinsynthesis- resistance-nodulationcelldivisioneffluxsystemsinP. inhibitingantibioticbelongingtothetetracyclineclass,andis baetica oneofthemostcommonlyusedantibioticsinaquaculture(Leal etal.2017). TheMexAB-OprMandMexEF-OprNsystemsaremembersofthe WhenP.baeticawasculturedonmediacontainingOT,itwas resistance-nodulationcelldivisionmultidrugeffluxpumpfam- able to grow in the presence of higher concentrations than P. ily(Poole 2011). BLASTp was used to compare the amino acid aeruginosaPAO1.TheminimuminhibitoryconcentrationofOT sequencesencodedbythemexEF-oprNandmexAB-oprMoperons required to inhibit the growth of P. baetica was determined as inP.aeruginosastrainPAO1withhomologuesinP.baetica. 4μgmL−1.TheabilityofP.baeticatotoleratehigherconcentra- MexAB-OprMisknowntoplayakeyroleinefflux-mediated tionsofOTthanalabstrainofPseudomonasmaybeduetotheuse resistance to a wide variety of compounds such as antibi- oftheantibioticinaquaculture,especiallyasanadditiveinfish otics and solvents in P. aeruginosa (Li, Zhang and Poole 1998; food.OTresistance(OTR)hasbeenobservedpreviouslyinbac- Poole2011).Similarly,theMexEF-OprNeffluxsubstratesinclude terialspeciescolonisingpelletisedfishfoodinChile,andalsoin antibiotics and the QS precursor 4-hydroxy-2-heptylquinoline halfofcommercialfishfoodsamplesanalysedina1995studyof (LamarcheandDe´ziel2011;Llanesetal.2011).Pseudomonasbaet- OTRinfishfarmsediments(Kerryetal.1995;Mirandaetal.2003). icasequencesproducingsignificantalignmenttoMexAB-OprM and MexEF-OprN were identified with between 69%–79% and 72%–88%identitytoP.aeruginosastrainPAO1,respectively.Inter- Glycerol-3-phosphateutilisationinP.baetica estingly,thearrangementofthemexAB-oprMoperoninP.baetica bearsmoresimilaritytoP.fluorescens,wheretheadjacentreg- A notable characteristic which differentiates P. baetica from ulatory gene encodes a member of the TetR regulator family, closely related Pseudomonas species is an inability to grow on unlike P. aeruginosa which encodes the MarR family regulator glycerol-3-phosphate(G3P)asasolecarbonsource(Lo´pezetal. MexRinthisposition.However,itshouldbenotedthatP.baet- 2012). Several other pseudomonads, such as P. fluorescens and icadoeshaveaMexRhomologuelocatedadjacenttoathirdef- P. aeruginosa can use G3P as sole carbon sources. However, P. fluxoperonwhichhassignificantlylesshomology(8%–31%)to baeticacanuseglycerolasasolecarbonsource.Thissuggested mexAB-oprM. thatP.baeticasuffersfromaspecificdeficitinG3Puptake,not metabolism.UptakeofG3Pisknowntooccurusingthespecific MFStransporterGlpT(Fann,BuschandMaloney2003). OutermembraneporinsinP.baetica ThenewlysequencedgenomeallowedustoexaminetheP. baeticagrowthdeficiencyonG3Pingreaterdetail.Remarkably,P. InadditiontotheMex-associatedporins,theP.baeticagenome baeticaappearstoharbouraglpThomologue,whichisquitesim- wassearchedforgenescodingforotheroutermembraneporins. ilartoGlpTfromotherpseudomonads(seeFigureS7,Support- There are 26 porin genes in P. aeruginosa involved in different ingInformation).ThestructureofGlpThasbeenelucidatedfor importantbiologicalfunctions:outermembranestability(OprF, Escherichiacoli,allowingustounderstandwhichresiduesareim- OprH) and nutrient uptake (OprB, OprP, and the porins of the portantforsubstraterecognition(Huangetal.2003).Allresidues OprDfamily),whichhaverecentlybeenreviewed(Chevalieretal. whichhavebeenidentifiedasimportantforfunctionalityinpre- 2017).TheP.baeticagenomerevealedonly17poringenes,but viousstudiesareunchangedinP.baetica.MutationofK80toala- themajorporinsdescribedinP.aeruginosaareconserved.The