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Systematic and Applied Microbiology
jo u r n al h om e p a g e :w w w . e l s e v i e r . d e / s y a p m
Pseudomonas gallaeciensis sp. nov., isolated from
crude-oil-contaminated intertidal sand samples after the Prestige oil spill
Magdalena Mulet
a, David Sánchez
a, Ana C. Rodríguez
a, Balbina Nogales
a,b, Rafael Bosch
a,b, Antonio Busquets
a, Margarita Gomila
a, Jorge Lalucat
a,b, Elena García-Valdés
a,b,∗aMicrobiologia,DepartamentdeBiologia,EdificiGuillemColom,UniversitatdelesIllesBalears,CampusUIB,07122PalmadeMallorca,Spain
bInstitutMediterranid’EstudisAvanc¸ats(IMEDEA,CSIC-UIB),CampusUIB,07122PalmadeMallorca,Spain
a r t i c l e i n f o
Articlehistory:
Received5July2017
Receivedinrevisedform26March2018 Accepted30March2018
Keywords:
Pseudomonas
Pseudomonaspertucinogenagroup Contaminatedsand
Oilspill Genome
a b s t r a c t
StrainsV113T,V92andV120havebeenisolatedfromsandsamplestakenattheAtlanticintertidalshore inGalicia,Spain,afterthePrestigeoilspill.Apreliminaryanalysisofthe16SrRNAandthepartialrpoD genesequencesindicatedthatthesestrainsbelongedtothePseudomonasgenus,buttheyweredistinct fromanyknownPseudomonasspecies.Theywereextensivelycharacterizedbyapolyphasictaxonomic approachandphylogeneticdatathatconfirmedthatthesestrainsbelongedtothePseudomonaspertu- cinogenagroup.Phylogeneticanalysisof16SrRNA,gyrBandrpoDgenesequencesshowedthatthethree strainswere99%similarandwerecloselyrelatedtomembersoftheP.pertucinogenagroup,withlessthan 94%similaritytostrainsofestablishedspecies;Pseudomonaspachastrellaewastheclosestrelative.The AverageNucleotideIndexbasedonblastvalueswas89.0%betweenV113TandtheP.pachastrellaetype strain,belowtheacceptedspecieslevel(95%).Thepredominantcellularfattyacidcontentsandwhole cellproteinprofilesdeterminedbyMALDI-TOFmassspectrometryalsodifferentiatedthestudiedstrains fromknownPseudomonasspecies.WethereforeconcludethatstrainsV113T,V92andV120representa novelspeciesofPseudomonas,forwhichthenamePseudomonasgallaeciensisisproposed;thetypestrain isV113T(=CCUG67583T=LMG29038T).
©2018ElsevierGmbH.Allrightsreserved.
Introduction
ThegenusPseudomonasrepresentsagroupofGram-negative bacteriawithintheGammaproteobacteriathatarenon-sporeform- ingrodsthataremotilebypolarflagella[29].Thebacteriaofthe genusPseudomonas are ubiquitous, metabolically versatile, and importantfortherecyclingofelementsintheenvironment.The genuswasfirstdescribedbyMigula(1894)andcurrentlycomprises alargenumberofspecies,andnewspeciesaredescribedcontin- uously.Atthetimeofthismanuscript’scomposition,167species withvalidnameshavebeendescribed(http://www.bacterio.cict.
fr/p/pseudomonas.html)[8,30].
∗ Corresponding authorat: Microbiologia, Departamentde Biologia, Edifici GuillemColom.UniversitatdelesIllesBalears,Crtra.ValldemosaKm7.5,Campus UIB,07122PalmadeMallorca,Spain.
E-mailaddress:elena.garciavaldes@uib.es(E.García-Valdés).
ThecharacterizationofPseudomonastype strainsbymultilo- cussequenceanalysis(MLSA),concatenatingthesequencesofthe 16SrDNA,gyrB,rpoDandrpoBgenes,permittedtheestablishment oftwomainlineages,PseudomonasfluorescensandPseudomonas aeruginosa,whichweredividedintoseveralgroups(G)andsub- groups(SG)[25,27].Inrecentyears,thePseudomonaspertucinogena Ghasexperiencedasignificantincreaseinthenumberofnewly describedspeciescomparedwiththeothergroups.In2004,this group wasrepresented only by P. pertucinogena[16]; today, it contains 16 species,thefollowing of whichhave beenrecently described:Pseudomonasaestusnigri[34,11],Pseudomonassalegens [3], Pseudomonassalina [39], Pseudomonasoceani [36,10], Pseu- domonas populi [4] and ‘Pseudomonassaudimassiliensis’ not yet validated[6].
