Modulation of the eps-ome transcription of bifidobacteria through simulation of human intestinal ...

12 Pages·2016·0.93 MB·English

FEMSMicrobiologyEcology,92,2016,fiw056
doi:10.1093/femsec/fiw056
AdvanceAccessPublicationDate:8March2016
ResearchArticle
RESEARCH ARTICLE
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Modulation of the -ome transcription D
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of bifidobacteria through simulation of human loa
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Chiara Ferrario1, Christian Milani1, Leonardo Mancabelli1, s://a
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Gabriele Andrea Lugli1, Sabrina Duranti1, Marta Mangifesta2, ad
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Alice Viappiani2, Francesca Turroni1, Abelardo Margolles3, ic
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Patricia Ruas-Madiedo3, Douwe van Sinderen4 and Marco Ventura1,∗ up
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1LaboratoryofProbiogenomics,DepartmentofLifeSciences,UniversityofParma,I-43124Parma,Italy, /fe
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2GenProbioLtd,43124Parma,Italy,3DepartmentofMicrobiologyandBiochemistryofDairyProducts,Dairy s
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ResearchInstituteofAsturias(IPLA-CSIC),Villaviciosa,Asturias,33300,Spainand4APCMicrobiomeInstitute /a
andSchoolofMicrobiology,NationalUniversityofIreland,Cork,Ireland rtic
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∗Correspondingauthor:LaboratoryofProbiogenomics,DepartmentofLifeSciences,UniversityofParma,ParcoAreadelleScienze11a,I-43124Parma, -ab
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Italy.Tel:[phone];Fax:[phone];E-mail:[email protected] tra
Orenperesseennttesnacelasrugmepmroapryo:rtEixoonpooflythsaecicnhtaerr-idspeseciniesbivfiadroiabbaiclitteyriiad,eonntiefioefdtahmeodnomgbinifiadnotbmacicteroribailaglegnrooumpesst.hatoccurinthegutofanimalsandhumans, ct/92
Editor:JulianMarchesi /4
/fiw
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ABSTRACT /21
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Bacterialexopolysaccharides(EPSs)aremono-oroligo-saccharideunitslinkedbyglycosidicbonds,forminghomo-or 4
7
hetero-polymers.Ingutcommensals,thesemacromoleculesareclaimedtoprotectbacterialcellsagainstgastrointestinal b
y
challengesandtobeinvolvedinmodulatingthecrosstalkbetweentheproducingbacteriumanditsgutenvironment.The g
u
predictedEPSarsenaloftheBifidobacteriumgenus,whichwedesignatehereastheeps-ome,consistsof9epsgeneclusters es
conservedamongdifferentbifidobacterialspeciesandafurther44uniqueepsloci,togetherrepresentingalargeproportion t o
n
oftheinter(sub)speciesvariabilityidentifiedamongbifidobacterialgenomes.Co-cultivationsofbifidobacterialspeciesin 1
0
mediasimulatingadultandinfanthumangutenvironmentsresultedinanincreasedtranscriptionofkeygenesforEPS A
p
biosynthesis,includingglycosyltransferase-encodinggenes,aswellasgenesspecifyingEPStransporterandpolymerase ril 2
functions,andsaccharidebiosynthesisandmodificationenzymes. 0
1
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Keywords:Bifidobacterium;exopolysaccharide;genomics;RNAseq;gutmicrobiota
INTRODUCTION
ducingcell.Manypolysaccharidesareproducedasextracellu-
lar macromolecules, such as capsular polysaccharides, which
Bacteria are able to produce different types of polysaccha-
formacohesivelayerorcapsulecovalentlylinkedtoothercell
rides,suchasglycogen,peptidoglycan,lipopolysaccharidesand
envelope-associatedstructures,andexopolysaccharides(EPSs)
lipoteichoic acids, each eliciting specific functions to the pro-
whichformaslimelayerthatislooselyattachedtothecelland
Received:8January2016;Accepted:7March2016
(cid:3)C FEMS2016.Allrightsreserved.Forpermissions,pleasee-mail:[email protected]
1
2 FEMSMicrobiologyEcology,2016,Vol.92,No.4
whichcanbereleasedintotheenvironment(Schmid,Sieberand gut environment influence the expression of eps genes in bi-
Rehm2015).AgivenEPSconsistsofrepeatedmono-oroligosac- fidobacteria by assaying their transcription through RNAseq
charidesubunitslinkedbyvaryingglycosidiclinkages,thereby analysis.
generatinghomo-orhetero-polysaccharides,thattogetherform
astructurallyverydiversecollectionofpolysaccharides(Ruas-
Madiedo2009;Notararigoetal.2013). MATERIALSANDMETHODS
Bacterial EPS, with proteins, lipids, nucleic acids and lipo-
oligosaccharidesmaycomposematricesofavarietyofbiofilms Strainsandcultureconditions
(Koo, Falsetta and Klein 2013). Biofilms are often associated
Bifidobacterium strains used in this study are listed in Table 1.
with chronic infections and infection relapses causing health
StrainswereroutinelygrownanaerobicallyindeMan,Rogosa,
problemsofgrowingimportance(Hall-Stoodley,Costertonand
Sharpe (MRS) medium (Scharlau), which was supplemented
Stoodley 2004; Fux et al. 2005). Conversely, it was demon-
with0.05%L-cysteine-HClandincubatedat37◦Cfor24h.
