Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance

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Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance
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Ahn, Sang-Joon
Qu, Ming-Da
Roberts, Elisha
Burne, Robert A.
Rice, Kelly C.
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Background: The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans. Results: Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/ repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant. Conclusions: Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans. Keywords: Stress, Oxygen, Competence, Cid/Lrg system, Streptococcus mutans
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The institutional Repository at the University of Florida community with research, news, outreach, and educational materials.
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Ahn et al. BMC Microbiology 2012, 12:187 http://www.biomedcentral.com/1471-2180/12/187; Pages 1-12
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doi:10.1186/1471-2180-12-187 Cite this article as: Ahn et al.: Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance. BMC Microbiology 2012 12:187.

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RESEARCHARTICLEOpenAccessIdentificationofthe Streptococcusmutans LytST two-componentregulonrevealsitscontribution tooxidativestresstoleranceSang-JoonAhn1,Ming-DaQu2,ElishaRoberts2,RobertABurne1andKellyCRice2*AbstractBackground: The S.mutans LrgA/Bholin-likeproteinshavebeenshowntoaffectbiofilmformationandoxidative stresstolerance,andareregulatedbyoxygenation,glucoselevels,andbytheLytSTtwo-componentsystem.Inthis study,wesoughttodetermineifLytSTwasinvolvedinregulating lrgAB expressioninresponsetoglucoseand oxygenationin S.mutans Results: Real-timePCRrevealedthatgrowthphase-dependentregulationof lrgAB expressioninresponseto glucosemetabolismismediatedbyLytSTunderlow-oxygenconditions.However,theeffectofLytSTon lrgAB expressionwaslesspronouncedwhencellsweregrownwithaeration.RNAexpressionprofilesinthewild-typeand lytS mutantstrainswerecomparedusingmicroarraysinearlyexponentialandlateexponentialphasecells.The expressionof40and136genesinearly-exponentialandlateexponentialphase,respectively,wasalteredinthe lytS mutant.Althoughexpressionof comYB ,encodingaDNAbinding-uptakeprotein,wassubstantiallyincreasedinthe lytS mutant,thisdidnottranslatetoaneffectoncompetence.However,a lrgA mutantdisplayedasubstantial decreaseintransformationefficiency,suggestiveofapreviously-unknownlinkbetweenLrgAand S.mutans competencedevelopment.Finally,increasedexpressionofgenesencodingantioxidantandDNArecombination/ repairenzymeswasobservedinthe lytS mutant,suggestingthatthemutantmaybesubjectedtoincreased oxidativestressduringnormalgrowth.Althoughtheintracellularlevelsofreactionoxygenspecies(ROS)appeared similarbetweenwild-typeand lytS mutantstrainsafterovernightgrowth,challengeofthesestrainswithhydrogen peroxide(H2O2)resultedinincreasedintracellularROSinthe lytS mutant. Conclusions: Overall,theseresults:(1)ReinforcetheimportanceofLytSTingoverning lrgAB expressioninresponse toglucoseandoxygen,(2)DefineanewroleforLytSTinglobalgeneregulationandresistancetoH2O2,and(3) UncoverapotentiallinkbetweenLrgABandcompetencedevelopmentin S.mutans Keywords: Stress,Oxygen,Competence,Cid/Lrgsystem, StreptococcusmutansBackgroundStreptococcusmutans isconsideredtheprimarycausativeagentofdentalcaries,andwhentransientlyintroducedintothebloodstreamfollowingdailydental hygienicpracticessuchastoothbrushingandflossing, thisbacteriumcanalsocausepotentiallylethalinfective endocarditis(IE)[1-4].Inbothinfectiousscenarios,the virulenceof S.mutans dependsuponitsabilitytoform biofilmsandtowithstandextremechangesin environmentalconditions,includingfluctuationsinoxygenation,shearstress,aswellasnutrientsourceand availability.Forexample,intheoralcavity, S.mutans mustbeabletorapidlyalteritsexpressionoftransportersandmetabolicenzymestocatabolizeavarietyof host-deriveddietarycarbohydrates.Internalizedcarbohydratesaremetabolizedthroughtheglycolyticpathway, resultingintheaccumulationofacidicend-productsin theenvironment,whichfavorsthegrowthof S.mutans andotheracid-tolerantcariogenicspecies.Repeated cyclesofacidificationcanleadtoanetdemineralization oftoothenamelandthedevelopmentofcaries.Sucrose, acommondietarysweetener,canalsobeutilizedby *Correspondence: kcrice@ufl.edu2DepartmentofMicrobiologyandCellScience,CollegeofAgriculturaland LifeSciences,UniversityofFlorida,Gainesville,FL32611,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Ahnetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Ahn etal.BMCMicrobiology 2012, 12 :187 http://www.biomedcentral.com/1471-2180/12/187

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S.mutans fortheproductionofextracellularpolysaccharides[5-8]thatfacilitatebacterialadhesionandbiofilmformation.Aerationhasalsobeenfoundtohavea profoundeffectoncarbohydratemetabolismandbiofilm formationby S.mutans [9-11].Itisthereforenotsurprisingthatthereisoverlapinthegeneticregulatorycircuitsresponsivetocarbohydratemetabolism,aeration/ oxidativestressresistanceandcontrolofbiofilmformationin S.mutans ,whichincludeCcpA[12-14],Rex[15], andFrp[16]. Morerecently,anemergingtrendinthestudyofbacterialbiofilmshasbeenafocusonthecontributionof bacterialcelldeathandautolysistobiofilmadherence, maturation,anddispersal.Ithasbeendemonstratedina widevarietyofbacteriathatdeathandlysisofasubpopulationofcellscanfacilitatebiofilmformationdueto thereleaseofDNAintotheextracellularenvironment (eDNA)[17-22].Likewise,celldeathandlysishavebeen implicatedindispersalofcellsfromamaturebiofilm [23-25].In Staphylococcusaureus ,theCid/Lrgsystem hasbeenshowntobeinvolvedintheregulationofcell death,autolysis,andbiofilmformation[17,21,26-28]. Characterizationof S.aureuscid and lrg mutantshas revealedthattheseoperonshaveopposingeffectsoncell deathandmureinhydrolaseactivity[27,29].These observations,combinedwiththefactthatLrgAand CidAsharestructuralfeatureswiththebacteriophage lambdafamilyofholinproteins[29],haveledtothehypothesisthatCidAandLrgAcontrolcelldeathandlysis inamanneranalogoustoeffectorandinhibitorholins, respectively[26,30].Bacteriophageholinsaresmall membraneproteinsthatoligomerizeinthecellmembrane,actingas “ molecularclocks ” thatregulatethetimingandlysisofthehostcellduringlyticinfection[31]. Forexample,thelambdaSholinregulatescelldeathand lysisbytheformationoflargelipid-excluding “ rafts ” that promotecytosolicleakageaswellasaccessofthephageencodedendolysin(mureinhydrolase)tothecellwall [32-34]. S.aureus CidAandLrgAhaverecentlybeen showntooligomerizeintohigh-molecular-masscomplexesinacysteinedisulfidebond-dependentmanner,a biochemicalfeaturealsos haredwithholinproteins [35].AlthoughthemoleculardetailsofhowCidand Lrgfunctiontocontrolcelldeathandlysishavenot yetbeencompletelyelucidated,thefactthat cid and lrg homologueshavebeenidentifiedinawidevariety ofbacterialandarchealgenomessupportsafundamental andconservedroleforthissystemincellphysiology [30,36]. Inpreviousworkitwasdeterminedthatexpressionof potential cidAB and lrgAB homologuesin S.mutans is highlyresponsivetocarbohydrateavailability[12,37]and oxygenation[11].Giventhepotentialimportanceof thesegenestobiofilmdevelopmentin S.mutans ,we previouslycharacterizedapanelof S.mutanscid and lrg isogenicmutantsandfoundthatasubsetofthesegenes didindeedinfluencebiofilmformation,productionof glucosyltransferases(enzymesthatsynthesizeextracellularglucanpolymersthatcontributetobiofilmadhesion), andoxidativestresstolerance[37].Inthisstudyitwas alsofoundthat,asdemonstratedpreviouslyin S.aureus [38,39],the S.mutans LytSTtwo-componentsystem wasrequiredforactivationof lrgAB expression,butnot cidAB expression[37].Geneshomologousto lytST appeartobepresentinmostGram-positiveorganismsthat contain lrgAB [30]andthesegenesareoftenlinkedto oneanother,inferringanimportantroleforthistwocomponentsysteminfine-tuning lrgABexpressioninresponsetoexternalenvironmentalsignals.Thereforein thisstudy,wesoughttodetermineifLytSTisinvolved inregulationof lrgAB expressioninresponsetoglucose andoxygenationin S.mutans ,andtoelaborateonthe contributionofLytSTtocellularhomeostasisandglobal controlofgeneexpression.ResultsEffectsofoxygenationandglucosemetabolismon S.mutanslrg and cid expressionTheLytSTtwo-componentregulatorysystemhasbeen showntopositivelyregulate lrgAB expressioninawidevarietyofbacteria,includingvariousstaphylococcal[38-40] and Bacillus species[41,42],aswellasin S.mutans [37]. TheconservednatureofthisregulationinGram-positive bacteria,combinedwiththeknowneffectsofLytSTand LrgABoncelldeath/lysis[29,38,39,43],biofilmdevelopment[21,37,38],andoxidativestressresistance[37],suggeststhatLytSTandLrgABarecentralregulatorsof physiologichomeostasis.However,littleisknownaboutthe environmentaland/orcellularcuestowhichLytSresponds. In S.aureus and B.anthracis ,ithasbeenshownthat lrgAB expressionisresponsivetodisruptionofcellmembranepotentialinaLytST-dependentmanner[41,44].However,we wereunabletodeterminewhe therthisregulationalso occursin S.mutans ,astreatmentwithmembrane-potential disruptingagents(gramicidin,carbonylcyanidem-chlorophenylhydrazone)didnothaveameasurableeffecton membranepotential,asassessedbystainingwithDIOC2(3)(datanotshown). Inpreviousstudies,itwasshownthatoxygenandglucosemetabolismhaveapronouncedeffecton lrg and cid expressionin S.mutans ,butthespecificroleofLytS, ifany,inthisregulationwasnotaddressed[11,37]. Therefore, S.mutans UA159anditsisogenic lytS mutant weregrownunderaerobicandlow-oxygenconditionsto exponential(EP)andstationary(SP)growthphasesin mediacontaining11mMor45mMglucose.Quantitativereal-timereversetranscriptasePCR(qRT-PCR)was performedonRNAisolatedfromculturesateachtimeAhn etal.BMCMicrobiology 2012, 12 :187 Page2of12 http://www.biomedcentral.com/1471-2180/12/187

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pointtoassesschangesin lrg expression(Figure1).In UA159,stationaryphase lrgAB expressionwasupregulated365-foldrelativetoexponentialphasewhengrown under11mMglucoseandlow-oxygenconditions (Figure1A) Althoughmutationof lytS resultedinaseverelossofstationaryphase lrgAB inductionincells grownin11mMglucose, lrgAB expressionwasnot completelyabolished.Whengrownunderaerobicconditionsand11mMglucose,stationaryphase lrgAB expressionwasupregulated2500-foldrelativetoexponential phaseinthewild-typestrain(Figure1A),confirming previously-publishedobservationsthataerobicgrowth promotes lrgAB expression[11].However,stationaryphase lrgAB expressionwasstillinduced216-foldinthe lytS mutantduringaerobicgrowth,suggestingthat(1) otheras-yet-unknownregulatorsalsocontributetothe positivecontrolof lrgAB expressionduringaerated growth,and(2)LytSTisapredominantregulatorof lrgAB expressionduringlowoxygengrowth,compared toaerobicgrowth.Underlow-oxygenandaeratedcultures,stationaryphaseinductionof lrgAB expression wasdramaticallyreducedwhengrownin45mMglucose,andsimilarlevelsofexpressionwereobservedin thewild-typeand lytS mutant(Figure1B),suggesting thatgrowthinhighlevelsofglucoseabrogatesoxygendependentregulationof lrgAB byLytST.Consistentwith previously-publisheddata[37],LytSdidnotappearto haveameasurableeffecton cidAB expressionunderany ofthegrowthconditionstestedhere(datanotshown). Insummary,LytST-dependentregulationof lrgAB expressionismuchmorepronouncedduringlow-oxygen growthandatlowglucoselevels.MicroarrayanalysisoftheLytSregulonBasedonthetranscriptionaldatapresentedabove,the effectsofLytSTregulationon lrgAB expressionaremost evidentwhile S.mutans isgrowingunderconditionsof low-oxygen(5%CO2)withalowerconcentrationofglucose.TobegintoexplorehowLytSTimpactscriticalphenotypesof S.mutans ,RNAexpressionprofilesinUA159 andthe lytS mutantwerecomparedusinganRNAmicroarrayapproach.RNAwasisolatedfromearlyexponential andlateexponentialgrowthph asesfromstaticplanktonic culturesgrowninBHI(containing11mMtotalglucose)at 37Cina5%CO2atmosphere(Additionalfile1:TableS1 andAdditionalfile2:TableS2).Atearlyexponentialgrowth phase,lossofLytSaffectedtheexpressionof40genes(12 upregulatedand28downregulated; P< 0.005;Additional file1:TableS1).Mostoftheupregulatedgenesinearlyexponentialphasedisplayedonl yamodestincreaseinexpressionandincludedgenesinvolvedinDNArepair,purine/ pyrimidinemetabolism,com petence,andanumberofunassignedandhypotheticalORFs.RNAtranscriptsthatwere stronglydown-regulatedgreat erthan10-foldincellslackingLytSduringearlyexponentialgrowthincludedthose annotatedasbacitracin/surfact in/gramicidinsynthesisproteins,transportandbindingproteins,andLrgAB.Incontrast,lossofLytSaffectedtheexpressionofamuchlarger numberofgenesinlateexponentialphase(136genestotal), with79upregulatedtranscrip tsand57downregulatedtranscripts( P <0.001;Additionalfile2:TableS2).Asidefrom dramaticallydecreased lrgAB expression,affectedgenes includedthoseinvolvedinaminoacidandco-factorbiosynthesis,carbohydrateandfatty acidmetabolism,stressadaptation,toxinproduction,DNArepair/recombination, EP low-O2 EP aer ob ic SP lo w-O2 SP aer o bic 0.1 1 10 100 Fold-change (relative to UA159 EP for each growth condition) EP low-O2 EP aerobic SP low O 2 SP aerobi c 0.1 1 10 100 1000 Fold-change (relative to UA159 EP for each growth condition) AB11 mMGlucose 45 mMGlucose UA159 lytS UA159 lytS Figure1 LytS-dependentexpressionof lrgAB in S mutans OvernightculturesweredilutedinTHYE,containingeither11mM( A )or45mM glucose( B )toanOD600=0.02andgrownat37Casstaticculturesat5%CO2( “ low-O2” )orasaerobicshakingculturesat250RPM( “ aerobic ” ). RNAwasharvestedatexponential(EP)andstationaryphase(SP).Reverse-transcription,real-timePCRreactions,anddeterminationofcopy numberwereperformedasdescribedpreviouslyusing lrgA and16S-specificprimers[37,77].Fold-changeexpressionof lrgAB and16Sundereach growthconditionwascalculatedbydividingthegenecopynumberofeachtestsamplebytheaveragegenecopynumberofUA159EP.Data wasthennormalizedbydividingeach lrgAB fold-changevaluebyitscorresponding16Sfold-changeexpressionvalue.Datarepresentthe averageof3biologicalreplicates.DarkgreybarsrepresentUA159andlightgreybarsrepresent lytS mutant.ErrorBarsrepresentthestandard error(SEM). Ahn etal.BMCMicrobiology 2012, 12 :187 Page3of12 http://www.biomedcentral.com/1471-2180/12/187