Proteobacteriahaveplayedakeyroleinthebacterialcommunity inthecontaminatedintertidalsandafterthePrestigeoilspill[1,2].
Thecharacterizationofthebacterialspeciespresentinthishabitat iscrucialtohaveacomprehensiveknowledgeofthemicroorgan- isms presentin this environment. Thispaperis focusedonthe https://doi.org/10.1016/j.syapm.2018.03.008
0723-2020/©2018ElsevierGmbH.Allrightsreserved.
genusPseudomonasandaputativenewspeciesinthegenus.Dur- ingastudyofthePseudomonasdiversityincrude-oilcontaminated intertidalsandsamplesafterthePrestigeoilspill(Galicia,Spain)in September2004,severalisolateswereconsideredrepresentatives ofpossiblenewspecies.Theseresultswereconfirmedbytheanal- ysisofpartialsequencesofsigma70subunitofRNApolymerase, encodedbytherpoDgene[26].Threeofthesestrains,V113T,V92 andV120,havebeencharacterizedtaxonomicallyinthepresent study.AnewPseudomonasspeciesisproposedintheP.pertucino- genaG,withthestrainV113Tasthetypestrain.
Materialsandmethods
Bacterialstrainsandgrowthconditions
StrainsV113TandV120havebeenisolatedfrom“BocadoRio”
beach(42◦5011.52N,9◦611.52W)fromburiedweatheredfuel.
StrainV92wasisolatedfromanunpollutedsandsamplefrom“Praia deSeda” beach(42◦4629.27N,9◦727.08W). Thestrainswere isolatedaftergrowthin mineralbasalmedium (MMB)[5] with naphthalene(V92andV113T)orhexadecane(V120)asenergyand carbonsources[26].Alistofthebacterialstrainsstudiedasrepre- sentativesofcloselyrelatedspeciesoftheP.pertucinogenagroup isprovided inTableS1[13,18,20,21,31,37,38].Allbacteriawere culturedat30◦ConLuria-Bertanimedium(LB)for24–48h.
DNAextraction,PCRamplification,DNAsequencingconditions
TheDNAextraction,PCRamplification,primersused,purifica- tionof theamplifiedproductsand DNA sequencingconditions, as well as the sequence analysis procedures, were previously described[28].
Primer sequences corresponding to enterobacterial repetitive intergenic consensus (ERIC) elements (ERIC 1R: 5-ATGTAAGCTCCTGGGGATTCAC-3 and ERIC2: 5- AAGTAAGTGACTGGGGTGAGCG-3) [35] and BOX elements (BoxA1R:5-CTACGGCAAGGCGACGCTGACG-3)[17]wereusedfor DNAfingerprinting.
Phylogeneticanalysis
Individualtreesbasedonthepartialsequencesofthe16SrRNA (1300nucleotides)and gyrB(801 nucleotides)geneshavebeen includedintheanalysis,togetherwiththerpoD(737nucleotides) gene sequence. An analysis of these three concatenated gene sequenceswas alsoperformed witha total of 2838nt [25].An update (until 2018) of the type strains of all species in the P.
pertucinogenaphylogeneticgroup,asdefinedby Muletandcol- laborators[27],wasincludedandcomparedinthepresentstudy (TableS1).TheJukes–Cantor(JC)[14],maximumlikelihood(ML) [9]andmaximumparsimony(MP)[22]algorithmswereusedfor thecomparisons.
Genomesequencingandanalysis
GenomicDNAwasisolatedfromstrainV113T,usingtheWizard GenomicDNAPurificationkit(Promega)accordingtothemanufac- turerinstructions.TheIlluminaHiSeq2000obtainedpaired-end libraryreadsweredenovoassembledusingtheNewblerAssem- bler v2.7 program (Roche). The draft genome was annotated usingtheNCBIProkaryoticGenomeAnnotationPipeline(PGAP).
TheWhole GenomeShotgunprojectfor strain V113T hasbeen deposited in DDBJ/ENA/GenBank under the accession number LMAZ00000000.Theversiondescribedinthispaperisthefirstver- sion,LMAZ01000000.Analysisandcomparisonofthefunctional annotationwasdoneusingtheKyotoEncyclopediaofgenesand
Genomes(KEGG AutomaticAnnotationServer[KAAS])[24].The presenceofplasmidshasbeenassessedmanuallyandalsowith thePlasmidFinderinsilicowebtool[7].
TherelatednessofthenovelspeciesPseudomonasgallaeciensis V113Tgenometowholegenomeshotgunsequencesofallspecies typestrainsintheP.pertucinogenaGavailableinpublicdatabases wasdeterminedbasedonthetetranucleotidefrequencycorrelation coefficients(TETRA),averagenucleotideidentity(ANI)usingthe BLASTNalgorithm(ANIb),andtheMUMMERultra-rapidaligning tool(ANIm)aswellasgenome-to-genomedistance(GGDC)meth- ods.ANIbandANImwerecalculatedusingtheJSpeciessoftware toolavailableatthewebpagehttp://www.imedea.uib.es/jspecies.