ninekilledheterologousG3P-Pitransportoftheproteinreconsti- oprFgenecodingforthemajorPseudomonasporinispresentinP. tutedintoproteoliposomes,andmutationofH165resultedina baeticaandthegenomiccontextaswell:estX-menG-cmaX-cfrX- transportratethatwasonly∼6%ofthatcatalysedbywild-type cmpX-sigX-oprF where EstX is an esterase, MenG a RNAse in- protein(Lawetal.2008).Thesedatasuggestthatthepresence hibitor,CmaXaCorA-likemagnesiumtransporter,CfrXaputa- ofa glpThomologdoesnotnecessarilyequate toG3Puptake, tiveanti-sigmafactor,CmpXamechano-sensitivechannel,and highlightingthenecessityofexperimentalvalidation.Thisre- SigX an ECH-sigma factor. OprF has a C-terminal periplasmic sultmaybeduetoalackofinductionoftheP.baeticaglpTgene domaininteractingwithpeptidoglycanandabarrelN-terminal uponexposuretoG3P. domainanditisamajorcontributortotheenvelopestability. The broad-spectrum bactericidal antibiotic, fosfomycin, is OtherconservedporingenesareoprGandoprH,whichcodefor takenupactivelyintobacterialcellsviaGlpTandtheglucose- smallporinswithonlyeightstrands.OprGbelongstotheOmpW 6-phosphate transporter UhpT, and inhibits the initial step in familyandhasbeenproposedtobeinvolvedinFe2+diffusionto cellwallsynthesis(Kahanetal.1974).InactivationofglpTwas theperiplasm(Catel-Ferreiraetal.2016).OprHisinducedbylow shown to confer increased fosfomycin resistance in P. aerugi- Mg2+andcontributestotheoutermembranestabilityviaitsin- nosa,withanapparentlackoffitnesscost.Theseauthorsiso- teractionwithLPS(Kucharskaetal.2015).AsinP.aeruginosa,P. lated 10 independent spontaneous mutants, obtained on LB- baeticaalsohasthreegenesfortheglucoseporinOprB,including fosfomycin,whichharbouredmutationsintheglpTgene.These oneintheconservedgltBFGKoprBoperon(Chevalieretal.2017). mutantsincludedseveraldeletions,frameshiftsandthreesingle Surprisingly,the P. baetica genome does not contain genes for aminoacidchanges,Gly137toAsp,Thr336toProandMet366toIle theOprOandOprPphosphateuptakeporinsofP.aeruginosa.The (Castan˜eda-Garc´ıaetal.2009).Indeed,mutationsintheStaphy- OprDfamilycomprises19membersinP.aeruginosasub-divided lococcusaureusglpTgenewererecentlyshowntobethemajor into two sub-families, the OccD being involved in the uptake determinantoffosfomycinresistanceinthisorganism(Xuetal. ofbasicaminoacidsandtheOccKfortheuptakeofnegatively 2017).Thehighfosfomycinresistanceandlowbiologicalfitness chargedcyclicmolecules(Erenetal.2012).Thecomparisonwith costresultingfromthelossofG3PuptakesuggeststhatP.baetica P.aeruginosaporinsviaBLASTPiscomplicatedbythefactthat mayhaveanevolutionaryadvantageinafosfomycin-richenvi- manymembersoftheOprDfamilyshowsignificantrelatedness. ronment.Futureworkwillbenecessarytoconfirmpreciselyhow Nevertheless,thesurveyoftheP.baeticagenomerevealedfive G3PuptakehasbeenlostinP.baetica. membersoftheOccDandsixoftheOccKfamily. Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 Beatonetal. 9 OsmoticstressresistanceofP.baetica SinceP.baeticaa390Twasoriginallyisolatedfromamarineaqua- culture scenario, we have investigated its genotypic and phe- notypic ability to overcome salt stress. Trehalose is a stress- relievingdisaccharidewhichaccumulateswithinthebacterial cellinresponsetostress.Ithasbeenwidelystudiedforitsrole asacompatiblesolutethatconfersprotectionagainstosmotic stress.Itisthoughtthattrehaloserehydratesthecellfollowing water loss, preventing the loss of cellular functions (De Smet et al. 2000). It has also been reported that trehalose is impor- tantforpathogenicityofPseudomonasspp.