Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable

15 Pages·2017·3.26 MB·English

FEMSYeastResearch,17,2017,fox025
doi:10.1093/femsyr/fox025
AdvanceAccessPublicationDate:2May2017
ResearchArticle
RESEARCH ARTICLE
Genome sequence of the highly weak-acid-tolerant D
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Zygosaccharomyces bailii IST302, amenable to genetic loa
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manipulations and physiological studies fro
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Margarida Palma1, Martin Mu¨nsterko¨tter2, Joa˜o Pec¸a1, Ulrich Gu¨ldener2,3 s://a
and Isabel Sa´-Correia1,∗ ca
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1InstituteforBioengineeringandBiosciences,DepartmentofBioengineering,InstitutoSuperiorTe´cnico, ic.o
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UniversidadedeLisboa,1049-001Lisbon,Portugal,2InstituteofBioinformaticsandSystemsBiology, p
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HelmholtzZentrumMu¨nchen,GermanResearchCenterforEnvironmentalHealth(GmbH),Ingolsta¨dter m
Landstrasse1,NeuherbergD-85764,Germanyand3ChairofGenome-orientedBioinformatics,TUMSchoolof /fe
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LifeSciencesWeihenstephan,TechnicalUniversityofMunich,85354Freising,Germany sy
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∗Correspondingauthor:InstituteforBioengineeringandBiosciences,DepartmentofBioengineering,InstitutoSuperiorTe´cnico,UniversidadedeLisboa, rtic
1049-001Lisbon,Portugal.Tel:[phone];Fax:[phone];E:mail:[email protected] le
Onesentencesummary:AnnotatedgenomesequenceofZygosaccharomycesbailiiIST302,ahaploidstrainamenabletogeneticmanipulationsand -ab
physiologicalstudies,andcomparativegenomicanalysiswithZ.bailiiandZ.bailii-derivedhybridstrains,SaccharomycescerevisiaeandZ.rouxii. stra
Editor:JensNielsen c
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ABSTRACT x
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Zygosaccharomycesbailiiisoneofthemostproblematicspoilageyeastspeciesfoundinthefoodandbeverageindustry /3
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particularlyinacidicproducts,duetoitsexceptionalresistancetoweakacidstress.Thisarticledescribestheannotationof 86
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thegenomesequenceofZ.bailiiIST302,astrainrecentlyproventobeamenabletogeneticmanipulationsandphysiological 5
0
studies.TheworkwasbasedontheannotatedgenomesofstrainISA1307,aninterspecieshybridbetweenZ.bailiianda b
y
closelyrelatedspecies,andtheZ.bailiireferencestrainCLIB213T.TheresultinggenomesequenceofZ.bailiiIST302is g
u
distributedthrough105scaffolds,comprisingatotalof5142genesandasizeof10.8Mb.ContrastingwithCLIB213T,strain es
IST302doesnotformcellaggregates,allowingitsmanipulationinthelaboratoryforgeneticandphysiologicalstudies. t o
n
ComparativecellcycleanalysiswiththehaploidanddiploidSaccharomycescerevisiaestrainsBY4741andBY4743, 0
2
respectively,suggeststhatZ.bailiiIST302ishaploid.Thisisanadditionaltraitthatmakesthisstrainattractiveforthe A
p
functionalanalysisofnon-essentialgenesenvisagingtheelucidationofmechanismsunderlyingitshightolerancetoweak ril 2
acidfoodpreservatives,ortheinvestigationandexploitationofthepotentialofthisresilientyeastspeciesascellfactory. 0
1
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Keywords:Zygosaccharomycesbailii;genomesequence;foodspoilageyeasts;weakacidtolerance;cellularaggregation
INTRODUCTION
This yeast species ability to cause spoilage results from its
remarkable capacity to tolerate stress induced by weak acids
Zygosaccharomycesbailiiisfoundamongthespoilageyeaststhat
widely used as fungistatic preservatives, such as acetic, ben-
frequentlyaffectacidicfoodsandbeverages,particularlymay-
zoicandsorbicacids(Ferreira,Loureiro-DiasandLoureiro1997;
onnaise, salad dressings, soft drinks, fruit concentrates and
Stratford et al. 2013). In fact, Z. bailii may proliferate in the
dairyproducts(reviewedinStratford2006;Sa´-Correiaetal.2014).
Received:9March2017;Accepted:27April2017
(cid:3)C FEMS 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedthe
originalworkisproperlycited.Forcommercialre-use,[email protected]
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2 FEMSYeastResearch,2017,Vol.17,No.4
presence of weak acid concentrations above those legally weakacidfoodpreservativesortotheinvestigationofZ.bailii
permitted in food products (Stratford 2006). Moreover, Z. bailii potentialascellfactory.
