CP006881熊猫直播频道

RESEARCHARTICLETranscriptionalRegulationandAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2IsolatedfromFecesoftheGiantPandaZiyaoZhou1,XiaoxiaoZhou2,JinLi1,ZhijunZhong1,WeiLi1,XuehanLiu1,FuruiLiu1,HuaiyiSu1,YongjiuLuo1,WuyangGu1,ChengdongWang3,HeminZhang3,DeshengLi3,TingmeiHe3,HualinFu1,SuizhongCao1,JinjiangShi1,GuangnengPeng1*1TheKeyLaboratoryofAnimalDiseaseandHumanHealthofSichuanProvince,CollegeofVeterinaryMedicine,SichuanAgriculturalUniversity,Ya'an,625014,PRChina,2ChengduCenterforAnimalDiseasePreventionandControl,Chengdu,610041,PRChina,3Ya'anBifengxiaBase,ChinaConservationandResearchCenterfortheGiantPanda,Ya'an,625007,PRChinaTheseauthorscontributedequallytothiswork.
*pgn.
sicau@163.
comAbstractInthegiantpanda,adaptationtoahigh-fiberenvironmentisafirststepfortheadequatefunctioningofintestinalbacteria,asthehighcellulosecontentofthegutduetothepanda'svegetarianappetiteresultsinaharshenvironment.
Asanexcellentproducerofseveralen-zymesandvitamins,Bacillussubtilisimpartsvariousadvantagestoanimals.
Inourpreviousstudy,wedeterminedthatseveralstrainsofB.
subtilisisolatedfrompandasexhibitedgoodcellulosedecompositionability,andwehypothesizedthatthisbacterialspeciescansurviveinandadaptwelltoahigh-fiberenvironment.
Toevaluatethishypothesis,weemployedRNA-SeqtechnologytoanalyzethedifferentiallyexpressedgenesoftheselectedstrainB.
subtilisHH2,whichdemonstratessignificantcellulosehydrolysisofdifferentcarbonsources(celluloseandglucose).
Inaddition,weusedbioinformaticssoftwareandresourcestoanalyzethefunctionsandpathwaysofdifferentiallyexpressedgenes.
Interestingly,com-parisonofthecelluloseandglucosegroupsrevealedthattheup-regulatedgeneswerein-volvedinaminoacidandlipidmetabolismortransmembranetransport,bothofwhichareinvolvedincelluloseutilization.
Conversely,thedown-regulatedgeneswereinvolvedinnon-essentialfunctionsforbacteriallife,suchastoxinandbacteriocinsecretion,possiblytoconserveenergyforenvironmentaladaptation.
TheresultsindicatethatB.
subtilisHH2trig-geredaseriesofadaptivemechanismsatthetranscriptionallevel,whichsuggeststhatthisbacteriumcouldactasaprobioticforpandasfedahigh-fiberdiet,despitethefactthatcellu-loseisnotaverysuitablecarbonsourceforthisbacterialspecies.
Inthisstudy,wepresentamodeltounderstandthedynamicorganizationofandinteractionsbetweenvariousfunc-tionalandregulatorynetworksforunicellularorganismsinahigh-fiberenvironment.
PLOSONE|DOI:10.
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0116935February6,20151/13OPENACCESSCitation:ZhouZ,ZhouX,LiJ,ZhongZ,LiW,LiuX,etal.
(2015)TranscriptionalRegulationandAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2IsolatedfromFecesoftheGiantPanda.
PLoSONE10(2):e0116935.
doi:10.
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0116935AcademicEditor:Yung-FuChang,CornellUniversity,UNITEDSTATESReceived:November1,2014Accepted:December16,2014Published:February6,2015Copyright:2015Zhouetal.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailabilityStatement:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.
Funding:ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(number31272620),SichuanProvincialDepartmentofScienceandTechnologySupportProgram(number2011NZ0060)andtheProgramforChangjiangScholarsandInnovativeResearchTeaminUniversity(numberIRT0848).
Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
IntroductionTheintestinalmicrobiota,ofwhichamajorcomponentisbacteria,greatlycontributestohostnutrition,metabolism,immunityandothercharacteristics[1–3].
However,thegutenviron-mentisnotanideallocationforintestinalbacteriabecausemostgrowthandreproductiongenesareinhibited[4].