strated that surface-associated EPSs (sEPSs) from lactic acid D
bacteria and bifidobacteria support colonization by gut com- For phenotypic characterization of eps producing strains, ow
m20e1n3;saTlusrr(oFnaninetinagl.,2H01a4ll).aFnudrthvearnmoSrine,dceoremnm2e0n1s2a;lHsEoPrnSsehtavale. rtruatchte,n10iu%mskriemd-mmiillkkp(oRwRMde)r,p1la%tegsluccoonssei,s1t.i5n%gaogfa0r.5a%ndy0e.a08stgel−x1- nload
ofrutheniumredperlitre(Moraetal.2002)wereused. e
been claimed to promote antitumor effects, to elicit anti- d
inflammatoryactivityandtolowerbloodcholesterol(Welman For co-cultivation experiments Bifidobacterium strains were fro
inoculated on either of two faecal media agar plates simulat- m
and Maddox 2003). EPS immune modulatory properties have ingthehumangutenvironment.InfantFecalMedium(IFM)was h
irMnidaezpmaorfatincBiuaalcnater2ro0bi1de0ee)sn. frsatguidliised(MfaozrmthaneiaPnSAetcaalp.s2u0l0a5r;pRoolyusnadccahnad- pfirceaptiaornesd.aBsrireeflpyo,rttheedbbaysaAlrmboeldeiyuametwaal.s(2p0r1e3p)awreidthasmrienpoorrtmedodbiy- ttps://ac
Arboleyaetal.(2013),withoutthesupplementationoftherecon- a
BifidobacteriaareGram-positivemicroorganismsbelonging d
to the Actinobacteria phylum, and represent typical residents stitutedformulamilk,andadding0.2%ofglucose.Fivegrams em
ofinfantstoolwasdilutedwithwater,withafinalconcentra- ic
of the gastro intestinal tract of mammals, insects and birds tionof5%w/vandsterilizedat121◦Cfor15min.Followingthis, .ou
(Ventura et al. 2007). Certain members of the genus Bifidobac- p
teriumcanexertspecifichealthbenefitsontheirhostsandare theinfantstoolsolutionwasaddedtothebasalmediaatafi- .co
nalconcentrationof1%w/v.HumanFecalMedium(HFM)was m
thereforeconsideredtobeprobioticmicroorganisms(FAO/WHO preparedaspreviouslyreportedbySalazaretal.(2009).Briefly, /fe
2006).Oneoftheproposedmechanismsbywhichbifidobacte- m
humanstoolwasdilutedwithwaterandsterilized.Thebasal s
riamediatesomeofthesehealthbenefitsistheproductionof e
sEPS (Ruas-Madiedo 2009; Fanning et al. 2012; Bottacini et al. HFMwasaddedwiththestoolsolutionatafinalconcentration c/a
2014b). of1%. rtic
Forallexperiments,anaerobicconditionswereachievedby le
biosFyrangthmeesnistairnymdeamtabeexrsisotfotnhethgeenguesneBtifiicdsobuanctdeerirulymin.gInstEhPiSs the use of an anaerobic cabinet (Ruskin), in which the atmo- -abs
context,a54.3kbgenecluster,designatedeps,whichencodes sphereconsistedof17%CO2,80%N2and2.99%H2. tra
c
predictedfunctionsresponsibleforsEPSbiosynthesis,wasiden- t/9
tifiedinthegenomeofBifidobacteriumanimalissubsp.lactisIPLA- 2/4
R1(Leivers etal. 2011)whilethree epsclusters,i.e.eps1,eps2a PhenotypiccharacterizationofsEPS-producing /fiw
and eps2b, have been described for B. breve UCC2003 (Fanning Bifidobacterium 05
etal.2012;Bottacinietal.2014a).Recently,Hidalgo-Cantabrana 6/2
et al. (Hidalgo-Cantabrana et al. 2014) identified eps clusters A phenotypic screening for sEPS-producing strains was per- 19
8
in 10 Bifidobacterium genomes, detecting high levels of in- formed using 48 Bifidobacterium strains, which include nega- 0
4
ter(sub)speciesepsvariability,inparticularwithrespecttothe tive controls (e.g., B. bifidum LMG11041, that does not present 7
b
number,functionalannotationandorganizationofthegenesof epsgenesinitsgenome)(Durantietal.2015)andpositivecon- y
g
agivenepscluster.Thisinsilicoanalysisrevealedtheexistence trols(e.g.B.biavatiiDSM23969,B.gallicumDSM20093andB.reuteri u
e
omfoalnecauplapramreanrtklyercfoonrsterarvciendggeepnselosceitinthnaetwmlyipguhbtlbisehuedsegfeunloamsae DprSeMvi2o3u9s75o,btsheartvsahtioownse)d(“Rmuausc-oMida”doierd‘roopety’acl.oluonnpyupbhliesnhoetdypdeastain). st on
sequences.Suchmarkersequencesincludegenespredictedto CulturesweregrownroutinelyonMRSbrothfor24hat37◦Cand 10
encode(i)theprimingglycosyltransferase(pGTF,whichcatal- thestrainabilitytogrowthinsuspensionwasevaluated.Values A
p
ysestheadditionofthefirstsaccharidicmoietytoalipophilic wereassignedasfollow:++highlyplanctonicgrowth,+planc- ril 2
membrane,membrane-associatedcarriermoleculesoastoini- tonicgrowth,–poorplanktonicgrowth/sedimentinggrowth,–– 0
1
tiateEPSrepeatingunitassembly)(Audyetal.2010),(ii)glycosyl cellssedimentduringgrowth. 9
transferases (GTFs), (iii) transporter enzymes (either so-called Subsequently,theyweresubmittedtolowcentrifugalforces
flippasesorABCtransporters),(iv)thesubunitpolymerization (3000 rpm) for 5 min at low temperature (4◦C), since sEPS in-
enzyme,(v)thechainlengthdeterminationproteinand(vi)vari- creasesviscosityandviscosityincreaseswhentemperaturede-
ouscarbohydrateprecursorbiosynthesis/modificationenzymes creases. Then supernatant turbidity was checked reading OD
(Audyetal.2010;Hidalgo-Cantabranaetal.2014). values at 600 nm using a Biophotometer (Eppendorf). A fur-
In this study, thanks to the wealth of data that has been therphenotypicevaluationforsEPS-producingstrainswasper-
generated on genomic sequences of members of the genus formedbygrowingbacteriaonrutheniumred-milk(RRM)agar
Bifidobacterium (Lugli et al. 2014; Milani et al. 2014, 2015a,b,c) plates.Rutheniumstainsthebacterialcellwall,producingpink
we molecularly characterized the genetic repertoire predicted coloniesfromnon-sEPS-producingstrains.Incontrast,forsEPS+
to be involved in EPS biosynthesis in the pan-genome of strains, the polymer prevents uptake of the stain and the
the genus Bifidobacterium. Moreover, we evaluated how differ- colonies appear white/transparent (Ruas-Madiedo and de los
ent environmental conditions reflecting the adult and infant Reyes-Gavilan2005).