isabletotoleraterelativelyhighethanolconcentrations(Kala-
thenos, Sutherland and Roberts 1995) and high temperatures MATERIALSANDMETHODS
(Martorelletal.2007),andtovigorouslyfermentsugars(Thomas
andDavenport1985),beingconsideredoneofthemostsignif- Strainsandgrowthmedium
icant threats in wine industry (Loureiro and Malfeito-Ferreira
2003). Like other members of the Zygosaccharomyces genus, Z. TheprototrophicstrainsZygosaccharomycesbailiiIST302(Palma
bailiiisfructophilic,i.e.prefersfructoseoverglucosewhenboth etal.2015),theneotypestrainZ.bailiiATCC58445T(=CLIB213T)
sugarsarepresentinthegrowthmedium(Sousa-Diasetal.1996; (Galeoteetal.2013)andstrainISA1307,aZ.bailii-derivedinter-
Pinaetal.2004).Therefore,acidicfoodandbeveragesthatcon- specieshybridstrain,wereusedinthiswork.Saccharomycescere-
tainfructoseareatparticularriskofspoilage,giventhatZ.bailii visiaeBY4741(genotypeMATa;his3(cid:2)1;leu2(cid:2)0;lys2(cid:2)0;ura3(cid:2)0)
specificgrowthratesandethanolproductionarehigherduring and BY4743 (MATa/α his3(cid:2)1/his3(cid:2)1 leu2(cid:2)0/leu2(cid:2)0 LYS2/lys2(cid:2)0
growthinthepresenceoffructosecomparedwithglucose.Al- met15(cid:2)0/MET15 ura3(cid:2)0/ura3(cid:2)0) were acquired from the Do
w
tphrooduughctsZ.cboanitliaiinisinugnathbilsestougdairreacrtelyamlsoetsaubsocleizpetisbulecrtoosesp,oaicliadgiec EriUchROYSPCDARgrFocwotlhlecmtieodni.uYmeatshtasttcraoinntsaiwnse,repemraliintetra,in2e%dignluscoolside nloa
d
bythisyeastspeciesgiventhat,atlowpH,sucrosecanbehy- (Merck,Kenilworth,NewJersey),1%yeastextract(DifcoLabo- ed
drolyzedinfructoseandglucose(PittandHocking2009).These ratories,Detroit,Michigan)and2%peptone(DifcoLaboratories, fro
traitsofZ.bailiiphysiologyresultinannuallossesofmillionsof Detroit,Michigan)and2%agar.Forsusceptibilityassays,yeast m
dollars(PittandHocking2009)makingthisspeciesaseriouscon- strains ISA1307, IST302 and BY4741 were cultured in MM4 http
cZe.rbnaiilniifhoaosdailnsdougsatriny.eRdeagtatrednlteisosnoffoirtsitascptiovtietyntaisalspinoiblaiogteeycheansot-, lwiqituhidoumteadmiuinmotahcaitdscoonrta(NinHs4,)p2SeOrl4it(rDei:f1c.o7Lgaybeoarsattonriiteros,geDnetbraosite, s://ac
logicalprocesses,beingproposedasanewcellfactoryforthe Michigan),20gglucose(Merck,Kenilworth,NewJersey),2.65g ad
productionoforganicacidsandheterologousproteins(Brand- (NH4)2SO4 (Merck), 20 mg methionine, 20 mg histidine, 60 mg em
uardietal.2004;Datoetal.2010). leucine and 20 mg uracil (all from Sigma-Aldrich, St. Louis, ic.o
AlthoughthereareplasmidvectorsavailableforZ.bailiige- Missouri). up
neticmanipulation(Branduardi,DatoandPorro2014),thege- .co
m
neticengineeringofthisspecieshasbeenhinderedbythelack TaxonomicidentificationofstrainIST302 /fe
ofstablehaploidstrains(MollapourandPiper2001;Rodrigues m
etal.2003)and,todate,onlyoneauxotrophicmutantwascon- ThenucleotidesequencesofthegenesfromstrainIST302en- sy
structed (Dato et al.2010). However, the parental strainof the codingtheRNApolymeraseIIlargestsubunitRpb1andtheRNA r/a
derivedmutant,previouslyconsideredasZ.bailii,wastaxonom- polymeraseIIsecond-largestsubunitRpb2,previouslyconsid- rtic
icallyreallocatedtoZ.parabailii(Suhetal.2013).Moreover,only ered relevant for the identification of Z. bailii species among le-a
recentlyitwaspossibletohaveaccesstothegenomesequence closely related species such as Z. parabailii and Z. pseudobailii bs
ofaZ.bailiistrain,thereferencestrainCLIB213T(Galeoteetal. (Suhetal.2013),werecomparedwiththenucleotidesequences tra
c
2013) and to Z. bailii-derived hybrid strain ISA1307 (Mira et al. ofRPB1andRPB2fromseveralZ.bailii,Z.parabailiiandZ.pseu- t/1
2014).Thishybridstrainhasbeenusedinseveralstudiesaim- dobailiistrainsasdescribedpreviously(Suhetal.2013).Thenu- 7/4
ingatunderstandingZ.bailiiphysiology,inparticularthemech- cleotidesequencesofRPB1andRPB2genesfromstrainIST302 /fo
anismsunderlyingaceticacidtoleranceandtoxicity(Sousaetal. wereobtainedbywhole-genomesequencing,asdescribedhere- x0
2
1998;Rodriguesetal.2001;Guerreiro,MiraandSa´-Correia2012; after. 5/3
Palmaetal.2015),butfollowingitsgenomesequencingandan- 78
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notationISA1307emergedasaninterspecieshybridbetweenZ. Cellcycleanalysisandquantificationof 3
5
bailiiandacloselyrelatedspecies(Miraetal.2014).Ontheother ZygosaccharomycesbailiiIST302totalgenomicDNAby 0 b
hand,Z.bailiiCLIB213T isextremelydifficulttomanipulatein y
flowcytometry g
thelaboratorymainlyduetoitsstrongaggregationphenotype u
e
awanneddre,Sftsoruragttfghoeirssdtre2ed0a1sao1s)n.b,MesotttrreaerinrresecpCernBetSslye6,n8ot5ua(rtNilvaCebYsoCroaf5t6toh3ry)eahsnapdsecbNieeCesYn(CJwa1mo7r6ek6s- Zg=ryo0gw.o6st±ahc0cm.h1a)e.rdAoimutoymctea,slaotbfa3i10li0◦iC7IS,cuTen3ll0tsi2lwmceeriledls-hewaxrepvroeensbetaentdtcihbaylcpcuheltnaustrreeifd(uOgiDna6tY0i0oPnnmD, st on 02
ingwithZ.bailiiIST302,whichwasfoundtobemoreamenableto washedtwicewithdistilledwaterandfixedatleastonenight A
p
geneticandphysiologicalmanipulationsthanstrainCLIB213T in 1 mL of 70 % ethanol (vol/vol). The remaining cell culture ril 2
(Palmaetal.2015,2017).Therefore,thesequencingofitsgenome wasinducedtoarrestattheG1phasewith8-hydroxyquinoline 0
1
sequencewasconsideredthenextstep. (Sigma-Aldrich, St. Louis, Missouri) at a final concentration of 9
Inthiswork,weprovidemolecularevidencessupportingthe 100μg/mLfor24horattheG2phasewithnocodazole(Sigma-
taxonomicidentificationofstrainIST302asZ.bailii,aswellas Aldrich,St.Louis,Missouri)atafinalconcentrationof5μg/mL
itsgenomesequenceandannotationandcomparativeanalysis for3h.Arrestedcellswereharvested,washedandfixedwith
withCLIB213Tandotherrelevantyeastspecies.Thisstudyalso ethanol,aspreviouslydescribed.Quantificationoftotalgenomic
envisagedtheconfirmationofthehaploidnatureofthegenome, DNAfromZ.bailiiIST302byflowcytometrywasperformedus-
thecharacterizationofrelevanttolerancephenotypesandthe ing a SYBR Green I-based staining protocol, as described be-
search for potential molecular targets involved in Z. bailii re- fore(Fortunaetal.2000).Cellsfixedwithethanolwerecollected
markableweakacidtoleranceandtheaggregationphenotype. bycentrifugation,washedwith50mMofsodiumcitratebuffer
Collectively, the information gathered in this study points Z. (pH7.5)andresuspendedin750μLofthissamebuffersupple-
bailiiIST302strainashighlyattractiveforthefunctionalanal- mentedwith250μLofRNAseA(1mg/mL)(Sigma-Aldrich,St.