Forthegiantpanda(Ailuropodamelanoleuca),oneofthemosthighlyendangeredmammals,withonly2,500to3,000individualssurvivedinwesternChina[5],ahigh-fibervegetariandietcombinedwithacarnivore-likegastrointestinalsystemresultsinaharshgutenvironment,assoftbambooshootsandstemsarethemajorfoodsourcesofthepandabutthisanimaldoesnotpossessarumenforfermentation[6,7].
Thus,adaptationtoahigh-fiberenvironmentisthefirststepfortheintestinalmicrobiotaofthegiantpandatoactasaprobiotic.
Althoughseveralrecentstudieshaveestablishedaframeworkforthecommunitycompositionandfunctionsofthepandaintestinalmicrobiotaviametagenomics[8–10],itisnotyetwellunderstoodhowasingleorganismcansurviveandadaptinthehigh-fibergutenvironmentofthegiantpanda.
Asavitalbacterialspeciesofthemammalianintestinalmicrobiome,Bacillussubtilisimpartsvariousadvantagestoanimalsduetoitsexcellentabilitytoproduceseveralenzymesandvita-mins[11,12].
Inourpreviousstudy[13,14],wedeterminedthatseveralB.
subtilisstrainsisolat-edfrompandasdemonstratedgoodcellulosedecompositioncapabilityaswellasothercontributions;thus,wespeculatedthatthisbacterialspeciescouldhelpthepandainseveralways,includingaidinginthedigestionofbambooinahigh-fiberintestinalenvironment.
Previously,glucosehadbeenrecognizedasthepreferredcarbonsourceformicrobes[15],whilemanybacterialspecieshadbeenshowntonotbeabletousecellulose.
OurpreviousresultsprovedthatB.
subtilishadabilitytosurviveinahigh-fiberenvironment,butthemecha-nismisstillunknown.
TheliteraturehasshownthattheadaptationofB.
subtilisindifferentenvironmentsoccursmainlythroughtranscriptionalregulation[16].
Thelevelsofmostbacterialstressadaptationmolecules,suchastheHfqprotein[17],smallRNAs[18]and16SrRNA[19],aredeterminedatthetranscriptlevel.
Therefore,weemployedRNA-Seqtechnologytocomparethedifferen-tiallyexpressedgenes(DEGs)oftheselectedB.
subtilisHH2strainwhenusingcelluloseandglucoseasprimarycarbonsources,andwethenanalyzedthefunctionsandpathwaysoftheDEGs.
Inthisstudy,werevealedmajortranscriptionalreconfigurationsinresponsetocelluloseadaptationaswellascertaincoordinatedchangesintheabundanceofB.
subtilisHH2.
Wethenproducedamodeltounderstandthedynamicorganizationoftheinteractionsbetweenvariousfunctionalandregulatorynetworksofunicellularorganismsinthegiantpandaintestine.
MaterialsandMethodsBacterialstrainandcultivationconditionsGlucosemediumwasmodifiedfromapreviousstudy[20]:70mmolK2HPO4,30mmolKH2PO4,25mmol(NH4)2SO4,0.
5mmolMgSO4,10μmolMnSO4,22mgferricammoniumcitrate,8gpotassiumglutamate,6gpotassiumsuccinate,1%glucose,0.
5mmolCaCl2,5μmolMnCl2,and1000mLofddH2OatpH=7.
2.
Thecellulosemediumwasformulatedinthesamewayastheglucosemedium,exceptthatthemaincarbonsourcewas1%sodiumcarboxymeth-ylcelluloseinsteadof1%glucose.
B.
subtilisHH2fromfreshfecesthatwascollectedfromhealthypandasattheYa'anBifengxiaBaseoftheChinaConservationandResearchCenterfortheGiantPanda(CCRCGP)andplacedinsterilesamplingbagsinourpreviousstudywasisolatedandidentified.
ThisAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2PLOSONE|DOI:10.
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0116935February6,20152/13CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
strainhasagoodabilitytodigestcellulose;thediameterofitscellulosehydrolysishalowas28.
00±0.
44mm(S1Fig.
).
Thestrainwasgrownin100mLofLBmediumat37°Cinashakerat150rpmfor24h.
Aftercultivation,1%ofthecellswasinoculatedintoglucoseandcellulosemediaandgrownat37°Cinashakerat150rpmuntilOD600~1.
RNAisolationandpreparationTotalRNAwasextractedusingthehotphenolmethod[21].
Inbrief,cellpelletswereresus-pendedandwashedonceinBufferA(50mMsodiumacetateand10mMEDTA,pH=5.
2).