Therecommendedspeciescut-offwas95%fortheANIbandANIm indices[32].GGDCwascalculatedusingthewebservicehttp://
ggdc.dsmz.de [23] and using therecommended BLAST method.
TheGGDCresultsshownarebasedontherecommendedformula 2whichisindependentofthegenomelengthandisthusrobust againsttheuseofincompletedraftgenomes.
Cellmorphologyandphysiologicaltests
Cellsize,morphologyandflagellarinsertionweredetermined bytransmissionelectronmicroscopyofcellsfromtheexponential growthphaseinLB.AHitachimodelH600electronmicroscopewas usedat75kV.Thesampleswerenegativelystainedwithphospho- tungsticacid(1%,pH7.0)aspreviouslydescribed[19].
The production of fluorescent pigments was tested onKing Bmedium (PseudomonasagarF, Difco), and pyocyanin produc- tionwastestedonKingAmedium(PseudomonasagarP,Difco).
ThestrainsV113T,V92andV120werecharacterizedphenotypi- callyusingAPI20NEstrips(bioMérieux),BiologGN2andGENIII MicroPlates(Biolog,Hayward,CA).Growthtemperatures(4,6,10, 15,18,25,30,37and42◦C)weredeterminedinLBmediumand growthinthepresenceofNaCl(0–15%w/v)andpHranges(4–11) wereobservedinNutrientBroth(Merck).
Chemotaxonomicanalysis
Thechemotaxonomicdataobtainedwithmatrix-assistedlaser desorption/ionisation time-of-flightmass spectrometry(MALDI- TOFMS)forstrainsV113T,V92andV120andtheircloselyrelated species type strains were obtained at the Scientific-Technical Services(UniversityofBalearicIslands,Spain)andanalysedaspre- viouslydescribed[34].
Wholecellfattyacidmethylesters(FAME)analysiswasper- formed at theSpanish Type Culture Collection, CECT, Valencia, Spain (http://cect.org/identificaciones) under high standardized conditions.Fattyacidswereextracted,preparedandanalysedas describedinRef.[34].
Resultsanddiscussion Phylogeneticanalysis
A preliminary analysis of the partialsequences of therpoD genecomparedamongallofthePseudomonastypestrainsrevealed thatstrainsV113T,V92andV120couldberepresentativesofa newspecies[26].Acompletephylogeneticanalysishasnowbeen accomplished.
Inallindividualandconcatenatedgenesequencetreesstud- ied,strainsV113T,V92andV120werelocatedinthesamebranch, independentfromtheothertype strainsin theP.pertucinogena group,withPseudomonaspachastrellaeCCUG46540Tbeingtheclos- esttypestraintothegroup(Figs.1and2).Highbootstrapvalues supportedthefollowingJCtreebranches:concatenatedandrpoD gene Trees100%, 16SrRNA geneTree 90%,and gyrB geneTree
Fig.1. Phylogenetictreebasedon16SrRNAgeneofPseudomonasgallaeciensisandphylogeneticallyclosemembersofPseudomonas.Distancematriceswerecalculatedby theJukes–Cantormethod.Dendrogramsweregeneratedbytheneighbour-joiningmethod.P.aeruginosaATCC10145Twasusedastheoutgroup.Thebarindicatessequence divergence.Percentagebootstrapvalueshigherthan50%(from1000replicates)areindicatedatthenodes.Filledcirclesindicatethatthecorrespondingnodeswerealso obtainedinthetreesgeneratedwiththemaximumlikelihoodandmaximumparsimonymethods.GenBankaccessionnumbersaregiveninparentheses.Accessionnumbers indicatedinboldareforsequencesdeterminedinthisstudy.
72%(rpoDandgyrBgeneTreedatanotshown).Thethreestrains were99%similarintheconcatenatednucleotidesequences,andat leastninedifferentnucleotidesweredetectedintheirconcatenated sequences.Theanalysisoftheconcatenatedsequencesshowedthat theclosesttypestraintostrainsV113T,V92andV120wasP.pachas- trellaeCCUG46540T(93.9,94.0and93.8%similarity,respectively).