Ferrarioetal. 3
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enomes. Isolation BumblebeedigestivetractIntestineofadultHumanfaecesRatfaecesFermentedmilkHoneybeehindgutFaecesoftamarinBrest-feedInfantfaecesBumblebeedigestivetractBumblebeedigestivetractBovinerumenInfantintestineFaecesofcommonmarmoAdultintestinePigletfaecesHoneybeehindgutRawcowmilkRabbitfaecesOralcavityAdultintestineChickencaecumInsectInfantfaecesIntestineofinfantAdultintestinePigfaecesRabbitfaecesBovinerumenSewageFermentedmare’smilkFaecesofgorillaInfantfaecesBovinerumenSwinefaecesPigcaecumChickenfaecesFaecesofmarmosetBovinerumenRabbitfaecesFaecesoftamarin -abstract/92/4/fiw056/2198
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bifidobacterial aStrains DSM22766ATCC15703LMG11039LMG10508DSM10140PRL2011DSM23969LMG11041DSM22767DSM19703LMG10736LMG13208DSM23973DSM16992LMG10510LMG18911LMG23609LMG10738LMG11405DSM20093LMG11586LMG11587DSM21854ATCC15697LMG13197LMG21814LMG11591LMG11341LMG11592DSM21395DSM27321LMG10505LMG11596LMG11571LMG21775LMG21816DSM23975LMG21811LMG14934DSM23967 47 by guest on
edin um 10 A
epsberofgeneticclustersidentifi mactinocoloniiformemadolescentismangulatummanimalisanimalissubsp.manimalislactissubsp.masteroidesmbiavatiimbifidummbohemicummbombimboummbrevemcallitrichosmcatenulatummchoerinummcoryneformemcrudilactismcuniculimdentiummgallicummgallinarummindicummkashiwanohensemlonguminfantissubsp.mlongumlongumsubsp.mlongumsuissubsp.mmagnummmerycicummminimummmongoliensemmoukalabensempseudocatenulatummpseudolongumglobosumsubsp.mpseudolongumpseudolongsubsp.mpsychraerophilummpullorummreuterimruminantiummsaecularemsaguini pril 2019
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1.Nu es bacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteribacteri
Table speci BifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifidoBifido
4 FEMSMicrobiologyEcology,2016,Vol.92,No.4
chonRRM WWWWTWWW anCollection IGfiddeeonbnoatmcitfieercisauetmqiougneennocufessthaoenfdethpaesv-a4oi8lmatbeylepoeifnsbtGirfiaeindnBosabbnaekclotwenergiriaengusteodthtoerBei--
wt ap trieveputativeepsclusters.Wedissectedthegenomesequences
evidence D600Gro Germany.JCMJ fktinohnreotrewhmpensol4rpoe8Gc,iTbthuFifisesdi(nflAogabcnacaeckstsienmsrigoioanrlleencgguiueolnmanorsbmmeorefsas:trhkNweePerpsr6eu9tht5ian4et5visv5eee,sqtNpiugPGeanT6tec9Fde5is4dt4oeo7nf)i.twdiFfieeuenlrld---
PhenotypicEPS SupernatantO 0.0880.1180.1080.0710.2140.1810.2550.111 andCellCultures, tspefoiunrfroybzmtuyeemnpilnsoiet-cksaap)en.loydTBlyghLcmeeAanSrieedbTrseiozn(h(aAstytuildifitcosrehncadhtaeeeuspnlpGszerTyctelmFcausulse.,rt,1sfle9oricp9srh7pawb)aiienssoreeseaslyerunoncstrhgheAtdeuhBssaiCisdsn/etqgmrtuaeaoenrldmrlsyiptfiihtnocoeraattptBeiieooirfirsnn--, Dow
m dobacteriumtypestraingenomesinordertoidentifyallpossible n
bh nis homologs. loa
psepsConservedclusterCellgrowt +1++11–++1++1++1++11– m.DSM,GermanCollectionofMicroorga cellssedimentduringgrowth. st(epdFPChEirrargaOeePettraAsSGdreceeplhspiolnesc)irfi-ctcdo4boieatd.eoymh1lonunemcfsetclio,eotpufiewipfforsaetsewetnndgedcrseaerogliuicrnefounetsnetfesgth(eseedtheoshdrtasfCetgttcphtOowePqh:an/GgaGeu/tuestsArirsnB.repePieeBTpefieridshr.rdfiabeofoedtoibsfbeootreyta.wbmcnrecalehdtatueceer.oatsdraeeitlwrcurleui(i.mprzauZusssrmoihkneos/na,vdgpsfodtioioespacefcreutrtlstiteewaehigdgsalsoaieenlzf.lrsnnuaoee2hctgedn/0eoefioc1dtw/wgut2raitsahi)oibrn.tenegnshgAeeresse/nnttf)fhhhe.caeoieeessrr- ded from https://academic.oup.co
eUniqueclusrer 1121 11ue Collection,Belgiu mentinggrowth,– Tbss(WuuyhilaetthesaoicdCrfkeirOzoneoLmttnOiafitlMcDa.al2Bat0grOi0keo6nHnv)3e.oo.Dr8tfsratieahmrnkevspH1eflo.epe5rrsms((gePHeeonwGndtTeaeess)dllwtrwhuaaannisttdhwamtcGihtahhayeiaedhpsvetraeefovdargelurbabalyntemdpmeanS2erI0aarGg0cmIi7q-ne)HugtiaMerrnreeMsdd-, m/femsec/artic
ContinuedTable1.(). aIsolationspeciesStrains BifidobacteriumscardoviiLMG21589BloodBifidobacteriumstellenboschenseDSM23968FaecesoftamarinBifidobacteriumstercorisDSM24849AdultfaecesBifidobacteriumsubtileLMG11597SewageBifidobacteriumthermoacidophilumporcinumsubsp.LMG21689PigletfaecesBifidobacteriumthermoacidophilumthermoacidophilumsubsp.LMG21395AnaerobicdigesterBifidobacteriumthermophilumJCM7027SwinefaecesBifidobacteriumtsurumienseJCM13495Hamsterdentalplaq aATCC:AmericanTypeCultureCollection,USA.LMG,BelgianCo-ordinatedCollectionofMicroorganisms-BacterialofMicroorganisms,Japan.+++bvalueswereassignedasfollow:highlyplanctonicgrowth,planctonicgrowth,–poorplanktonicgrowth/sediccolonycolour.P:pink,W:white,T:transparent. aCCBiitfBaDtoaaBawxRTpiwiRnnnniaoo.dnnsliruilNONSoaagfilnfiastumbadHt5MatAssdL-ettadoAAthfhrel0hecnoOxoFldtcoeo1sLlwere0esubepMvlriMnetobe6RMxfaaiaiaoealmeμrat8b9arlxNt,tttcBeytsoGsrer9ticilitLretnmiaAoccOaveleemcct21emrMrostnarnemcc(aahaei,1steifiBnwwttroounGusleeBspt4ecdue.ipRcinlunfdm.0acea1tasodobraaatuNogiko5lacts3sagwiottnclsbnafite-tm2Aslswwaisrt,io4nilaedcdo0ouvultsnppoetw◦eiipatrur8reochrntbCslndriefarlimlauheolimloiyeneevyiatamcBBwtrmanntohs.ient’tuiL.uetnsirsrhsMeniushafisodMclD(ptaliddt,eBittgotlhtednRnnrieaSicG.iwBcv3Ts-eormbSnftseMacuR.acta7E1aarebeatl.oouotld◦1Nkelrdps2prcauvrserCutlmo0aTene0eitletehacdmslwa4scoenu0htefatdsleht.1tDroniuse9srebestgienii,cceariraS3oeaywSmrristeoBispfite,totn2mtiMunaoiv.rrn.yBldmiw4–aosrtaeacCnmfipop2.s8ieo.stnhairstnoiseia4n<0eyncbQoensghel2n8a◦ooAoassbt1luevieuC24gsevri-Tcpeinndanatr0u9wka,cdeto−useCroletl0)oeluliruae3iracelnftoCwertn0l(n(aeciemtrieQyitfADdn1un(toetratewitQoeaa5,ileisgmoSanlsralmncfiebs7anieinMnsngwcaRlueeir0o0LceoouccsduegeilnNl23.ueeMnnaoouer-4nlel1,trttritAttcnlgt.lnr,nGeiiu8uecatBt-1iuTofli)Uo5tmtftee1ra.rmv0emaeureue4endx1sKaac3cgidgs)seeLs0tdansth)rCruieanodM3eoatiineganafedtctFrsc9dticndrricnynGaeocpUi,cooogadta,oup.odlm1Beuniwrmototwnlomor0oAn.rfaif.1gdRunonrrdr5whItdf0itoltFiiNFunle0it.e−nhneei0tntomeinMml5ssA1ihndd0gegerr---,.,, le-abstract/92/4/fiw056/2198047 by guest on 10 April 2019
Ferrarioetal. 5
USA)analysis.RNAconcentrationandpuritywasthenevaluated grown on RRM plates. In addition, 16 strains displayed inter-
byPicodropmicrolitreSectrophotometer(Picodrop,UK). mediate phenotypic traits that did not allow a clear assess-
ment of their ability to produce EPS (Table 1). However, phe-
notypicmethodsarenotalwayscapableofdiscriminatingEPS-
RNAseqanalysisperformedbytheMiSeqIllumina
producing strains since their production is highly sensitive to
ForRNAsequencing,10μgoftotalRNAwastreatedtoremove theinvitroconditionsused,whichmaynotbeuniversallyvalid
ribosomal RNA by the MICROBExpress Bacterial mRNA purifi- foralltestedstrains.Forthisreason,wedecidedtocharacter-
cation kit (Life technologies, USA), followed by purification of izethepresenceandcompositionofepsgeneclustersofthese
the rRNA-depleted sample by ethanol precipitation. RNA was bifidobacteria.
further processed according to manufacturer’s instructions.