Aftercollectingthecellsbycentrifugation,thepelletswereresuspendedinBufferAcontaining1%SDSandimmediatelyaddedtohotphenol.
Afterincubationat65°Cfor5minutesfollowedbycentrifugationfor10minutesat4°C,theRNA-containingsupernatantsweretransferredtoanewtubeforethanolprecipitation,washedandthendissolvedinDEPC-treatedwater.
TheRNAwasfurtherpurifiedwithtwophenol-chloroformtreatmentsandthentreatedwithRQ1DNase(Promega)toremoveDNA.
ThequalityandquantityofthepurifiedRNAweredeter-minedbymeasuringtheabsorbanceat260nm/280nm(A260/A280)usingSmartspecPlus(BioRad).
TheintegrityoftheRNAwasfurtherverifiedby1.
5%agarosegelelectrophoresis.
cDNAlibraryconstructionandsequencingRibosomalRNAswereremovedfromtheRNAsamples(10μg)usingaRiboMinusrRNAdepletionkit(Ambion),andtheresultingsampleswereusedtopreparedirectionalRNA-Seqlibraries[22,23].
ThepurifiedmRNAsweretheniron-fragmentedat95°Cfollowedbyendre-pairand5'adaptorligation.
Then,reversetranscriptionwasperformedusingRTprimerscon-taininga3'adaptorsequenceandarandomizedhexamer.
ThecDNAswerepurifiedandamplified,andall200-500-bpPCRproductswerepurified,quantifiedandstoredat-80°Cuntiltheywereusedforsequencing.
Forhigh-throughputsequencing,thelibrarieswerepreparedfollowingthemanufacturer'sinstructions,andtheIlluminaGAIIxsystemwasusedtocollectdatafrom80-ntsingle-endsequencing(ABlifeInc.
;Wuhan,China).
AlignmentofreadstothegenomeAfterobtainingthesequencingdata,therawdatawerescreened,whichincludedremovaloftwo-N-containingreads,removalofthesequenceadaptor,andidentificationofcleanreadswithlengthsofmorethan16ntafterremovaloflow-qualityvalues.
Becausethe16SrRNAgeneofB.
subtilisHH2hasmaximalhomologywithBacillus_subtilis_PY79(CP006881.
1)whencomparedwithallB.
subtilisgenomesintheNCBIdatabase(beforeNovember11,2013),wedecidedtouseBacillus_subtilis_PY79asthereferencegenomeforB.
subtilisHH2.
Weuti-lizedTophat[24]with2-ntmismatchestoalignoursequencingdatatothereferencegenomeofB.
subtilisHH2(ftp://ftp.
ncbi.
nlm.
nih.
gov/genomes/Bacteria/Bacillus_subtilis_PY79_uid229877/).
UsingtheRPKMs(readsperkilobaseofagenepermillionreads)[25],weeliminatedthedeviationsduetothelengthsofdifferentgenes.
AnalysisofdifferentiallyexpressedgenesToperformdifferentialgeneexpressionanalysis,weappliedthesoftwareedgeR[26],whichisspecificallyusedtoanalyzethedifferentialexpressionofgenesusingRNA-Seqdata.
Todeter-minewhetheragenewasdifferentiallyexpressed,theanalysisresultswerebasedonthefoldchange(FC2orFC-2)andP-value(P0.
01).
TopredictgenefunctionandcalculatetheAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2PLOSONE|DOI:10.
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0116935February6,20153/13functionalcategorydistributionfrequency,KEGGandGeneontology(GO)analyseswereem-ployedusingDAVIDbioinformaticsresources[27].
ResultsandAnalysisCelluloseisnotahighlysuitablecarbonsourceforB.
subtilisHH2B.
subtilisHH2wasfirstculturedinmediasupplementedwithcelluloseorglucoseastheprimarycarbonsource.
WefoundthatthegrowthofHH2wassignificantlyinhibitedinthecelluloseme-diumcomparedwiththeglucosemedium(Fig.
1).
Inaddition,whenthebacterialculturereachedOD600~1,weobservedboththecellulose-andglucose-grownbacterialsamplesunderalightmi-croscope.
Bacterialsporesweremoreapparentinthecellulosemediumthanintheglucosemedi-um(Fig.
2),whichindicatedthatcelluloseisnotasuitableenergysourceforthisbacterialstrain.
ProfileoftheRNA-SeqdataAftercultivation,weextractedtotalRNAfromthebacteriatoanalyzethetranscriptionalregulationinthedifferentcarbonenvironmentsvianextgenerationsequencing(NGS).
