Beaton, Ainsley and Lood, Cédric and Cunningham-Oakes, Edward and MacFadyen, Alison and ...

13 Pages·2017·2.4 MB·English

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:[phone];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
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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
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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.
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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
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Beatonetal. 5
Figure1.PlacementofnovelbaeticagenomeinthegenusPseudomonasbaseduponANI,andsharedk-mersA)ANIheatmapgeneratedthePython3modulepyani.
Thesequencedbaeticagenome,Pseudomonassp.Irchel3E19andreferencegenomesfromthecloselyrelatedkoreensissubcladeweresubjecttoANIanalysis.B)A
Mash-basedtreegeneratedfromreferencegenomes(n=86)from19speciesclades,comprisingtheentiregenusPseudomonas.Thistreewasgeneratedbasedupon
theJaccardindex,calculatedfromsharedk-mers.CellvibriojaponicumUeda107wasusedasoutgroup.
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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-
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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 – –
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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.
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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).
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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). DoroskyRJ,YuJM,PiersonLS,IIIetal.Pseudomonaschlororaphis
produces two distinct R-tailocins that contribute to bacte-
Conflictofinterest.Nonedeclared.
rialcompetitioninbiofilmsandonroots.ApplEnvironMicrob
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