The yield of rRNA depletion was checked by Tape station Identificationofeps-omeinbifidobacteria
2200 (Agilent Technologies). Then, 500 ng of rRNA-depleted
RNAs was fragmented using Bioruptor NGS ultrasonicator All48Bifidobacteriumgenomesarepredictedtoharbouratleast D
(Diagenode,USA)followedbysizeevaluationusingTapestation one eps cluster, except for B. bifidum (Table 1). As shown in ow
2200(AgilentTechnologies).Awholetranscriptomelibrarywas Fig. 1, no common structure organization was identified in nlo
constructed using the TruSeq Stranded RNA LT Kit (Illumina). theepsbiosynthesisclustersofbifidobacteria,whichconfirmed ad
e
SampleswereloadedintoaFlowCellV3150cycles(Illumina) whathadbeenpublishedpreviously(Hidalgo-Cantabranaetal. d
as reported by the technical support guide. The reads were 2014).Furthermore,thereisaconsistentinterspeciesvariabil- fro
m
depleted of adapters, quality filtered (with overall quality, ity in terms of cluster length as well as the number and pre- h
qualitywindowandlengthfilters)andalignedtotheBifidobac- dictedfunctionsofthegenesencompassingtheidentifiedeps ttp
tCeoriuunmtsroefferreeandcseogveenrloamppeisngthOrRouFsghweBrWeApe(rLfioramndedDuusribnignH2T0S0e9q). lfioecdi.inThteheepespsclloucsutesrsofraBn.mgeodngionliesnizsee(fBrMomONni1n7e33g-eBnMeOsNid1e7n4t2i-, s://ac
a
(http://www.huber.embl.de/users/anders/HTSeq/doc/overview. remnant) to 55 genes found in the eps region of B. den- d
e
html) and analysis of the RPKM values was performed using tium (BDP1816-BDP1871). The presence of several genes en- m
theformulaRPKM=numReads/(geneLength/1000∗totalNum- coding an ABC transporter, glucose-1-phosphate thymidylyl- ic.o
u
Reads/1000000)(Mortazavietal.2008). transferase, fused dTDP-4-keto-L-rhamnose reductase/dTDP- p
TovalidategenetranscriptionlevelsfoundbyRNAseqanaly- 4-keto-6-deoxyglucose-3,5-epimerase and dTDP-glucose 4,6- .co
m
sis,weselected10randomsignificantlydifferentiallyexpressed dehydratase,displayingaDNAsimilarityrangingfrom66.3%to /fe
genes and determined their transcription levels by qRT-PCR 99.3%,wasconservedamongallgenomes,withtheexceptionof m
s
experiments.Experimentalproceduresandstatisticalanalyses B.asteroides,B.angulatum,B.catenulatum,B.pullorumandB.den- e
c
werereportedpreviouslybyDurantietal.(2014). tium(Fig.1). /a
Comparison among all the bifidobacterial eps clusters, re- rtic
le
Nucleotidesequenceaccessionnumbers vInefaolremdatthioene)xbiastseendcoenocfonminmeoenpseprseggeionnestic(Foigrg.aSn1iaz,aStiuopnpso.Crtoinng- -abs
AllRNAseqrawdatafromthisstudyweresubmittedtotheNCBI servedepsclustersvariedinsizeaswellasthepredictedfunc- tra
c
Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) tionoftheepsgenes(Fig.2). t/9
underaccessionno.SRP066713. Apart from these nine relatively conserved eps regions, 2/4
we identified 44 unique gene cassettes for EPS biosynthesis /fiw
that were shown to be exclusively present in one bifidobac- 0
RESULTSANDDISCUSSION terial genome but not in any of the other analysed genomes 56/2
PhenotypicevaluationofsEPS-producingbifidobacteria (Table1).Inaddition,phenotypiccharacteristicssuggestthatnot 19
8
allidentifiedepsclusters(conservedornot)arefunctional.Asan 0
4
Phenotypic characterization of all the type strains of the cur- example,theB.pullorumLMG21816genomecontainsthecon- 7
b
rentlyrecognized48(sub)speciesharbouringtheBifidobacterium servedclustereps2,thoughthestrainexhibitsanEPS-negative y
g
genus(Table1)revealedthat15bifidobacterialstrainsshowed phenotypeunderconditionswehavetested(Table1).Analysing u
e
asutcyhpaicsaBl.EgPaSll-icpurmodDuScMin2g0p0r9o3fialne.dIBn.rpeaurtteirciuDlaSrMp2o3s9i7t5iv,earceounntraobllse, tInhfeogrmenaetsioinn),ththeeepasb2secnlucseteorfotfhBe.ppGulTloFrucmou(lFdigs.uSg1gae,sStsupthpoatrttinhge st on
tocompactatlowcentrifugalforces,showinghigherODvalues cluster is incomplete and therefore explains the apparent ab- 10
(Table1).Moreover,coloniesgrownonRRMplatesshoweda‘mu- senceofEPSproductionobservedforthisstrain(Table1).Sim- A
p
coid’character.AsimilarprofilewasdetectedforB.adolescen- ilarobservationswerenotedforstrainsB.saguiniDSM23967,B. ril 2
tisATCC15703,B.catenulatumDSM16992,B.cuniculiLMG10738,B. saeculareLMG14934andB.minimumLMG11592. 0
1
thermacidophilumsubsp.thermacidophilumLMG21395,B.ruminan- The G+C content of the identified eps gene cassettes was 9
tiumLMG21811,B.boumLMG10736,B.stellenboschenseDSM23968, compared to the average G+C content of the 48 genomes
B.moukalabenseDSM27321,B.thermophilumJCM7027,B.thermaci- (Table S1, Supporting Information). Only in a small number
dophilumsubsp.porcinumLMG21689,B.bohemicumDSM22767,B. of cases (B. cuniculi eps3 and eps4 and B. subtile eps9) the
actinocoloniformeDSM22766andB.bombiDSM19703strains. G+C content of eps clusters were highly similar (difference
In contrast, a strain used as negative control, such as B. less than 1.1%) to that of the corresponding genomes, while
bifidum LMG11041, exhibited a precipitating phenotype when for the other eps loci, a lower G+C value (maximum dif-
grown in liquid medium and associated low OD value of the ference of 15.2%) was detected. Furthermore, in silico analy-
medium following centrifugation, and slightly pink colonies ses aimed at exploring possible acquisition events of eps loci
whencultivatedinRRM.Suchphenotypeswerealsoobserved through HGT, revealed that about 39% of the identified eps
forB.pullorumLMG21816,B.saguiniDSM23967andB.minimum lociseemtohaveundergoneHGTtransfer(TableS2,Support-
LMG11592strains.Other10bifidobacterialstrainsshowedasus- ingInformation).Predictingthedonorsoftheseputativealien
pendedgrowthorhighODvalues,butawhitephenotypewhen genes indicated a preferential origin from members of the
6 FEMSMicrobiologyEcology,2016,Vol.92,No.4
D
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Figure1.Presence/absenceofEPSbiosynthesisgenes.Eachrowrepresentedthepresence(red)orabsence(black)ofgenesdetectedin(sub)speciesofBifidobacterium
involvedinEPSbiosynthesis.Uniquegeneswerenotconsidered.Functionofprotein-encodinggeneswereindicatedontheleft,legendwasreportedonthebottom
ofthefigure,whilenameof(sub)speciesusedwerelistedatthetop.