Weob-tained20,231,277and24,807,479fragmentsfromthecelluloseandglucosegroups,respective-ly.
Afterscreening,14,733,930and20,066,967cleanreadsweregeneratedfromthetwogroups,respectively.
WethenanalyzedwhetherthesereadsmatchedthereportedreferencegenomeintheGenBankdatabaseusingblastn(E-value1e-5).
Wefoundthatmorethan75%ofthereadscouldbemappedtothereferencegenome,ofwhichapproximately50%wereuniquelymappedFig1.
ThegrowthcurvesofB.
subtilisHH2exposedtodifferentcarbonsources.
B.
subtilisHH2wasculturedincelluloseorglucosemediumfollowing1%inoculationat37°Cinashakerat150rpm;theOD600wasmeasuredeveryhour.
Eachgraphrepresentsthemeanofthreeindependentbiologicalreplicatesgrownonthreedifferentdays.
Theerrorbarsrepresentthestandarddeviations(SDs)oftheopticaldensityateachtimepoint.
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0116935February6,20154/13reads,representingacombinedsequencecoverageof250X(Table1).
Inthisstudy,thedetectedexpressedgenes(mappedreadsnumber10)comprised81.
67%(3,279/4,278)ofthereadsforthecellulosegroupand92.
69%(3,779/4,278)ofthereadsfortheglucosegroup.
Thetotalnum-berofdetectedgenesinthetwosampleswas4,134,whichaccountedfor96.
63%oftheB.
subti-lisgenes,indicatingthatthegenedetectioninthisstudylargelyreachedsaturation.
Thecorrelationcoefficient(R2)fortheexpressionofthesamegeneinthetwosampleswas0.
739,whichdemonstratedthatpartsofgenesexhibitedchangesinexpressionlevels.
Whencomparingthecellulosegroupwiththeglucosegroup,thenumberofsignificantlydown-regu-latedgenes(bymorethan10times)was164,whichwassignificantlygreaterthanthenumberofup-regulatedgenes(23genes),illustratingthattheseorganismsmaysignificantlyreducetheexpressionofsomegenesandlosepartialfunctionasawaytocopewiththeimpactsofanex-tremeenvironment[28].
AccordingtoGOfunctionalanalysis,23gene-functionclusterswereenrichedamongtheup-regulatedgeneswhencomparingthecellulosegrouptotheglucosegroup,and22clustersamongthedown-regulatedgeneswereenriched(S1Table).
Interesting-ly,amongtheclusterswiththetop10enrichmentscores,thefunctionsoftheup-regulatedgenes(clusters)weremainlyassociatedwithcelluloseutilization,whereasmostofthedown-regulatedgeneswereassociatedwithnon-essentialfunctionsforbacteriallife,indicatingare-ductioninenergyconsumptiontopermitenvironmentaladaptation(Table2).
CarbonmetabolismgeneexpressionissignificantlyalteredinbothgroupsGenerally,carbonmetabolism-relatedgeneclusterexpressionisalteredinbacteriainconjunc-tionwithchangesinnutritionalfactors.
Wefoundthatthemostsignificantlyup-regulatedFig2.
Bacteriaunderthelightmicroscope.
(A)B.
subtilisHH2wasculturedinglucosemediumuntilOD600~1.
(B)B.
subtilisHH2wasculturedincellulosemediumuntilOD600~1.
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g002Table1.
MappingofcleanreadsintheB.
subtilisgenome.
SampleInputreadsTotalmappedUniquemappedMultiplemappedCellulose14,733,93011,293,808(76.
65%)5,314,734(47.
06%)5,979,074(52.
94%)Glucose20,066,96716,316,254(81.
31%)10,742,645(65.
84%)5,573,609(34.
16%)doi:10.
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0116935February6,20155/13clustersinthecellulosegroupwereinvolvedinthemetabolismofaminoacids,lipids,galactose,andketonebodies,whichindicatesthatwhenadirectlyusedcarbonsource(usuallybeta-D-glucose)intheenvironmentislimiting,anorganismmayconsumeotherintracellularsub-stancesforsupplementation.
Cellulasecomponentgenes,suchasthebeta-glucanasegeneU712_19750,weresignificantlyhighlyexpressedinthecellulosegroupcomparedtotheglu-cosegroup.