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proteinsynthesis,transcrip tionalregulation,andcompetence,aswellasmultiplehypotheticaland/orunassigned ORFs(Additionalfile2:TableS2andFigure2).Asubsetof geneswasdifferentiallyexpressedasafunctionoftheloss ofLytSinbothearlyexponentialandlateexponential growthphases(Additionalfile1:TableS1andAdditional file2:TableS2).Theseincludedmanygenesencodedby the S.mutans genomicislandTnSMu2[45](SMU.1335c, 1339-1342,1344c-1346,1354c,1360c,1363c,1366c), ssbA comYB ,and lrgAB .Giventhatthesegeneswereregulated byLytSinbothgrowthphasesexamined,itispossiblethat theyareunderthedirectcontrolofLytST.Tovalidatethe microarraydata,qRT-PCRwasperformedonlateexponentialphasewild-typeand lytS mutantRNAtoassessexpressionof14oftheaffectedgenes.AsshowninTable1,the expressionratios( lytS mutant/wild-type)foreachgene obtainedbyreal-timePCRwe resimilartothemicroarray results.Interestingly,expressionratiosofthesegeneswere allcloseto1.0whencomparingexpressionbetweenthe wild-typestrainanda lrgAB mutant(Table1),indicating thatthedifferentialexpressionpatternsobservedinthe lytS 0246 8101214161820 Mobil and extrachromosomal element functions Signal transduction Protein fates Regulatory functions Amino acid biosynthesis Cell envelopment DNA metabolism Purines,pyrimidines,nucleosides and nucleotides Unassigned Biosynthesis of cofactors,prosthetic groups,carriers Central intermediary metabolism Transcription Energy metabolism Fatty acid and phospholipid metabolism Hypothetical Cellular processes Transport and binding proteins Unknown Protein synthesis UPregulated DOWN regulatedNumber of g enes Figure2 DistributionoffunctionsofgenesaffectedbylossofLytSatlateexponentialphase. StatisticalanalysiswascarriedoutwithBRB arraytools(http://linus.nci.nih.gov/BRB-ArrayTools.html/)withacutoff P valueof0.001.The136genesdifferentiallyexpressedat P 0.001are groupedbyfunctionalclassificationaccordingtotheLosAlamos S.mutans genomedatabase(http://www.oralgen.lanl.gov/). Table1Real-timePCRvalidationofRNAmicroarray resultsMicroarrayReal-timepcr lytS mutant lytS mutant lrgAB mutant (SMU.1985) comYA(comYB) 22.99276.84490.8163 SMU.1967 ssbA 5.58034.10760.8791 (SMU.1515) vicR(vicX) 2.67641.76471.0267 SMU.924 tpx 2.41483.61681.058 SMU.1739 fabF 2.24432.03331.084 SMU.1666 livG 2.11833.43311.009 SMU.80 hrcA 0.49530.61071.0204 SMU.1424 pdhD 0.47690.40311.2004 SMU.580 xseA 0.298490.54091.1398 SMU.1600 celB 0.21860.28251.2979 SMU.113 pfk 0.15970.1761.3578 SMU.82 dnaK 0.15230.26520.9907 SMU.1344 fabD 0.02230.0121.0637 SMU.1341 grs 0.00080.01211.1027Resultsareexpressedinfold-change(mutant/wild-type).Ahn etal.BMCMicrobiology 2012, 12 :187 Page4of12 http://www.biomedcentral.com/1471-2180/12/187

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mutantwerenotaconsequenceofdown-regulated lrgAB expression.InvestigationoftheeffectofLytSTandLrgABon competenceInanalyzingthemicroarraydatainAdditionalfile1: TableS1andAdditionalfile2:TableS2,itappearedthat thegenemosthighlyupregulatedinresponsetolossof LytSinbothphasesofgrowthwas comYB (SMU.1985), ahomologueofthe B.subtiliscomGB genethatencodes partofanABCtransporteressentialforDNAbindinguptakeduringcompetencein S.mutans [46].Interestingly,a comYB mutantof S.mutans wasshowntobe unaffectedincompetencesignaling,butshowedreduced biofilmformation,whichwasthoughttobeafunction ofitsinabilitytobindbiofilmmatrixeDNA[47].Sincethe lytS mutantdisplayedanincreasein comYB expression (Additionalfile1:TableS1andAdditionalfile2:TableS2), wehypothesizedthatthisstra inmaydisplayalterationsin itsabilitytoformbiofilmand/oritstransformabilityduring geneticcompetence.However,the lytS mutantdidnotdisplayanyappreciabledifferenceinitsabilitytoformstatic biofilminthepresenceofglucoseorsucrose(datanot shown),andlikewise,didnotdisplayadifferenceinitsabilitytouptakeplasmidDNAinaquantitativecompetence assay,relativetothewild-typestrain(Figure3).Since lrgAB expressionissostronglyregulatedbyLytST,theabilityof isogenic lrgA lrgB ,and lrgAB mutantstouptakeplasmid DNAviacompetencewasalsoassessed(Figure3).Ofall themutantstested,the lrgA mutantwasthemostseverely impairedinitsabilitytouptakeplasmidDNArelativeto theparentalstrain,displayinga26-and24-folddecreasein transformationefficiencyinthepresenceandabsenceof competence-stimulatingpep tide(CSP),respectively (Figure3),suggestingthatLrgAissomehowinvolved ingenetictransformationinaCSP-independentmanner.Thisfindinghasparticul arsignificanceconsideringthatLrgABhasbeenlinkedtoregulationofcell deathandlysisin S.aureus [21,29]and S.mutans [37], andthesephysiologicalprocessesarealsoextremely importantduringnaturalco mpetence.Itisinteresting tonotethat,similartothecompetenceresultsdescribed here,the lrgA mutantwaspreviouslyshowntodisplay decreasedglucose-dependentbiofilmformationand decreasedglucosyltransferaseproduction,whereasthe lrgB and lrgAB mutantsbehavedinamannersimilarto theparentalstrain[37].Thesephenotypicpatternssuggest thatthepresenceofLrgBalone,ratherthanthelackof LrgA,mayberesponsibleforthebiofilmandcompetence phenotypesobservedinthe lrgA mutant.EffectofLytSTonoxidativestresstolerancePreviously,ourinvestigationsdisclosedastronglinkbetweenoxidativestresstoleranceandtheCid/Lrgsystem [37],aroleforthesegenesthathadnotbeendescribed inotherorganisms.Specifically,wefoundthat lrgAB lrgB cidAB ,and cidB mutantsexhibitedreducedgrowth inthepresenceofparaquat,andgrowthof lrgAB cidAB and cidB mutantsonBHIagarplatesinaerobicconditionswasalmostcompletelyinhibited[37].Itisthereforeinterestingtonotethatinthe lytS microarray results(Additionalfile2:TableS2),genesencodingantioxidantandDNArepair/recombinationenzymeswere significantlyupregulatedinthe lytS mutantinlate exponentialphase.Theseincluded yghU and tpx ,encodingtheputativeanti-oxidantenzymesglutathione S-transferaseandthiolperoxidase,respectively,aswell as recJ ,whichencodesasingle-strandedDNAexonucleaseproteinthatfacilitatesDNArepairinresponseto oxidativestress[48-51].Tofurtherinvestigatetheeffect of lytS and lrgAB onoxidativestresstolerance,wild-type, lytS ,and lrgAB mutantsweregrownasplanktonicstatic BHIculturesinaerobicatmosphereandinthepresence andabsenceofH2O2(Figure4).Whenchallengedwith H2O2,UA159experiencedanincreasedlagphaseof growth,andtheoverallODoftheculturewas10-25% lessthantheuntreatedcultureuntil20hgrowth.Under theseassayconditions,the lrgAB mutantdisplayeda UA159-p A T 2 8 lytS-pAT2 8 lrgB-pAT28 lr gAB-pAT28 UA159-pOri23 lr gA-pOr i23Transformation efficiency (% Transformants)1e-7 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0P = 0.008 P = 0.008+CSP -CSP Figure3 TransformationefficienciesofUA159andisogenic lytS and lrg mutants. TocomparetheabilityofUA159anditsisogenic lytS lrgA lrgB ,and lrgAB mutantstotakeupexogenously-addedplasmid DNA,aquantitativecompetenceassaywasperformedonn=4-6 biologicalreplicatesofeachstrainasdescribedinMethods[83]. PlasmidpAT28[encodingspectinomycinresistance;[84]wasusedto assesstransformationefficiencyinUA159, lytS lrgB ,and lrgAB mutants.Becausethe lrgA mutantwasgeneratedwitha spectinomycin-resistancecassette[37],plasmidpORi23[encoding erythromycinresistance;[85]]wasusedtoassesstransformation efficiencyinUA159and lrgA mutant.Transformationefficiencies (Yaxis)inthepresence(greybars)andabsence(whitebars)ofCSP areexpressedasthepercentageoftransformants(CFU/mlonBHI+ selectiveantibiotic)amongtotalviablecells(CFU/mlonBHI).Error barsrepresentSEM.Bracketswith P valuesdenotestatisticallysignificantdifferencesbetweentwosamples(Mann – WhitneyRank SumTest). Ahn etal.BMCMicrobiology 2012, 12 :187 Page5of12 http://www.biomedcentral.com/1471-2180/12/187

