h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Phylogenetic
MLSA
and
phenotypic
analysis
identification
of
three
probable
novel
Pseudomonas
species
isolated
on
King
George
Island,
South
Shetland,
Antarctica
Felipe
Vásquez-Ponce,
Sebastián
Higuera-Llantén,
María
S.
Pavlov,
Sergio
H.
Marshall,
Jorge
Olivares-Pacheco
∗PontificiaUniversidadCatólicadeValparaíso,FacultaddeCiencias,InstitutodeBiología,LaboratoriodeGenéticaeInmunología
Molecular,Valparaíso,Chile
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received13August2017 Accepted14February2018 Availableonline15March2018 AssociateEditor:VâniaMelo
Keywords: Pseudomonas
Antarctica
P.fluorescenscomplex
MultilocusSequenceAnalysis
a
b
s
t
r
a
c
t
Antarcticaharborsagreatdiversityofmicroorganisms,includingbacteria,archaea, microal-gaeandyeasts.ThePseudomonasgenusisoneofthemostdiverseandsuccessfulbacterial groupsdescribedtodate,butonlyeightspeciesisolatedfromAntarcticahavebeen char-acterized.Here,wepresentthreepotentiallynovelspeciesisolatedonKingGeorgeIsland. Themostabundantisolatesfromfourdifferentenvironments,weregenotypicallyand phe-notypicallycharacterized.Multilocussequenceanalysisand16SrRNAgeneanalysisofa sequenceconcatenateforsixgenes(16S,aroE,glnS,gyrB,ileSandrpoD),determinedoneofthe isolatestobeanewPseudomonasmandeliistrain,whiletheotherthreearegoodcandidates
fornewPseudomonasspecies.Additionally,genotypeanalysesshowedthethreecandidates
tobepartofanewsubgroupwithinthePseudomonasfluorescenscomplex,togetherwith theAntarcticspeciesPseudomonasantarcticaandPseudomonasextremaustralis.Wepropose termingthisnewsubgroupP.antarctica.Likewise,phenotypicanalysesusingAPI20NEand BIOLOG®corroboratedthegenotypingresults,confirmingthatallpresentedisolatesform
partoftheP.fluorescenscomplex.PseudomonasgenusresearchontheAntarcticcontinentis
initsinfancy.Tounderstandthesemicroorganisms’roleinthisextremeenvironment,the characterizationanddescriptionofnewspeciesisvital.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:jorge.olivares@pucv.cl(J.Olivares-Pacheco).
Introduction
Antarctica is arguably one of the planet’s harshest environments.1Intensecold,lowhumidity,alimited availabil-ityofliquidwater,aswellasperiodsofhighsolarradiation and extended total darkness, mean that the continent
https://doi.org/10.1016/j.bjm.2018.02.005
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
represents a relatively inhospitable environment for the development of the life.1 Nevertheless, Antarctica is now consideredtohostaveryhighdiversityofmicroorganisms, including bacteria, archaea, yeastsand microalgae.2,3 As a resultofintenseselectivepressuresthroughouttheir evolu-tionaryhistory,allofthesemicroorganismsarefullyadapted totheAntarcticconditions.4,5Theseadaptationsareofgreat interest to the biotechnology industry, since they harbor thepossibilityofobtainingbio-productsthatcouldbeused invarious processesofconservation atlowtemperatures.6 There is therefore an increasing need to investigate the diversityandtoexploitthepotentialofthemicroorganisms ofthecoldcontinent.
AbacterialgenusthathasappearedfrequentlyinAntarctic researchisPseudomonas.Oneofthe mostwell-studied bac-terial genera, it ispresent in most environments and can beconsidered oneof the mostsuccessful bacterialgroups onEarth.7 Pseudomonas’successismainlybased onitslow nutritionalrequirementsandgreatmetabolicdiversity,which allowsittousenumerousorganiccompoundsasasourceof bothcarbonandenergy.8,9Inaddition,thesebacteriaproduce alargeamountofsecondarymetabolites,whichareessential fortheirsurvivaland evenenablesomespeciestodegrade aromatic and halogenatedcompounds.10,11 Basedon these characteristics,thebacteriaofthegenusPseudomonas repre-sentaninterestingalternativeforproductionofdifferentand biotechnologicallyusefulmolecules.
Despite being extremely well-studied on a global level, little is known about the species diversity of the genus
Pseudomonas in Antarctica. To date, only eight species
havebeen describedon thiscontinent: Pseudomonas
antarc-tica,Pseudomonas meridiana,Pseudomonasproteolytica,12
Pseu-domonasguineae,13Pseudomonasextremaustralis,14Pseudomonas
deceptionensis,15 Pseudomonas prosekii16 and, most recently,
Pseudomonasgregormendelii.17 Inorder tofurtherour
under-standingofthisgenus’biogeographicdistribution,aswellas itsfunctionaspartoftheecosystemoftheextreme Antarc-ticenvironment, itisofutmostimportancetoidentifyand describenewAntarcticspeciesofPseudomonas.Withthisin mind,thepresentstudyaimstocarryoutaphylogeneticand phenotypic characterizationoffour bacterial isolates origi-natingfrom differentenvironments onKingGeorgeIsland, Antarctica. Specifically,we setthe followingspecific objec-tives:(1)anevaluationofthegrowthrateofdifferentstrains atdifferenttemperatures;(2)theamplificationand sequenc-ingofthe16SrRNAgene;(3)theamplificationandsequencing ofthegenesaroE,glnS,gyrB,ileSandrpoD,allofwhichwere analyzedbothindividuallyand,aftersequenceconcatenation, by Multilocus Sequence Analysis (MLSA), a robust, high-resolutiontechnique forthe identification ofnovel species andtheconfirmationofspeciesidentityofnewisolates18–20; and (4)a phenotypiccharacterization usingAPI20 NE and BIOLOG GN2. Basedon the both the phylogenetic analysis of16S-rRNA geneand the MLSAresults,as wellas onthe phenotypiccharacterization,weshowthatthefourisolates belongtothegenusPseudomonasandspecificallytotheP.
fluo-rescenscomplex,andthatthreeofthemmightrepresentnovel
species,whileonestraincouldbeclassifiedwithinthespecies
P.mandelii.