(Freeman,Chenand Beattie2010;Djonovic´ etal.2013).Bacteriapossessthreemain pathwaysfortrehalosebiosynthesis:OtsAB,TreYZandTreS. TheOtsABpathwayisthemostcommonrouteoftrehalose biosynthesis in bacteria. OtsA catalyses the synthesis of tre- halose6-phosphatefromUDP-glucoseorADP-glucoseandglu- cose6-phosphate.ThephosphategroupisthenremovedbyOtsB togivetrehalose(DeSmetetal.2000).TheTreYZandTreSpath- waysproducetrehaloseusingdifferentsubstrates.TreYconverts reducingmaltosylunitsofmaltooligosaccharidestogiveα-1,1 terminal moieties which are then cleaved by TreZ to produce Figure3.StresstoleranceandgrowthcharacteristicsofP.baeticaa390T.Graph trehalose.TheTreSpathwayisomerisesmaltoseintotrehalose, ofendpointgrowthmeasuredasOD600after24hincubationat30◦CinPseu- catalysedbythetrehalosesynthaseTreS,butitsutilityinvivois domonasminimalmedia(L-glutamine5%,K2HPO41.5%,MgSO40.2%,20mMglyc- erolorothercarbonsourceasindicated)withpHorsaltconcentrationadjusted context-dependent(Miahetal.2013). asindicated.Datarepresentedmean±SD.∗P<0.05,∗∗P<0.01(determinedby Comparative genomics have shown that in common with two-wayt-test). other Pseudomonas spp., P. baetica lacks the genes for the OtsA/OtsBpathway.However,P.baeticapossessestheenzymes oftheTreYZandTreSpathways. ofbothgenomesusingSEEDviewershowedthatP.baeticahasa AlthoughTreSisknowntobecapableofgeneratingtrehalose homologueoftheOsmYgenewhichhasaproteinmodification insomebacteria,itispredictedthatthefunctionofTreSinP. function during osmotic stress whereas P. fluorescens does not baeticaistocatabolisetrehalose.Phenotypicobservationssug- (Me´tris,GeorgeandRopers2017). gestthattheabilityofP.baeticatoutilisetrehaloseasasolecar- bonsourceisdependentonotherconditions.InastudybyLo´pez IronuptakegenesinP.baetica et al., it was reported that P. baetica was unable to utilise tre- halose.Becauseofitsimportancetoosmoticstress,werepeated The P. baetica genome was searched for genes coding for theOmniloganalysisfortrehaloseandobservedanintermedi- siderophorebiosynthesisanduptakeproteinsaswellasthose atephenotypeandfurtheranalysisondefinedmediashowsthat involved in Fe2+ uptake. As expected, genes for the biosyn- P.baeticaiscapableofutilisingtrehalose. thesis and uptake of the fluorescent siderophore pyoverdine ThetreSgeneisfusedwiththemaltokinase-encodingpep2 arepresent(34intotal)andarescatteredinfourgenomicloci gene(Chandra,ChaterandBornemann2011),consistentwitha as it is usually the case for fluorescent pseudomonads (Ravel TreS/Pep2fusionproteinconvertingtrehaloseintoα-maltose1- andCornelis2003).Pseudomonasbaeticaalsoproducesasecond phosphate(M1P)(Miahetal.2013).M1Pisexpectedtobeacted siderophore/metallophore,yersiniabactin,whichwasdescribed uponbyGlgEandGlgBtoproduceaglycogen-likeα-glucan,given in Yersinia pestis and in other Enterobacteriaceae (Chaturvedi thatthetreS/pep2,glgEandglgBgenesareclustered. etal.2012).Yersiniabactinsharessomebiosynthesisstepswith OtherPseudomonasspp.suchasP.fluorescensandP.syringae pyochelin,thesecondsiderophoreofP.aeruginosaandisableto possessanadditionalhomologueofanunfusedtreSgeneem- bindcopperwithhighaffinity.Ithasbeenalsodescribedinsome phasisingtheimportanceoftrehaloseproductionintheseor- P.syringaeandP.avellanaestrains(JonesandWildermuth2011; ganisms.Interestingly,thisadditionalhomologueisabsentinP. MarcellettiandScortichini2015).Interestingly,whilemostpy- baetica.P.fluorescensandP.syringaelikelybenefitfrombeingable