SimilarobservationswerenotedforU712_19485andU712_19495,genesthaten-codetwosubunitsofthelichenin-specificphosphotransferaseenzyme,whichhasbeenconfirmedtobeinvolvedinthedigestionofcellobiose,asecondaryproductofcellulosecataly-sis[29].
Tofurtherutilizecellobiose,variousformsofglucosidase(suchasU712_19480andU712_03600)arealsoup-regulatedtocreatethefinalproductbeta-D-glucose.
Incontrast,theexpressionofglucosemetabolism-relatedgeneswassignificantlydecreasedinthecellulosegroupcomparedtotheglucosegroup,particularlyatthestartofglucosemetab-olism.
Inaddition,theexpressionoftheUDP-glucose6-dehydrogenasetuaD(U712_17840),anecessaryenzymeinthepentosephosphatepathway[30],wasdecreasedbymorethan16-fold.
Furthermore,atthebeginningofglucosemetabolism,theexpressionofgluconatekinase(U712_20270)wasdecreased9-foldinthecellulosegroupcomparedtotheglucosegroup;andatthesametime,thedownstreamproductionofglucose6-phosphate,whichisthesubstrateforthepentosephosphatepathway,wasdecreased[31].
S2Tableshowsthedifferentialexpres-sionofvariousgenesthatareimportantforcarbonmetabolisminthetwosamples.
Theexpressionofmostnon-essentialgenesisdecreasedinthecellulosegrouptoreduceenergyconsumptionApartfromglucosemetabolismgenes,thedown-regulatedgenesinthecellulosegroupwereinvolvedinchemotaxis,secretionoftoxinsandbacteriocins,motility,polymercompoundTable2.
GOtermanalysisofDEGs(top10enrichmentscores).
DifferentiallyexpressedgeneclusterDescriptionEnrichmentScoreDown-regulatedCluster1Toxin,peptide,antibioticandbacteriocinmetabolicprocesses3.
96Down-regulatedCluster2Chemotaxis,taxisandlocomotorbehavior2.
59Down-regulatedCluster3Flagellarassemblyandmotility2.
29Down-regulatedCluster4Membraneandtransmembrane2.
00Down-regulatedCluster5Flagellarassemblyandbacterialagellumproteinexport1.
98Down-regulatedCluster6Cellularmacromolecularcomplexassembly1.
28Down-regulatedCluster7Aniontransport1.
14Down-regulatedCluster8ABCtransporters0.
79Down-regulatedCluster9Cellwallbiogenesis/degradation0.
78Down-regulatedCluster10Metal-binding0.
68Up-regulatedCluster1Aminoacidmetabolism1.
44Up-regulatedCluster2Transmembrane1.
23Up-regulatedCluster3Carbohydratetransport1.
23Up-regulatedCluster4Oxidoreductaseandelectroncarrieractivity1.
02Up-regulatedCluster5Cellwallmacromoleculecatabolicprocesses0.
74Up-regulatedCluster6Proteintransportandlocalization0.
72Up-regulatedCluster7Calciumionbindingandsubstratebinding0.
56Up-regulatedCluster8Aminoglycanandpolysaccharidecatabolicprocesses0.
55Up-regulatedCluster9Aminoacidtransmembranetransporteractivity0.
52Up-regulatedCluster10Sporulation0.
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0116935February6,20156/13assembly,andcomplexproteinassembly.
Theexpressionofthesubtilosin-AassemblygeneU712_18825wassharplydecreasedby~187-fold.
Inadditiontothedecreasedexpressionofgenesinvolvedintheassemblyofcomplexsubstances,thedown-regulationofcertainnon-es-sentialgenesreducedtheenergyconsumptionofthecell.
Forexample,multiplevitalgenesintheflagellarassemblygenecluster(Fig.
3)weresignificantlydecreasedinthecellulosegroup.
Inaddition,theexpressionofnearlyeverystructuralproteingenewithinthetypeIIIsecretionsystem(T3SS)oftheflagellarassemblygeneclusterwasdown-regulated.
Furthermore,theex-pressionofaseriesofflagellargeneswasdecreased,indicatingthatmotilityandinfectionabili-tyweredecreasedduringnutritionaldeficiency.
Giventheinhibitionofvariousnon-essentialmetabolicgenesaswellasofgenesinvolvedinnon-essentialfunctionsinthecellulosegroup,itisapparentthatB.
subtilisHH2triggersaseriesofmechanismsthatconserveenergytobeusedforadaptationtoahigh-fiberenvironment.