Similarresultswithhighbootstrap values wereobtainedwhen MLandMPalgorithmswereused.Commonnodebranchesofthe threetreesareindicatedinFig.2.Theintragroupaveragesimilar- ityvaluecalculatedformembersoftheP.pertucinogenagroupwas 85.0±3.6%(TableS2).The94%similaritybetweenstrainV113Tand P.pachastrellaeaffiliatedthisstrainwiththeP.pertucinogenagroup;
however,thissimilarityislower than97%,thethresholdestab- lishedtodiscriminateamongspeciesinthegenusPseudomonasby MLSAofthreeconcatenatedgenesequences[25].StrainsV113T, V92andV120couldnotbeaffiliatedwithanyPseudomonasspecies previouslydescribedandshouldbeconsideredrepresentativesof anewspecies.
Phenotypiccharacteristics
Strain V113T was a Gram-negative, rod shaped bacterium (0.9–2.0mlongand0.6mwide)thatwasmotilebymeansof a singlepolarflagellum (Fig. S1). StrainsV113T, V92and V120 werepositiveforcatalaseandoxidaseactivities.Afterincubation for48hat30◦ConLBplates,colonieswereround,convex,beige incolour,brightandwithentiremargins(1–4mmofdiameter).
StrainsV113T,V92andV120wereabletogrowinLBmediumat 6–37◦C.Nogrowthwasdetectedat4◦C or42◦C. Theoptimum growthwasbetween25–30◦C. GrowthwasobservedonNutri- entBrothin thepresenceof 2–13%NaCl(w/v),optimum 4-8%, andthebacteriatoleratedapHrangingfrom5to10(optimum 6)(Table1).StrainsV113T,V92andV120andalltheotherstrains
intheP.pertucinogenagroupfailedtoproduceeitherfluorescent pigmentsorpyocyaninwhenculturedfor24–48hat30◦ConKing BorKingAmedium.Thedifferentialphenotypiccharacteristicsin theAPI20NE,BiologGN2andGENIIItestsareindicatedinTable1.
StrainsV113T,V92andV120sharedtheabilitytoassimilatecapric acid,adipicacid,malicacid;positiveforutilizationofl-alanine, l-glutamic acid, glucuronamide, methyl pyruvate, l-lactic acid, Tween40,-hydroxy-d,l-butyricacid,acetoaceticacid,propionic acid,aceticacid,Tween80,sebacicacid,l-asparagineandpositive forthesensitivityof1%sodiumlactate,fusidicacid,d-serine,trole- andomycin,rifamycinSV,lincomycin,guanidineHCl,niaproof4, vancomycin,tetrazoliumviolet,tetrazoliumblue,lithiumchloride, potassiumtellurite, aztreonam,sodiumbutyrate.Other14tests werevariable,indicatingahighintraspeciesdiversity.Therestof testswerenegativeorweak.Incontrast,P.pachastrellaewasunable tousel-alaninamide,d-alanineandl-proline.P.aestusnigriwas unabletoassimilateglucose,arabinoseandmalicacid,andnegative forl-arabinose,cis-aconiticacid,l-alaninamideandd-alanine.
P.oceanicouldbedifferentiatedfromalltypestrainsanalyzedin thisstudybytheabilitytousel-fucose,gelatin,l-phehylalanineand
␣-ketovalericacidandwasnegativefortheutilizationofD-lactic acid.RegardingtheoxidationtestsperformedwithBiologGN2and GENIII,thetypestrainsoftheP.pertucinogenagroupandV113T,V92 andV120showedalimitedabilitytousecarbonsources,withneg- ativeorweakresultsforalargenumberofsubstratesaspreviously published[15,33,34].
Chemotaxonomicanalysis
Theprotein profiles obtainedshowedthat thethree strains, V113T,V92andV120,werecloselyrelated(100%similarity).TheP.
pachastrellaetypestrainwasthecloseststrain(95%similarity),and thesimilaritiestotheotherstrainsintheP.pertucinogenagroup
Fig.2.Phylogenetictreebasedonconcatenatedsequencesofthe16SrRNA,gyrBandrpoDgenesofPseudomonasgallaeciensisandphylogeneticallyclosemembersof Pseudomonas.DistancematriceswerecalculatedbytheJukes–Cantormethod.Dendrogramsweregeneratedbytheneighbour-joiningmethod.Pseudomonasaeruginosa ATCC10145Twasusedastheoutgroup.Thebarindicatessequencedivergence.Percentagebootstrapvalueshigherthan50%(from1000replicates)areindicatedatthe nodes.Filledcirclesindicatethatthecorrespondingnodeswerealsoobtainedinthetreesgeneratedwiththemaximum-likelihoodandmaximumparsimonymethods.
GenBankaccessionnumbersaregiveninparenthesesinthefollowingorder:16SrRNA,gyrBandrpoDgenes.Accessionnumbersindicatedinboldareforsequences determinedinthisstudy.