ysisofnon-essentialZ.bailiigenesandtheelucidationofmech- Louis,Missouri).After1hofincubationat50◦C,50μLofpro-
anisms underlying its high tolerance to acetic acid and other teinaseK(20mg/mL)(NZYtech,Lisbon,Portugal)wereaddedto
Palmaetal. 3
at37◦Cinasolutioncontaining0.05MEDTA,1mg/mLofpro-
teinaseK(NZYtech,Lisbon,Portugal),1mg/mLRNAse(Sigma-
Aldrich, St. Louis, Missouri) and 1 % sodium-N-lauryl sarcosi-
nate. The plugs were washed with EDTA 50 mM at 37◦C, 100
rpm,during15minsandincubatedatroomtemperaturefor1
hinTEbuffer,pH8.0.PFGEwasperformedinaCHEF-Pharma-
ciaLKBGeneNavigatorsystem.PFGEgelswererunin0.5%Tris
borate–EDTAbufferat13◦Cwithavoltageof3V/cmandswitch
timesof600sfor48hand300sfor96hina1%pulsed-fieldgel
agarose(NZYtech,Lisbon,Portugal).
Genomesequencing,assemblyandannotation
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ZygosaccharomycesbailiiIST302genomesequencingandassem- o
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blywasperformedasdescribedpreviously(Miraetal.2014)using nlo
Figure1.NucleotidevariationinRBP1andRBP2genesfromstrainIST302when awhole-genomeshotgunapproachthatexploredpaired-endIl- ad
comparedwiththepartialsequenceofRBP1andRBP2fromZ.bailii,Z.parabailii luminasequencing(IlluminaHiseq2000system,CDGenomics, ed
andZ.pseudobailiistrains.NucleotidevariationswerecalculatedusingEMBOSS New York, USA). Short reads were assembled using SOAPden- fro
WaterlocalalignmenttoolfromEMBL-EBIpackages.Valuesarepresentedasper- m
ovo (http://soap.genomics.org.cn) (Li et al. 2010). The obtained
centageofnucleotidevariationsandarethemeanofresultsobtainedbycom- h
paringRBP1andRBP2sequencesfromstrainIST302(ZBIST˙2664andZBIST4878, scaffoldsweresequentiallyorderedbasedontheirlevelofsyn- ttp
rpeasrpabeacitliiviealnyd)wtwitohstthreaiirnhsoomfZo.lopgsesufdroobmailsiiixacsctroaridnisngoftoZ.Sbuahiliei,tsael.v(e2n01s3tr).ainsofZ. tbeanilyii-tdoetrhiveedgehnyobmriedssotfraZi.nroIuSxAii13C0B7Sa7n3d2TZ,.S.bcaeirlieivCisLiaIBeS221838Tc.,TZo. s://ac
a
predictgenesonthescaffolds,twoabinitiogenepredictoral- d
e
gorithmswereused:GeneMark-SandGenMark-ESversion2.3 m
thecellsuspensionandthemixturewasincubatedat50◦Cfor (Ter-Hovhannisyan et al. 2008). Gene models differently pre- ic.o
anotherhour.Cellsweresubsequentlystainedovernightat4◦C dictedbythealgorithmsweremanuallycuratedbasedonthe up
usingSYBR(cid:2)R GreenIatafinalconcentrationof500-folddilu- structureobtainedforZ.rouxiiCBS732T,S.cerevisiaeS288c,Z. .co
tionofthecommercialSYBR(cid:2)R GreenI(LifeTechnologies,Carls- bailii-derived hybrid strain ISA1307 and Z. bailii CLIB 213T ho- m/fe
bad,California).TritonX-100wasaddedtothestainedsamples mologs.ThegenomeswereanalyzedusingthePEDANTsystem m
atafinalconcentrationof0.25%(v/v).Sampleswerevortexed, (Walter et al. 2009) to allow comparative feature analysis. To sy
sonicatedinaBransonSonifier250(3-4pulseswithatoutput avoidmisleadingorthologinformationbasedonsimilarityand r/a
powerof3and30%dutycycle)andanalyzedinaFACScalibur bidirectionalbesthits,QuartetS(Yuetal.2011)wasappliedto rtic
le
(BectonDickson,FranklinLakes,NewJersey)flowcytometer.A retrieveareliableorthologmatrix,whichwasusedforcompar- -a
b
minimumof50000cellspersamplewereacquiredatlowflow ativeanalysisofthegenesencodingmultidrug/multixenobiotic s
rateusingCellQuestsoftware(BectonDickson,FranklinLakes, resistance(MDR/MXR)transporters,ofthegenesinvolvedinlife tra
c
NewJersey).Analysisoftheacquireddatawasperformedus- cycleandmeiosisandofthegenesinvolvedincellularaggre- t/1
ingtheFlowJo(cid:2)R v10.0.8software.Thefluorescenceintensities gation in Z. bailii IST302, Z. bailii CLIB 213T, Z. rouxii CBS732T 7/4
ofthecellcyclepeakG0/G1forS.cerevisiaeBY4741andBY4743 andS.cerevisiaeS288c.Theidentificationofthecentromereswas /fo
x