Materials
and
methods
Strainsandcultureconditions
Different Antarctic environments on King George island, South Shetland archipelago,were sampled inorder to iso-latespecificallybacteriamembersofthePseudomonasgenus. ThesesampleswerecollectedonAustralsummerseasonon January 2011. Samples ofseawater from the Fildes bay at 5mdepth (62◦1150.3 S; 58◦5450.3 W);freshwater from a summerlakeonFildespeninsula(62◦109.2S;58◦5529W); marinesedimentsfromFildesbay(62◦1328.8S;58◦5842.7 W)andsoilfromtheFildesPeninsula(62◦109.2S;58◦5529 W);weretakeninordertoisolatebacteriafromall environ-mentaldiversity.10Lofwatersampleswerefirstfilteredby a0.1mmfiltersandthenthesampleswereconcentratedina 0.2mfilter.Thisfilterwasthenresuspendedin3mLofsaline buffer (0.85%NaCl).5gofsedimentsandsoilsampleswere resuspended in5mLofsalinebuffer solution(0.85% NaCl). 100LofthesolutionwereplatedontoNutrientAgar(BDTM
DIFCOTM,NewJersey,NJ,USA)andincubatedat4,10,15and
25◦Cfor72h.Inordertoisolatebacteriaofthegenus
Pseu-domonas,allcolonieswerereplatedonPseudomonasIsolation
Agar(PIA)(SIGMA–Aldrich,LosÁngeles,CA,USA)and incu-batedat4,10,15and25◦Cfor72h.Allobtainedcolonieswere 16SrRNAgenesequenced.Themostabundantisolateofeach environmentwasselectedanddesignedasaparticularstrain. GrowthratesofallselectedstrainswereestimatedinLysogeny Broth(LB)(BDTM DIFCOTM,NewJersey,NJ,USA)at4,10,15,
25,30and37◦C.Alltestswereperformedin100mLflasksin 20mLmediumandatconstantagitationrateof240rpm,in duplicatesandonthreedifferentdays(makingforatotalof sixsamples).ThestrainP.fluorescensA506(ATCC® 31948TM)
servedasacontrolforthephenotypiccharacterization.
Amplificationofthe16SrRNAgeneandphylogenetic analysis
GenomicDNAwasextractedfromallstrainsusingthe Gene-JETGenomicDNAPurificationKit(ThermoFisherScientific, Waltham, MA, USA) following the manufacturer’s instruc-tions. The 16S rRNA gene was amplified by PCR using universalprimers27F(5-AGAGTTTGATCMTGGCTCAG-3)and 1492R(5-GGTTACCTTGTTACGACTT-3).AllPCRproductswere sequenced by Sanger method for five times (Forward and reverse).TheobtainedsequencesweredepositedinGenBank (NCBI)(TableS1).ThesequenceswerethenBLASTed21against the16SrRNAGenBankdatabase(NCBI).Sixty-one16SrRNA genesequencesofthegenusPseudomonaswereselectedfor the phylogeneticanalysis.The16SrRNAgeneofEscherichia colistrainMG1655K12wasusedasanoutgroup.Sequences ofallmembersofthegenusPseudomonaswereobtainedfrom the databaseonthe websitewww.pseudomonas.com22 and alignedusingClustalW(usingstandardparameters)as imple-mentedinthesoftwareMEGA7.0.14.23Gapsweretreatedwith the partialdeletionmethod. Amaximumlikelihood phylo-genetic treewas constructedusing theTamura-Nei model. Bootstrapvalueswerecalculatedbasedon1000replicas.
MLSA
MLSAwasperformedonsixhousekeepinggenes:16S-rRNA,
aroE (Shikimato 5-dehydrogenase), glnS (glutaminyl-tRNA transferase), gyrB (DNA gyrase subunit B), ileS (isoleucine-tRNAtransferase),andrpoD(RNApolymerasesigmafactor). PCRprimersweretakenfromAndreanietal.24Forallgenes, PCRconsisted ofan initialdenaturalization stepof10min at95◦C,followed by30cycles of45s at95◦C;45s at55◦C and 1minat72◦C,and afinal extensionstepof10minat 72◦C.All PCR productswere sequencedbySanger method forfivetimes(Forwardand reverse).Thepartialsequences obtained for the genes aroE, glnS, gyrB, ileS and rpoD of strainsIB20,12B3and6A1weredepositedinGenBank(NCBI) (TableS1).ThecorrespondingsequencesforstrainKG01were takenfromitswholegenomesequence,whichwehave pub-lishedpreviously.25Theobtainedsequenceswereindividually BLASTed21 againstGenBankinorder toidentifythe homo-logueswithgreatestsequenceidentity.Forthephylogenetic analysis,weconcatenated thepartialgenesequences after themultisequencealignmentinthefollowingorder:
16S-aroE-glnS-gyrB-ileS-rpoD, resulting in a single sequence. For the
samegenes,wetooktheorthologuesfrom51ofthespecies thataremostrepresentativeofthegenusPseudomonas,whose sequenceswereobtainedfrom thedatabaseonthewebsite
www.pseudomonas.com.22Asanoutgroup,weusedthe
con-catenateoftheorthologuesfromthestrainE.coliK12MG1655. Thephylogeneticanalysiswascarriedoutinthesamewayas forthe16SrRNAgene.
Phenotypiccharacterization
ThephenotypiccharacterizationoftheAntarcticisolatesIB20, 12B3,6A1 and KG01,aswell asofthe controlstrain P.
flu-orescensA506(ATCC®31948TM),wascarriedoutusingAPI20
NE(Biomerieux,L’Etoile,France)andBIOLOGGN2(BIOLOG®, Hayward,CA,USA).ForbothAPI20NEandBIOLOGassays, weusedpreviouslyisolatedcoloniesthathadbeengrownfor 24hat25◦C onLBagar plates(BDTM DIFCOTM,New Jersey,
NJ,USA).Thesecolonieswerere-suspendedinsalinesolution (0.85%NaCl)andinoculatedaccordingtothemanufacturer’s instructions.Oxidaseproductionwasdeterminedusing Oxi-daseReagentDroppers(Becton,DickinsonandCompany,New Jersey,NJ, USA)followingthemanufacturer’s specifications. Allphenotypicexperimentswereperformedintriplicateusing sixcoloniesinallrounds.