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dramaticgrowthdefectinboththepresenceandabsenceofH2O2.Itisinterestingtonotethatthisaerobic growthdefectwasalsopreviouslyobservedwhenthe lrgAB mutantwasgrowninaerobicatmosphereonBHI agarplates[37].The lytS mutantdisplayedanincreased lagingrowthrelativetoUA159whenculturedinthe presenceofH2O2,butODvalueswerecomparableto thewild-typestrainby16hgrowth.TheseresultssuggestthattheLytSTregulonimpactstheabilityofcellsto growunderconditionsofoxidativestress. Thecell-permeablefluorescentdyeCM-H2DCFDA (InvitrogenMolecularProbes)wasalsousedtoassess intracellularROSinUA159andthe lytS mutant (Figure5).Thisfluorescentcompoundisoxidizedinthe presenceofH2O2andotherreactiveoxygenspecies (ROS)andisconsideredageneralindicatorofintracellularoxidativestress[52,53].Thisanalysisrevealedthat stationary-phaseculturesofthewild-typeand lytS mutantstrainshadsimilar “ endogenous ” intracellularlevels ofROS(Figure5,lightgreybars).WhenstationaryphasecellsfromeachstrainwereloadedwithCMH2DCFDAandthenchallengedwith5mMH2O2(Figure5,darkgreybars),agreaterincreaseinfluorescencewasobservedinthe lytS mutantrelativetoUA159 ( P =0.009,Mann – WhitneyRankSumTest),suggesting thatlossofLytShasanimpactontheabilityofthecells todetoxifyH2O2and/orotherintracellularROS.DiscussionThetranscriptomeanalysespresentedinthisstudyhave revealedthattheLytSTtwo-componentsystemhasa widespreadeffectongeneexpressionin S.mutans .A muchhighernumberoftranscriptswereaffectedbythe lytS mutationinlateexponentialphaseandthemagnitudeofchangesinexpressionwasgreater(n=136genes, Additionalfile2:TableS2)relativetoearly-exponential phase(n=40genes,Additionalfile1:TableS1),where mostgenesexhibitedonlyamodest(1-2fold)changein expression.Thesedifferencesingeneexpressionpatternsareunlikelytobeanindirectfunctionofaltered lrgAB expressioninthe lytS mutant,asexpressionof lytS -regulatedgeneswasunalteredinan lrgAB mutant relativetothewild-typestrain(Table1).Takentogether, theseobservationssuggestthatLytSTexertscontrol overitstranscriptomeinagrowth-phasedependent manner,andtoourknowledge,thisisthefirststudythat hascomparedthescopeofLytSTregulationatdifferent phasesofgrowth.Interestingly,RNAmicroarraystudies of lyt mutantshavealsobeenperformedin S.aureus [38], S.epidermidis [40],and B.subtilis [42].Aswehave observedherein S.mutans ,aglobaleffectofLytSTon geneexpressionwasalsonotedin S.aureus and S.epidermidis [38,40].In S.aureus ,LytSTappearedtoexert Hours of growth 051015 20 OD 600 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 UA159 lytS lrgAB UA159 + 1.0 mM H 2 O 2 lytS + 1.0 mM H 2 O 2 lrgAB + 1.0 mM H 2 O 2 Figure4 H2O2challengeassayofUA159, lytS and lrgAB mutants. CulturesofUA159, lytS ,and lrgAB mutants(n=6biological replicatesperstrain)weregrowninthepresence(opensymbols) andabsence(filledsymbols)of1.0mMH2O2for20hat37C (aerobicatmosphere)inaBiotekmicroplatereader.OD600measurementsofeachwellwererecordedat2hintervals.Black circlesrepresentUA159,redtrianglesrepresent lytS mutant,blue squaresrepresent lrgAB mutant.ErrorbarsrepresentSEM. UA159 lytS UA159 + H2O2 lytS + H2O2 Average RFU/OD 600 0 2000 4000 6000 8000 10000 P = 0.009 Untreated H2O2-treated Figure5 MeasurementofintracellularROSinUA159and lytS mutantbyCM-H2DCFDAstaining. Cellswereharvestedfrom20h BHIculturesofUA159andisogenic lytS mutantgrownat37C5% CO2(n=3-6biologicalreplicateseach),resuspendedinHBSS containing5 MCM-H2DCFDA,andincubatedat37Ctoloadthe cellswithstain.After60minincubation,cellsuspensionswere centrifuged,washedonceinHBSSbuffer,andthenresuspendedin HBSSbufferalone(lightgreybars)orinHBSScontaining5mM H2O2(darkgreybars).Eachsuspensionwastransferredtowellsofan optically-clear96wellplate,andincubatedat37Cinamicroplate reader.Cellfluorescence(asmeasuredbyrelativefluorescenceunits; RFU)andtheOD600ofeachwellwasrecordedafter30min incubation.RFUmeasurementsareexpressedperOD600ofeachwell toaccountforanysubtlevariationsincelldensity.Errorbars representSEM.Bracketswith P valuesdenotestatistically-significant differencesbetweentwosamples(Mann – WhitneyRankSumTest). Ahn etal.BMCMicrobiology 2012, 12 :187 Page6of12 http://www.biomedcentral.com/1471-2180/12/187

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primarilypositiveeffectsongeneexpressioninexponential phasewhenaerobicculturesweregrowninmediacontainingexcess(35mM)glucose,asonly7geneswerefoundto beupregulatedinthe lytS mutant[38].In S.epidermidis ,a largenumberofgeneswereup-ordown-regulatedasa functionofthepresenceofLytSTduringexponential phaseduringaerobicgrowthinmediumcontaining 12mMglucose[40].Incontrast,mutationof lytS only appearedtoaffecttheexpressionof lytST itselfandgenes encoding lrgAB and cidAB homologuesin B.subtilis [42]. However,duetothedifferencesingrowthconditionsused (glucoselevelsand/orcultureaeration)andthediffering metabolicpathwayspresentintheseorganisms,itisdifficulttoestablishdirectcorrelationsbetweenthesestudies andthe S.mutans microarrayresultspresentedhere. Asdemonstratedpreviously[37],expressionof lrgAB wasalsoshowntobetightlycontrolledbytheLytST two-componentsystemin S.mutans inthisstudy.Specifically,wehavefoundthatLytST-dependentexpression of lrgAB isregulatedinpartbyglucosemetabolismand oxygenin S.mutans (Figure1).Furthermore,controlof lrgAB expressionbyLytSTappearstobehighlygrowthphasedependent: lrgAB expressioninthe lytS mutant exhibitedonlyamodestdecreaseinexpressioninearly exponentialphase(0.49relativetoUA159,Additional file1:TableS1),whereas lrgAB expressionwasdownregulatedsome200-foldinthe lytS mutantatlateexponentialphase(Additionalfile2:TableS2).Alternatively, itispossiblethatcontrolof lrgAB expressionbyLytST isrelatedtohigherglucoseavailabilityduringearlyexponentialphase.Althoughdetailedmechanisticstudies havenotyetbeenperformed,thereismountingevidence thattheseproteinsarecriticalforoxidativestressresistancein S.mutans :(1) lrgAB expressionishighlyregulatedbyoxygen([11]andthisstudy);(2)a lrgAB mutant wasdefectiveinaerobicgrowthonBHIagarplates[37]; (3)a lrgAB mutantdisplayedadecreasedgrowthratein thepresenceofparaquat(asuperoxide-generatingagent) relativetothewild-typestrain[37];and(4)a lrgAB mutantdisplayedastronggrowthdefectduringstatic planktonicaerobicgrowthinBHIinthepresenceand absenceofH2O2challenge(thisstudy).Interestingly,a linkbetweenLrgABandoxidativestresswasalso demonstratedin S.aureus ,where lytSR and lrgAB expressionwereupregulated2-5foldinresponsetoazurophilicgranuleproteins,H2O2,andhypochlorite[54]. InagreementwitharoleforLrgABinoxidativestress resistance,severalLytST-regulatedgenesidentifiedin thisstudyhavealsobeenimplicatedinbacterialoxidativestressresponses.Upregulatedpotentialoxidative stressgenesinclude yghU ,aputativeanti-oxidantenzyme[50], tpx ,apredictedthiolperoxidase[55],and recJ ,asingle-strandedDNAexonucleaseproteinthat facilitatesDNArepairinresponsetooxidativestress [51].Conversely,severalgenesbelongingtotheTnSMu2 genecluster(SMU.1334c – SMU.1359)weredownregulatedinthe lytS mutant.Thesegenesareannotatedas encodingaseriesofgeneproductsinvolvedinbacitracin andgramicidinsynthesis[56],butmorerecentlyhave beenshowntoberesponsiblefornonribosomalpeptide andpolyketide(NRP/PK)biosynthesisofapigmentthat enhancesaerobicgrowthandtolerancetoH2O2challengein S.mutans UA159[45].Thealteredexpression ofoneormoreofthesegenesmayexplain,inpart,the increasedROSaccumulationthatwasobservedinthe lytS mutantwhenchallengedwithH2O2(Figure5).Furthermore,itwaspreviouslyfoundthatatwo-component systemresponsibleforpositiveregulationoftheNRP/ PKgeneswaslocatedontheTnSMu2genomicislandof UA140butnotinUA159[45].Thisobservation, combinedwiththemicroarrayresultsperformed here(Additionalfile1:TableS1andAdditionalfile2: TableS2)suggestthatLytSTmayhavetakenoversome oftheregulatoryfunctionsofthisnon-core-genome two-componentsystemthatismissinginUA159. Interestingly,H2O2hasalsobeenshowntobeapotent stimulatorofcompetenceandeDNAreleasein S.sanguinis [57], S.gordonii [57,58],and S.pneumoniae [59]. AlthoughtheeffectsofH2O2on S.mutans competence, celllysis,andeDNAreleasehavenotbeendirectlymeasured,ithasbeenshownthatgrowthunderaerobicconditionspromotescompetencein S.mutans [47],and thatexpressionofcompetence-relatedgenesisupregulatedduringaerobicgrowth[11].Theresultspresented herehavedemonstratedthatexpressionof comYB ,a geneencodingacomponentoftheDNA-bindinguptake systemin S.mutans [47]wasupregulated2-foldinearly exponentialphaseand22-foldinlateexponentialphase inthe lytS mutant(Additionalfile1:TableS1and Additionalfile2:TableS2).Thesignificanceofhighlevel comYB expressioninthe lytS mutantatlateexponentialphaseisunclear,giventhatmaximal S.mutans competencedevelopsinactively-growingpopulations [60,61].Accordingly,upregulationof comYB expression didnotcorrelatewithincreasedtransformabilityofthe lytS mutantundertheconditionstestedinthisstudy (Figure3).However,itwasfoundthatthe lrgA mutant displayedasignificantreductionincompetence.Ithas beenrecentlyreportedthatonlyasubpopulationof S.mutans culturelysesinresponsetoCSP,andthislysis eventiscontrolledinpartbytheCipBbacteriocinand theCipIimmunityprotein[62].Subsequentmicroarray analysisofa cipI (immunityprotein)mutantshowedthat both lytST and lrgAB expressionwerehighlyupregulated inthe cipI mutant[63].Theseresults,combinedwith thefactthatLrgA/Bhasbeenshowntobeinvolvedin regulatingcelllysisandeDNAreleasein S.aureus [21,29],lendsstrongsupporttotheideathatLrgAplaysAhn etal.BMCMicrobiology 2012, 12 :187 Page7of12 http://www.biomedcentral.com/1471-2180/12/187

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animportantroleduringcompetence,possiblybyalteringmembranepermeabilityorbymodulatingmurein hydrolaseactivity. The S.mutanscomY operonconsistsofninecotranscribedgenes,ofwhichthefirsteightgenesareeitheressentialtoorsignificantlyaffectcompetence[46]. Theninthgeneofthisoperonispredictedtoencode acetatekinase(AckA),anenzymethatcatalyzesthe inter-conversionofacetyl-phosphateandacetate[46,64]. Formicro-organismswithaninefficientorincomplete TCAcyclesuchas S.mutans ,AckA-mediatedconversionofacetyl-phosphatetoacetateisthoughttobea criticalmechanismofgeneratingATP[reviewedin[65]]. Since ackA ( comYI )waspreviouslyfoundtobeupregulatedin S.mutans duringaeratedgrowth[11],itispossiblethatLytSTisinvolvedintheregulationofenergy generationthroughthephosphateacetyltransferase (Pta)-AckApathwayduringaerobicgrowthand/orduringoxidativestress.Inthisrespect,ithasrecentlybeen reportedthatan S.mutanspta mutantwasmoresusceptibletobothacidandoxidativestresses[66]. Theabilityof S.mutans tocombatH2O2stressiscriticalforitssurvivalintheoralcavity,yetH2O2detoxifyingmechanismsandtheirregulationhavenotbeen extensively-characterizedinthisorganism,limitedprimarilytotheScnRKandVicRKtwo-componentsystems [67,68], ropA [69], brpA [70], luxS [71]andgenomicislandTnSMu2[45].H2O2hasbeenshowntohavepotent antibacterialeffectson S.mutans [72],anditisthought thatH2O2producedbyotheroralstreptococcalspecies servesasanantagonistagainst S.mutans .Forexample, S.sanguinis andS .gordonii havebeenshowntoproduce H2O2viapyruvateoxidaseunderaerobicgrowthconditions,andthisH2O2productionallowsthemtocompete effectivelyagainst S.mutans whenco-culturedunder aerobicgrowthconditions[57].Itisthereforepossible thatthe S.mutans LytSTregulonmediatesapleiotropic protectiveresponseagainsttheseH2O2-producingniche competitors.On-goingandfuturestudiesbyourgroup willfocusonexperimentaltestingofthishypothesis.ConclusionsInsummary,theLytSTtwo-componentsystemhasbeen showntohaveapleiotropiceffectongeneexpressionin S.mutans .Thisiscongruentwithmicroarrayanalysesof lytS mutantsin S.aureus [38]and S.epidermidis [40].However,unlikeinotherorganisms,wehavebeenabletoidentifyapatternofLytS-mediatedgeneexpressionthat suggestsaroleforthisreguloninrespondingtooxidative/ H2O2stress.Althoughwehavenotyetbeenabletoidentify theexternalsignaltowhichLytSresponds,itislikelylinked toanoxidativestress-sensingmechanism,suchasH2O2mediatedmembranedamage(ie. lipidperoxidation)viaits largenumberoftransmembranedomains,oroxygen/ROS interactionswithitspredictedcytoplasmicGAFdomain,a ubiquitoussignalingdomaint hathasbeenshowntodetect changesintheredoxstateofboundironoroxygenin Mycobacteriumtuberculosis [73-75].EstablishingmechanisticlinksbetweentheLytSTregulon,H2O2resistance,and competenceregulationwillprovidevaluablenewinsights into S.mutans survivalandvirulenceintheoralcavity.MethodsBacterialstrains,media,andgrowthconditionsForallexperiments,frozenglycerolstocksof S.mutans UA159anditsisogenic lytS (SAB111; lytS ::NPKmr), lrgA (SAB113; lrgA ::NPSpr), lrgB (SAB119; lrgB :: NPEmr),and lrgAB (SAB115; lrg AB :: Kmr)mutants [createdpreviouslyin[37]werefreshlystreakedforisolationoneitherToddHewittYeastExtract(THYE)or BrainHeartInfusion(BHI),containingselectiveantibioticasappropriate:kanamycin(Km) – 1000 g/ml, erythromycin(Em) – 10 g/ml,spectinomycin(Sp)1000 g/ml).WiththeexceptionofSAB115( lrgAB mutant),allmutantswerecreatedusingnon-polar(NP) antibiotic-resistancemarkers[37].Unlessotherwiseindicated,all S.mutans culturesweregrownasstaticculturesinBHIorTHYEbrothat37Cand5%CO2.Analysisof lrgAB expressionTomeasuretheeffectsofoxygenandglucoseon lrg expression,overnightTHYEculturesofUA159andthe lytS mutant(n=3biologicalreplicateseach,grownat0 RPM,37Cand5%CO2)wereeachinoculatedtoan OD600=0.02intoTHYEcontainingeither11mMor 45mMglucose.For “ lowO2” cultures,2Lcultureflasks eachcontaining400mlmediaweregrownat0RPM, 37C,and5%CO2.Foraerobiccultures,500mlculture flaskseachcontaining100mlmediaweregrownat37C and250RPM.TotalRNAwasisolatedfromallcultures sampledatexponential(EP;OD600=0.2 – 0.4)andstationary(SP;OD600=1.4 – 1.7)growthphase,withan RNeasyMinikit(Qiagen)andFASTPREP(MPBiomedicals)usingpreviously-describedmethods[76].Real-time reverse-transcriptasePCRanddataanalysisusing lrgA and16Sprimerswasperformedusingpreviously describedprimers[37]andmethods[77].Fold-change expressionof lrgA and16Sundereachgrowthcondition (11mMlow-O2,11mMaerobic,45mMlow-O2, 45mMaerobic)wascalculatedbydividingthegene copynumberofeachtestsamplebytheaveragegene copynumberofUA159EP.Datawasthennormalized bydividingeach lrgA fold-changeexpressionvaluebyits corresponding16Sfold-changeexpressionvalue.RNAmicroarrayanalysisofUA159and lytS mutantToassesstheeffectofLytSonglobalgeneexpression, overnightBHIculturesofUA159and lytS mutant(n=3Ahn etal.BMCMicrobiology 2012, 12 :187 Page8of12 http://www.biomedcentral.com/1471-2180/12/187