wereusedtobuildacalibrationcurveinordertoestimatethe performedmanuallybasedonpreviouslyidentifiedpointcen- 0
2
genomesizeofZ.bailiiIST302strain.Fluorescenceintensitiesfor tromeresequencesandonthecollinearconservationofflank- 5/3
thehybridstrainISA1307wereusedforcomparisonpurposes. inggenesofhemiascomycetousyeasts,inparticularofZ.rouxii 78
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(Pribylovaetal.2007;Soucietetal.2009). 3
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The genome sequence and annotation of Z. bailii IST302 0
KaryotypingofZygosaccharomycesbailiiIST302strain b
has been deposited in ENA (http://www.ebi.ac.uk/ena/ y
g
DNA for pulsed field gel electrophoresis (PFGE) was prepared data/view/FUGC01000001-FUGC01000105). u
e
iSanttr2ap5ilnu0sgrspISmaTs3i0np2rYeavPniDodugCsrLloyIwBdt2he1s3mcTreibwdeieudrme(Mc.uYalertiainvsagtteceleedlloasvnwedrenLrieygdhhatalrlavt2e03s00te6◦Cd). Susceptibilityassays st on 02
bycentrifugationinordertoobtainacellpelletof∼50μLvol- SusceptibilityofZ.bailiiIST302,ISA1307hybridstrainandS.cere- A
p
uanmde.reCseulslppeenlldeetsdwiner1e00wμasLhoefd0t.w05icMewEDitThA0,.0p5HM8.0E.DPTlAug,spHwe8r.e0 vseissisaeedBbYy47s4p1ottoassseavyesr.aYlegarsotwctehllisnwhiebrietogrryowconminpoMunMd4smweadsiuams- ril 20
1
formedbymixingthesuspensionofcellswithatotalof50μL attheappropriatepHuntilexponentialphase(OD600nmof0.5± 9
ofaZymolyasesolution[46μLSCEbuffer(1Msorbitol,0.1M 0.05)andthenreinoculatedatanOD600nm of0.05,in50mLof
sodiumcitrate,and60mMEDTA),1.5μLzymolyase(NZYtech, freshmedium.WhentheculturesreachedanOD600nm of0.5±
Lisbon,Portugal)and2.5μLβ-mercaptoethanol]wasaddedto 0.05,cellswereresuspendedinsterilewatertoobtainsuspen-
eachsamplefollowedbytheadditionof300μLoflow-melting sionswith∼5×105 cell/mL.Thesecellsuspensionsandthree
pointagarosesolution(1%lowmeltingpointagarosein10mL subsequentdilutions(1:5;1:10;1:20)wereappliedas4μLspots
EDTA 0,125 mM, pH 7.0). The mixture was pipetted intowells onMM4platessupplementedwithadequateconcentrationsof
fromtheplugmoldsandplacedat4◦Cduring30min.Theplugs acetic,benzoic,formic,lactic,propionicorsorbicacids;ofthe
werethencarefullyremovedandthenincubatedovernightat herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-
37◦C,50rpm,inETBbuffer(EDTA0.05M,pH8.0,Tris-HCl0.1M, chlorophenoxyaceticacid(MCPA)oralachlor;andofthefungi-
pH8.0and5%(vol/vol)β-Mercaptoethanol).Afterthisincuba- cidesitraconazole,fluconazole,miconazole,tioconazole,clotri-
tionstep,plugswerewashedthreetimesinTEbuffer(10mM mazoleormancozeb.Agarplateswereincubatedat30◦Cfor3
Tris,pH8.0and1mMEDTA,pH8.0)andincubatedovernight days.DuetothedifferentpKaoftheacids,yeastsusceptibility
4 FEMSYeastResearch,2017,Vol.17,No.4
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Figure2.ZygosaccharomycesbailiiIST302ismoretoleranttoweakacidfoodpreservativesandherbicides,butnottoantifungals,whencomparedwithS.cerevisiae
BY4741.GrowthofZ.bailiiIST302,S.cerevisiaeBY4741andthehybridstrainISA1307wascomparedbyspotassaysinMM4mediumsupplemented,ornot,withthe
appropriateconcentrationsof(A)weakorganicacids,(B)herbicidesand(C)antifungaldrugs.Threecontrolplatesareshown,withandwithoutthesolventsused
todissolvethedifferentdrugswhennecessary.Cellsuspensionswith∼5×105cells/mL(lanea)andsubsequentdilutionsof1:5,1:10and1:20(lanesb,candd,
respectively)werespottedontothesurfaceofMM4solidmedium.Theimagesshownweretakenafter3daysofincubationat30◦Candarerepresentativeofatleast
threeindependentexperiments.
Palmaetal. 5
centrifugationat8000rpmandsuspendedin0.05MEDTA(pH
8.0),followedbyvigorousagitation.Thesecellswereobserved
onaZeiss(cid:2)R Axioplanmicroscope(×400magnification).Cells
suspendedin0.05MEDTA(pH8.0)weresubsequentlywashed
twicewithsterilewater,suspendedin0.01MCaCl2,vigorously
mixedandfinallyobservedunderthemicroscope.