Table1
DifferentialphenotypiccharacteristicsofPseudomonasgallaeciensisfromrelatedPseudomonastypestrains.Strains:P.gallaeciensis(1.V113T,2.V92,3.V120),4.P.pachastrellae CCUG46540T,5.P.aestusnigriCECT8317T,6.P.oceaniDSM100277T.
Characteristics 1 2 3 4 5 6
Temperature(◦C) 6–37 10–37 6–37 7–41 6–37 4–41
NaCl(%)(w/v) 2–13 2–12 2–12 0–10a 2–10b 0–10c
pH 5–10 6–10 5–9 ND 6–10b 6–10c
Reductionofnitrate(API20NEtest) − + − − − −
Growthon(API20NEtest)
Glucose w + + + − +
Arabinose w w w + − W
Malicacid + + + + − W
Trisodiumcitrate + − + + − +
BiologGENIIItest
l-Fucose − − − − − +
Minocycline − w − + − −
Gelatin − − − − − +
BiologGN2test
␣-Cyclodextrin − w − + − −orwc
l-Arabinose + w w + − −orwc
cis-Aconiticacid + w + + − −orwc
␣-Ketovalericacid − − − − − +c
d-Lacticacid + + + + + −c
l-Alaninamide + w + − − +c
d-Alanine + w + − − +c
l-Phenylalanine − − − − − +c
l-Proline + w + − + +c
Putrescine w w − − − +c
Positive(+),negative(−),weak(w)andnotdetermined(ND).Unlessotherwiseindicateddatawereobtainedinthisstudy.a,b,cdatatakenfromaRomanenkoetal.[33],
bSanchezetal.[34]andcWangandSun[36].Oxidaseandcatalasetestswerepositiveforallstrains.
Table2
Cellularfattyacidcomposition(%)ofPseudomonasgallaeciensis,anditsclosest relatedspeciestypestrainsofthegenusPseudomonas.
Fattyacid(%) 1 2 3 4 5 6
C12:0 9.3 8.7 10.1 10.1 8.9 8.5
C14:0 1.1 0.9 1.2 1.6 0.9 1.3
C16:0 14.8 12.8 14.6 16.9 16.2 20.5
C18:0 1.0 0.9 0.8 0.6 0.7 1.1
C10:03-OH 5.0 4.4 5.7 3.9 5.3 4.9
C12:03-OH 4.2 4.1 4.8 5.1 4.7 3.8
C17:0cyclo 0.0 0.0 0.0 0.0 1.3 0.8
Summedfeatures3 29.3 26.7 32.3 34.5 25.3 24.4 Summedfeatures8 33.4 35.1 30.0 26.9 34.5 30.9 Allstrainswereculturedontryptonesoyagar(TSA)andincubatedat28◦Cfor48h.
Strains:P.gallaeciensis(1.V113T;2.V92;3.V120),4,P.pachastrellaeCCUG46540T, 5.P.aestusnigriCECT8317T,6.P.oceaniDSM100277T.DataforP.aestusnigriCECT 8317TwerefromSanchezetal.[34].Summedfeaturerepresentsgroupsoftwoor morefattyacidsthatcouldnotbeseparatedbytheMicrobialIdentificationSys- tem.SummedFeature3,C16:17cand/orC16:16c;Summedfeature8,C18:17c and/orC18:16c.
werelowerthan80%.IntheMALDI-TOFMSanalysis,V113T,V92 andV120showedthreecommonm/zpeaks(supermassvalues) thatwerenotpresentinP.pachastrellae,theclosesttypestrain:
4808m/z,6901m/zand9617m/z(Fig. S2).Thesedatafurther supportedtheconclusionthatthesestrainsrepresentedadistinct species that was separated from all other species of the Pseu- domonasgenus,evenatthelevelofexpressionofthemostabundant cellularproteins.
Fatty acid profiles were similar in strains V113T, V92 and V120 (Table 2). Strains V113T, V92 and V120 exhibited very similarprofiles, and summed feature8 (C18:1 ω6c and/orC18:1
ω7c, 35.1–30.0%), summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 32.3–26.7%), C16:0 (14.8–12.8%), C12:0 (10.4–8.7%),C10:0 3- OH(5.7–4.4%)andC12:03-OH(4.8–4.1%)werethemostabundant fattyacids.ThefattyacidprofilesofstrainsV113T,V92andV120 weresimilar to those of species of theP. pertucinogenagroup, andtheyhadthethreefattyacidstypicallypresentinthegenus Pseudomonas,which areC10:0 3-OH, C12:0 2-OH andC12:0 3-OH, accordingtoPalleroni[29].