Results
SelectionofmostabundantPseudomonasisolatesbased on16SrRNAsequence
Atthefirst-roundselectioninnutrientagar,morethan6000 colonieswereobtainedinalltemperaturesused.Allcolonies werereplatedinPIAmediuminordertoselectonlymembers
ofthePseudomonasgenus.Only268coloniesgrowinPIAinall
temperaturesforallenvironments selected.These colonies were16SrRNAgenesequencedandthemostabundant iso-latesforenvironmentwere usedinthis work.Isolatefrom
Table1–Theoptimumgrowthtemperatureofthe Antarcticbacterialisolatesis25◦C.Thetableshowsthe doublingtimesofthefourAntarcticbacterialstrainsat differenttemperatures(N/G=nogrowth).
Doublingtime(min)+SD
4◦C 10◦C 15◦C 25◦C 30◦C 37◦C
IB20 341±38 118±21 65±6 43±8 75±11 N/G
12B3 412±46 132±16 77±4 56±6 89±11 N/G
6A1 381±26 158±18 69±8 49±9 78±8 N/G
KG01 376±31 141±22 72±10 48±5 92±6 N/G
seawaterwasnamedIB20;fromfreshwater12B3;frommarine
sediments6A1andfromsoilKG01.
TheAntarcticbacterialisolatesarepsychrotolerant,with
optimumgrowthat25◦C
In order to determine the optimum growth conditions for
theAntarcticisolates,weperformedgrowthexperimentsat
different temperatures. Growth curveanalysis and the
cal-culationofdoublingtimesclearlyshowedthattheoptimum
growth temperature is 25◦C for all of the Antarctic
iso-lates(Table1).Whilenogrowth couldbedetectedat37◦C,
all isolatesgrew well at 4◦C,as istypical of psychrotoler-antbacteria.26Theoptimumgrowthtemperatureof25◦Cis
sharedbyPseudomonasstrainsacrosslatitudes.Onthe Antarc-tic South Shetland Islands, however, this temperature has notbeenreachedforthepast25,000years,27suggestingthat members of the genus Pseudomonas might have colonized Antarcticabeforeitbecameapolarcontinent.
Phylogeneticanalysisbasedon16SrRNAgeneandMLSA
In order to determine the genus ofthe Antarctic bacterial isolates, we sequenced the 16S rRNA gene. The obtained sequences were deposited in GenBank(NCBI; Table S1). In afirststep,thesequenceswereBLASTedagainstNCBI’s16S rRNAGenBank.21ForisolatesIB20,12B3andKG01,sequence identitywithanyspeciescontainedinthedatabasewasbelow 97%,suggestingthat allthreeisolates couldrepresentnew bacterialspecies.28However,queryingthesequenceofstrain 6A1revealeda99.9%sequenceidentitywiththestrainP.
man-deliiJR-1,meaningthattheisolatemightinfactbelongtothat
species.Ontheotherhand,thephylogenetictree(Fig.1)shows all ofthese strainsas part oftheP. fluorescens lineage and withinthat,theP.fluorescenscomplex,oneofthemostdiverse andubiquitouslineagesofthegenus.29 Withregardstothe otherbacteriaofthegenusPseudomonasthathavebeen iso-latedinAntarctica,itcanbeseenthatbothstrainsofP.guineae
formalineage,whichisseparatefromtheonesthathavebeen described forP. aeruginosaandP.fluorescens.However,these resultsareinsufficienttodrawconclusions.
Inordertoexpandontheresultsofthe16SrRNAgene anal-ysis,wepartiallysequencedthefivehousekeepinggenesaroE,
glnS,gyrB,ileSandrpoD. Eachoneofthesegeneswas then
BLASTedagainsttheGenBanknucleotidedatabase(NCBI).21 Theresultsarelisted inTable2.Withtheexception ofthe strain6A1,wherefouroffivegenesshowextremelyhigh sim-ilarities(≤98%)demonstratingthatthis isthefirststrainof
P. exremorientalis KMM3447 P. tolaasii PMS117 P. simiae MEB105 P. fluorescens BRIP 34879 P. poae RE1114 P. trivialis IHBB745 P. orientalis CFML96-170 P. antarctica CMS35 P. meridiana CMS38 P. fluorescens BS2 P. fluorescens SBW25 P. extremaustralis 14-3 P. veronii CIP104663 P. marginalis ATCC10844 P. proteolytica CMS64 P. fluorescens LBUM223 P. fluorescens SS101 P. fluorescens A506 P. brassicacearum DF41 P. brassicacearum NFM421 P. frederiksbergensis SI8 P. libaniensis DSM17149 P. synxantha BG33R 93 12B3* IB20* KG 01* 6A1* P. mandelii JR1 P. lini DSM768 P. prosekii AN2801 P. gregormendelii CCM8506S P. protegens PF5 P. protegens CHA0
P. synringae pv. syringae B728a
P. syringae pv. tomato DC3000 100 P. syringae pv. actinidiae CFBP7286 P. deceptionensis DSM26521 P. putida KT2440 P. putida HB3267 P. putida GB-1 P. putida BIRD-1 P. putida F1 P. putida ND6 P. putida KG4 P. putida S16 P. putida W619 P. stutzeri DSM 10701 P. mendocina NK-01 P. mendocina ymp P. aeruginosa PA14 P. aeruginosa LESB58 P. aeruginosa ATCC 14886 P. aeruginosa M18 P. aeruginosa B136-33 P. aeruginosa PAO1 P. aeruginosa NCGM 1900 P. denitrificans ATCC 13867 P. entomophila L48 100 P. guineae M8 P. guineae X14 100 E. coli K12 MG1655 0.01 88 91 99 86 100 97 75 84 98 93 76 P . fluorescens comple x P . fluorescens lineage P . aer uginosa lineage
Fig.1–Maximum-likelihoodphylogenetictreebasedon
thepartial16SrRNAgenesequencesofmembersofthe
Pseudomonasgenus.The16SsequenceofE.coliK12was
usedasanoutgroup.Thetreeshowsallbacteriaofthe genusPseudomonasisolatedinAntarcticatodate(bold). Isolatespresentedinthisstudyareshowninboldand underlined.ItcanbeseenthatthemajorityoftheAntarctic bacteriaarepartoftheP.fluorescenslineage,withthe exceptionoftheP.guineaeisolates,whichseemtobepart ofaseparatelineage.