RESULTSANDDISCUSSION
TaxonomicidentificationofstrainIST302
ThemoleculardifferencesregisteredamongZygosaccharomyces
bailiiandcloselyrelatedspecies(Suhetal.2013),togetherwith
D
thegenomiccomplexityofthehybridstrainISA1307(Miraetal. o
w
2014) that was first considered as Z. bailii, support the notion n
oftheremarkablegenomicdiversityamongZ.bailiiandZ.bailii loa
d
closelyrelatedspecies.Thisfactrecommendedthecarefulex- e
d
aminationofthetaxonomicpositionandgenomiccharacteri- fro
zationofIST302strain.Thepreliminarytaxonomicidentifica- m
tionofthisstrainasZ.bailiiwasbasedonthecomparisonofthe http
large subunit 26S ribosomal DNA partial sequence with other s
DNAsequencesfromZ.bailiistrainsusingtheBasicLocalAlign- ://a
c
ment Search Tool (BLAST) of the National Center for Biotech- a
d
nology Information (NCBI) (Palma et al. 2015). However, con- em
sideringthereallocationofyeaststrainsformerlyidentifiedas ic
.o
Z.bailiitothenewspeciesZ.parabailiiandZ.pseudobailii(Suh u
p
etal.2013),wehavealsocomparedthepartialsequencesofRBP1 .c
o
and RBP2 genes from strain IST302 with their homologs from m
severalstrainsofthethreespecies,asrecommendedbySuhetal. /fe
m
(2013).Resultsshowthatnucleotidesequencevariationpercent- s
y
ageislowwhenRBP1andRBP2nucleotidesequencesfromstrain r/a
IST302arecomparedwiththecorrespondingsequencesfromZ. rtic
bailiistrains(0.1%–0.7%),whereassignificantsequencevariation le-a
Figure3.KaryotypeprofilesofZ.bailiiIST302andCLIB213Tstrains.Separationof isobservedcomparedwithZ.parabailii(3.8%–4.1%)andZ.pseu- bs
thechromosomeswasperformedbyPFGEasdescribedinMaterialsandMethods dobailii(5.8%–6.8%)strains(Fig.1).Collectively,theseresultscon- tra
c
section.ThesizeofZ.bailiiIST302andCLIB213Tchromosomeswasestimated firmIST302strainasZ.bailii. t/1
fromacomparisonwiththesizeofthechromosomesfromHansenulawingeithat 7
wereusedasthemolecularsizestandards(M). ToleranceofZygosaccharomycesbailiiIST302toweak /4/fo
x
acids,drugsandpesticides 02
toacetic,benzoic,sorbicandpropionicacidswastestedinMM4 5/3
mediumatpH4.0,whereassusceptibilitytoformicandlactic The tolerance of Z. bailii IST302 to several compounds was 7
8
acidswastestedinMM4atpH3.5. assessed by spot assays (Fig. 2). Compared with Saccha- 63
5
romyces cerevisiae BY4741, Z. bailii IST302 is much more 0
b
SporulationofZygosaccharomycesbailiiIST302 taonlderafnotrmtioc waceiadks),actoidsth(eacleiptiocp,hbileicnzowiec,aksoarcbiidc, hperrobpicioidneics y gu
e
ZygosaccharomycesbailiiIST302cellsweregrowninapresporu- 2,4-D (2,4-D-dichlorophenoxyacetic acid) and MCPA (methyl- s
lationmedium(0.8%yeastextract,0.3%peptone,10%glucose chlorophenoxyaceticacid)andalsototheherbicidealachlor,but t on
and2%agar)during2daysbeforebeingtransferredtosporula- nottotheantifungalstested(Fig.2AandB).Ingeneral,Z.bailii 02
tionmedia(0.3%maltextract,0.5%peptoneand2%agar).The IST302isslightlymoretoleranttoweakacidsthanthehybrid A
p
fsocrompeat(i×o1n0o0f0smpoargensifiwcaastioobns)earfvteedro2nanadZe4isdsay(cid:2)RsAofxiionpcluabnamtioincroa-t sictaranitnlyISmAo1r3e07to.Ilenrtaenretsttoinagllly,ththeeahnytibfruindgsatlradirnuIgSsAt1e3s0te7disthsiagnniZf-. ril 20
30◦C. bailiiIST302andS.cerevisiaeBY4741(Fig.2C).Thestandardiza- 19
tionofthenumberofviable/totalcellswasnotpossibleforZ.
bailiiCLIB213Tduetothedifficultytocalculateviablecellcon-
Microscopicobservationofthemorphologyofyeast
centration or culture opticaldensity. Forthisreason,only the
coloniesandcells
susceptibilityofZ.bailiiIST302,thehybridstrainISA1307andS.
YeastcellswereplatedinYPDwithatargeteddensityof20–50 cerevisiaeBY4741toseveralcompoundscouldbecomparedun-
CFU/plate.ColonieswereobservedandphotographedinaCarl derequivalentexperimentalconditions.
ZeissStemi2000-Cstereomicroscopeafter4–5daysofgrowthat
30◦C. EstimationoftotalDNAcontentandkaryotypingof
CellsofZ.bailiiIST302,Z.bailiiCLIB213TandofISA1307hy-
ZygosaccharomycesbailiiIST302
bridstrainwereculturedinYPDmediumfor24handusedtoin-
oculateafreshYPDmediumwithaninitialOD600nmof0.05±0.01. The chromosomal profiles of Z. bailii IST302 and of the refer-
After 24 h of growth, cells were harvested in 1.5 mL tubes by ence strain Z.bailii CLIB 213T were compared by PFGE (Fig. 3).
6 FEMSYeastResearch,2017,Vol.17,No.4
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ttp
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://a
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Figure4.CellcycleanalysishistogramsofS.cerevisiaeBY4741(haploid)(A)andBY4743(diploid)(B),ofthehybridstrainISA1307(C)andofZ.bailiiIST302(D–F). /3
7
FluorescentintensitiesofG0/G1peaksofthecellcyclehistogramswereestimatedbyflowcytometry.Themeanfluorescencevalues(FL1-H)obtainedforS.cerevisiae 86
haploid(A)anddiploid(B)strainswereusedtobuildacalibrationcurvethatwasusedtoestimatethesizeofthegenomeofZ.bailiiIST302(D).Zygosaccharomycesbailii 35