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biologicalreplicatesperstrain)weredilutedtoan OD600=0.02inBHI,andgrownasstaticculturesat37C and5%CO2.TotalRNAwasisolatedfromeachculture atearly-exponential(OD600=0.15)andlateexponential phase(OD600=0.9),usingpreviously-publishedmethods [77].RNAmicroarrayanalysiswasperformedusing S.mutans UA159microarraysprovidedbyTheInstitute forGenomicResearch,andpreviously-describedmethodsanddataanalysis[11,70,78].Inbrief,2 gtotalbacterialRNAwasusedineachreverse-transcriptionand cDNAlabelingreaction(performedasdescribedin [70,78]),andasinglepreparationfromeachculturewas hybridizedpermicroarrayslideinaMauihybridization chamber(BioMicroSystems,SaltLakeCity,UT).The resultingmicroarrayslideswerescanned,analyzed,and normalizedusingTIGRSpotfindersoftware(http://www. tigr.org/software/),andin-slidereplicateanalysiswas performedwiththeTIGRmicroarraydataanalysissystem(MIDAS;http://www.tigr.org/software/).Statistical analysiswascarriedoutwithBRBarraytools(http:// linus.nci.nih.gov/BRB-ArrayTools.html/)withacutoff P value<0.005fortheearlyexponential-phasedataand P <0.001forthelateexponentialphasedata.Tovalidate themicroarrayresults,real-timequantitativeRT-PCR wasperformedonasubsetofthedifferentially-expressed genes,asdescribedpreviously[77,79].Allreal-timePCR primersweredesignedwithBeaconDesigner4.0software(PremierBiosoftInternational,PaloAlto,CA),and standardcurvesforeachgenewerepreparedaspublishedelsewhere[80].Themicroarraydataobtainedfrom thesestudieshavebeendepositedtoNCBI ’ sgeneexpressionomnibus(GEO)[81](GEOAccession#GSE39470) andcomplywithMIAMEguidelines[82].QuantitativecompetenceassaysTocomparetheabilityofUA159anditsisogenic lytS lrgA lrgB ,and lrgAB mutantstotakeupexogenouslyaddedplasmidDNA,aquantitativecompetenceassay wasperformedonn=4-6biologicalreplicatesofeach strainusingapreviously-publishedprotocol[83]with thefollowingmodifications:Overnightculturesofeach strainweredilutedtoanOD600=0.02inBHI,andgrown ina96-wellplatetoanOD600=0.15priortoadditionof 500ngplasmidDNAwithandwithout100ngCSP. PlasmidpAT28(encodingspectinomycinresistance; [84])wasusedtoassesstransformationefficiencyin UA159, lytS lrgB ,and lrgAB mutants.Becausethe lrgA mutantwasgeneratedwithaspectinomycin-resistance cassette[37],plasmidpORi23[encodingerythromycin resistance;[85]]wasusedtoassesstransformationefficiencyinUA159and lrgA mutant.After2.5hincubation inthepresenceofplasmidDNA+/-CSP,cultureswere seriallydilutedandplatedonBHIagarwithandwithout selectiveantibiotic.CFU/mlofeachculturewere enumeratedafter48hgrowthat37Cand5%CO2,and transformationefficiencieswerecalculatedasthepercentageoftransformants(CFU/mlonBHI+selective antibiotic)amongtotalviablecells(CFU/mlonBHI).H2O2assaysToassessoftheabilityofUA159, lytS, and lrgAB mutantstogrowinthepresenceofH2O2,overnightculturesofeachstrain(n=6biologicalreplicates)wereeach diluted40-foldintoBHI.1mlaliquotsofeachdiluted culturewereeitheruntreatedorchallengedwith1mM H2O2.Aliquotsofeach(500 lperwell,2wellstotal) werethenimmediatelytransferredtoanoptically-clear 48-welltissuecultureplate(Costar3548),whichwas incubatedfor20hat37C(aerobicatmosphere)ina BiotekSynergymicroplatereader.OD600measurements ofeachwellwererecordedat2hintervals.OxidativestressmeasurementsToassessintracellularoxidativestressinUA159and lytS mutant,singleisolatedcoloniesofeachstrain(n=3-6 biologicalreplicatesperstrain)wereinoculatedintoculturetubescontaining4mlBHI,andgrownin “ low-O2” conditions(37C,0RPM,5%CO2).After20hgrowth, 21mlaliquotsofeachculturewereharvestedbycentrifugationinamicrocentrifuge(3minat13,000RPM). Theculturesupernatantswerediscarded,andcellpellets wereeachresuspendedin1mlHanksBuffer(HBSS) containing5 Mchloromethyl2 0 ,7 0 -dichlorofluorescein diaceate(CM-H2DCFDA;InvitrogenMolecularProbes), acell-permeablefluorescentcompoundthatisoxidizedinthepresenceofH2O2andotherreactiveoxygen species(ROS)andisconsideredageneralindicator ofcellularoxidativestress[52,53].Cellsuspensions wereincubatedat37Cfor60minto “ load ” thecells withCM-H2DCFDA,followedbycentrifugation(3min at13,000RPM).Supernatantswerediscarded,andcell pelletswerewashedoncewithHBSSpriortoresuspensionin1mlHBSSorin1mlHBSScontaining 5mMH2O2.Eachcellsuspensionwastransferred intotriplicatewells(200 lperwell)ofanoptically-clear 96wellplate(Costar3614),andtheplatewastransferred toaBiotekSynergymicroplatereader.Fluorescence inrelativefluorescenceunits(RFU;using492-495nm excitationand517-527nmemission)andOD600readingsofeachwellwererecordedafter30minincubationat37C.StatisticalanalysisAllstatisticalanalyses,unlessotherwiseindicated,were performedusingSigmaplotforWindows11.0software (Build11.0.0.75,SystatSoftware,Inc.).Ahn etal.BMCMicrobiology 2012, 12 :187 Page9of12 http://www.biomedcentral.com/1471-2180/12/187

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AdditionalfilesAdditionalfile1:TableS1. Genesdifferentiallyexpressedbylossof LytSatearly-exponentialphase(P<0.005). Additionalfile2:TableS2. Genesdifferentiallyexpressedbylossof LytSatlateexponentialphase(P<0.001). Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors'contributions SJAcarriedouttheRNAmicroarrayexperimentsandassociateddataanalysis, performedallreal-timePCRstudies,participatedintheconceptionand designofthestudy,andhelpeddraftthemanuscript.MDQcarriedoutallof theRNAisolationsforcomparingtheeffectsofglucoseandoxygenationon lrgAB expression.ERoptimizedandcarriedoutallofthequantitative competenceassays.RABparticipatedinthedesignandcoordinationofthe study,andhelpeddraftthemanuscript.KCRparticipatedintheconception anddesignofthestudy,performedtheH2O2assays,intracellularROS measurements,anddraftedthemanuscript.Allauthorsreadandapproved thefinalmanuscript. Acknowledgements ThisworkwassupportedbyaUniversityofFloridaHHMI-ScienceforLife UndergraduateResearchAwardtoM.D.Q.,NIH-NIDCRgrantsR03DE019179 (KCR)andR01DE13239(RAB).WethankChristopherBrowngardtfor technicalassistanceineditingmicroarraydata. Authordetails1DepartmentofOralBiology,CollegeofDentistry,UniversityofFlorida, Gainesville,FL32611,USA.2DepartmentofMicrobiologyandCellScience, CollegeofAgriculturalandLifeSciences,UniversityofFlorida,Gainesville,FL 32611,USA. Received:29May2012Accepted:21August2012 Published:1September2012 References1.DeonarineB,LazarJ,GillMV,CunhaBA: Quadri-valvularendocarditis causedby Streptococcusmutans ClinMicrobiolInfect 1997, 3 (1):139 – 141. 2.BiswasS,BowlerIC,BunchC,PrendergastB,WebsterDP: Streptococcus mutans infectiveendocarditiscomplicatedbyvertebraldiscitisfollowing dentaltreatmentwithoutantibioticprophylaxis. 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83.SeatonK,AhnSJ,SagstetterAM,BurneRA: Atranscriptionalregulatorand ABCtransporterslinkstresstolerance,(p)ppGpp,andgenetic competencein Streptococcusmutans JBacteriol 2011, 193 (4):862 – 874. 84.Trieu-CuotP,CarlierC,Poyart-SalmeronC,CourvalinP: Apairof mobilizableshuttlevectorsconferringresistancetospectinomycinfor molecularcloningin Escherichiacoli andingram-positivebacteria. NucleicAcidsRes 1990, 18 (14):4296. 85.QueYA,HaefligerJA,FrancioliP,MoreillonP: Expressionof Staphylococcus aureus clumpingfactorAin Lactococcuslactis subsp. cremoris usinga newshuttlevector. InfectImmun 2000, 68 (6):3516 – 3522.doi:10.1186/1471-2180-12-187 Citethisarticleas: Ahn etal. : Identificationofthe Streptococcusmutans LytSTtwo-componentregulonrevealsitscontributiontooxidative stresstolerance. BMCMicrobiology 2012 12 :187. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Ahn etal.BMCMicrobiology 2012, 12 :187 Page12of12 http://www.biomedcentral.com/1471-2180/12/187



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Table S2 Genes differentially expressed by loss of LytS at late exponential phase ( P 0.001) Functional group Gene symbol Description Fold change ( lytS /wild type) Amino acid biosynthesis Pyruvate family SMU.233 ilvC ketol acid reductoisomerase 3.6018586 SMU.1023 pycB oxaloacetate decarboxylase 0.2798236 SMU.157 cysE putative serine acetyltransferase, serine O acetyltransferase 1.8680243 Biosynthesis of cofactors, prosthetic groups, and carriers SMU.1296 yghU putative glutathione S transferase YghU 8.961394 SMU.1084 hemK putative protoporphyrinogen oxidase 2.2752837 SMU.582 ispA, fps putative farnesyl diphosphate synthase 0.4890623 SMU.841 putative aminotransferase 2.077611 Cell envelope SMU.1688 dltD putative extramembranal protein, DltD protein 2.7890333 SMU.455 pdp2x putative penicillin binding protein 2X 2.4351256 SMU.1039c waaR, waaJ, kdt putative lipopolysaccharide glycosyltransferase 0.3236446 Cellular processes Adaptation to atypical conditions SMU.924 tpx thiol peroxidase 2.4148275 Cell division SMU.15 ftsH putative cell division protein FtsH 2.0504131 SMU.1003 gid tRNA (uracil 5 ) methyltransferase Gid 1.7732082 SMU.713 ftsW putative cell division protein FtsW 1.7183926 SMU.1324 ftsX putative cell division protein FtsX 2.6479981 Chaperones SMU.1954 groEL chaperonin GroEL 0.5289494 SMU427 copZ putative copper chaperone 0.3809382 SMU.82 dnaK molecular chaperone DnaK 0.1523051 Detoxification SMU.1286c yitG, blt putative permease, multidrug efflux protein 3.5693367 Toxin production and resistance SMU.1339 bac C putative bacitracin synthetase 0.0193854 SMU.1342 bacA1 putative bacitracin synthetase 1, BacA 0.0128161 SMU.1341c grs, mycB putative gramicidin S synthetase 0.0086318 SMU.1340 bacA2 putative surfactin synthetase 0.0307009 Central intermediary metabolism SMU.636 nagB putative N acetylglucosamine 6 phosphate isomerase 3.0522759 SMU.1322 budC acetoin reductase 1.7732552 SMU.1687 ppaC putative manganese dependent inorganic pyrophosphatase 1.7436224 DNA metabolism SMU.1967 ssb A single stranded DNA binding protein 5.5803232 SMU.1472 recJ putative single strand DNA specific exonuclease RecJ 4.5589829 Energy metabolism ATP proton motive force interconversion SMU.1528 atpB F0F1 ATP synthase subunit beta 2.582669