Two-componentsystems(TCSs)andATP-bindingcassette(ABC)transportersareimportantregulatorysystemsforhigh-fiberenvironmentadaptionWhenbacteriaexperiencestress,theysignaltotheorganismthatanappropriatephysiologicalresponseisrequired.
TCSsandABCtransportershavebeenproposedtobeunitsthatpartici-pateinthecommonphysiologicalprocessofsignaltransductionandsubstancetransport[32,33].
InB.
subtilis,theyts,yvcandyxdgeneclustersencodeanABCtransporterfromsub-family9andacoupledTCSfromtheOmpRfamily[32],andthetranscriptionfactorofthissystemcanactivatetranscriptionbybindingDNAandinteractingwithRNApolymerase[34].
Wefoundthattheexpressionofeachgeneinthethreeclusters,aswellasthoseofthecorre-spondingABCtransporters,wasdecreasedbymorethanhalfinoursimulatedintestinalenvi-ronment.
Giventheinvolvedsignalingcascade,thereducedexpressionoftheOmpRfamilymayregulatetheinhibitionofmanygenes.
Furthermore,theexpressionofseveralzinc-bindinglipoproteinsandzincmetalloproteaseswasdecreasedinthecellulosegroup,whichcouldnega-tivelyaffecttheexpressionofmanyenzymes,storageproteins,transcriptionfactors,andpro-teinsinvolvedinreplication[35].
Therefore,TCSsandABCtransportersbothregulateenergyconservationinresponsetoahigh-fiberenvironment.
Conversely,TCSsandABCtransportersarealsowidelyinvolvedinnutrientabsorption.
InB.
subtilis,ATP-drivenuptakesystemspreferprimarycarbonandenergysources[36].
Severalki-nasesinTCSpathwaysinvolvedinthemetabolismofsomeaminoacidswereobservedtobeup-regulatedincellulosemedium,e.
g.
,GlnT(U712_01235)andYesM(U712_03510),whichin-dicateschangesintheexpressionofcarbonmetabolismgenes.
Severalhigh-affinityABCtrans-portersfordifferentsugarscatalyzethetransportofsugar-oligomerstoanevengreaterdegreethanpeptidetransporters,whichpermitsorganismstothriveinnutrient-poorenvironments[37,38].
Inthisstudy,theexpressionofmanyoligosaccharideandpolyoltransportersinvolvedincellulosehydrolyzatetransportwereincreasedinthecellulosegroup;forexample,theexpressionofthegeneU712_03520,whichencodestheputativeABCtransportersubstrate-bindingproteinyesO,wasincreased11.
26-foldcomparedtotheglucosegroup.
Itislikelythattheexpressionofmanypeptide-andcalcium-bindingABCtransporterproteingenes(suchasU712_06760,U712_06745andU712_08240)wasenhancedtoimprovethetransportofcellulosehydrolyzates.
StructuralmembraneandtransporterproteinsarekeyfactorsforadaptationtostressAsthefirstorganelletoexperiencepressureandthekeyorganelleforadaptationtoaharshenvironment,thecellmembranemakesvitalcontributionstocombattheeffectsofenvironmentalAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2PLOSONE|DOI:10.
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KEGGanalysisofflagellarassembly(bsu02040)inB.
subtilis.
Yellowboxesindicatesignificantlydown-regulatedgenesinthecellulosegroup,andgrayboxesindicateup-regulatedgenes(noneinthisfigure).
Greenindicatesagroupofproteins.
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0116935February6,20158/13aggression,suchasinitiatingthesecretionofseveralproteinsandexchangingintracellularandex-tracellularsubstances[38].
Inthisstudy,membraneandtransmembranegeneswereenrichedinboththeup-anddown-regulatedgeneclustersaccordingtoGOanalysis(S1Table);however,ontheindividuallevel,thespecificsofthisenrichmentwerecompletelydifferent.
Theup-regulatedclustersprimarilyconsistedoftransporterproteins,includingcarbohydratetransporters,sugartransporters,andsymporters,indicatingthatcellulosehydrolyzatemayrequireagreateramountofenergytobedeliveredintothecell.
Incontrast,structuralproteins,suchasplasmamembrane,cellmembrane,andintrinsicmembraneproteins,wereprimarilydown-regulated.
Thedown-regulationofstructuralmembranegenesincreasesthepermeabilityofthecellmembrane,allow-ingtheeasytransportofcellulosehydrolyzatesintothebacteria.