Ferrarioetal. 7
(a)
B
0B. thermacidophilum subsp. thermacidophilum.5B. thermophilumB. thermacidophilum subsp. porcinumB. ruminantiumB. subtileB. psychraerophilumB. callitrichosB. indicumB. coryneformeeeppB. brevess78B. gallinarumB. minimum. longumsubspB. longum B. saeculareB. reuteriepB. tsurumienses6B. bombiB. saguiniB. kashiwanohenseB. longum subsp. infantisB. bohemicumB. actinocoloniiformeB. magnumB. gallicumB. crudilactisB. mongolienseB. boumB. stellenboschenseB. scardoviisubspB. longum . suisepB. merycicums5B. pseudocatenulatumB. adolescentisB. biavatiiB. pseudolongum . pseudolongumsubspB. pseudolongum subsp. globosumB. cuniculieeppB. choerinumss34. lactisB. animalis subspB. animalis . animalissubspB. stercorisB. pullorumB. moukalbenseeeppB. dentiumss12B. catenulatum B. angulatum. asteroides APGUPPTRAOroorchlBntiayhllymakyiCncelmmnstsori-rano ptesnafcrgwoyruonaci-lsnszsnnhtgaerfca salseafttyppruerinicioooasrndsoersnnfcaetcsees etn eyuirbrdoxalrsis nspotdoerssoearyrrsntni svbtfhaieoetressasyissne/ct (hhpeaGsiinTs Fle)ngth determination Downloaded from h
eps9 Trascriptional regulator ttp
(b) Flippasewzx s
://a
c
eps1 BDP_1816 BDP_1871 a
d
eps2 BDP_2030 BDP_2058 em
eps3 BL12_1287 BL12_1328 ic
.o
eps4 BPSG_1548 BPSG_1565 u
p
eps5 BMERY_1870 BMERY_1860 .co
m
eps6 BISA_1708 BISA_1728 /fe
eps7 BLSL_0353 BLSL_0363 m
s
eps8 BCAL_1657 BCAL_1682 e
c
eps9 BPORC_1087 BPORC_1148 /artic
le
Figure2.Phylogeneticrepresentationofepsclustersofbifidobacteria.Panel(a)representedatreegeneratedfromhierarchicalclusteringbasedonthepresence/absence -a
oftheclustersoforthologousgenesinepsbifidobacterialcassettes.Panel(b)depictedphysicalmapsofarepresentativestrainforeachofthenineepsclusters.Genes b
s
werecategorizedaccordingtotheirfunctionswithdifferentlycolouredarrows. tra
c
t/9
Lactobacillaceae and Lachnospiraceae families (Table S2, Sup- 4,withtheexceptionofB.magnumandB.gallicumwhichwere 2
/4
porting Information), which are known to share the same showntocontainuniqueepsclusters. /fiw
ecological niche of bifidobacteria (Jandhyala et al. 2015). An- Among the conserved eps-key genes, we observed the 0
5
other key sign supporting the putative HGT evolution of occurrenceofgenescodingforenzymesinvolvedinrhamnose 6
eps clusters in bifidobacteria is represented by the pres- biosynthesis, showing high DNA similarity (above 80%) with /21
9
enceofISelements/transposase-encodinggeneswithinmany glucose-1-phosphate thymidylyltransferase, as well as with 8
0
of the eps loci here identified. A small number of eps dTDP-4-dehydrorhamnose 3,5-epimerase and with dTDP- 4
7
loci(BISU2462-BISU2478,BPORC1120-BPORC1152,BIGA0877- glucose4,6-dehydrataseofGardnerellavaginalisandLactobacillus b
y
BIGA0888) were predicted to be flanked by genes encod- spp.Moreover,amongtheepsloci,weidentifiedgenespredicted g
u
ing transposases and insertion sequences, whereas oth- to encode rhamnosyltransferases, galactofuranosyltransferase es
ers (BLSL1763-BLSL1791, BBRE1189-BBRE1205, BIGA0341- (e.g.B.crudilactis,BCRU1303)andmannosyltransferases(B.saec- t o
n
BIGA0359),containedsuchmobileelementswithintherespec- ulare,BSAE1664).Thisindicatesthepresenceofaheterogenic 1
0
tiveepscluster.Thepresenceofsuchelementssuggestspossible EPS composition among bifidobacterial species with different A
p
HBiGfidToboafcctoermiupmleoterootrhpearrbtiaaclteeprisalgespneeccielus.stersbetweendifferent etocodloifgfiecraelnotreignivnisr,onpemrhenaptss.bAescamuesentoiofnaedmoalbeocvuel,artraadnaspptoarttioonf ril 2
0
the undecaprenyl-associated oligosaccharidic subunit across 19
thecytoplasmicmembraneiscarriedoutbyaso-calledflippase
Phylogeneticanalysesofepsclustersinbifidobacteria protein or by an ABC-type transporter (Hidalgo-Cantabrana et
al.2014).Weidentifiedflippase-liketransportencodingsystems
Atreegeneratedfromthehierarchicalclusteringbasedonthe in eps clusters 1, 2 and 3, while ABC transporters were more
presence/absence of the clusters of orthologous genes in the abundantinbothconservedanduniqueEPScassettes(Fig.1).