overdinegeneshavetheirclosesthomologsinP.koreensis,all, tocontrolthesynthesisoftrehaloseundermorevariedcondi- butone,yersiniabactingenesofP.baeticashowacloseidentity tions. withtheyersiniabactingenesfromP.azotoformans,whichcould WehypothesisedthatP.baeticawascapableofcausingdis- beindicativeofhorizontalgenetransfer.Thegenomecontains easeinmarinefishbecauseithadgreatersalttolerancethan 27 genes coding for a TonB-dependent receptor, including the otherpseudomonads;however,thisprovedtobeincorrect.Our fpvAgenefortheferripyoverdineuptake,andtwofyuAgenesfor datashowthatallofthepseudomonadstestedherearecapable theuptakeofferri-yersiniabactin.However,twoofthesegenes ofgrowthinNaClconcentrationsrelevanttothemarineenvi- aretruncatedandaprobableframeshiftisevidentforonerecep- ronment(seeFiguresS8andS9,SupportingInformation). torgene.Noteworthyisthepresenceofsixgenesannotatedas Inadditiontosalttoleranceexperiments,P.baeticaandP.flu- codingforaTonBprotein.Asamatterofcomparison,onlythree orescenswerealsotestedfortheirabilitytogrowinminimalme- arepresentinP.aeruginosa(Cornelis,MatthijsandvanOeffelen dia with the addition of Instant Ocean which has similar lev- 2009). Another surprising discovery is the absence of feoABC elsofmosttraceelements,nutrientsandalkalinitytoseawater genesfortheuptakeofFe2+inP.baetica.Thisfunctionisprobably samples(Fig.3).PseudomonasbaeticagrowswellinInstantOcean securedbytheproductsoftheefeUOABoperon,firstdescribedin whileP.fluorescenshasminimalgrowthsuggestingthatP.baetica E.coliO157:H7,whichisinvolvedintheuptakeofferrousironin mayhavesomeadaptationtothemarineenvironment.Analysis theperiplasmwhereitgetsre-oxidized(Caoetal.2007). Downloaded from https://academic.oup.com/femsle/article-abstract/365/9/fny069/4951603 by University of Strathclyde user on 25 April 2018 10 FEMSMicrobiologyLetters,2018,Vol.365,No.9 DISCUSSION Arndt D, Grant JR, Marcu A et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res Whole-genomesequencinghasbecomeubiquitousinmicrobi- 2016;44:W16–21. ology and access to the technology is now widespread. Here, BankevichA,NurkS,AntipovDetal.SPAdes:anewgenomeas- weshowthatcombiningtworelativelycheapanduser-friendly semblyalgorithmanditsapplicationstosingle-cellsequenc- methods(IonTorrentSemiconductorandOxfordNanoporeMin- ing.JComputBiol2012;19:455–77. IONsequencing)enablesgenomeassemblyofarelativelylarge BertelliC,LairdMR,WilliamsKPetal.IslandViewer4:expanded bacterialgenometoafinishablestate.Ourphylogeneticanaly- predictionofgenomicislandsforlarger-scaledatasets.Nu- sesshowthatP.baeticaiscloselyrelatedtoP.koreensisintheP. cleicAcidsRes2017;45:W30–5. fluorescensgroup.However,ourdatashowthattheP.fluorescens Blin K, Wolf T, Chevrette MG et al. antiSMASH 4.0— grouptaxonomyisinneedofreview.Giventhecloserelation- improvements in chemistry prediction and gene cluster shipofP.baeticaa390TtoP.irchel3e19,weproposethat3e19is boundaryidentification.NucleicAcidsRes2017;45:W36–41. astrainofP.baetica.Althoughbothareaquaticisolates,Irchel BravoJI,LozanoGL,HandelsmanJ.DraftGenomesequenceof 3e19wasisolatedfromfreshwaterandP.baeticaa390Tisma- FlavobacteriumjohnsoniaeCI04,anisolatefromthesoybean rine(Lo´pezetal.2012;Butaite˙ etal.2017).Ourdatashowthat rhizosphere.GenomeAnnounc2017;5:e01535–16. theabilitytogrowinmarinesaltconcentrationsiscommonin Butaite˙ E, Baumgartner M, Wyder S et al. Siderophore cheat- thegenusPseudomonas.This,combinedwiththeoriginofIrchel ing and cheating resistance shape competition for iron in 3e19suggeststhatP.baeticaa390Tisnotamarine-restrictedor- soilandfreshwaterPseudomonascommunities.NatCommun