hybridstrainISA1307wasusedforcomparisonpurposes(C).ZygosaccharomycesbailiiIST302cellswerearrestedinG0/G1with8-hydroxyquinoline(E)orinG2/Mwith 0 b
nocodazole(F)inordertoconfirmtheG0/G1andG2/Mpeaksanddeterminetheploidyofthestrain. y
g
u
e
Karyotypingshoweddifferencesinthenumberandsizeofthe (Mira et al. 2014), was also included in this analysis for com- s
chromosomesbetweenZ.bailiiIST302andCLIB213Tstrains.Fol- parison purposes. Zygosaccharomyces bailii CLIB 213T was ex- t on
lowingtheseparationofchromosomesbyPFGE,strainIST302 cludedfromthisanalysisduetoitsstrongcellularaggregation 02
originatedatotalofsevenDNAbands,whereasonlysixDNA phenotype. As anticipated, the fluorescent light intensity of A
p
bandsareobservedforCLIB213T.Basedonthemolecularsize G0/G1orG2/MphasecellsfromthereferencehaploidstrainS. ril 2
of chromosomes from the marker species Hansenula wingei, it cerevisiae BY4741 is half the fluorescent light intensity of the 0
1
waspossibletoestimatethetotalDNAcontentofZ.bailiiIST302 diploid BY4743 strain, since strain BY4741 possesses half the 9
andCLIB213T as11.65and9.5Mb,respectively.Giventhatthe DNAcontentofstrainBY4743(Fig.4AandB).Flowcytometry
DNAcontentcalculatedfromCLIB213T islowerthantheDNA datafromstrainISA1307(Fig.4C)showstwofluorescencepeaks
sequenced(Galeoteetal.2013),itispossiblethattwocomigrat- (G0/G1 and G2/M) with a DNA content similar to one of the
ingchromosomescouldhavenotbeenseparatedusingthePFGE diploidS.cerevisiaeBY4743(Fig.4B).Interestingly,strainIST302
conditionstested.Infact,thechromosomalDNAbandof1.8Mb (Fig.4D)showstwofluorescencepeaks(G0/G1andG2/M)witha
is more intense than expected for a single band and possibly DNAcontentsimilartotheoneobservedforS.cerevisiaeBY4741
representstwochromosomesofthesamesize. haploidstrain(Fig.4A).Giventheunexpectedhighproportion
Flowcytometrywasalsousedtoestimatethegenomesize of Z. bailii IST302 cells in G2/M phase, it was decided to treat
of Z. bailii IST302, as well as its ploidy. Saccharomyces cere- cellswith8-hydroxyquinolineandnocodazoleinordertoarrest
visiae BY4741 was used as the haploid reference strain and S. cells inG0/G1 (Fig. 4E) and in G2/M (Fig. 4F) cell cycle phases,
cerevisiae BY4743 as the diploid reference. The hybrid strain respectively,thusconfirmingthefluorescentpeakcorrespond-
ISA1307,whosegenomesizeandploidywererecentlyestimated ing to each phase. Results suggest that Z. bailii IST302 has a
Palmaetal. 7
Table1.GenomeassemblystatisticsofZ.bailiiIST302.
IST302 ISA1307(Miraetal.2014) CLIB213T(Galeoteetal.2013)
Totalreads 13024866 120000000 50868918
No.ofscaffolds 105 154 56
Coverage ×120 ×600 ×250
N50(bp) 432084 232974 932251
Maximumcontiglength(bp) 1149457 806952 1686157
Minimumcontiglength(bp) 1051 2160
Averagecontiglength(bp) 102599 137280
Assemblysize(bp) 10772966 21141152 10361356
D
o
Table2.ComparisonofZ.bailiiandZ.bailii-relatedstrainsgenomefeatures. w
n
lo
a
No.ofchromosomes Ploidy Genome AverageGC Totalno. d
e
Strain separatedbyPFGE ratio size(Mb) content(%) ofCDS d
fro
Z.bailiiIST302 7 ∼n ∼11.0 42.2 5142 m
Z.bailiiCLIB213T 6 nd ∼10.4 42.5 5084 http
Z.bailiihybridstrainISA1037 13 ∼2n 22.0 42.4 9931 s://a
c
nd.Notdetermined ad
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5
/3
7
8
6
3
5
0
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s
t o
n
0
2
A
Figure5.MultigenomealignmentofgenomicregionsofZ.bailiiIST302,Z.bailiiCLIB213TandZ.rouxiiCBS732T.ZygosaccharomycesbailiiIST302ispresentedasthe p
centralreferencestraintowhichtheothersarecompared.Conservedsyntenyblocksarepresentedinshadedboxes.Theimagewasobtainedusingthemultigenome ril 2
alignmentGbrowsesyn(McKay,VergaraandStajich2010). 0
1
9
haploidDNAcontent(Fig.4D).Basedonthefluorescentinten- allsequencecoverageof×120.Asummaryofgenomeassembly
sityofG0/G1subpopulationfromS.cerevisiaehaploidanddiploid statisticsanditscomparisonwithZ.bailiiCLIB213T andtheZ.
strains,thesizethegenomeofZ.bailiiIST302wasestimatedas bailii-derivedinterspecieshybridISA1307ispresentedinTable1.
having∼11Mb.Thelifecycleofthestrainwillbediscussedfur- Thesumofallscaffoldssizeis10772966bp,whichcorresponds
therinthiswork. to98%ofthegenomesizeestimatedbyflowcytometryand93%
ofthegenomesizeestimatedbyPFGE.TheannotationofZ.bailii
IST302genomesequencewasbasedonthecombinationoftwo
AssemblyandannotationofZygosaccharomycesbailii
abinitiogenepredictoralgorithmsandthegenestructureofZ.
IST302genomesequence
rouxiiCBS732T,S.cerevisiaeS288c,Z.bailii-derivedhybridstrain
ThegenomesequenceofZ.bailiiIST302wasobtainedbypaired- ISA1307andZ.bailiiCLIB213T.Atotalof5142geneswerepre-
end Illumina sequencing. A total of 13 million reads were ac- dictedtobeencodedinthegenomeofZ.bailiiIST302(Table2),
quiredandassembledinto105scaffolds,resultinginanover- whereas5084putativeprotein-codinggeneswerepredictedto
8 FEMSYeastResearch,2017,Vol.17,No.4
Table3.ORFsidentifiedinthegenomesofZ.bailiiIST302,CLIB213TandZ.rouxiiCBS732TencodingthehomologsofS.cerevisiaegenesinvolved
inmatingtype.