epsbifidobacterialcassettes,revealedtheexistenceofseveneps We have furtherpredicted theevolutionof theepsarsenal
phylogeneticgroups(Fig.2).Thehierarchicalclusteringtreeis of bifidobacteria using BlastGraph (Ye et al. 2013). This analy-
differentfromthephylogenetictreebasedontheidentifiedcore sis allows the generation of a tree based on information re-
genes of eps clusters (Fig. S1b, Supporting Information). The gardingthepresenceorabsenceofCOGfamiliesineachofthe
identifiedclusteringoftheepslocireflectsthephylogeneticre- bifidobacterial strains assayed with the dollo-parsimony algo-
latednessoftheanalysedstrains.Forexample,membersofthe rithm (Farris 1977). Notably, this analysis predicted the early
B.pseudolongumgroupwereshowntoshareepsclusters3and acquisition of the pGTF (undecaprenyl-phosphate galactose
8 FEMSMicrobiologyEcology,2016,Vol.92,No.4
E.coli
B. indicum
B. coryneforme
B. asteroides
B. actinocoloniiforme
B. bohemicum
B.bombi
B. crudilactis
B. psychraerophilum
B. mongoliense
B. subtile
B. minimum
B. gallicum
B. magnum
B. animalis subsp.lactis
B. animalis subsp.animalis1333
B. pseudolongum subsp.pseudolongum
B. pseudolongum subsp.globosum
B. choerinum D
B. cuniculi ow
B. boum n
B. thermophilum lo
B. thermacidophilum subsp.porcinum ad
B. thermacidophilum subsp.thermacidophilum e
BB.. gsaaellciunlaarruem d fro
B. pullorum m
B. angulatum h
BB.. mdeenrtyiucimcum ttps
BB.. amdooulkeasclaebnetinsse ://a
c
B. stercoris a
B. ruminantium 32 de
B. kashiwanohense m
B. pseudocatenulatum ic
B. catenulatum .o
B. scardovii u
GLoaisns B. biavatii p.c
ABC transporter B. callitrichos o
Acyltransferase B. stellenboschense m
CFRlahippapsmuanslaoers epobliyossaycnctahreidsies bgieonseysnthesis protein BB.. rseaugtueirnii /fem
Glycosylhydrolases B. longum subsp.suis se
Glycosyltransferase B. longum subsp.infantis c
Membrane protein B. longum subsp.longum /a
PRCohslpayDmsan(copcsGhyaTlrtirFda)en spfeorlaysmeerase BB.. bbirfeidveum rticle
Other B. tsurumiense -a
b
s
tra
Figure3.Reconstructionofgenegainandlosseventsamongbifidobacteria.AtreewasconstructedusinginformationrelatedtothepresenceorabsenceofCOGsfor c
thewholeBifidobacteriumpan-genome.EachnodewasmarkedbyapiediagramshowingtheacquiredCOGs(ingreen)andtheCOGsderivedfromthepreviousnode t/9
2
(inred). /4
/fiw
0
5
phosphotransferaseCspD)familymemberduringtheevolution paringcompletegenomesequencesavailableindatabaseswith 6/2
oftheBifidobacteriumtaxon(Fig.3),whichiscrucialfortheini- thegenomesofthebifidobacterialtypestrainsanalysedinthis 1
9
tialstepoftheEPS-unitsynthesis(Audyetal.2010).Inparticular, study. 80
4
thepGTF-encodinggeneappearstohavebeenacquiredbybifi- Comparative genome analysis of B. adolescentis strains re- 7
b
dobacterialspeciesbelongingtoB.longum,B.bifidum,B.adolescen- vealedhighintra-speciesgenomevariability(Durantietal.2016, y
g
tis,B.pullorumandB.boumphylogeneticgroups(Luglietal.2014). in press). The presence of similar eps clusters in the pan- u
e
Interestingly,B.bifidumLMG11041,whichistheonlyBifidobac- genomeofB.adolescentistaxonwasdetectedforB.adolescentis s
teriumtaxonwithoutepsgenes,seemstohavelostpGTF,GTFs, ATCC15703andLMG10733,whilethechromosomesofB.adoles- t on
membraneproteins,polysaccharidebiosynthesisproteins,ABC centisLMG18897andB.adolescentis22Ldonotencompass any 10
transportersandgenesrelatedtorhamnosebiosynthesisinthe predictedepslocus,asreportedpreviously(Durantietal.submit- A
p
lastevolutionaryevent(Fig.3).Furthermore,explorationofthe ted).RegardingB.breve,twomainepsclusterswereidentified. ril 2
functional evolution of this genus showed that bifidobacterial Theseencompasstheeps8cluster,whichwasidentifiedasEPS 0
1
speciesisolatedfrominsects(B.bombi,B.bohemicum,B.actino- cluster2byBottacinietal.(2014a),beingpresentinallB.breve 9
coloniiforme,B.asteroides,B.coryneformeandB.indicum)acquired strainsanalysed.IntheB.longumpan-genome,similarlytothe
epsgenesonlyinthelastnode. eps-ome of the B. adolescentis species, a high variability in the
geneticcompositionofepsclusterswasdetected,withthepres-
enceofsimilarepsclustersdetectedinB.longumsubsp.longum
Theeps-omeintra-speciesvariability LMG13197andJCM1217(Fig.S1c,SupportingInformation).
Notably,noepsgeneclusterswereidentifiedintheB.bifidum
Theintra-specieseps-omevariabilitywasexploredanalysingthe pan-genome,whileinB.animalissubsp.lactispan-genomethe
pan-genome of Bifidobacterium taxa that are publicly available genesencodingforepsenzymeswereshowntobehighlycon-
(Fig. S1c, Supporting Information), such as B. bifidum (Duranti served.
etal.2015),B.breve(Bottacinietal.2014a),B.adolescentis(Duranti Suchfindingshighlightthatthereisahighdiversityamong
etal.submitted),B.longum(O’Callaghanetal.2015)andB.ani- eps clusters but this is taxa dependent, probably as a conse-
malissubsp.lactis(Milanietal.2013).Datawereanalysedcom- quencetoenvironmentalstressandadaptivemechanisms.
Ferrarioetal. 9
Theeps-omemodulationinmono- an upregulation (>2-fold of induction, P < 0.05) of genes en-
andmulti-associationsexperiments codingglycosyltransferases(BSTER1563,BCAT0973,BDP2058,
BIANG1470), and ABC transporters (BIANG1485, BIANG1493,
Inordertoevaluatehowco-cultivationofBifidobacteriumstrains BSTER1559)(Fig.4c).