Therefore,withaseriesofadjustments,thecellmembranecouldbecomemoreadaptedtocellulosemedium.
Sporulationisalast-resortresponsetopressureandissuppresseduntilalternativeresponsesproveinadequateWhenalternativeresponsesproveinadequatetorelievestress,sporulationisthefatechosenbymostBacillusspecies[39].
Weobservedincreasedsporulationinthecellulosegroup,bothunderthelightmicroscope(Fig.
1)andatthetranscriptionallevel(U712_12580,encodingthesporulationinhibitorSda,wasdecreasedmorethan16-fold).
However,amongthesevenstages(0-VI)ofsporulationproteingenes(clusters),whichareproteinsthatdeterminethesporula-tionform,onlystageIIandstageIIIsporulationproteinswereobservedtobesignificantlyup-regulatedinthecellulosegroup.
Incontrast,theexpressionofproteinsinvolvedinthefiveotherstagesofsporulationwasnotgreatlychanged.
Therefore,celluloseremainsanacceptablecarbonsourceforB.
subtilisHH2,andthisstraincanadaptwelltoahigh-fiberenvironmentthroughaseriesofalterationsintranscriptionalregulation.
AregulatorymodelofB.
subtilisHH2inahigh-fiberenvironmentAsdescribedabove,werevealedmajortranscriptionalreconfigurationsinresponsetocelluloseadaptationaswellascertaincoordinatedchangesintheabundanceofB.
subtilisHH2inahigh-fiberenvironment.
Tosummarizetheseadaptationmechanisms,weproposearegulatorymodelofB.
subtilisHH2forhigh-fiberenvironmentaladaptationandcellulosedigestion.
Inthismodel,utilizationofandadaptationtocelluloserequireatleastfourfunctionalclassesofproteins,including(i)membraneproteinsandmembrane-associatedsignalchannels,(ii)en-zymesthatcatalyzecellulosehydrolysis,(iii)proteinsencodedbyoperonsthatdecreasecellularenergyandnutrientconsumption,and(iv)proteinsinvolvedinsporulation.
Thecellulardeg-radationofandadaptationtocelluloseconsistoffivesteps.
First,whenbacteriaaregrownonamediumwithcelluloseasaprimarycarbonsource,ionchannel-coupledreceptorsinthecellmembranearestimulatedbycelluloseandsendsignalstotheassociatedtransductionsystems.
Asaresult,cellulasecomponentsareexpressed,secreted,assembledandtransportedtothecellsurface,whichhydrolyzesthecellulose.
Cellobioseandglucan,bothproductsofcellulosehydrolyzation,aretransportedintothecellthroughchannelsforhydrolyzatesandABCtrans-portersforfurtherutilization.
Simultaneously,uponpressuresignals,bacteriapartiallyreducethesynthesisofnon-essentialproteinstosaveenergy.
Theexpressionofsporulationgenesalsopartiallyincreasestoaddresspotentialcontinuingharshpressures.
DiscussionB.
subtilisiswidelyusedasaprobioticandfoodadditivewithmammalianapplicationsduetoitsexcellentabilitytosecreteavarietyofantimicrobialsubstancesthatmaintaintheintestinalmicroflorabalance[40]andimprovethedigestibilityofforagedfoodsinthegastrointestinalAdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2PLOSONE|DOI:10.
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0116935February6,20159/13tract[41].
AnumberofstudieshaveexaminedB.
subtilisresistance[16,19],butthereisstillarelativedearthofresearchonenvironmentalcelluloseadaptationmechanisms.
Ahigh-fiberenvironmentismostcharacteristicoftheherbivorousanimalgutenvironment,particularlyforthegiantpanda,whichhasnorumenforthefermentationofvegetation.
Inadditiontothege-nomicpotentialoftheintestinalmicrobiota,understandingbacterialadaptationmechanismsforacelluloseenvironmentwillhelpustobetterclarifytheinteractionsbetweenintestinalbac-teriaandtheirpandahost.
ThroughphenotypeexperimentofB.
subtilisHH2isolatedfrompandasandgrownondif-ferentcarbonsources,wedemonstratedthatcelluloseisnotaverysuitablecarbonsourceforB.
subtilisHH2.
Howeverbasedonourtranscriptionalpathwayanalysis,wefoundthatthisbacte-riumcanadaptwellviaaseriesofregulatorynetworksandthatthedifferentiallyexpressedgenesclusteredintotwomaincategories:celluloseutilizationandhigh-fiberenvironmentad-aptation.