P.mandeliiisolatedinAntarctica;allthreestrainsshowedlow
similaritiesinatleastfourgenes(≥96%).Theseresultsconfirm thatIB20,12B3andKG01areseriouscandidatestobeclassify asanovelspeciesofthePseudomonasgenus.
Concatenatedsequenceanalysisindicatesthatthe Antarcticstrainscouldformanovelsub-groupofthe fluorescenscomplex
The sequences of the genes 16S, aroE, glns, gyrB, ileS and
rpoDwereconcatenatedtoformasinglesequenceof4150bp
Table2–Percentageidentityvaluesobtainedby BLASTingthefivehousekeepinggenesofthefour Antarcticisolates.Thelettersrepresentthedifferent speciesandstrainsofPseudomonas,towhichthe percentageidentityrefers.
aroE glnS gyrB ileS rpoD
IB20 ≤90%g,h,i ≤95%h,i,j ≤94%i,k,l ≤96%h,m,n ≤99%o,p,q
12B3 ≤91%g,h,i ≤96%h,i,j ≤95%i,s,t ≤96%h,m,n ≤99%o,p,q
6A1 ≤94%a,b,c ≤98%a,b,y ≤98%a,b,d ≤98%a,b,e ≤99%a,b,f
KG01 ≤88%m,u,v ≤94%w,p,q ≤94%i,x,w ≤96%i,h,n ≤99%p,q,w
a=P. mandelii JR-1, b=P. fluorescens NCIMB11764, c=P.
chloro-rapis subsp aurantiaca JD37, d=P. fluorescens PS1, e=P. mandelii
DSM17967T,f=P.fluorescensFW300-N2E3,g=P.fluorescensSBW25,
h=P.antarcticaPAMC27494,i=P.fluorescens LBUM636,j=P.
fluo-rescensUK4,k=P.reactansUSB131,l=P.reactansUSB20,m=P.trivialis
IHBB745,n=P.fluorescensKENGFT3,o=P.fluorescensA506,p=P.
fluo-rescensPICF7,q=P.fluorescensPCL1751,r=P.fluorescensNCIMB11764,
s=P.yamanorum8H1,t=P.putidaMG2010,u=P.fluorescensL111,v=P.
fluorescens L321, w=P. azotoformansS4,x=P. reactansUSB94 y=P.
koreensisD26.
andcomparedagainstaconcatenateoforthologousgenesof
thegenusPseudomonas.Aconcatenateofthesamegenesof
E.colistrainK12MG1655wasusedasanoutgroup.Sequences
werealigned,andaphylogenetictreewasconstructedusing
the maximumlikelihood modelwith1000bootstraps
repli-cas(Fig.2).ThetwomajorlineagesofthegenusPseudomonas,
thatofP.fluorescensandthatofP.aeruginosa,canbeclearly
distinguishedfromoneanother.Likewise,withintheP.
fluo-rescenslineage,threesubgroupscanbeclearlydistinguished;
theserepresenttheP.fluorescenscomplex,theP.syringaegroup
andtheP.putidagroup.ItcanbeseenthatallAntarctic
bac-teriaofthegenusPseudomonasdescribedtodate(inbold)are
partoftheP.fluorescenscomplex.TogetherwithP. antarctica
PAMC27494andP. extremaustralis14-3,thestrainsdescribed here appearto forma newsubgroupwithinthis complex, which wehavecalledthe P.antarctica subgroup.Thestrain 6A1,ontheotherhand,formsaclusterwithP.mandeliiJR-1, confirming that it belongs to that particular species. Like-wise,P.deceptionensis,thoughpartoftheP.fluorescenscomplex, belongstoanother,asyetundescribed,subgroup.
Phenotypicanalysis
The resultsofthe MLSAanalysishad already classified all strainsaspartoftheP.fluorescenscomplex.Toconfirmthis, acomprehensivephenotypicanalysisoftheAntarcticstrains was carried out using BIOLOG GN2 and API 20 NE. The phenotypic analysesaresummarized inTable3. Itis note-worthythatAPI20NEanalysesclassifiedallisolateswithin thespeciesP.fluorescens.Itis,however,knownthatthe res-olution of this technique is not particularly high, since it classifiesmostmembersoftheP.fluorescenscomplexwithin thisspecies.29BIOLOGanalysesyieldedsignificantdifferences betweenthe Antarcticisolatesandthe controlstrainP.
flu-orescensA506(ATCC® 31948TM).Basedon thesedifferences,
threeofthefournewisolatescanbeconsidered good can-didatesfornewspecies.Thefollowingspecificfindings are worth mentioning.Thestrain KG01isthe onlyoneunable toassimilated-mannose,l-proline,putrescine,pyruvicacid
Pseudomonas sp. 12B3 Pseudomonas sp. IB20 Pseudomonas sp. KG 01 Pseudomonas sp. 6A1 100 P. extremaustralis 14-3 P. antarctica PAMC 27494 100 P. tolaasii PMS117 P. simiae MEB105 P. trivialis IHBB745 P. fluorescens SBW25 P. fluorescens BRIP34879 P. poae RE*1-14 P. fluorescens BS2 P. fluorescens A506 P. lini DSM768 P. fluorescens LBUM223 P. Libaniensis DSM17149 P. orientalis CFML96-170 P. frederiksbergensis SI8 P. mandelii JR1 R P. brassicacearum DF41 P. brassicacearum NFM421 P. protegens CHA0 P. protegens Pf-5
P. syringae pv. syringae B728a
P. syringae pv. actinidiae CFBP7286 P. syringae pv.tomato str. DC3000 P. putida W619 P. putida KT2440 P. putida HB3267 P. putida S16 P. putida KG4 P. putida GB-1 P. putida F1 P. putida BIRD-1 P. putida ND6 P. entomophila L48 P. stutzeri DSM 10701 P. mendocina NK-01 P. mendocina ymp P. denitrificans ATCC 13867 P. aeruginosa RP73 P. aeruginosa B136-33 P. aeruginosa PA14 P. aeruginosa M18 P. aeruginosa PAO1 P. aeruginosa ATCC 14886 P. aeruginosa LESB58 P. aeruginosa NCGM 1900 E. coli K12 MG1655 100 100 100 77 99 99 77 100 98100 93 100 96 100100 99 100 100 100 100 99 100 98 P. antarctica subgroup p. fluorescens subgroup P . fluorescens comple x P . fluorescens lineage P. madelii subgroup P. corrugata subgroup P. protegens subgroup P . syr ingae g roup P . putida g roup P . aer u ginosa lineage 0.01 P. deceptionensis DSM26521 79
Fig.2–Maximum-likelihoodphylogenetictreebasedon
theconcatenatedsequencesofthegenes
16S-aroE-glnS-gyrB-ileS-rpoD,showingtheevolutionary
relationshipamongselectedmembersofthePseudomonas
genus.ThebacteriaisolatedinAntarcticaareshownin bold,andisolatespresentedinthisstudyareshowninbold andunderlined.ThetreeclearlyshowsthelineagesofP.