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SMU.1527 atpC F0F1 ATP synthase subunit epsilon 2.1918756 Biosynthesis and degradation of polysaccharides SMU.1432c bgc putative endoglucanase precursor 2.1643653 Fermentation SMU.1424 pdhD putative dihydrolipoamide dehydrogenase 0.4769444 SMU.1423 pdhA putative pyruvate dehydrogenase, TPP dependent E1 component alpha subunit 0.4088209 SMU.1421 pdhC branched chain alpha keto acid dehydrogenase subunit E2 0.1532662 SMU.1011 citG putative CitG protein 0.2764563 Glycolysis/gluconeogenesis SMU.99 fbaA fructose bisphosphate aldolase 2.4059349 SMU.113 pfk putative fructose 1 phosphate kinase 0.1597532 SMU.1489 lacX LacX 0.1118628 Sugars SMU.887 galT galactose 1 phosphate uridylyltransferase 0.4829715 SMU.1535 phsG glycogen phosphorylase 0.3388341 SMU.495 gldA glycerol dehydrogenase 0.1858757 SMU.1490 lacG 6 phospho beta galactosidase 0.1020705 Fatty acid and phospholipid metabolism SMU.1739 fabF 3 oxoacyl (acyl carrier protein) synthase II 2.2443808 SMU.962 mmgC putative dehydrogenase 2.7906811 SMU.1734 accA acetyl CoA carboxylase subunit alpha 2.660529 SMU.1735 accD acetyl CoA carboxylase subunit beta 2.2984387 SMU.1344c fabD putative malonyl CoA acyl carrier protein transacylase 0.0223155 Hypothetical SMU.958 Hypothetical protein 4.4670806 SMU.1587c Hypothetical protein 2.8223919 SMU.2105 Hypothetical protein 2.365655 SMU.614 Hypothetical protein 2.1191022 SMU.1946 Hypothetical protein 1.941314 SMU.1360c Hypothetical protein 0.1569922 Mobile and extrachromosomal element functions SMU.1354c putative putative transposase 0.2418496 SMU.1363c tpn putative transposase 0.0278486 Protein fate Degradation of proteins, peptides, and glycopeptides SMU.395 pepX x prolyl dipeptidyl aminopeptidase 0.4433766 Protein folding and stabilization SMU.83 dnaJ heat shock protein DnaJ (HSP 40) 0.2918908 SMU.81 grpE heat shock protein GrpE (HSP 70 cofactor) 0.2638283 Protein modification and repair SMU.755 lgt prolipoprotein diacylglyceryl transferase 1.7790999 Protein synthesis Protein modification SMU.1051 nifS iron sulfur cofactor synthesis protein 0.5142172 Ribosomal proteins: synthesis and modification SMU.2000 rplQ 50S ribosomal protein L17 6.3196395 SMU.2002 rs11 30S ribosomal protein S11 4.9743901 SMU.2003 rpsM 30S ribosomal protein S13 4.7967458 SMU.2003c rpmJ 50S ribosomal protein L36 4.3377373

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SMU.957 rplJ 50S ribosomal protein L10 3.7847357 SMU.960 rplL 50S ribosomal protein L7/L12 3.5678142 SMU.2032 rpsB 30S ribosomal protein S2 3.1319002 SMU.169 rplM 50S ribosomal protein L13 2.9645129 SMU.170 rpsI 30S ribosomal protein S9 2.9612505 SMU1626 rplA 50S ribosomal protein L1 2.6580663 SMU.2026c rps10, rpsJ 30S ribosomal protein S10 2.2157606 SMU.1200 rpsA 30S ribosomal protein S1 2.2048536 SMU.1627 rplK 50S ribosomal protein L11 2.2026091 Translation factors SMU.2004 infA translation initiation factor IF 1 3.1577867 SMU.359 fus, tetO elongation factor G 2.1290956 Other SMU.1606 smpB SsrA binding protein 2.1903478 tRNA aminoacylation SMU.1311 asnC asparaginyl tRNA synthetase 2.2366516 SMU.1770 valS valyl tRNA synthetase 2.1066599 SMU.1586 thrS threonyl tRNA synthetase 1.9637127 Other SMU.1606 smpB SsrA binding protein 2.1903478 Purines, pyrimidines, nucleosides, and nucleotides SMU.944 thyA thymidylate synthase 3.1234637 SMU.1086 kith thymidine kinase 2.2807132 SMU.580 xseA exodeoxyribonuclease VII large subunit 0.2949093 Regulatory functions DNA interactions SMU.80 hrcA heat inducible transcription repressor 0.4953722 SMU.953c putative transcriptional regulator/aminotransferase 0.4070108 SMU.584 argR, ahrC putative arginine repressor 0.3546557 General regulatory SMU.105 scrR, lacI putative transcriptional regulator, repressor of sugar transport operon 0.490465 Other SMU.1515 covX hypothetical protein 2.6764309 Signal transduction PTS SMU.1600 celB cellobiose phosphotransferase system IIB component 0.218699 SMU.114 putative PTS system, fructose specific IIBC component 0.1936115 SMU.115 putative PTS system, fructose specific IIA component 0.1914331 SMU.1598 celC cellobiose phosphotransferase system IIA component 0.1738983 SMU.1596 celD cellobiose phosphotransferase system IIC component 0.1633683 Two component systems SMU.1129 ciaR putative response regulator CiaR 0.4817681 SMU.1037c phoR putative histidine kinase 0.3980582 SMU.577 lytS putative histidine kinase LytS 0.0048108 Transcription SMU.611 deaD, rheA ATP dependent RNA helicase 2.2659316 SMU.2001 rpoA DNA directed RNA polymerase subunit alpha 5.5173626 SMU.1745c putative transcriptional regulator 2.763516

PAGE 4

SMU.1599 celR putative transcriptional regulator, possible antiterminator 0.1755442 Transport and binding proteins SMU.568 putative amino acid ABC transporter, ATP binding protein 2.3434772 SMU.1666 livG putative branched chain amino acid ABC transporter, ATP binding protein 2.1183644 SMU.1325 ftsE putative ABC transporter, ATP binding component 1.7886366 SMU.1366c putative ABC transporter, ATP binding protein 0.0185912 SMU.1668 livH putative branched chain amino acid ABC transporter, permease protein 2.264163 SMU.1667 livM putative branched chain amino acid ABC transporter, permease protein 2.004054 SMU.879 msmF multiple sugar binding ABC transporter, permease protein MsmF 0.4471718 SMU.1365c ylbB Permease 0.0161324 SMU.1963 levQ putative sugar binding periplasmic protein 0.4323656 SMU.880 msmG multiple sugar binding ABC transporter, permease protein MsmG 0.4254579 SMU.1985 comYB ABC transporter ComYB 22.9927435 Unassigned SMU.367 Streptococcus specific protein; similar to glucan binding protein 1.8993877 SMU.1511c putative acetyltransferase; ; possible transcriptional repressor 1.8718513 SMU.587 lipase/acylhydrolase with GDSL like domain 0.5174147 SMU.400 putative secreted esterase 0.4787778 SMU.1040c ydfG putative oxidoreductase, short chain dehydrogenase/reductase 0.4780691 SMU.583 hlyX putative hemolysin 0.2493708 SMU.1345c ituA putative peptide synthetase MycA 0.1495599 SMU.1346 bacT putative thioesterase BacT 0.0393511 SMU.575c lrgA holin like protein LrgA 0.0049531 SMU.574c lrg lrgB like family protien 0.004215 Unknown SMU.1545c Conserved hypothetical protein 4.7357545 SMU.508 Conserved hypothetical protein, possible hydrolase 4.4179662 SMU.509 Conserved hypothetical protein 3.7447991 SMU.2046c Conserved hypothetical protein 3.0173063 SMU.1506c Conserved hypothetical protein, possible multidrug efflux pump 3.0151856 SMU.516 Conserved hypothetical protein, N6 adenine specific DNA methylase signature domain 2.9309334 SMU.260 Conserved hypothetical protein 2.7516912 SMU.159 Conserved hypothetical proteinRibonuclease III family protein 2.6082643 SMU.1083c Conserved hypothetical protein 2.5998184 SMU.1621c Conserved hypothetical protein 2.2656552 SMU.239c Conserved hypothetical protein, putative transporter 2.0467063 SMU.1623c Conserved hypothetical protein, predicted s1 RNA binding domain 1.8833143 SMU.298 Conserved hypothetical protein, predicted CoA binding protein 1.7995944 SMU.588 Conserved hypothetical protein 0.3923297 SMU.1022 citX2 Conserved hypothetical protein, possible CitXG protein 0.2912856