S.cerevisiaeS288C Biologicalfunction Z.bailiiIST302 Z.bailiiCLIB213T Z.rouxiiCBS732T
MF(ALPHA)1/2 Matingpheromonealpha-factor ZBIST1222 (Severalgapsinthe ZYRO0G08184g
(YPL187W/YGL089C) genomesequenceat
thislocation)
MFA1/2 (YDR461W/ Matingpheromonea-factor ZBIST2952 - -
YNL145W)
HMRA1/2 (YCR097W/ Silencedcopyofa1/a2atHMR - - ZYRO0C18348g
YCR096C)
HMLALPHA1/2 SilencedcopyofALPHA1/2atHML - - ZYRO0F15818g
(YCL066W/
D
YCL067C) o
w
MATALPHA1 Transcriptionalco-activatorinvolvedin ZBIST5098 BN86000122gl ZYRO0F15840g n
lo
(YCR040W) regulationofmatingtypespecificgene a
d
expression e
d
MATALPHA2 Transcriptionalrepressorofa-specificgenesin - - - fro
(YCR039C) haploids m
MATA1 Homeodomainproteininvolvedin - - - h
transcriptionalregulationofmatingtypespecific ttps
genes ://a
MATA2 Proteinofunknownfunction;identicaltothe - - - ca
d
C-terminaloftheMATalpha2protein e
m
HO(YDL227C) Site-specificendonuclease ZBIST2381 BN86001090gn ZYRO0C10428g ic
.o
u
p
.c
o
m
be encoded in the genome of Z. bailii CLIB 213T (Galeote et al. Information)revealedthatthisstrainholdshomologsofS.cere- /fem
2013).About80%oftheannotatedgenesarelocatedinthefirst visiaematingpheromonealpha-anda-factors,MF(ALPHA)1and sy
25scaffoldsand47%ofthescaffoldscontainzerooronegene MFA1, respectively, and a homolog of S. cerevisiae MATALPHA1 r/a
annotated. thatcodesforatranscriptionalco-activatorofmatingtypespe- rtic
le
Asexpected,ahighdegreeofconservationisobservedbe- cific genes. Neither MATALPHA2 nor MATA1/2 genes were de- -a
tweenZ.bailiiIST302andCLIB213Tstrains.However,thecom- tected in the genome sequence of this strain, suggesting that bs
parisonoftheseZ.bailiistrainswiththetaxonomicallyrelated this haploid strain is of alpha mating type. Remarkably, al- tra
c
yeast species Z. rouxii reveals numerous chromosomal rear- thoughahomologoftheendonucleaseencodinggeneHO,re- t/1
7
rangements(Fig.5). sponsible for mating type switching in S. cerevisiae, is present /4
Atotalofsevencentromere-likesequenceswereidentified inZ.bailiiIST302genome,thesilentcassettesHML/HMRwere /fo
x
inthegenomesequenceofIST302(Supplementaryfile1,Sup- not identified in the genome sequence of this strain, neither 0
2
portingInformation),basedonpreviouslyidentifiedpointcen- in CLIB 213T (Table 3). In the context of evolution of hemias- 5/3
tromeresequencesandonthecollinearconservationofflank- comyceteyeasts,theabsenceofsilentcassettesisunexpected 78
6
inggenesofhemiascomycetousyeasts,inparticularofZ.rouxii giventhatZ.rouxii,acloserelativeofZ.bailii,possessesthose 3
5
(Pribylovaetal.2007;Soucietetal.2009).Thisresultisinagree- cassettes(Butleretal.2004;Fabreetal.2005;Watanabe,Uehara 0
b
mentwiththesevenchromosomalbandsidentifiedduringthe and Mogi 2013). Although the hypothesis that HML/HMR cas- y
g
karyotyping analysis (Fig. 3). Interestingly, seven centromere- settes were not sequenced cannot be discarded, the fact that u
e
loifkeCLsIeBq2u1e3nTcesstrawiner(eSuaplspoleimdeennttiafireydfiilne2t,hSeugpepnoormtinegsIenqfouremncae- tqhueeyncweesreofnCoLtIiBde2n1t3iTfie(dGainleottheisetstauld.y20n1o3r) ianndthIeSAge1n30o7m(eMsirea- st on
tion),thusreinforcingthattheseparationofthechromosomes etal.2014)stronglysuggeststhatZ.bailiiisunabletoundergo 02
byPFGE(Fig.3)wasnotcompletefortwoofthechromosomes mating type switching. Other genes involved in meiosis are A
p
thatpossesshighlysimilarsizes.Itisalsolikelythat,giventhe also absent from the genome sequence of IST302, as well as ril 2
differentsizesofbothZ.bailiistrainschromosomesandthecon- in CLIB 213T (Supplementary file 3), as, for example, the ho- 0
1
servationofcollinearity,severalgeneticeventsmighthaveoc- mologsofS.cerevisiaegeneIME1thatcodesforamasterregula- 9
curredleadingtodifferentchromosomesizes.Strain-specificdi- torofmeiosis,andofothergenesinvolvedinchromosomecohe-
versityofchromosomecopynumberand/orsizehasalsobeen sion/recombination(SPO13,SPO22,MER3,REC104,MLH2,MSH4
reported in other yeast species, such as Z. rouxii (Solieri et al. and MSH5), spore assembly (MCP54, SMA1, SPO20, SPO74 and
2008),S.cerevisiae(Zhu,SherlockandPetrov2016),Debaryomyces SPO77)orintheformationofthesynaptonemalcomplex(ZIP2,
hansenii(Jacquesetal.2010),Candidaalbicans(Rustchenko-Bulgac ZIP3,SPO16).Thesegenesarealsoabsentfromthegenomese-
1991), C. glabrata (Muller et al. 2009) and Dekkera bruxellensis quenceofthehybridstrainISA1307(Miraetal.2014).Thelife
(HellborgandPiskur2009). cycle of Z. bailii and Z. bailii-related strains has been investi-
gated based on their ability to sporulate when grown in spe-
cificmedia(MollapourandPiper2001;Rodriguesetal.2003).The
ThelifecycleofZygosaccharomycesbailiiIST302
peculiarformationofmitotic,butnotmeiotic,sporeswasde-
The analysis of Z. bailii IST302 genes involved in life cycle scribedbothindiploidZ.bailiistrains(MollapourandPiper2001)