aeruginosaandP.fluorescens.Likewise,theP.fluorescens
complexanditssubgroupscanbeclearlydistinguished.All AntarcticPseudomonascanbeclassifiedwithintheP.
fluorescenscomplex.Amongthem,P.deceptionentisseems
toformaseparatesubgroup,andtheisolatePseudomonas
sp.6A1appearsinabranchoftheP.mandeliisubgroup. TogetherwiththespeciesP.antarcticaandP.
extramaustralis,theisolatesPseudomonassp.IB20,
Pseudomonassp.12B3andPseudomonassp.KG01formthe
newsubgroupP.antarctica.
methyl ester, l-serine, and d,l-␣-glycerol phosphate, while being theonly strainable to assimilate adipicacid and p-hydroxyphenylaceticacid.IB20isthe onlystrainunable to assimilated-arabitolandpropionicacid;however,itistheonly oneabletoassimilatel-phenylalanine(weakly),formicacid (weakly)andl-alaninamide.12B3istheonlystrainunableto
assimilate malonicacid,urocanicacid and inosine.Finally, the strain 6A1 is the only one capable of assimilating N-acetyl-d-glucosamine,d-serine(weakly)anddextrin(weakly), whilebeingunabletoassimilateadonitol,d-galacturonicacid, d-glucuronic acid and l-ornithine. Additionally, this strain is unable to hydrolyze l-arginine and gelatin, thereby dif-fering significantly from the other strains studied. Finally, all fourAntarctic strainsareable toassimilated-trehalose, d-galactonic acid lactone, glycyl-l glutamic acid and d,l-carnitine,whilethecontrol,P.fluorescensA506(ATCC31948®
TM),oneofthemostrepresentativestrainsoftheP.fluorescens
complex,isnot.Nevertheless,inthegreatmajorityof phe-notypictests,theresultsfortheAntarcticstrainsaresimilar tothoseforthecontrol,includingtheinabilitytoassimilate nitrate,therebycorroboratingthattheAntarcticisolatesare
partoftheP.fluorescenscomplex.
Discussion
Inthepresentstudy,wedescribefourdifferentAntarctic iso-lates,which weclassified aspartofthegenusPseudomonas
using both molecular and phenotypic strategies. 16S rRNA geneanalysis,MLSAandphenotypictestsusingBIOLOGand API20NEallsuggestthattheAntarcticisolatesPseudomonas
sp.KG01,Pseudomonassp.IB20andPseudomonassp.12B3are serious candidatestoclassifyasnewspeciesofthe genus. Likewise,wedeterminedthattheisolatePseudomonassp.6A1 isthefirst strainofthe speciesP.mandeliitobeisolatedin Antarctica.
TherehasbeenlittleresearchonthegenusPseudomonas
ontheAntarcticcontinent,andonlyeightspecieshavebeen described in the past 20 years. Recent research has thor-oughlyrevisedthetaxonomyandclassificationofthegenus
Pseudomonas,29 dividingitintotwomajorlineages:thatofP.
aeruginosa, which includes almost exclusively bacteria
iso-lated fromclinicalsettings,andthatofP.fluorescens,which containsmostofthegenus’environmentalbacteria, includ-ing thespeciesP.putidaandP.syringae,bothofwhichhave beenwidelystudied.Oneofthemostdiversegroupswithin thislineageiscalledthe“P.fluorescenscomplex”.Ithasbeen furthersubdividedintoninesubgroups,P.protegens,P.
chloro-raphis,P. corrugata,P. koreensis,P. jessenii,P.mandelii, P. fragi,
P.gessardiiandP.fluorescens.29AllAntarcticstrainsstudiedto
datehavebeenclassifiedwithinthiscomplex.Thisisinline withourfindings forthethreecandidatesfornewspecies,
Pseudomonassp.KG01,Pseudomonassp.IB20andPseudomonas
sp. 12B3.Likewise, the isolate Pseudomonas sp.6A1, classi-fiedinthisstudy asastrainofthespeciesP. mandelii,also formspartofthiscomplex.Anoriginalcontributionofour work is that with these three potentially new species, we have identified a novel subgroup, which we have calledP.
antarctica,since it alsoincorporatesthe strains P. antarctica
PAMC27494andP.extremaustralis14-3.Thesewerepreviously considered to be part of the subgroup P. fluorescens.12 The hugely diverse P. fluorescens complex has a wide environ-mentaldistribution, and duetotheir versatilemetabolism, mostofitsmembersplaykeyrolesattheecologicallevel.30,31 It is worth mentioning that some of the species of the P.
Table3–PhenotypiccharacterizationoftheAntarcticbacterialisolates.Thephenotypiccharacterizationwasperformed usingBIOLOGGN2andAPI20NE.+=positivetest,−=negativetest,W=weakreaction,a=API20NE,b=BIOLOG,*=P. fluorescensA506(ATCC® 31948TM).