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1471-2180-12-187
ji 1471-2180
fm
dochead Research article
bibl
title
p Identification of the it Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance
aug
au id A1 snm Ahnfnm Sang-Jooninsr iid I1 email SAHN@dental.ufl.edu
A2 QuMing-DaI2 mdq1990@gmail.com
A3 RobertsElishae.roberts345@gmail.com
A4 Burnemi ARobertRBURNE@dental.ufl.edu
A5 ca yes RiceCKellykcrice@ufl.edu
insg
ins Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, FL 32611, USA
Department of Microbiology and Cell Science, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611, USA
source BMC Microbiology
issn 1471-2180
pubdate 2012
volume 12
issue 1
fpage 187
url http://www.biomedcentral.com/1471-2180/12/187
xrefbib pubidlist pubid idtype doi 10.1186/1471-2180-12-187pmpid 22937869
history rec date day 29month 5year 2012acc 2182012pub 192012
cpyrt 2012collab Ahn et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Stress
Oxygen
Competence
Cid/Lrg system
Streptococcus mutans
abs
sec
st
Abstract
Background
The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans.
Results
Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (Hsub 2O2) resulted in increased intracellular ROS in the lytS mutant.
Conclusions
Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.
bdy
Background
Streptococcus mutans is considered the primary causative agent of dental caries, and when transiently introduced into the bloodstream following daily dental hygienic practices such as toothbrushing and flossing, this bacterium can also cause potentially lethal infective endocarditis (IE) abbrgrp
abbr bid B1 1
B2 2
B3 3
B4 4
. In both infectious scenarios, the virulence of S. mutans depends upon its ability to form biofilms and to withstand extreme changes in environmental conditions, including fluctuations in oxygenation, shear stress, as well as nutrient source and availability. For example, in the oral cavity, S. mutans must be able to rapidly alter its expression of transporters and metabolic enzymes to catabolize a variety of host-derived dietary carbohydrates. Internalized carbohydrates are metabolized through the glycolytic pathway, resulting in the accumulation of acidic end-products in the environment, which favors the growth of S. mutans and other acid-tolerant cariogenic species. Repeated cycles of acidification can lead to a net demineralization of tooth enamel and the development of caries. Sucrose, a common dietary sweetener, can also be utilized by S. mutans for the production of extracellular polysaccharides
B5 5
B6 6
B7 7
B8 8
that facilitate bacterial adhesion and biofilm formation. Aeration has also been found to have a profound effect on carbohydrate metabolism and biofilm formation by S. mutans
B9 9
B10 10
B11 11
. It is therefore not surprising that there is overlap in the genetic regulatory circuits responsive to carbohydrate metabolism, aeration/oxidative stress resistance and control of biofilm formation in S. mutans, which include CcpA
B12 12
B13 13
B14 14
, Rex
B15 15
, and Frp
B16 16
.More recently, an emerging trend in the study of bacterial biofilms has been a focus on the contribution of bacterial cell death and autolysis to biofilm adherence, maturation, and dispersal. It has been demonstrated in a wide variety of bacteria that death and lysis of a subpopulation of cells can facilitate biofilm formation due to the release of DNA into the extracellular environment (eDNA)
B17 17
B18 18
B19 19
B20 20
B21 21
B22 22
. Likewise, cell death and lysis have been implicated in dispersal of cells from a mature biofilm
B23 23
B24 24
B25 25
. In Staphylococcus aureus, the Cid/Lrg system has been shown to be involved in the regulation of cell death, autolysis, and biofilm formation
17
21
B26 26
B27 27
B28 28
. Characterization of S. aureus cid and lrg mutants has revealed that these operons have opposing effects on cell death and murein hydrolase activity
27
B29 29
. These observations, combined with the fact that LrgA and CidA share structural features with the bacteriophage lambda family of holin proteins
29
, have led to the hypothesis that CidA and LrgA control cell death and lysis in a manner analogous to effector and inhibitor holins, respectively
26
B30 30
. Bacteriophage holins are small membrane proteins that oligomerize in the cell membrane, acting as “molecular clocks” that regulate the timing and lysis of the host cell during lytic infection
B31 31
. For example, the lambda S holin regulates cell death and lysis by the formation of large lipid-excluding “rafts” that promote cytosolic leakage as well as access of the phage-encoded endolysin (murein hydrolase) to the cell wall
B32 32
B33 33
B34 34
. S. aureus CidA and LrgA have recently been shown to oligomerize into high-molecular-mass complexes in a cysteine disulfide bond-dependent manner, a biochemical feature also shared with holin proteins
B35 35
. Although the molecular details of how Cid and Lrg function to control cell death and lysis have not yet been completely elucidated, the fact that cid and lrg homologues have been identified in a wide variety of bacterial and archeal genomes supports a fundamental and conserved role for this system in cell physiology
30
B36 36
.In previous work it was determined that expression of potential cidAB and lrgAB homologues in S. mutans is highly responsive to carbohydrate availability
12
B37 37
and oxygenation
11
. Given the potential importance of these genes to biofilm development in S. mutans, we previously characterized a panel of S. mutans cid and lrg isogenic mutants and found that a subset of these genes did indeed influence biofilm formation, production of glucosyltransferases (enzymes that synthesize extracellular glucan polymers that contribute to biofilm adhesion), and oxidative stress tolerance
37
. In this study it was also found that, as demonstrated previously in S. aureus
B38 38
B39 39
, the S. mutans LytST two-component system was required for activation of lrgAB expression, but not cidAB expression
37
. Genes homologous to lytST appear to be present in most Gram-positive organisms that contain lrgAB
30
and these genes are often linked to one another, inferring an important role for this two-component system in fine-tuning lrgAB expression in response to external environmental signals. Therefore in this study, we sought to determine if LytST is involved in regulation of lrgAB expression in response to glucose and oxygenation in S. mutans, and to elaborate on the contribution of LytST to cellular homeostasis and global control of gene expression.
Results
Effects of oxygenation and glucose metabolism on S. mutans lrg and cid expression
The LytST two-component regulatory system has been shown to positively regulate lrgAB expression in a wide variety of bacteria, including various staphylococcal
38
39
B40 40
and Bacillus species
B41 41
B42 42
, as well as in S. mutans
37
. The conserved nature of this regulation in Gram-positive bacteria, combined with the known effects of LytST and LrgAB on cell death/lysis
29
38
39
B43 43
, biofilm development
21
37
38
, and oxidative stress resistance
37
, suggests that LytST and LrgAB are central regulators of physiologic homeostasis. However, little is known about the environmental and/or cellular cues to which LytS responds. In S. aureus and B. anthracis, it has been shown that lrgAB expression is responsive to disruption of cell membrane potential in a LytST-dependent manner
41
B44 44
. However, we were unable to determine whether this regulation also occurs in S. mutans, as treatment with membrane-potential disrupting agents (gramicidin, carbonyl cyanide m-chlorophenylhydrazone) did not have a measurable effect on membrane potential, as assessed by staining with DIOC2 (3) (data not shown).In previous studies, it was shown that oxygen and glucose metabolism have a pronounced effect on lrg and cid expression in S. mutans, but the specific role of LytS, if any, in this regulation was not addressed
11
37
. Therefore, S. mutans UA159 and its isogenic lytS mutant were grown under aerobic and low-oxygen conditions to exponential (EP) and stationary (SP) growth phases in media containing 11 mM or 45 mM glucose. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed on RNA isolated from cultures at each time point to assess changes in lrg expression (Figure figr fid F1 1). In UA159, stationary phase lrgAB expression was upregulated 365-fold relative to exponential phase when grown under 11 mM glucose and low-oxygen conditions (Figure 1A). Although mutation of lytS resulted in a severe loss of stationary phase lrgAB induction in cells grown in 11 mM glucose, lrgAB expression was not completely abolished. When grown under aerobic conditions and 11 mM glucose, stationary phase lrgAB expression was upregulated 2500-fold relative to exponential phase in the wild-type strain (Figure 1A), confirming previously-published observations that aerobic growth promotes lrgAB expression
11
. However, stationary-phase lrgAB expression was still induced 216-fold in the lytS mutant during aerobic growth, suggesting that (1) other as-yet-unknown regulators also contribute to the positive control of lrgAB expression during aerated growth, and (2) LytST is a predominant regulator of lrgAB expression during low oxygen growth, compared to aerobic growth. Under low-oxygen and aerated cultures, stationary phase induction of lrgAB expression was dramatically reduced when grown in 45 mM glucose, and similar levels of expression were observed in the wild-type and lytS mutant (Figure 1B), suggesting that growth in high levels of glucose abrogates oxygen-dependent regulation of lrgAB by LytST. Consistent with previously-published data
37
, LytS did not appear to have a measurable effect on cidAB expression under any of the growth conditions tested here (data not shown). In summary, LytST-dependent regulation of lrgAB expression is much more pronounced during low-oxygen growth and at low glucose levels.
fig Figure 1caption LytS-dependent expression of lrgAB in S.text
b LytS-dependent expression of lrgAB in S.mutans. Overnight cultures were diluted in THYE, containing either 11 mM (A) or 45 mM glucose (B) to an OD600 = 0.02 and grown at 37°C as static cultures at 5% CO2 (“low-O2”) or as aerobic shaking cultures at 250 RPM (“aerobic”). RNA was harvested at exponential (EP) and stationary phase (SP). Reverse-transcription, real-time PCR reactions, and determination of copy number were performed as described previously using lrgA and 16S-specific primers 37B77 77. Fold-change expression of lrgAB and 16S under each growth condition was calculated by dividing the gene copy number of each test sample by the average gene copy number of UA159 EP. Data was then normalized by dividing each lrgAB fold-change value by its corresponding 16S fold-change expression value. Data represent the average of 3 biological replicates. Dark grey bars represent UA159 and light grey bars represent lytS mutant. Error Bars represent the standard error (SEM).
graphic file 1471-2180-12-187-1
Microarray analysis of the LytS regulon
Based on the transcriptional data presented above, the effects of LytST regulation on lrgAB expression are most evident while S. mutans is growing under conditions of low-oxygen (5% CO2) with a lower concentration of glucose. To begin to explore how LytST impacts critical phenotypes of S. mutans, RNA expression profiles in UA159 and the lytS mutant were compared using an RNA microarray approach. RNA was isolated from early exponential and late exponential growth phases from static planktonic cultures grown in BHI (containing 11 mM total glucose) at 37°C in a 5% CO2 atmosphere (Additional file supplr sid S1 1: Table S1 and Additional file S2 2: Table S2). At early exponential growth phase, loss of LytS affected the expression of 40 genes (12 upregulated and 28 downregulated; P < 0.005; Additional file 1: Table S1). Most of the upregulated genes in early exponential phase displayed only a modest increase in expression and included genes involved in DNA repair, purine/pyrimidine metabolism, competence, and a number of unassigned and hypothetical ORFs. RNA transcripts that were strongly down-regulated greater than 10-fold in cells lacking LytS during early exponential growth included those annotated as bacitracin/surfactin/gramicidin synthesis proteins, transport and binding proteins, and LrgAB. In contrast, loss of LytS affected the expression of a much larger number of genes in late exponential phase (136 genes total), with 79 upregulated transcripts and 57 downregulated transcripts (P < 0.001; Additional file 2: Table S2). Aside from dramatically decreased lrgAB expression, affected genes included those involved in amino acid and co-factor biosynthesis, carbohydrate and fatty acid metabolism, stress adaptation, toxin production, DNA repair/recombination, protein synthesis, transcriptional regulation, and competence, as well as multiple hypothetical and/or unassigned ORFs (Additional file 2: Table S2 and Figure F2 2). A subset of genes was differentially expressed as a function of the loss of LytS in both early exponential and late exponential growth phases (Additional file 1: Table S1 and Additional file 2: Table S2). These included many genes encoded by the S. mutans genomic island TnSMu2
B45 45
(SMU.1335c, 1339-1342, 1344c-1346, 1354c, 1360c, 1363c, 1366c), ssbA, comYB, and lrgAB. Given that these genes were regulated by LytS in both growth phases examined, it is possible that they are under the direct control of LytST. To validate the microarray data, qRT-PCR was performed on late exponential phase wild-type and lytS mutant RNA to assess expression of 14 of the affected genes. As shown in Table tblr tid T1 1, the expression ratios (lytS mutant/wild-type) for each gene obtained by real-time PCR were similar to the microarray results. Interestingly, expression ratios of these genes were all close to 1.0 when comparing expression between the wild-type strain and a lrgAB mutant (Table 1), indicating that the differential expression patterns observed in the lytS mutant were not a consequence of down-regulated lrgAB expression.
suppl
Additional file 1
Table S1. Genes differentially expressed by loss of LytS at early-exponential phase (P< 0.005).
name 1471-2180-12-187-S1.docx
Click here for file
Additional file 2
Table S2. Genes differentially expressed by loss of LytS at late exponential phase (P< 0.001).
1471-2180-12-187-S2.docx
Click here for file
Figure 2Distribution of functions of genes affected by loss of LytS at late exponential phase.
Distribution of functions of genes affected by loss of LytS at late exponential phase. Statistical analysis was carried out with BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html/) with a cutoff P value of 0.001. The 136 genes differentially expressed at P ≤0.001 are grouped by functional classification according to the Los Alamos S. mutans genome database (http://www.oralgen.lanl.gov/).
1471-2180-12-187-2
table
Table 1
Real-time PCR validation of RNA microarray results
tgroup align left cols 6
colspec colname c1 colnum 1 colwidth 1*
c2 2
char c3 3
c4 4
c5 5
c6
thead valign top
row
entry nameend namest
. rowsep
Microarray
Real-time pcr
lytS
mutant
lytS
mutant
lrgAB
mutant
tfoot
Results are expressed in fold-change (mutant/wild-type).
tbody
(SMU.1985)
comYA (comYB)
22.9927
6.8449
0.8163
SMU.1967
ssbA
5.5803
4.1076
0.8791
(SMU.1515)
vicR (vicX)
2.6764
1.7647
1.0267
SMU.924
tpx
2.4148
3.6168
1.058
SMU.1739
fabF
2.2443
2.0333
1.084
SMU.1666
livG
2.1183
3.4331
1.009
SMU.80
hrcA
0.4953
0.6107
1.0204
SMU.1424
pdhD
0.4769
0.4031