ganismandislikelytobeanopportunistpathogenofmarine 2017;8:414. fish,possiblyassociatedwithhighfishdensitiesinaquaculture CaoJ,WoodhallMR,AlvarezJetal.EfeUOB(YcdNOB)isatripar- facilities.Ontheotherhand,thepresenceofaT3SSINBOTH tite,acid-inducedandCpxAR-regulated,low-pHFe2+trans- P.baeticaA390TandIrchel3E19(Prof.RolfKummerli,personal porterthatiscrypticinEscherichiacoliK-12butfunctional communication)suggeststhattheseorganismsarenotmerely inE.?coliO157:H7.MolMicrobiol2007;65:857–75. benignenvironmentalstrains.TheP.baeticagenomeshowsev- Castan˜eda-Garc´ıa A, Rodr´ıguez-Rojas A, Guelfo JR et al. The idenceofgenomeplasticityandcontainsavarietyofgenomic glycerol-3-phosphate permease GlpT is the only fos- islandsandprophagesasisthecasewithotherpseudomonads fomycin transporter in Pseudomonas aeruginosa. J Bacteriol includingP.aeruginosa(Klockgetheretal.2011).Thediverseiron 2009;191:6968–74. acquisitionandmembranetransportsystemsencodedintheP. Catel-FerreiraM,MartiS,GuillonLetal.Theoutermembrane baeticagenomeareconsistentwithanorganismthatishighly porinOmpWofAcinetobacterbaumanniiisinvolvediniron adaptabletoarangeofenvironments.Wefindnogenomicevi- uptakeandcolistinbinding.FEBSLett2016;590:224–31. denceofknownQSsystemsfoundinotherpseudomonads,but Ca´rcamo-Oyarce G, Lumjiaktase P, Ku¨mmerli R et al. Quorum ourobservationsthatP.baeticaiscapableofproteasesecretion sensing triggers the stochastic escape of individual cells andswarmingsuggestthatitmayengageinsocialbehaviour. fromPseudomonasputidabiofilms.NatCommun2015;6:5945. Chandra G, Chater KF, Bornemann S. Unexpected and SUPPLEMENTARYDATA widespread connections between bacterial glycogen and trehalosemetabolism.Microbiology2011;157:1565–72. SupplementarydataareavailableatFEMSLEonline. Chaturvedi KS, Hung CS, Crowley JR et al. The siderophore yersiniabactinbindscoppertoprotectpathogensduringin- FUNDING fection.NatChemBiol2012;8:731–6. Chevalier S, Bouffartigues E, Bodilis J et al. Structure, function WorkinNPT’slabhasbeenfundedbyBiotechnologyandBio- andregulationofPseudomonasaeruginosaporins.FEMSMicro- logicalSciencesResearchCouncilgrant BB/K019600/1andChief biolRev2017;41:698–722. Scientists Office grant TCS/16/24. AB is supported by a PhD Connor TR, Loman NJ, Thompson S et al. CLIMB (the Cloud StudentshipfundedbytheIndustrialBiotechnologyInnovation Infrastructure for Microbial Bioinformatics): an online re- CentreandtheUniversityofStrathclyde.TSwassupportedby source for the medical microbiology community. Microb a PhD studentship funded by the Iraqi Government. MG was Genom2016;2:e000086. supportedbyapostdoctoralfellowshipfromFWOVlaanderen CornelisP,MatthijsS,vanOeffelenL.Ironuptakeregulationin (12M4618N). CL was supported by an SB PhD fellowship from Pseudomonasaeruginosa.Biometals2009;22:15–22. FWOVlaanderen (1S64718N).JBwassupportedbyagrantfrom De Smet KA, Weston A, Brown IN et al. Three pathways Fundac¸a˜oparaaCieˆnciaeaTecnologia (SFRH/BD/104095/2014). for trehalose biosynthesis in mycobacteria. Microbiology NPTwouldliketopersonallythankallofthecontributorstothis 2000;146:199–208. paperwhohavemadethisnovelcollaborationanopenandplea- Djonovic´ S, Urbach JM, Drenkard E et al. Trehalose biosynthe- surableexperience.AMandEDweresupportedbytheBiotech- sispromotesPseudomonasaeruginosapathogenicityinplants. nologyandBiologicalSciencesResearchCouncil-fundedSouth PLoSPathog2013;9:e1003217. WestBiosciencesDoctoralTrainingPartnership (BB/M009122/1). 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