may stimulate transcription of genes involved in EPS synthe- The genomes of B. dentium LMG11596 and B. catenulatum
sis, we investigated the transcriptome of mono- and multi- LMG11043harbouraparticularconservedepscluster,i.e.called
association of Bifidobacterium strains simulating the microbial eps1, whose transcription was shown to be significantly en-
consortium occurring in their natural environment, using an hanced(from2-to16-fold,P<0.05)whenthesestrainswere
RNAseqapproach.Asareferencecondition,bifidobacteriawere grown in faecal medium. The closely related strains B. adoles-
cultivatedinmono-associationonMRSplates.Twodistincthu- centisATCC15703andB.pseudocatenulatumLMG10505,whichbe-
mangutenvironmentsweresimulated,i.e.theadultandinfant longtotheB.adolescentisphylogeneticgroup(Luglietal.2014),
gut,wheredifferentbifidobacterialspeciesarecommonlyiden- revealedtranscriptionalincreaseofthegenesbelongingtoeps5
tified. These include adult-type bifidobacterial strains such as (from2-to27-fold,P<0.05),representingasharedepsgeneclus- D
B.adolescentisATCC15703,B.catenulatumDSM16992,B.gallicum ter, when cultivated on faecal medium (Fig. 4c). Finally, tran- ow
DSM20093,B.angulatumLMG11039,B.dentiumLMG11405andB. scription of eps8 genes of B. breve LMG13208 was stimulated nlo
stercorisDSM24849(Luglietal.2014),andinfant-typebifidobac- (from2-to3-fold,P<0.05)whenthestrainwascultivatedin ad
terialstrains,suchasB.bifidumLMG11041,B.pseudocatenulatum faecalmedium(Fig.4c). ed
LMG10505,B.breveLMG13208andB.kashiwanohenseDSM21854 Otherepsgeneclustersweredetectedthroughinsilicoanal- fro
(LugInliettraanl.s2c0ri1p4t)o.me experiments, HFM- or IFM-grown mono- yussiesdoinf mthoengoe-naonmdemsubltei-loasnsgoincigattioontehxepebrifiimdoebnatsct(ewriiathl tshtreaeinxs- m http
aassssoocciiaattiioonnss,wwehreilecoHmFMpa-roerdItFoMt-hgerorewfnermenucletiM-aRssSo-gcriaotwionnmswoneore- cpeoprtteiodnaboofvBe.).bTifiradunmsc,riwpthioicnhodfotehsesneoctlucastreryrsewpsasgseingensi,ficaasnrtely- s://ac
evaluatedusingHFM-orIFM-grownmono-associationsasrefer- enhanced(from2-to16-fold,P<0.05)bythecultivationinboth ad
enceconditions. faecalmedia(HFMandIFM)(Fig.S2,SupportingInformation). em
foldThinedduicffteiorenn,tPb<ifid0.o0b5a)cttrearniaslcsritpratiionnsaelxihnicbrieteadsessigonfifipcaarntitc(u>la2r- Itnhatthhisarcboonutresxttw,woeepfsoucnuisqeudeocnlusBt.eprsse(uBdPoScEat1e2n6u4la-tBuPmSELM12G8180a5n0d5 ic.oup
genesdependingondifferentstimuliapplied(Fig.4a).Growthon BPSE1306-BPSE1326).Weobservedanoverexpressionofgenes .co
ftaheecnalu-mbabseerdomfgeedniaeswtarsansshcorwibnedtoinparollmsptrtaiannsetneshtaendcienmmenotnoo-f sBpPeScEif1y3in09g)faonrdpsoulygsaarcocrhaacryidlteraAnBsCfetrraasnessp(oBrPtSeErs1(2B7P8S,EBP1S3E0812a7n9d) m/fem
association, while multi-associations (in both MRS and faecal (Fig.S2,SupportingInformation). se
media) highlighted an increased number of genes transcribed c/a
forB.pseudocatenulatumLMG10505andBbreveLMG13208forthe rtic
infant-typestrains,andforB.dentiumLMG11405andB.stercoris CONCLUSIONS le
-a
DSM24849fortheadulttype-bifidobacteria(Fig.4a).Thesegenes b
s
werefunctionallycategorizedincarbohydrateandaminoacid Amonghumangutcommensals,bifidobacteriaareconsidered tra
metabolismandinhousekeepingfunctionslikeDNAreplication, keyplayers,especiallyduringthefirststagesoflife(Turronietal. ct/9
orstressresponse,inbothmono-andmulti-associationsexper- 2012;Venturaetal.2014;Milanietal.2015).Theprecisemecha- 2
/4
iments(Fig.4b). nismsbywhichbifidobacteriaestablishthemselveswithintheir /fiw
Transcription of eps gene clusters of bifidobacterial strains hostarenotfullyunderstood,butEPShasbeenproposedasan 0
5
cultivated in mono- or multi-association experiments, was importantplayer(Fanningetal.2012). 6
showntobeenhancedfrom2-to27-foldascomparedtoMRS EachBifidobacteriumtaxonharboursatleastoneepscluster, /21
9
mono-associations. In this context, eps genes were induced withtheexceptionofB.bifidumspecies.Amongallbifidobacte- 8
0
whenbifidobacteriawereco-cultivatedonfaecalmedia(Fig.4a). rial(sub)species,nineepsclustersbasedoncommonepsgenetic 47
In MRS multi-associations a total number of six to seven eps organizationswererecognized.Notably,theeps1andeps2clus- b
y
loci were overexpressed, respectively, for all infant and adult tersarefoundinbifidobacterialtaxacommonlyisolatedfrom g
u
type strains (condition A, Fig. 4a). In the case of faecal me- the human intestine. Whereas the eps3 and eps4 clusters are es
dia mono-associations (condition B, Fig. 4a) the expression of identified in the genomes of bifidobacterial (sub)species com- t o
n
onlyoneepsgenewasenhancedfortheinfant-typebifidobac- monlyrecoveredfromthegutofvariousanimals(Table1).More- 1
0
teria(BBRE0054,encodingaglycosyltransferase)and27forthe over,thehighvariabilityinthenumberofgenesandstructural A
p
aCd,uFilgt-.ty4ap)esshtorawineds.tIhFeMhaignhdeHstFnMummbueltri-oafsseopscilaotcioinbsei(ncgon2d3itainond oprlograinnigzathtieonpaonb-sgeernveodmienstohfemepasnylobciifi,dwoabsaicdteenritaifilteadxaal.sobyex- ril 20
15 genes, respectively, transcriptionally upregulated. The ob- Inthisstudy,wedevelopedaco-cultivationstrategyinvolving 19
tained transcriptome data were confirmed by real-time quan- variousbifidobacterialstrainsonfaeces-basedmediumsimulat-
titativePCR(qRT-PCR)involvingkeyfunctionalgenesidentified ingthehumangutenvironmentofboththeinfantandtheadult.
fromRNAseq(TableS3,SupportingInformation). Thedevelopmentofafaecalmediasimulatingintestinalcondi-
Asanexample,weselectedB.stercorisDSM24849eps2,which tionswithitscomponents(e.g.carbonsourcessuchasstarch,
represents a conserved eps locus (Fig. 4c). When we assayed arabinogalactansandpectins,glycoproteinlikemucinandbile
the transcriptome of this strain, we observed a significant in- salts)allowedabifidobacterialco-cultivationstrategytoinves-
creaseintranscriptionoftheeps2locus(>2-fold,P<0.05)when tigate the eps gene-expression modulated by intestinal condi-
this strain was cultivated in multi-associations (see Fig. 4c). tions.Thisisastraightforwardapproachbasedonastandard-
Theeps2geneclusterwasalsoidentifiedinthegenomesofB. izedmedium,whichdoesnotrequirecomplicatedandexpen-
dentium LMG11596, B. catenulatum LMG11043 and B. angulatum siveequipment.However,thismodelislimitedbysubstratede-
LMG11039(Figs4candS1a,SupportingInformation).Analyses pletionandtheaccumulationoftheendproductsofbacterial
ofthetranscriptomesofthesestrainscultivatedinHFMrevealed metabolism.