Forcelluloseutilization,strainHH2notonlyincreasedtheexpressionofcellulasebutalsoofaseriesofenzymecomponentsthathydrolyzecellulose,aswellassomeABCtransport-ers,whichservedassupport.
Whereasithasbeenshownthatorganismscanselectivelyexpresssomegeneshighlyforstressadaptation[39,42],inthisstudy,itwasobservedthatmanygenes(clusters),suchasseveralproteinkinases,wereup-regulatedbutthattheexpressionofmostnon-essentialgenesweredown-regulatedtoconserveenergy.
Interestingly,theexpressionoftheHfqprotein(U712_09100),severalsporulationkinasesandgenesofproteinsinvolvedinsporulationwasdecreasedinthecellulosegroup.
TheHfqproteinisanRNA-bindingproteinassociatedwithsmallregulatoryRNAs(sRNAs)andhasmanyfunctionsinpressureadaptation[17].
SporulationisthefinaladaptationbythegenusBacillustorelievestress,andthedecreaseintheexpressionofthesegenesindicatesthatcellu-losemaybeanacceptablecarbonsourceforB.
subtilisHH2.
Asanintestinalprobiotic,B.
subtilisHH2'scelluloseutilizationabilitycouldaidpandasindigestingbamboo.
Thebacterialmetabolitesubstances,suchaspolypeptidesandlipids,whichcouldbedigestedbythehostwereproducedincellulosedecomposing.
Inadditiontoitsnutri-tionaleffects,HH2alsocontributestomaintainingtheintestinalmicroflorabalanceinthehost,sincethesubstancesforantimicrobialeffectandimmunestimulationwerecontinuousproducinginahighcelluloseenvironment.
Basedonthegeneexpressiondata,wefoundthatalthoughHH2decreasingsomeantimicrobialpeptidesexpressioninthecellulosemediumcomparedtothatinglucosemediummayreduceitsantimicrobialfunctions,mostofbacitracinexpressioncouldstillbedetectedinthecellulosegroup.
Suchasflagellin(U712_17730)andaseriesofsurfactincomponents,whichareinvolvedinimmunestimulationandresistancetopathogencolonization[40],weredetectedmorethan2,000reads.
Thus,webelievethatB.
sub-tilisHH2canstillexertmostprobioticfunctionsbothinnutritionalandantimicrobialeffectsinahigh-fiberenvironmentwithinanimals.
Insummary,thisstudyrevealedmajorchangesintranscriptionalregulationsinresponsetocelluloseadaptationofB.
subtilisHH2ondifferentcarbonsources,asdetectedbyRNA-Seq.
Theseresultsdemonstratethatthisbacteriumcouldplaypartoffunctionsasaprobioticforpandasinahigh-fiberenvironment,althoughcelluloseisnotaverysuitablecarbonsourceforthisstrain.
Wealsodemonstratedamodelforunderstandingthedynamicorgani-zationandinteractionsofthevariousfunctionalandregulatorynetworksforunicellularor-ganismsinahigh-fiberenvironment.
Asawell-characterizedbacteriumandaGram-positivelaboratorymodel,thetranscriptionalregulationofB.
subtilisHH2inahigh-fiberenviron-mentwillbeareferenceforotherintestinalbacteria.
Therefore,theseresultsrepresentanim-portantcontributiontotheresearchontheprotectionbytheintestinalmicrobiotaofthepanda.
AdaptationtoaHigh-FiberEnvironmentinBacillussubtilisHH2PLOSONE|DOI:10.
1371/journal.
pone.
0116935February6,201510/13SupportingInformationS1Fig.
CellulosehydrolysishalosofB.
subtilisHH2.
Thisstrainhasagoodabilitytodigestcellulose;thediameterofitscellulosehydrolysishalowas28.
00±0.
44mm.
(TIF)S1Table.
GOtermanalysisofdifferentiallyexpressedgenes(DEGs).
(XLSX)S2Table.
Theexpressionlevelsofselectedimportantcarbon-metabolismgenes.
(DOCX)AuthorContributionsConceivedanddesignedtheexperiments:ZYZXZZJZGP.
Performedtheexperiments:ZYZXZJLWL.
Analyzedthedata:ZYZXZWLXL.
Contributedreagents/materials/analysistools:FLHSYLWGCWHZDLTH.
Wrotethepaper:ZYZXZHFSCJSGP.
Samplecollectpermis-sion:HZDL.
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