TotalDNAfingerprinting
TotalDNAfingerprintingbyERICandBOXPCRsshowedthatiso- latesV113T,V92andV120wererepresentativesofthreedifferent strains(Fig.S3).ThethreeP.gallaeciensisstrainshadsixidentical bandsintheERIC-PCR(425bp,525bp,1150bp,1200bp,1350bp and approximately2500bp);strain V113T had three additional bands(750bp,925bpandapproximately1900bp)andV120had fiveadditionalbands(475bp,600bp,850bp,1300bpand1900bp).
IntheBOX-PCR,strainV113Thadtwoadditionalbands(425bpand 830bp),V92hadthree(400bp,600bpand800bp)andV120had four(200bp,475bp,775bpand1100bp).TheDNAprofileswere alsodifferentfromthoseofthetypestrainsofP.pachastrellaeand P.aestusnigri.
GeneraltaxonomicgenomefeaturesofstrainV113T
Thedraftgenomewasassembledin18scaffolds(71contigs) withan average of 75× coverage. No plasmids weredetected.
Themaincharacteristicsofthewhole-genomesequenceofstrain V113T aredepictedinTable3.Allgenomerelatednessvaluesof strainV113TcalculatedbythealgorithmsANIb,ANImandGGDC againstthespeciestypestrainsoftheP.pertucinogenagroupwere clearlybelowtheestablishedcut-offforeach(ANIb:95%,ANIm:
95%,GGDC:70%),confirmingthatstrainV113Trepresentsanovel genomicspeciesinthegroup.TheresultsareshowninTableS3.
Table3
GenomiccharacteristicsofPseudomonasgallaeciensisV113Tsp.nov.
Characteristics V113T
GeneBankID LMAZ00000000
Genomesize(bp) 4,246,542
No.scaffolds/contigs 18/71
N50scaffold/contigsize 509,657/154,985
Largestcontigsize 1,749,682
Q40PlusBases(%) 4,232,689
Q39MinusBases(%) 502
GC-content(%) 61,2%
Totalgenes 3905
Protein-codinggenes(CDS) 3806
No.hypotethicalproteins 1321
RNAgenes(clusters) 1
tRNAs 46
Pseudogenesa 49
Mobilome:
Integrases 2
Transposases 11
aThenumberoftotalPseudogenesindicatedincludesgeneswithambiguous residues,frameshiftedgenes,incompletegenes,geneswithinternalstopsorother multipleproblems.
Insightsfromthegenomesequence
Threethousandeight-hundredandsixofthe3,905totalgenes werepredictedasprotein-codinggenes(CDS)in thegenomeof strainV113T.Genomeanalysisallowedthepredictionofseveral relevantmetabolictraits.Nitrogencouldbeassimilatedbyreduc- ingnitratetonitriteandammonia,andsulfateandsulfurreduced tosulfide for biosynthesis. Ferricenterobactin was detectedas siderophoreforironadquisition.Sugarsmightbemetabolizedby theglycolyticorthepentosephosphatepathways.
Thestrainswereisolatedfromahighlypollutedareaaftera crude-oilspill.Therefore,thepresenceofgenesforthebiodegra- dationofaromaticandlinealhydrocarbonswasstudied.Acomplete setof16 geneswitha regulatoryprotein oftheFisfamily was detectedforthedegradationpathwayofmonoaromatics,relatedto thecatabolismoftoluene,benzeneandphenol.Amulticomponent phenolhydroxylase(P0,P1,P2,P3,P4andP5)predictedtopro- ducecatecholor4-methylcatechol,whichmightbemetacleaved bycatechol2,3-dioxigenasetoproducepyruvateandacetyl–CoA.
Thissetofgeneswas97-100%identicaltothecorrespondinggenes oftheclose-relatedspeciesP.aestusnigriandP.pachastrellaetype strains[11,12].Acompletesetofgenesrelatedtoalkanedegra- dationpathwayweredetected.Thealkane1-monooxygenaseand therubredoxinconductingtothecorrespondingfattyacidwere 98%identicaltothehomologousproteinsinP.pachastrellae.
Genesrelatedwiththeinteractionwiththeenvironment,like thoserelatedtosecretionsystems,biofilmformationormotility werestudied in detail. Types IIand VI secretionsystems were detected,butnoevidenceofTypesI,III,IVorVwasfound.Addition- ally,systemsrelatedtoquorumsensingpathways(BarA/Uvry/CsrA system),tobiofilmformation(GacA/Rsmpathway)and involved inalginatebiosynthesishavebeenfound.Genesrelatedtoefflux pumpsasmexH,mexIandoprMinvolvedinbetalactamresistance havebeenfoundandalsothemultidrugresistanceeffluxpumpmtd genes.Togetherwithflagellation,genesfortwitchingmotilityand swarmingwerepresent.