1.2004
SMU.580
xseA
0.29849
0.5409
1.1398
SMU.1600
celB
0.2186
0.2825
1.2979
SMU.113
pfk
0.1597
0.176
1.3578
SMU.82
dnaK
0.1523
0.2652
0.9907
SMU.1344
fabD
0.0223
0.012
1.0637
SMU.1341
grs
0.0008
0.0121
1.1027
Investigation of the effect of LytST and LrgAB on competence
In analyzing the microarray data in Additional file 1: Table S1 and Additional file 2: Table S2, it appeared that the gene most highly upregulated in response to loss of LytS in both phases of growth was comYB (SMU.1985), a homologue of the B. subtilis comGB gene that encodes part of an ABC transporter essential for DNA binding-uptake during competence in S. mutans
B46 46
. Interestingly, a comYB mutant of S. mutans was shown to be unaffected in competence signaling, but showed reduced biofilm formation, which was thought to be a function of its inability to bind biofilm matrix eDNA
B47 47
. Since the lytS mutant displayed an increase in comYB expression (Additional file 1: Table S1 and Additional file 2: Table S2), we hypothesized that this strain may display alterations in its ability to form biofilm and/or its transformability during genetic competence. However, the lytS mutant did not display any appreciable difference in its ability to form static biofilm in the presence of glucose or sucrose (data not shown), and likewise, did not display a difference in its ability to uptake plasmid DNA in a quantitative competence assay, relative to the wild-type strain (Figure F3 3). Since lrgAB expression is so strongly regulated by LytST, the ability of isogenic lrgA, lrgB, and lrgAB mutants to uptake plasmid DNA via competence was also assessed (Figure 3). Of all the mutants tested, the lrgA mutant was the most severely impaired in its ability to uptake plasmid DNA relative to the parental strain, displaying a 26- and 24-fold decrease in transformation efficiency in the presence and absence of competence-stimulating peptide (CSP), respectively (Figure 3), suggesting that LrgA is somehow involved in genetic transformation in a CSP-independent manner. This finding has particular significance considering that LrgAB has been linked to regulation of cell death and lysis in S. aureus
21
29
and S. mutans
37
, and these physiological processes are also extremely important during natural competence. It is interesting to note that, similar to the competence results described here, the lrgA mutant was previously shown to display decreased glucose-dependent biofilm formation and decreased glucosyltransferase production, whereas the lrgB and lrgAB mutants behaved in a manner similar to the parental strain
37
. These phenotypic patterns suggest that the presence of LrgB alone, rather than the lack of LrgA, may be responsible for the biofilm and competence phenotypes observed in the lrgA mutant.
Figure 3Transformation efficiencies of UA159 and isogenic lytS and lrg mutants.
Transformation efficiencies of UA159 and isogenic lytS and lrg mutants. To compare the ability of UA159 and its isogenic lytS, lrgA, lrgB, and lrgAB mutants to take up exogenously-added plasmid DNA, a quantitative competence assay was performed on n = 4-6 biological replicates of each strain as described in Methods B83 83. Plasmid pAT28 [encoding spectinomycin resistance; B84 84 was used to assess transformation efficiency in UA159, lytS, lrgB, and lrgAB mutants. Because the lrgA mutant was generated with a spectinomycin-resistance cassette 37, plasmid pORi23 [encoding erythromycin resistance; B85 85] was used to assess transformation efficiency in UA159 and lrgA mutant. Transformation efficiencies (Y axis) in the presence (grey bars) and absence (white bars) of CSP are expressed as the percentage of transformants (CFU/ml on BHI + selective antibiotic) among total viable cells (CFU/ml on BHI). Error bars represent SEM. Brackets with P values denote statistically-significant differences between two samples (Mann–Whitney Rank Sum Test).
1471-2180-12-187-3
Effect of LytST on oxidative stress tolerance
Previously, our investigations disclosed a strong link between oxidative stress tolerance and the Cid/Lrg system
37
, a role for these genes that had not been described in other organisms. Specifically, we found that lrgAB, lrgB, cidAB, and cidB mutants exhibited reduced growth in the presence of paraquat, and growth of lrgAB, cidAB, and cidB mutants on BHI agar plates in aerobic conditions was almost completely inhibited
37
. It is therefore interesting to note that in the lytS microarray results (Additional file 2: Table S2), genes encoding antioxidant and DNA repair/recombination enzymes were significantly upregulated in the lytS mutant in late exponential phase. These included yghU and tpx, encoding the putative anti-oxidant enzymes glutathione S-transferase and thiol peroxidase, respectively, as well as recJ, which encodes a single-stranded DNA exonuclease protein that facilitates DNA repair in response to oxidative stress
B48 48
B49 49
B50 50
B51 51
. To further investigate the effect of lytS and lrgAB on oxidative stress tolerance, wild-type, lytS, and lrgAB mutants were grown as planktonic static BHI cultures in aerobic atmosphere and in the presence and absence of H2O2 (Figure F4 4). When challenged with H2O2, UA159 experienced an increased lag phase of growth, and the overall OD of the culture was 10-25% less than the untreated culture until 20 h growth. Under these assay conditions, the lrgAB mutant displayed a dramatic growth defect in both the presence and absence of H2O2. It is interesting to note that this aerobic growth defect was also previously observed when the lrgAB mutant was grown in aerobic atmosphere on BHI agar plates
37
. The lytS mutant displayed an increased lag in growth relative to UA159 when cultured in the presence of H2O2, but OD values were comparable to the wild-type strain by 16 h growth. These results suggest that the LytST regulon impacts the ability of cells to grow under conditions of oxidative stress.
Figure 4H2O2 challenge assay of UA159, lytS and lrgAB mutants.
H2O2challenge assay of UA159, lytS and lrgAB mutants. Cultures of UA159, lytS, and lrgAB mutants (n = 6 biological replicates per strain) were grown in the presence (open symbols) and absence (filled symbols) of 1.0 mM H2O2 for 20 h at 37°C (aerobic atmosphere) in a Biotek microplate reader. OD600 measurements of each well were recorded at 2 h intervals. Black circles represent UA159, red triangles represent lytS mutant, blue squares represent lrgAB mutant. Error bars represent SEM.
1471-2180-12-187-4 The cell-permeable fluorescent dye CM-H2DCFDA (Invitrogen Molecular Probes) was also used to assess intracellular ROS in UA159 and the lytS mutant (Figure F5 5). This fluorescent compound is oxidized in the presence of H2O2 and other reactive oxygen species (ROS) and is considered a general indicator of intracellular oxidative stress
B52 52
B53 53
. This analysis revealed that stationary-phase cultures of the wild-type and lytS mutant strains had similar “endogenous” intracellular levels of ROS (Figure 5, light grey bars). When stationary-phase cells from each strain were loaded with CM-H2DCFDA and then challenged with 5 mM H2O2 (Figure 5, dark grey bars), a greater increase in fluorescence was observed in the lytS mutant relative to UA159 (P = 0.009, Mann–Whitney Rank Sum Test), suggesting that loss of LytS has an impact on the ability of the cells to detoxify H2O2 and/or other intracellular ROS.
Figure 5Measurement of intracellular ROS in UA159 and lytS mutant by CM-H2DCFDA staining.
Measurement of intracellular ROS in UA159 and lytS mutant by CM-H2DCFDA staining. Cells were harvested from 20 h BHI cultures of UA159 and isogenic lytS mutant grown at 37°C 5% CO2 (n = 3-6 biological replicates each), resuspended in HBSS containing 5 μM CM-H2DCFDA, and incubated at 37°C to load the cells with stain. After 60 min incubation, cell suspensions were centrifuged, washed once in HBSS buffer, and then resuspended in HBSS buffer alone (light grey bars) or in HBSS containing 5 mM H2O2 (dark grey bars). Each suspension was transferred to wells of an optically-clear 96 well plate, and incubated at 37°C in a microplate reader. Cell fluorescence (as measured by relative fluorescence units; RFU) and the OD600 of each well was recorded after 30 min incubation. RFU measurements are expressed per OD600 of each well to account for any subtle variations in cell density. Error bars represent SEM. Brackets with P values denote statistically-significant differences between two samples (Mann–Whitney Rank Sum Test).
1471-2180-12-187-5
Discussion
The transcriptome analyses presented in this study have revealed that the LytST two-component system has a widespread effect on gene expression in S. mutans. A much higher number of transcripts were affected by the lytS mutation in late exponential phase and the magnitude of changes in expression was greater (n = 136 genes, Additional file 2: Table S2) relative to early-exponential phase (n = 40 genes, Additional file 1: Table S1), where most genes exhibited only a modest (1-2 fold) change in expression. These differences in gene expression patterns are unlikely to be an indirect function of altered lrgAB expression in the lytS mutant, as expression of lytS-regulated genes was unaltered in an lrgAB mutant relative to the wild-type strain (Table 1). Taken together, these observations suggest that LytST exerts control over its transcriptome in a growth-phase dependent manner, and to our knowledge, this is the first study that has compared the scope of LytST regulation at different phases of growth. Interestingly, RNA microarray studies of lyt mutants have also been performed in S. aureus
38
, S. epidermidis
40
, and B. subtilis
42
. As we have observed here in S. mutans, a global effect of LytST on gene expression was also noted in S. aureus and S. epidermidis
38
40
. In S. aureus, LytST appeared to exert primarily positive effects on gene expression in exponential phase when aerobic cultures were grown in media containing excess (35 mM) glucose, as only 7 genes were found to be upregulated in the lytS mutant
38
. In S. epidermidis, a large number of genes were up- or down-regulated as a function of the presence of LytST during exponential phase during aerobic growth in medium containing 12 mM glucose
40
. In contrast, mutation of lytS only appeared to affect the expression of lytST itself and genes encoding lrgAB and cidAB homologues in B. subtilis
42
. However, due to the differences in growth conditions used (glucose levels and/or culture aeration) and the differing metabolic pathways present in these organisms, it is difficult to establish direct correlations between these studies and the S. mutans microarray results presented here.As demonstrated previously
37
, expression of lrgAB was also shown to be tightly controlled by the LytST two-component system in S. mutans in this study. Specifically, we have found that LytST-dependent expression of lrgAB is regulated in part by glucose metabolism and oxygen in S. mutans (Figure 1). Furthermore, control of lrgAB expression by LytST appears to be highly growth-phase dependent: lrgAB expression in the lytS mutant exhibited only a modest decrease in expression in early exponential phase (0.49 relative to UA159, Additional file 1: Table S1), whereas lrgAB expression was down-regulated some 200-fold in the lytS mutant at late exponential phase (Additional file 2: Table S2). Alternatively, it is possible that control of lrgAB expression by LytST is related to higher glucose availability during early exponential phase. Although detailed mechanistic studies have not yet been performed, there is mounting evidence that these proteins are critical for oxidative stress resistance in S. mutans: (1) lrgAB expression is highly regulated by oxygen (
11
and this study); (2) a lrgAB mutant was defective in aerobic growth on BHI agar plates
37
; (3) a lrgAB mutant displayed a decreased growth rate in the presence of paraquat (a superoxide-generating agent) relative to the wild-type strain
37
; and (4) a lrgAB mutant displayed a strong growth defect during static planktonic aerobic growth in BHI in the presence and absence of H2O2 challenge (this study). Interestingly, a link between LrgAB and oxidative stress was also demonstrated in S. aureus, where lytSR and lrgAB expression were upregulated 2-5 fold in response to azurophilic granule proteins, H2O2, and hypochlorite
B54 54
.In agreement with a role for LrgAB in oxidative stress resistance, several LytST-regulated genes identified in this study have also been implicated in bacterial oxidative stress responses. Upregulated potential oxidative stress genes include yghU, a putative anti-oxidant enzyme
50
, tpx, a predicted thiol peroxidase
B55 55
, and recJ, a single-stranded DNA exonuclease protein that facilitates DNA repair in response to oxidative stress
51
. Conversely, several genes belonging to the TnSMu2 gene cluster (SMU.1334c – SMU.1359) were downregulated in the lytS mutant. These genes are annotated as encoding a series of gene products involved in bacitracin and gramicidin synthesis
B56 56
, but more recently have been shown to be responsible for nonribosomal peptide and polyketide (NRP/PK) biosynthesis of a pigment that enhances aerobic growth and tolerance to H2O2 challenge in S. mutans UA159
45
. The altered expression of one or more of these genes may explain, in part, the increased ROS accumulation that was observed in the lytS mutant when challenged with H2O2 (Figure 5). Furthermore, it was previously found that a two-component system responsible for positive regulation of the NRP/PK genes was located on the TnSMu2 genomic island of UA140 but not in UA159
45
. This observation, combined with the microarray results performed here (Additional file 1: Table S1 and Additional file 2: Table S2) suggest that LytST may have taken over some of the regulatory functions of this non-core-genome two-component system that is missing in UA159.Interestingly, H2O2 has also been shown to be a potent stimulator of competence and eDNA release in S. sanguinis
B57 57
, S. gordonii
57
B58 58
, and S. pneumoniae
B59 59
. Although the effects of H2O2 on S. mutans competence, cell lysis, and eDNA release have not been directly measured, it has been shown that growth under aerobic conditions promotes competence in S. mutans
47
, and that expression of competence-related genes is upregulated during aerobic growth
11
. The results presented here have demonstrated that expression of comYB, a gene encoding a component of the DNA-binding uptake system in S. mutans
47
was upregulated 2-fold in early exponential phase and 22-fold in late exponential phase in the lytS mutant (Additional file 1: Table S1 and Additional file 2: Table S2). The significance of high-level comYB expression in the lytS mutant at late exponential phase is unclear, given that maximal S. mutans competence develops in actively-growing populations
B60 60
B61 61
. Accordingly, upregulation of comYB expression did not correlate with increased transformability of the lytS mutant under the conditions tested in this study (Figure 3). However, it was found that the lrgA mutant displayed a significant reduction in competence. It has been recently reported that only a subpopulation of S. mutans culture lyses in response to CSP, and this lysis event is controlled in part by the CipB bacteriocin and the CipI immunity protein
B62 62
. Subsequent microarray analysis of a cipI (immunity protein) mutant showed that both lytST and lrgAB expression were highly upregulated in the cipI mutant
B63 63