and meiosis (Table 3 and Supplementary file 3, Supporting and in the hybrid strain ISA1307 (Rodrigues et al. 2003). Dato
Palmaetal. 9
et al. (2008) also described the presence of binucleate cells in
early-stationary phase. We investigated the ability of Z. bailii
IST302toformsporesand,surprisingly,whengrowninasporu-
lation medium we observed the formation of protrusions be-
tweentwocells(Fig.6B–F)beforethedevelopmentoftwomitotic
sporespercell(Fig.6DandE),apparentlywithouttheoccurrence
ofkaryogamyaspreviouslydescribed(MollapourandPiper2001;
Rodriguesetal.2003).Interestingly,afterconjugationandforma-
tionoftwosporespercell,oneofthecellsseemstodonateeach
ofthetwosporestotheothercell(Fig.6G–I)beforesporerelease
(Fig.6J).Altogether,ourresultssuggestthatZ.bailiiIST302hap-
loidcellscannotundergomatingtypeswitching,butundernu-
trientdeprivationareabletoformvegetativespores.Thisstudy
D
providedadditionalinformationfortheunderstandingofZ.bailii o
w
andZ.bailii-relatedstrainslifecyclecomplexity,althoughmore nlo
studiesinvolvingotherstrainsarerequiredtodisclosethepecu- a
d
liarlifecycleofthisspecies. ed
fro
m
h
GenesencodingMDR/MXRtransporters ttp
s
Having in mind that when S. cerevisiae cells are exposed to ://a
c
a
weak acids their ability to decrease the counterions’ intra- d
e
cellular accumulation is essential to cope with their deleteri- m
ous effects (reviewed by Piper et al. 2001; Mira, Teixeira and ic.o
Sa´-Correia2010),wesearchedfortransportersassociatedwith up
multidrug/multixenobiotic resistance (MDR/MXR)belonging to .co
m
the ATP-binding cassette (ABC) superfamily and to the Major
Facilitator Superfamily (MFS) (Higgins 2007; Sa´-Correia et al. /fem
2009)proposed,orhypothesized,toberesponsiblefortheac- sy
tiveeffluxofweakacidcounterionsorothertoxicants.Forthis, r/a
homologs of several S. cerevisiae ABC and MFS proteins pre- rtic
le
viously implicated in the MDR/MXR phenomenon were iden- -a
tified in the genome of Z. bailii IST302 (Tables 4–6). Two ho- bs
mologs of S. cerevisiae ABC protein Pdr12, implicated in the tra
c
Figure 6.MicroscopicobservationofZ.bailiiIST302sporulation.Zygosaccha- active efflux of propionate, sorbate or benzoate anions (Piper t/1
romycesbailiiIST302cellsweregrowninapre-sporulationmedium(A)for2days etal.1998;Holyoaketal.1999),wereidentifiedinthegenome 7/4
andtransferredtoasporulationmedium(B–J).Theformationofsporeswasob- of Z. bailii IST302 and in Z. rouxii CBS 732T as well (Ta- /fo
servedonaZeiss(cid:2)RAxioplanmicroscope(×1000magnification)after2(B-D)and ble 4). Moreover, a total of four homologs of the Pdr18/Snq2 x0
4(E-J)daysofincubationat30◦C. 2
paralog pair were identified in Z. bailii IST302 and ISA1307 5/3
7
8
Table4.ORFsidentifiedinthegenomesofZ.bailiiIST302,CLIB213TandZ.rouxiiCBS732TencodingthehomologsofS.cerevisiaeMDR/MXR 6
3
transportersofABCsuperfamily.Thehomologswereorganizedaccordingtothetaxonomicclusterspreviouslydefined(Seretetal.2009). 5
0
b
y
ABCfamily Total g
u
e
s
S.cerevisiaeS288c PDR5 SNQ2 PDR12 PDR11 YOL075c ADP1 10 t o
PDR15 PDR18 AUS1 n
0
PDR10 2
A
Z.bailiiIST302 ZBIST3521 ZBIST0908 ZBIST5124 - - ZBIST3422 12 p
ZBIST4874 ZBIST0076 ZBIST5081 ril 2
0
ZBIST4875 ZBIST4998 1
9
ZBIST4873 ZBIST3502
ZBIST4996
Z.bailiiCLIB213T BN86000584gq BN86007778gc BN86000100gt - - BN86001948gi 9
BN86004478gf BN86004852gg
BN86004500gf
BN86004456gf
BN86000276gc
Z.rouxiiCBS732T ZYRO0D17710g ZYRO0B14762g ZYRO0F08888g - ZYRO0B05588g ZYRO0E04092g 10
ZYRO0D11858p ZYRO0A04114g ZYRO0F08866p
ZYRO0D11836p
ZYRO0D11880g
10 FEMSYeastResearch,2017,Vol.17,No.4
s
molog Total 12 21 19 21
mMFS.Theho Others BIST0204BIST4991BIST0576BIST3170BIST4947BIST5091 N86007888gdN86000232gcN86000188gtN86000122gtN86000276gbN86007470g YRO0E10230gYRO0G15422gYRO0E10054gYRO0F02090gYRO0D00286gYRO0F02178p
fro ZZZZZZ BBBBBB ZZZZZZ
A1family LR1 3176317729853175317131733879 00232gr00254gr00166gk00210gq00144gu00188gr00232gj E09966gE09922pF04642gE09988gE09900gB16808pA13618g
eDH F BISTBISTBISTBISTBISTBISTBIST N860N860N860N860N860N860N860 YRO0YRO0YRO0YRO0YRO0YRO0YRO0 D
h ZZZZZZZ BBBBBBB ZZZZZZZ o
t w
of 4 n
DR/MXRtransporters YHK8TPO -- -- ZYRO0G16302g- loaded from https://a
M b g ca
ae 0g 90 de
S.cerevisiof TPO1 ZBIST1684ZBIST1685 BN8600681 ZYRO0B009 mic.oup.co
s m
olog 8gh 46g /fem
TCBS732encodingthehom´dSa-Correia2013). DHA1family HOL1TPO2TPO3 BIST3441ZBIST0758 N86002432giBN8600436 YRO0E02288gZYRO0G196 syr/article-abstract/17/4/fo
xiian Z B Z x0
TZ.rouandLIB213uslydefined(Dias 1QDR3 ZBIST0934 BN86008350gc ZYRO0A03564gZYRO0F08228g 25/3786350 by gu
Z.bailiiesofIST302,Cnomicclustersprevio QDR1AQRQDR2 ZBIST5047-4184ZBIST BN86001244gg- ZYRO0A01474g- est on 02 April 2019
mo
Table5.ORFsidentifiedinthegenowereorganizedaccordingtothetax S.cerevisiaeDTR1S288c Z.bailii2327IST302ZBIST TZ.bailiiBN86005512gCLIB213 TZ.rouxiiCBS732ZYRO0F14652g