Test(Activeingredients) Detail KG01 IB20 6A1 12B3 P.fluorescens*
d-mannosea,b Assimilation − + + + +
l-Prolineb Assimilation − + + + +
l-Serineb Assimilation − + + + +
Putrescineb Assimilation − + + + W
PyruvicAcidMethylEsterb Assimilation − + + + W
d,l-␣-GlycerolPhosphateb Assimilation − + W + −
d-Arabitolb Assimilation + − + + +
PropionicAcidb Assimilation + − W + +
Adonitolb Assimilation + + − + −
d-GalacturonicAcidb Assimilation + + − +
-d-GlucuronicAcidb Assimilation + + − +
-l-Ornithineb Assimilation W + − +
-Gelatine,bovineorigena Hydrolysis + + − +
-MalonicAcidb Assimilation + W + − +
UrocanicAcidb Assimilation + + + − +
Inosineb Assimilation + + + − +
Adipicacida Assimilation + − − − −
p-HydroxyPhenylaceticAcidb Assimilation + − − − +
l-Alaninamideb Assimilation − + − − −
l-Phenylalanineb Assimilation − W − − −
FormicAcidb Assimilation − W − − −
N-Acetyl-Dglucosamineb Assimilation − − + − +
d-Serineb Assimilation − − W − −
Dextrin Assimilation − − W − −
d-Trehaloseb Assimilation + + + + −
d-GalactonicAcidLactoneb Assimilation + + + + −
Glycyl-LglutamicAcidb Assimilation W + + + −
d,L-Carnitineb Assimilation + W + + −
SuccinicAcidMono-Methyl-Esterb Assimilation − + − + −
SuccinamicAcidb Assimilation − + − + −
Glucuronamideb Assimilation − + − + −
Glycyl-LasparticAcidb Assimilation − + − W −
i-Erythritolb Assimilation − + − W − Xylitolb Assimilation − W − + − Thymidineb Assimilation − + − + − 2-Aminoethanolb Assimilation − + − + W Tween40b Assimilation − + + + + L-Leucineb Assimilation − + W + W d-Fructoseb Assimilation − W + + + l-Threonineb Assimilation − + W − −
Hydroxy-lProlineb Assimilation − − W + +
Uridineb Assimilation − W − + +
d-Sorbitolb Assimilation + − − + −
producing secondarymetabolites, including antibioticsand
fungicides,whichhelpprotectplantsfrominfectionsbyfungi
andpathogenicbacteria.32,33Inaddition,ithasbeenreported
thatcertainsubgroupsofthiscomplexcanactinthe biore-mediation of heavy metals and pesticides.34 Furthermore, several members of the complex play a vital role in the carboncycle.30 Ithasbeensuggestedthatthe useof mem-bersofthesubgroupP.fluorescensasplantgrowth-promoting rhizobacteria(PGPR)inplantsofagronomicalinterestcould reduce CO2 emissions, thereby mitigating global warming
andtheresultingclimatechange.35Onthisbackground,the biotechnological potential of the three candidates for new species is obvious. Future studies are needed to analyze theirpossible use asplantgrowth-promoting rhizobacteria (PGPR), as well as their use in biocontrol and bioremedia-tion.
Itisworthnotingthatthefourisolateswerecollectedfrom differentenvironments,includingseawater,marinesediment and freshwater, suggesting that the P. fluorescens complex mighthaveanotablecapacityofcolonizationofdiverse envi-ronmentsintheAntarcticcontinent.Thisdoesnotseemto beuncommoninextremeenvironments,and astudy from the Himalayas, another environment ofextreme cold,also reportedthe P.fluorescenscomplextobehighlyrepresented within the genus Pseudomonas.36 Of 336 specimens of the genusPseudomonas,308isolatesbelongedtothiscomplex.Of note,thetemperaturesofthesamplingsitesrangedfrom2to 11◦CintheHimalayas,thusconfirmingthatmembersofthis complexaretolerantofthecold.Futurestudiesexamininga largenumberofAntarcticisolatesareneededtodeterminethe abundanceofthiscomplexontheAntarcticcontinentandto elucidatetheecologicalroleplayedbythesebacteria.
Duetothehighvolumeofwholegenomesequencingdata andmetagenomicanalyses,thedescriptionofnewbacterial speciesisturningincreasinglymorecomplex.MLSAisarobust techniquetodeterminewhetheraparticularisolatebelongs toapreviouslydescribedspecies,orwhetheritrepresentsa newspecies.Several studies haveshown thatthe study of concatenatedsequences ofcarefully selectedhousekeeping genesisapowerfulandreliabletooltoidentifynewspecies withinagenus.37,38Here,wesuggestthat,inadditionto 16S-rRNA,the genesaroE, gyrB,ileS,glnS and rpoDare idealfor useinMLSAinthegenusPseudomonas,sincetheycombine allnecessaryfeatures:theyare“housekeeping”genes,theyare protein-coding,andtheyarenottransmittedhorizontally.37,39 AlthoughpreviousstudieshavesuggestedthatthegeneileS
isnotagoodcandidatetobeusedinMLSA,40 inthisstudy, itprovedtobehighlyefficient.Ontheotherhand,thegene
rpoBiscommonlyusedinMLSA;29,41–44however,despitetrying severalsetsofprimers,wefailedtoobtainauniqueproduct suitableforsequencingfromourAntarcticbacterialisolates. Thegenewasthereforeexcludedfromtheanalyses.Oneofthe strengthsofourworkisthatweperformedMLSAona concate-nateofsixgenes(16S,aroE,gyrS,glnS,ileSandrpoD),asopposed tothethreeorfourgenesusedinmoststudiescharacterizing membersofthegenusPseudomonas.Thisresultedina signif-icantlymorerobustanalysis.Withoutdoubt,oneofthemain problemsofthisstudylayintheshortageofavailable
Antarc-tic Pseudomonas sequences. Sequences for all genes (aroE,
glnS,gyrB,ileSandrpoD)wereonlyavailableforthestrainsP.
antarcticaPAMC27494,P.extremaustralis14-3andP.
deceptionen-sisDSM26521.ThephylogenetictreeobtainedthroughMLSA couldthereforebefurthercomplementedwhenthesequences ofother species becomeavailablein the database. For the 16SrRNAgene,however,sequencesfromallAntarcticspecies wereavailable.