10 FEMSMicrobiologyEcology,2016,Vol.92,No.4
(a) (c) EPS2 A B C
Infant-type bifidobacteria BDP_2027
EPS1 A B C BBDDPP__22002289
B 18L9BM. bG1iA5Af41i51d0u4m21 02 CBB. p1s7eL2uMdoG11Ac7A61a90te5n04u53la1tumCB 3L09MB.G b11Ar5A6e38v2e08342 CBB. 4k0Da4sShMi1wA4A23a31n8o5h14e6n1seC BBBBBBBBBBBDDDDDDDDDDDPPPPPPPPPPP___________11111111111888888888881111222222267890123456 BBBBBBBBBBBDDDDDDDDDDDPPPPPPPPPPP___________22222222222000000000003333333333401234567890 AAATTTECCCCCCP111555777S000333___5111555665109 A B C
0 6 1 0 BDP_1827 BDP_2041 ATCC15703_1558
B 0 0 0 C B 0 0 11 C B 1 1 6 C B 0 0 6 C BBBDDDPPP___111888223890 BBBDDDPPP___222000444234 AAATTTCCCCCC111555777000333___111555555765
BA. aTdCoClAe1s5c7Ae0n3tdisult-tyBp.L ecMa btGeAni1fu1il0ad4tu3ombacteriLBaM. gGaAl1l1ic5u9m6 BBBBBBBBBBDDDDDDDDDDPPPPPPPPPP__________1111111111888888888833333334431245789126 BBBBBBBBBBDDDDDDDDDDPPPPPPPPPP__________2222222222000000000044444555555678901234 AAAAAAAAATTTTTTTTTCCCCCCCCCCCCCCCCCC111111111555555555777777777000000000333333333_________111111111555555555555544445431098762 Download
85 19 55 BDP_1843 BDP_2055 ATCC15703_1545 e
BDP_1844 BDP_2056 BPSE_1518 d
5 0 1 BBDDPP__11884456 BBDDPP__22005578 BBPPSSEE__11551290 fro
B B213B1. deA10ntiu1m115C CB B139B5. stA2e0rco0r8i2sC CB B18B57. anA00gula1t8u7mC C BBBBBBBBBDDDDDDDDDPPPPPPPPP_________111111111888888888444555555789012345 BBBBBBBBBCDDDCCCCCAAAAAAPPP___TTTTTT222______000000000566999999901777777543102 BBBBBBBBPPPPPPPPSSSSSSSSEEEEEEEE________11111111555555552222222212345678 m https://aca
LMGA11405 DSMA24849 LMGA11039 BBBDDDPPP___111888555786 BBBCCCAAATTT___000999666879 BBBPPPSSSEEE___111555233901 dem
BDP_1859 BCAT_0966 ic
117 252 103 BDP_1860 BCAT_0965 .o
BDP_1862 BCAT_0964 u
BDP_1863 BCAT_0963 p
0 1 3 BDP_1864 BCAT_0962 .c
B 258 A 186 CB 61 A 202 CB 366 A 68 C BBDDPP__11886656 BBCIAANTG__09146915 om
01 30 00 BBBDDDPPP___111888666789 BBBIIIAAANNNGGG___111444999031 EPS8 A B C /fem
B 7 3 C B 0 9 C B 7 1 C BDP_1871 BIANG_1489 s
(Ibnf)ant-type bifidobacteria Adult-type bifidobacteria BBBBBBCCCCCCAAAAAATTTTTT______111111666666444444012456 BBBBBBIIIIIIAAAAAANNNNNNGGGGGG______111111444444888888748653 BBBBBBBBBBRRRRREEEEE_____00000000004444401234 ec/article
[S] [S] BBCCAATT__11664487 BBIIAANNGG__11448821 BBBBRREE__00004456 -ab
[[[[[[HQR[EFPI]]]]]]] [[[[[[HQR[EFPI]]]]]]] BBBBBBBBBBBBCCCCCCCCCCCCAAAAAAAAAAAATTTTTTTTTTTT____________111111111111666666666666555555555666123456789023 BBBBBBBBBBBBIIIIIIIIIIIIAAAAAAAAAAAANNNNNNNNNNNNGGGGGGGGGGGG____________111111111111444444444444877777777776098765432109 BBBBBBBBBBBBBBBBRRRRRRRREEEEEEEE________00000000000000004455555489012347 stract/92/4/fiw056
[[GC]] [[GC]] BBBCCCAAATTT___111666666456 BBBSSSTTTEEERRR___111555776109 /219
[[[[[[[[[[M[AKDVNUOLTJ]]]]]]]]]]] [[[[[[[[[[MAKVUO[DNLTJ]]]]]]]]]]] BBBBBBBBBBBBBBBBBBBCCCCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAAAATTTTTTTTTTTTTTTTTTT___________________1111111111111111111666666666666666666666777777778888888867801234567123456789 BBBBBBBBBBBBBBBBBBSSSSSSSSSSSSSSSSSSTTTTTTTTTTTTTTTTTTEEEEEEEEEEEEEEEEEERRRRRRRRRRRRRRRRRR__________________111111111111111111555555555555555555666666666555555555876543210987654321 0 Color scale 5 8047 by guest on 10 April 2
BCAT_1689 BSTER_1550 0
0 20 40 60 0 20 40 60 BBCCAATT__11669901 BBSSTTEERR__11554498 19
A B C A B C BBSSTTEERR__11554465
BSTER_1544
Figure4.Transcriptionalmodulationofbifidobacterialgenesbysimulatedhumanadultorinfantgutenvironments.Thedifferentconditionsappliedwerereportedas
follows:conditionAisMRSmulti-association,conditionBisIFMorHFMmono-association,andconditionCisIFMorHFMmulti-association.ForAandBconditions,the
referenceconditionwasMRSmono-association,whileforconditionCthereferenceconditionwereHFM-orIFM-grownmono-associations.Panel(a)representedVenn
diagramsoftheobservedtranscriptomeofthe10bifidobacterialtaxachosenasrepresentativeofinfant-andadult-typeBifidobacterium.Largediagramsrepresented
totalnumberofgenesupregulatedinthemono-andmulti-associationstested.SmalldiagramsshowedthenumberoflociinvolvedinEPSbiosynthesisthatexhibit
increasedtranscription.Panel(b)depictedfunctionalannotationoftheaveragetranscribedgenesofinfantandadult-typebifidobacteriacultivatedunderA,BandC
conditionsaccordingtotheCOGcategory.EachCOGfamilywasidentifiedbyaone-letterabbreviation(NationalCenterforBiotechnologyInformationdatabase).Panel
(c)illustratedtheexpressionofdifferentepslociinBifidobacteriumstrainsinvolvedinmulti-associationsexperiments.ConservedEPS-associatedclusters,namedeps1
(forB.catenulatumLMG11043andB.dentiumLMG11405),eps2(forB.catenulatumLMG11043,B.dentiumLMG11405,B.angulatumLMG11039andB.stercorisDSM24849),
eps5(forB.adolescentisATCC15703andB.pseudocatenulatumLMG10505)andeps8(B.breveLMG13208),werereported.Intheheatmaprepresentation,whitespecifiedan
observedincreaseintranscriptionallevels,blackindicatedadecreaseinobservedtranscriptionallevels.Eachrowshowedaseparatetranscript,whileeachcolumn
indicatedaseparatecondition.