. These results, combined with the fact that LrgA/B has been shown to be involved in regulating cell lysis and eDNA release in S. aureus
21
29
, lends strong support to the idea that LrgA plays an important role during competence, possibly by altering membrane permeability or by modulating murein hydrolase activity.The S. mutans comY operon consists of nine co-transcribed genes, of which the first eight genes are either essential to or significantly affect competence
46
. The ninth gene of this operon is predicted to encode acetate kinase (AckA), an enzyme that catalyzes the inter-conversion of acetyl-phosphate and acetate
46
B64 64
. For micro-organisms with an inefficient or incomplete TCA cycle such as S. mutans, AckA-mediated conversion of acetyl-phosphate to acetate is thought to be a critical mechanism of generating ATP [reviewed in
B65 65
]. Since ackA (comYI) was previously found to be upregulated in S. mutans during aerated growth
11
, it is possible that LytST is involved in the regulation of energy generation through the phosphate acetyltransferase (Pta)-AckA pathway during aerobic growth and/or during oxidative stress. In this respect, it has recently been reported that an S. mutans pta mutant was more susceptible to both acid and oxidative stresses
B66 66
.The ability of S. mutans to combat H2O2 stress is critical for its survival in the oral cavity, yet H2O2 detoxifying mechanisms and their regulation have not been extensively-characterized in this organism, limited primarily to the ScnRK and VicRK two-component systems
B67 67
B68 68
, ropA
B69 69
, brpA
B70 70
, luxS
B71 71
and genomic island TnSMu2
45
. H2O2 has been shown to have potent antibacterial effects on S. mutans
B72 72
, and it is thought that H2O2 produced by other oral streptococcal species serves as an antagonist against S. mutans. For example, S. sanguinis and S. gordonii have been shown to produce H2O2 via pyruvate oxidase under aerobic growth conditions, and this H2O2 production allows them to compete effectively against S. mutans when co-cultured under aerobic growth conditions
57
. It is therefore possible that the S. mutans LytST regulon mediates a pleiotropic protective response against these H2O2-producing niche competitors. On-going and future studies by our group will focus on experimental testing of this hypothesis.
Conclusions
In summary, the LytST two-component system has been shown to have a pleiotropic effect on gene expression in S. mutans. This is congruent with microarray analyses of lytS mutants in S. aureus
38
and S. epidermidis
40
. However, unlike in other organisms, we have been able to identify a pattern of LytS-mediated gene expression that suggests a role for this regulon in responding to oxidative/H2O2 stress. Although we have not yet been able to identify the external signal to which LytS responds, it is likely linked to an oxidative stress-sensing mechanism, such as H2O2-mediated membrane damage (ie. lipid peroxidation) via its large number of transmembrane domains, or oxygen/ROS interactions with its predicted cytoplasmic GAF domain, a ubiquitous signaling domain that has been shown to detect changes in the redox state of bound iron or oxygen in Mycobacterium tuberculosis
B73 73
B74 74
B75 75
. Establishing mechanistic links between the LytST regulon, H2O2 resistance, and competence regulation will provide valuable new insights into S. mutans survival and virulence in the oral cavity.
Methods
Bacterial strains, media, and growth conditions
For all experiments, frozen glycerol stocks of S. mutans UA159 and its isogenic lytS (SAB111; ΔlytS::NPKmsup r), lrgA (SAB113; ΔlrgA::NPSpr), lrgB (SAB119; ΔlrgB::NPEmr), and lrgAB (SAB115; ΔlrgAB::ΩKmr) mutants [created previously in
37
were freshly streaked for isolation on either Todd Hewitt Yeast Extract (THYE) or Brain Heart Infusion (BHI), containing selective antibiotic as appropriate: kanamycin (Km) – 1000 μg/ml, erythromycin (Em) – 10 μg/ml, spectinomycin (Sp) 1000 μg/ml). With the exception of SAB115 (lrgAB mutant), all mutants were created using non-polar (NP) antibiotic-resistance markers
37
. Unless otherwise indicated, all S. mutans cultures were grown as static cultures in BHI or THYE broth at 37°C and 5% CO2.
Analysis of lrgAB expression
To measure the effects of oxygen and glucose on lrg expression, overnight THYE cultures of UA159 and the lytS mutant (n = 3 biological replicates each, grown at 0 RPM, 37°C and 5% CO2) were each inoculated to an OD600 = 0.02 into THYE containing either 11 mM or 45 mM glucose. For “low O2” cultures, 2 L culture flasks each containing 400 ml media were grown at 0 RPM, 37°C, and 5% CO2. For aerobic cultures, 500 ml culture flasks each containing 100 ml media were grown at 37°C and 250 RPM. Total RNA was isolated from all cultures sampled at exponential (EP; OD600 = 0.2 – 0.4) and stationary (SP; OD600 = 1.4 – 1.7) growth phase, with an RNeasy Mini kit (Qiagen) and FASTPREP (MP Biomedicals) using previously-described methods
B76 76
. Real-time reverse-transcriptase PCR and data analysis using lrgA and 16S primers was performed using previously described primers
37
and methods
77
. Fold-change expression of lrgA and 16S under each growth condition (11 mM low-O2, 11 mM aerobic, 45 mM low-O2, 45 mM aerobic) was calculated by dividing the gene copy number of each test sample by the average gene copy number of UA159 EP. Data was then normalized by dividing each lrgA fold-change expression value by its corresponding 16S fold-change expression value.
RNA microarray analysis of UA159 and lytS mutant
To assess the effect of LytS on global gene expression, overnight BHI cultures of UA159 and lytS mutant (n = 3 biological replicates per strain) were diluted to an OD600 = 0.02 in BHI, and grown as static cultures at 37°C and 5% CO2. Total RNA was isolated from each culture at early-exponential (OD600 = 0.15) and late exponential phase (OD600 = 0.9), using previously-published methods
77
. RNA microarray analysis was performed using S. mutans UA159 microarrays provided by The Institute for Genomic Research, and previously-described methods and data analysis
11
70
B78 78
. In brief, 2 μg total bacterial RNA was used in each reverse-transcription and cDNA labeling reaction (performed as described in
70
78
), and a single preparation from each culture was hybridized per microarray slide in a Maui hybridization chamber (BioMicro Systems, Salt Lake City, UT). The resulting microarray slides were scanned, analyzed, and normalized using TIGR Spotfinder software (http://www.tigr.org/software/), and in-slide replicate analysis was performed with the TIGR microarray data analysis system (MIDAS; http://www.tigr.org/software/). Statistical analysis was carried out with BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html/) with a cutoff P value < 0.005 for the early exponential-phase data and P < 0.001 for the late exponential phase data. To validate the microarray results, real-time quantitative RT-PCR was performed on a subset of the differentially-expressed genes, as described previously
77
B79 79
. All real-time PCR primers were designed with Beacon Designer 4.0 software (Premier Biosoft International, Palo Alto, CA), and standard curves for each gene were prepared as published elsewhere
B80 80
. The microarray data obtained from these studies have been deposited to NCBI’s gene expression omnibus (GEO)
B81 81
(GEO Accession #GSE39470) and comply with MIAME guidelines
B82 82
.
Quantitative competence assays
To compare the ability of UA159 and its isogenic lytS, lrgA, lrgB, and lrgAB mutants to take up exogenously-added plasmid DNA, a quantitative competence assay was performed on n = 4-6 biological replicates of each strain using a previously-published protocol
83
with the following modifications: Overnight cultures of each strain were diluted to an OD600 = 0.02 in BHI, and grown in a 96-well plate to an OD600 = 0.15 prior to addition of 500 ng plasmid DNA with and without 100 ng CSP. Plasmid pAT28 (encoding spectinomycin resistance;
84
) was used to assess transformation efficiency in UA159, lytS, lrgB, and lrgAB mutants. Because the lrgA mutant was generated with a spectinomycin-resistance cassette
37
, plasmid pORi23 [encoding erythromycin resistance;
85
] was used to assess transformation efficiency in UA159 and lrgA mutant. After 2.5 h incubation in the presence of plasmid DNA +/- CSP, cultures were serially diluted and plated on BHI agar with and without selective antibiotic. CFU/ml of each culture were enumerated after 48 h growth at 37°C and 5% CO2, and transformation efficiencies were calculated as the percentage of transformants (CFU/ml on BHI + selective antibiotic) among total viable cells (CFU/ml on BHI).
H2O2 assays
To assess of the ability of UA159, lytS, and lrgAB mutants to grow in the presence of H2O2, overnight cultures of each strain (n = 6 biological replicates) were each diluted 40-fold into BHI. 1 ml aliquots of each diluted culture were either untreated or challenged with 1 mM H2O2. Aliquots of each (500 μl per well, 2 wells total) were then immediately transferred to an optically-clear 48-well tissue culture plate (Costar 3548), which was incubated for 20 h at 37°C (aerobic atmosphere) in a Biotek Synergy microplate reader. OD600 measurements of each well were recorded at 2 h intervals.
Oxidative stress measurements
To assess intracellular oxidative stress in UA159 and lytS mutant, single isolated colonies of each strain (n = 3-6 biological replicates per strain) were inoculated into culture tubes containing 4 ml BHI, and grown in “low-O2” conditions (37°C, 0 RPM, 5% CO2). After 20 h growth, 2 × 1 ml aliquots of each culture were harvested by centrifugation in a microcentrifuge (3 min at 13,000 RPM). The culture supernatants were discarded, and cell pellets were each resuspended in 1 ml Hanks Buffer (HBSS) containing 5 μM chloromethyl 2′,7′-dichlorofluorescein diaceate (CM-H2DCFDA; Invitrogen Molecular Probes), a cell-permeable fluorescent compound that is oxidized in the presence of H2O2 and other reactive oxygen species (ROS) and is considered a general indicator of cellular oxidative stress
52
53
. Cell suspensions were incubated at 37°C for 60 min to “load” the cells with CM-H2DCFDA, followed by centrifugation (3 min at 13,000 RPM). Supernatants were discarded, and cell pellets were washed once with HBSS prior to resuspension in 1 ml HBSS or in 1 ml HBSS containing 5 mM H2O2. Each cell suspension was transferred into triplicate wells (200 μl per well) of an optically-clear 96 well plate (Costar 3614), and the plate was transferred to a Biotek Synergy microplate reader. Fluorescence in relative fluorescence units (RFU; using 492-495 nm excitation and 517-527 nm emission) and OD600 readings of each well were recorded after 30 min incubation at 37°C.
Statistical analysis
All statistical analyses, unless otherwise indicated, were performed using Sigmaplot for Windows 11.0 software (Build 11.0.0.75, Systat Software, Inc.).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SJA carried out the RNA microarray experiments and associated data analysis, performed all real-time PCR studies, participated in the conception and design of the study, and helped draft the manuscript. MDQ carried out all of the RNA isolations for comparing the effects of glucose and oxygenation on lrgAB expression. ER optimized and carried out all of the quantitative competence assays. RAB participated in the design and coordination of the study, and helped draft the manuscript. KCR participated in the conception and design of the study, performed the H2O2 assays, intracellular ROS measurements, and drafted the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
This work was supported by a University of Florida HHMI-Science for Life Undergraduate Research Award to M. D. Q., NIH-NIDCR grants R03 DE019179 (KCR) and R01 DE13239 (RAB). We thank Christopher Browngardt for technical assistance in editing microarray data.
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epdcx:valueString Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance
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Abstract
Background
The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans.
Results
Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant.
Conclusions
Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.
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Ahn, Sang-Joon
Qu, Ming-Da
Roberts, Elisha
Burne, Robert A
Rice, Kelly C
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BioMed Central Ltd
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Sang-Joon Ahn et al.; licensee BioMed Central Ltd.
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BMC Microbiology. 2012 Sep 01;12(1):187
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Table S1 Genes differentiall y expressed by loss of LytS at early exponential phase ( P 0.00 5 ) Functional group Gene symbol Description Fold change ( lytS /wild type) Cellular processes Chaperones SMU.956c clpL, clpE putative Clp like ATP dependent protease, ATP binding subunit 1.6060089 SMU.1955c groES co chaperonin GroES 0.7375962 Pathogenesis SMU.1396 gbpC glucan binding protein C, GbpC 0.6540701 Protein and peptide secretion SMU.589 putative DNA binding protein 0.7800888 Toxin production and resistance SMU.1339 bacD putative bacitracin synthetase 0.0302466 SMU.1340 bacA2 putative surfactin synthetase 0.0750438 SMU.1341c putative gramicidin S synthetase 0.0120763 SMU.1342 bacA1 putative bacitracin synthetase 1, BacA 0.021351 DNA metabolism SMU.1967 ssbA single stranded DNA binding protein 1.3516108 Energy metabolism Sugars SMU.1004 gtfB glucosyltransferase I 0.5220086 Fatty acid and phospholipid metabolism SMU.1335c putative enoyl (acyl carrier protein) reductase 0.6312763 SMU.1344c putative malonyl CoA acyl carrier protein transacylase 0.0077153 Hypothetical SMU.55 hypothetical protein 1.2114844 SMU.618 hypothetical protein 0.8050277 SMU.1360c hypothetical protein 0.2861994 Mobil and extrachromosomal element functions SMU.767 putative transposase 0.7201993 SMU.1354c putative transposase 0.3185797 SMU.1363c tpn putative transposase 0.0875149 SMU.1379 tpn putative transposase 0.4902598 Protein fate SMU.539c hopD, c omC signal peptidase type IV 1.8597887 Purines, pyrimidines, nucleosides, and nucleotides SMU.32 purF, purB amidophosphoribosyltransferase 1.3723331 SMU.34 purM phosphoribosylaminoimidazole synthetase 1.3693086 SMU.35 purN phosphoribosylglycinamide formyltransferase 1.4040296 Signal transduction PTS SMU.1957c levG, ptnD putative PTS system, mannose specific IID component 0.7169777 SMU.1958c levF putative PTS system, mannose specific IIC component 0.7095883 SMU.1960c levE putative PTS system, mannose specific IIB component 0.6800983

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Two component systems SMU.577 lytS putative histidine kinase LytS 0.0214838 Transport and binding proteins SMU.1365c ylbB permease 0.0184631 SMU.1366c putative ABC transporter, ATP binding protein 0.025025 SMU.1985 comYB ABC transporter ComYB 2.2386703 SMU.1987 comYA putative ABC transporter, ATP binding protein ComYA, late competence gene 2.1892837 Unassigned SMU.574c lrg lrgB like family protein 0.4989436 SMU.575c lrgA holin like protein LrgA 0.484686 SMU.1345c putative peptide synthetase MycA 0.024107 SMU.1346 bacT putative thioesterase BacT 0.0131318 Unknown SMU.53 Conserved hypothetical protein 1.2504286 SMU.1327c Conserved hypothetical protein, 4Fe 4S binding domain 0.5220086 SMU.1349 Conserved hypothetical protein 2.0185992 SMU.1956c Conserved hypothetical protein 0.7068102 SMU.1982c Conserved hypothetical protein 2.044082