Themobilomerepresentsoneofthemaincontributorstobac- terial intraspeciesvariability. The prophage contributiontothe bacterialgenome is highly variable. A clusterof 28 genes, six- teenbacteriophagestructuralproteins,and12hypoteticalproteins relatedtophageswerefoundinscaffold3(ASB5811015to11150).
Additionally,11transposases and2integraseswerefoundscat- teredinthechromosome.
Table4
ProtologueforPseudomonasgallaeciensissp.nov.
Taxonumber TA00074
Speciesname Pseudomonasgallaeciensis
Genusname Pseudomonas
Specificepithet gallaeciensis
Speciesstatus sp.nov.
Speciesetymology N.L.fem.adj.gallaeciensis,pertainingtoGalicia,Spain,wherethetypestrainwasisolated
Designationofthetypestrain V113
Straincollectionnumbers CCUG67583,LMG29038
16SrRNAgeneaccessionnumber FN995250
Alternativehousekeepinggenes rpoD[FN994225],gyrB[LN876645]
Genomeaccessionnumber LMAZ00000000
Genomestatus draft
Genomesize 4246542
GCmol% 61.2
Countryoforigin Spain
Regionoforigin Galicia
Other Lari ˜nomunicipality
Dateofisolation 01September2004
Sourceofisolation SandcontaminatedbyPrestigecrudeoil
Samplingdate 01September2004
Geographiclocation PraiadaSedabeach
Latitude 42◦4629.2N
Longitude 9◦727.1W
Depth 0
Altitude(alti) 0
Numberofstrainsinstudy 3
Sourceofisolationofnon-typestrains PraiadaSedabeach,Lari ˜nomunicipality,Galicia,Spain Growthmedium,incubationconditions
usedforstandardcultivation
Luria-Bertanimedium(LB)at30◦C
Gramstain NEGATIVE
Cellshape rod
Cellsize(lengthordiameter) 0.9–2.0mlongand0.6mwide
Motility Motile
Ifmotile Flagellar
Ifflagellated Onepolarflagellum
Sporulation(restingcells) None
Colonymorphology Round,convex,colourbeige,brightandwithentiremargins(1–4mmdiameter)onLBplatesafter incubationfor48hat30◦C
Temperaturerange 6–37
Lowesttemperatureforgrowth 6
Highesttemperatureforgrowth 37
Temperatureoptimum 25–30
LowestpHforgrowth 5
HighestpHforgrowth 10
LowestNaClconcentrationforgrowth 2
HighestNaClconcentrationforgrowth 13
Salinitycategory Moderatehalophile(optimum7–15%NaCl)
PositivetestswithBIOLOG l-Alanine,l-glutamicacid,glucuronamide,methylpyruvate,l-lacticacid,Tween40,
-hydroxy-d,l-butyricacid,acetoaceticacid,propionicacid,aceticacid,Tween80,sebacicacid, l-asparagine,1%sodiumlactate,fusidicacid,d-serine,troleandomycin,rifamycinSV,lincomycin, guanidineHCl,niaproof4,vancomycin,tetrazoliumviolet,tetrazoliumblue,nalidixicacid,lithium chloride,potassiumtellurite,aztreonam,sodiumbutyrate
NegativetestswithBIOLOG Negativeallstrains:d-maltose,d-trehalose,d-cellobiose,gentiobiose,sucrose,turanose, stachyose,d-raffinose,␣-d-lactose,d-melibiose,b-methyl-d-glucoside,d-salicin, N-acetyl-d-glucosamine,N-acetyl--d-mannosamine,N-acetyl-d-galactosamine,
N-acetyl-neuraminicacid,␣-d-glucose,d-mannose,d-fructose,d-galactose,3-methylglucose, d-fucose,l-fucose,l-rhamnose,inosine,d-sorbitol,d-mannitol,d-arabitol,myo-Inositol, d-glucose-6-PO4,d-fructose-6-PO4,d-asparticacid,d-serine,gelatin,glycyl-l-proline,l-arginine, l-asparticacid,l-histidine,l-pyroglutamicacid,l-serine,pectin,d-galacturonicacid,d-galactonic acidlactone,d-gluconicacid,d-glucuronicacid,mucicacid,quinicacid,d-saccharicacid, p-hydroxy-phenylaceticacid,d-lacticacidmethylester,d-malicacid,gamma-amino-butyricacid,
␣-keto-butyricacid,formicacid,sodiumbromate,␣-cyclodextrin,i-erythritol,lactulose,d-psicose, xylitol,d-glucosaminicacid,itaconicacid,␣-ketovalericacid,malonicacid,l-alanyl-glycine, glycyl-l-asparticacid,l-histamine,hydroxy-l-proline,l-leucine,l-ornitine,l-phenylalanine, l-threonine,d,l-carnitine,urocanicacid,uridine,thymidine,phenylethylamine,2,3-butanediol, glycerol,d,l-␣-glycerolphosphate,glucose-1-phosphateWeakornegative:dextrin,glycogen,