Molecular identification by MLSA has proven to be an effective tool to determine whether a bacterial isolate belongs to a particular species. However, in addition to phylogenetic,phenotypic or serologicalanalyses, the most recommended method to discriminate between bacterial species,and tocharacterizenewbacterial species,is DNA-DNA hybridization.45 This method does, however, have limitations:it takes long to develop and relies on special-izedexpertise,it doesnotdefine thephylogenetic distance betweenspecies,anditisnotcumulative.42Afastandrobust method ofclassification forthe genotypic characterization basedonthesequencesofprotein-codinggenes,MLSAhas allowedresearcherstoclassifyadiverseandpreviously unde-scribed group ofprokaryotesobtained from metagenomics analyses,manyofwhichcannotbecultured.28,42Ithas there-foreevenbeenarguedthatDNA-DNAhybridizationanalysis may no longer be necessary for the description ofa new species.42,43,46
Inconclusion,ourresultsstronglysuggestthatthe Antarc-ticisolatesPseudomonassp.KG01,Pseudomonassp.IB20and
Pseudomonassp.12B3are candidatesfornewspeciesofthe
genusPseudomonas,thatallofthembelongtotheP.fluorescens
complex,andthat,togetherwiththeAntarcticstrainsP. antarc-ticaPAMC27494andP.extremaustralis14-3,theyformpartofthe newsubgroup“P.antarctica”.Futurestudiesincluding metage-nomicsstudies,are neededtodeterminetheabundanceof
membersofthegenusPseudomonasinAntarcticaandto elu-cidatetheirpossibleecologicalroleonthecoldcontinent.
Funding
Thiswork wassupportedbytheDirección deInvestigaciónof thePontificia UniversidadCatólica deValparaíso[ProjectDI 037.431/2015],byanINACHPhDscholarship[DT02-13],andby
theProgramadeInvestigaciónAsociativa[PIUAs-PUCV
037.293-2015].
Conflicts
of
interest
Theauthorsdeclarethattheyhavenoconflictofinterest.
Acknowledgments
WewouldliketothankDr.MarceloGonzálezoftheChilean AntarcticInstitute(INACH)forprovidingtheAntarctic bacte-rialisolates.
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2018.02.005.
r
e
f
e
r
e
n
c
e
s
1.CowanDA,TowLA.EndangeredAntarcticenvironments.
AnnuRevMicrobiol.2004;58:649–690.
2.SmithJ,TowL,StaffordW,CaryC,CowanD.Bacterial
diversityinthreedifferentAntarcticcolddesertmineral
soils.MicrobEcol.2006;51(4):413–421.
3.TytgatB,VerleyenE,ObbelsD,etal.Bacterialdiversity
assessmentinAntarcticterrestrialandaquaticmicrobial
mats:acomparisonbetweenbidirectionalpyrosequencing
andcultivation.PLOSONE.2014;9(6):e97564.
4.LoperenaL,SoriaV,VarelaH,etal.Extracellularenzymes
producedbymicroorganismsisolatedfrommaritime
Antarctica.WorldJMicrobiolBiotechnol.2012;28(5):2249–2256.
5.CavicchioliR.MicrobialecologyofAntarcticaquaticsystems.
NatRevMicrobiol.2015;13(11):691–706.
6.Martínez-RosalesC,FullanaN,MustoH,Castro-SowinskiS.
AntarcticDNAmovingforward:genomicplasticityand
biotechnologicalpotential.FEMSMicrobiolLett.
2012;331(1):1–9.
7.AnzaiY,KimH,ParkJY,WakabayashiH,OyaizuH.
Phylogeneticaffiliationofthepseudomonadsbasedon16S
rRNAsequence.IntJSystEvolMicrobiol.2000;50Pt
4(4):1563–1589.
8.RojoF.CarboncataboliterepressioninPseudomonas:
optimizingmetabolicversatilityandinteractionswiththe
environment.FEMSMicrobiolRev.2010;34(5):658–684.
9.PalleroniNJ.ThePseudomonasstory.EnvironMicrobiol.
2010;12(6),1377-1383.10.
10.PuchałkaJ,OberhardtMA,GodinhoM,etal.Genome-scale
reconstructionandanalysisofthePseudomonasputida
KT2440metabolicnetworkfacilitatesapplicationsin
11.SpiersAJ,BucklingA,RaineyPB.ThecausesofPseudomonas
diversity.Microbiology.2000:2345–2350.
12.ReddyGSN,MatsumotoGI,SchumannP,StackerbrandtE,
ShivajiS.PsychrophilicpseudomonadsfromAntarctica:
Pseudomonasantarticasp.nov.,Pseudomonasmeridianasp.nov. andPseudomonasproteolyticasp.nov.IntJSystEvolMicrobiol.
2004;54(3):713–719.
13.BozalN,MontesMJ,MercadéE.Pseudomonasguineaesp.nov.,
anovelpsychrotolerantbacteriumfromanAntarctic
environment.IntJSystEvolMicrobiol.2007;57(11):2609–2612.
14.LópezN,PettinariMJ,StackebrandtE,etal.Pseudomonas
extremaustralissp.nov.,apoly(3-hydroxybutyrate)producer
isolatedfromanAntarcticenvironment.CurrMicrobiol.
2009;59(5):514–519.
15.CarriónO,Mi ˜nana-GalbisD,MontesMJ,MercadéE.
Pseudomonasdeceptionensissp.nov.,apsychrotolerant
bacteriumfromtheAntarctic.IntJSystEvolMicrobiol.
2011;61(Pt10):2401–2405.
16.KosinaM,BartákM,Maˇsla ˇnováI,etal.Pseudomonasprosekii
sp.nov.,anovelpsychrotrophicbacteriumfromAntarctica.
CurrMicrobiol.2013;67(6):637–646.
17.KosinaM, ˇSvecP, ˇCernohlávkováJ,etal.Descriptionof
Pseudomonasgregormendeliisp.nov.,anovelpsychrotrophic
bacteriumfromJamesRossIsland,Antarctica.CurrMicrobiol.
2016;73(1):84–90.