␣-hydroxybutyricacid,adonitol,glycyl-l-glutamicacid,putrescine,2-aminoethanol VariabletestswithBIOLOG Minocycline,citricacid,␣-ketoglutaricacid,l-malicacid,bromo-succinicacid,nalidixicacid,
acetoaceticacid,l-arabinose,mono-methyl-succinate,cis-aconiticacid,succinicacid,succinamic acid,l-alaninamide,d-alanine,l-proline
PositivetestswithAPI CAP,ADI,MLT
NegativetestswithAPI(APIN) NEGATIVE:TRP,GLU,ADH,URE,ESC,GEL,PNPG,MNE,MAN,NAG,MAL,GNT,PAC,WEAK:ARA VariabletestswithAPI(APIV) NO3,GLU(fermentation),CIT
Commercialkitsused BIOLOGGENIII,BIOLOGGN2,API20NE
Energymetabolism Chemoorganotroph
Table4(Continued)
Oxidase Positive
Catalase Positive
Negativetests FluorescenceonKingAandKingBagar
Majorfattyacids Summedfeature8(C18:1ω6cand/orC18:1ω7c,35.1–30.0%),summedfeature3(C16:1ω7cand/or C16:1ω6c,32.3–26.7%),C16:0(14.8–12.8%),C12:0(10.4–8.7%),C10:03-OH(5.7–4.4%)andC12:03-OH (4.8–4.1%)
Biosafetylevel 1
Habitat Beachsand(http://purl.obolibrary.org/obo/ENVO00002138)
Bioticrelationship Free-living
Knownpathogenicity None
Conclusion
Thechemotaxonomic datasupportedbyMALDI-TOFandcell fatty acid methyl esters of the strains V113T, V92 and V120 clearlylocatethesestrainsinthePseudomonasgenus.Thegenomic sequencesof thehousekeepinggenes studied(16SrRNA, rpoD, gyrB)indicatethatthethreestrainsarerepresentativeofanew species. The ANIb genome analysis confirms this presumption.
StrainsV113T,V92andV120wereisolatedfromdifferentsitesof theintertidalcoastandaremembersofanewbacterialspeciesable topersistinacontaminatedenvironment.
Consideringthephylogenetic,chemotaxonomicandphenotypic characteristicspresented,weproposeanewspecies,P.gallaecien- sissp.nov.,withP.gallaeciensisV113Tasthetypestrain.Thefull descriptionofthenewtaxonisshowninTable4togetherwiththe descriptionoftheclosestspeciesinTableS4asobtainedfromthe DigitalProtologuewebsite(http://imedea.uib-csic.es/dprotologue/
)inwhichthenewspecieswasregisteredunderreferenceTA00074.
Note
TheGenBank/EMBL/DDBJaccessionnumbersforthenucleotide sequencesreportedin thisstudyare asfollows: LN876648and LN881559 (strain P. gallaeciensis V120 and Pseudomonas yang- mingensisDSM24213Tforthe16SrRNA),LN876645, LN876646, LN876647,LN881554,LN881555,LT724117andLT837808(strain P.gallaeciensisV113T,V92,V120,P.salegensCECT24213T,P.yang- mingensisDSM24213T,P.salinaJCM19469TandP.populiCCTCC AB2013069TforthegyrBgene),LN881556,LN881557,LN881558, LT724118andLT837807(strainP.salegensCECT8338T,P.yangmin- gensisDSM24213T,PseudomonasformosensisJCM18415T,P.salina JCM19469TandP.populiCCTCCAB2013069TfortherpoDgene).
Acknowledgements
WeareindebtedtoDr.M.Teuberfor correctingtheetymol- ogy.Financial supportwas obtainedfromthe SpanishMINECO throughprojectCGL2015-70925,withFondoEuropeodeDesarrollo Regional(FEDER)co-funding.MargaritaGomilawassupportedby apostdoctoralcontractfromtheConselleriad’Innovació,Recerca iTurismedelGoverndelesIllesBalearsandtheEuropeanSocial Fund.Allauthorsweresupportedbyfundsforcompetitiveresearch groupsfromtheGovernmentoftheBalearicIslands(withFEDER co-funding).D.Sánchezwastherecipientofapre-doctoralfellow- shipfromtheConselleriad’Interior,DireccióGeneraldeRecerca, DesenvolupamentTecnològiciInnovaciódelGoverndelesIlles Balears(FPI09)andtheEuropeanSocialFund(ESF).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttps://doi.org/10.1016/j.syapm.2018.03.
008.
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