18.SchleiferKH.Classificationofbacteriaandarchaea:past,
presentandfuture.SystApplMicrobiol.2009;32(8):533–542.
19.SchleiferK-H,AmannR,Rosselló-MóraR.Taxonomyinthe
ageofgenomics.Introduction.SystApplMicrobiol.
2015;38(4):207–208.
20.TindallBJ,Rosselló-MóraR,BusseH-J,LudwigW,KämpferP.
Notesonthecharacterizationofprokaryotestrainsfor
taxonomicpurposes.IntJSystEvolMicrobiol.2010;60(Pt
1):249–266.
21.AltschulSF,GishW,MillerW,MyersEW,LipmanDJ.Basic
localalignmentsearchtool.JMolBiol.1990;215(3):403–410.
22.WinsorGL,LamDKW,FlemingL,etal.Pseudomonas
GenomeDatabase:improvedcomparativeanalysisand
populationgenomicscapabilityforPseudomonasgenomes.
NucleicAcidsRes.2011;39(suppl1):D596–D600.
23.KumarS,StecherG,TamuraK.MEGA7:Molecular EvolutionaryGeneticsAnalysisVersion7.0forBigger Datasets.
24.AndreaniNA,MartinoME,FasolatoL,etal.Trackingtheblue:
aMLSTapproachtocharacterisethePseudomonasfluorescens
group.FoodMicrobiol.2014;39:116–126.
25.PavlovMS,LiraF,MartínezJL,OlivaresJ,MarshallSH.Draft
genomesequenceofAntarcticPseudomonasspStrainKG01
withfullpotentialforbiotechnologicalapplications.Genome
Announc.2015;3(4.).
26.BölterM.Ecophysiologyofpsychrophilicand
psychrotolerantmicroorganisms.CellMolBiol
(Noisy-le-grand).2004;50(5):563–573.
27.KejnaM.AirtemperatureonKingGeorgeIsland,South
ShetlandIslands,Antarctica.PolPolarRes.1999;20(3):183–201.
28.GeversD,CohanFM,LawrenceJG,etal.Opinion:
re-evaluatingprokaryoticspecies.NatRevMicrobiol.
2005;3(9):733–739.
29.Garrido-SanzD,Meier-KolthoffJP,GökerM,etal.Genomic
andgeneticdiversitywithinthePseudomonasfluorescens
ComplexVinatzerBA,ed.PLOSONE.2016;11(2):e0150183.
30.RemoldSK,Purdy-GibsonME,FranceMT,HundleyTC.
PseudomonasputidaandPseudomonasfluorescensspecies
grouprecoveryfromhumanhomesvariesseasonallyandby
environment.PLOSONE.2015;10(5):
e0127704.
31.LiuX,EcarnotM,KontroMH.Thephysicochemical
conditionsofisolationsourcedeterminetheoccurrenceof
Pseudomonasfluorescensgroupspecies.AnnMicrobiol.
2015;65(4):2363–2377.
32.GrossH,LoperJE.Genomicsofsecondarymetabolite
productionbyPseudomonasspp.NatProdRep.
2009;26(11):1408–1446.
33.WellerDM,LandaBB,MavrodiOV,etal.Roleof
2,4-diacetylphloroglucinol-producingfluorescent
Pseudomonasspp.inthedefenseofplantroots.PlantBiol.
2007;9(1):4–20.
34.WasiS,TabrezS,AhmadM.UseofPseudomonasspp.forthe
bioremediationofenvironmentalpollutants:areview.
EnvironMonitAssess.2013;185(10):8147–8155.
35.NieM,BellC,WallensteinMD,PendallE.Increasedplant
productivityanddecreasedmicrobialrespiratoryClossby
plantgrowth-promotingrhizobacteriaunderelevatedCO2.
SciRep.2015;5:9212.
36.NegiYK,PrabhaD,GargSK,KumarJ.Geneticdiversity
amongcold-tolerantfluorescentPseudomonasisolatesfrom
IndianHimalayasandtheircharacterizationforbiocontrol
andplantgrowth-promotingactivities.JPlantGrowthRegul.
2010;30(2):128–143.
37.KonstantinidisKT,TiedjeJM.Towardsagenome-based
taxonomyforprokaryotes.JBacteriol.2005;187(18):
6258–6260.
38.MartensM,DawyndtP,CoopmanR,GillisM,DeVosP,
WillemsA.Advantagesofmultilocussequenceanalysisfor
taxonomicstudies:acasestudyusing10housekeeping
genesinthegenusEnsifer(includingformerSinorhizobium).
IntJSystEvolMicrobiol.2008;58(Pt1):200–214.
39.MuletM,LalucatJ,García-ValdésE.DNAsequence-based
analysisofthePseudomonasspecies.EnvironMicrobiol.
2010;12(6):1513–1530.
40.AndreaniNA,MartinoME,FasolatoL,etal.Trackingtheblue:
a{MLST}approachtocharacterisethePseudomonas fluorescensgroup.FoodMicrobiol.2014;39:116–126.
41.GomilaM,Pe ˜naA,MuletM,LalucatJ,García-ValdésE.
PhylogenomicsandsystematicsinPseudomonas.Front
Microbiol.2015;6:214.
42.BennasarA,MuletM,LalucatJ,García-ValdésE.
Pseudo-MLSA:adatabaseformultigenicsequenceanalysis
ofPseudomonasspecies.BMCMicrobiol.2010;10:118.
43.GlaeserSP,KämpferP.Multilocussequenceanalysis(MLSA)
inprokaryotictaxonomy.SystApplMicrobiol.
2015;38(4):237–245.
44.Alvarez-PérezS,deVegaC,HerreraCM.Multilocussequence
analysisofnectarpseudomonadsrevealshighgenetic
diversityandcontrastingrecombinationpatterns.PLOSONE.
2013;8(10):e75797.
45.StackebrandtE,FrederiksenW,GarrityGM,etal.Reportof
thead-hoccommitteeforthere-evaluationofthespecies
definitioninbacteriology.IntJSystEvolMicrobiol.2002;52(Pt
3):1043–1050.
46.GeversD,CoenyeT.Phylogeneticandgenomicanalysis.Man
