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Proteomic analysis of the gene DR1709 mutant in Deinococcus radiodurans

By Debbie Freeman,2014-02-18 12:51
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Proteomic analysis of the gene DR1709 mutant in Deinococcus radioduransof,in,the,gene

    Proteomic analysis of the gene DR1709

    mutant in Deinococcus radiodurans NuclearScienceandTechniques20(2OO9)331334

    ProteomicanalysisofthegeneDR1709mutantin

    Deinococcusradiodurans

    TIANShuangqiQINGuangyongLIYongliangCHANGShenghe

    /onBeamBio

    engineeringLab,SchoolofPhysics,~engzhouUniversity,Zhengzhou450052,China AbstractDR1709isapredictedMntransporterinDeinococcusradiodurans(D.radioduFans).Themensuration

    methodtoevaluateproteinviabilitywitlltwo

    dimensionalelectrophoresisinD.radioduransandthemutantswas

    establishedinthisstudy.TheresultsshowedthatafterDR1709wasdisrupted.

    eexpressionsofDR1120

    (acetokinase),DR1691(heatshockprotein),DR1485(putativelipase),DR2095(putativec-typecytochrome)and

    otherthreehypotheticalproteins(DR0124,DR0047andDR2474)wererepressed.Howevertheexpressionof

    DR1794(putativenosX)wasinduced.Phenomenaabovesuggestedthattheincreasedradiationsensitivityofthe

    mutantcellsmaybeattributedtonotonlytheprotectionofgeneDR1709,butalsotheproteins'differentexpressions

    betweenthewildtypeandthemutantmightalsoplayimportantrolesinprotectingD.radioduransfromirradiation.

    AlthoughDR2095wasahomologueofc-typecytochrome,ithasnorealiticfunctions. KeywordsDeOCOCCUSradiodurans.DR1709.Proteomics

    1Introduction

    De'inococcusradioduFansrD.radiodurans)shows extremeresistancetothelethalandmutageniceffects ofionizingradiations.andUVorother

    electromagneticwavesaswell,whichcausephysical andchemicaldamagetoDNA【卜引.Different

    mechanismsoftheresistancesofthemicroorganism againstthedeleteriouseffectshavebeensuggested[1.

    Recently,itwasfoundthattheradiationresistancewas determinedbythelevelofoxidativeproteindamage inducedduringtheirradiation,andMnionscould protectproteinagainstoxidativestressinD. radtodurans[91.Butthemechanismisvettobe c1fied.

    DR1709wasapredictedMn2+transportergenein D.radiodurans.WiththedisruptedDR1709gene.the mutantcellshadamuchlesssurvivalratethanwild type,whentreatedwithradiation[1o.However.itwas

    notsurewhethertheothergenesassignedtheirrolesto thislesssurvivalrate.Inthispaper,wereportthe proteomicanalysisinanattempttosolvethese SupportedbyNationalProject973(No.2004CB719604) Correspondingauthor.E-mailaddress:shchang@zzu.edu.CT1 Receiveddate:2o08.1214

    problems.Theresultsshowedthatninegeneswere closelyrelatedwithDR1709atleast.Theirpossible rolescontributingtotheirradiationinD.radiodurans werealsodiscussed.

    2Materialsandmethods

    2.1Bacterialstrainandgrowthconditions D.radioduransstrainR1wasagenerousgiftfrom

    InstituteofNuclear-AgriculturalSciences,Zhejiang University.TheDR1709mutantstrainwas constructedatourlaboratory.TheD.radioduransRl andmemutantcellsweregrownat31cICinTGYbroth (0.5%bactotryptone,0.1%glucose.and0.3%bacto yeastextract)I[11,12].

    2.2Reagentsandapparatus

    ProteanIEFdeviceandtheseconddimensional electrophoresisapparatus,ReadyStripIPGStrip, BioLyteAmpholyte,tributylphosphine,ureaand CHAPSwerefromBioRad(Hercules,CA,USA).

    Theotherchemicalsofmolecularbiologygradewere 332TIANShuangqieta1./NuclearScienceandTechniques20(2009)331334

    obtainedfromSigmaChemica1.LvsisbufferAf9 mol/Lurea,4%massconcentrationCHAPS,1%mass concentrationDTT.0.5%CAandacocktailof proteaseinhibitormixture),LysisbufferB(40mmol/L TrisHCl.7mo1/Lurea,2mol/Lthiourea,4%CHAPS, 40in/iloD1Tr)andrehydrationbufferf8mo1/Lurea, 2%CHAPS.0.5%Gbuffer,0.002%bromphenol blue)werepreparedaccordingtoReadyStripGStrip InstrucfionManual(Catalog#1632099.BioRad).

    Theproteininhibitorsmixture(35~g/mLPMSF,0.3 mg/mLED11A,0.7rtg/rnLpepstatin,0.5pg/mL leupepfin)wasmadetopreventproteinsfrombeing degraded.

    2.3Extractionofsolubleproteins

    Cellswerecollectedbycentrifugationafterculturing for48h.Thepelletsweresuspendedinphosphate buffer(1mol/L,pH=7.0),andthesuspensionwas

    mixedwithfourtimesthevolumeofLysisbufferAin a50mLtubewithacocktai1ofproteaseinhibitor mixture.ThecellsinthemixtureweresonicatedatOC byhalf-timeintermittentsonication(Branson,Sonifier 450)for1h.Thehomogenateswerecentrifugedat 12000xgfor20minat4.C.Thesupematantswere collectedintoa1.5mLmicrotubeasasolubleprotein fraction.Acetoneoffourvolumeswasmixedwiththe supernatants,soastoavoidcarotenoids.Themixture wascentrifugedat32000xgfor20minat4.C. Sedimentatedproteinwassuspendedwithfour volumesofLysisbufferA.rI10talproteinconcentration wasmeasuredaccordingtomemodifiedBradford"J.

    Thesolubleproteinswerestoredat-70~Cfor two.dimensionalgelelectrophoresis.

    2.4Two.dimensionalgelselectrophoresis Theprocedureofthe2Dgelelectrophoresiswas

    carriedoutaccordingtothemodifiedstandard method.Foreachchanne1.350ggofpurified samplewasdilutedwithrehydrationbufferf8mol/L urea,2%CHAPS,0.5%Gbuffer,0.002%

    bromphenolblue)andloadedontotheIPGstrip(24 cm.pH4._7:AmershamBiosciences,USA)andthe isoelectricfocusingwasperformed(50V12h:200V 2h;500lh;150030min;8000V,1h;10000

    6h.).Thestripsweretreatedwithequilibrationbuffer I(6mol/Lurea,30%glycerol,2%SDS,50mmol/L Tris-HC1.1%DT0.002%bromphenolblue)and equilibrationbufferII(6mol/Lurea,30%glycerol,

2%SDS,50mmol/LTrisHCl,4%iodoacetamide,

    0.002%bromphenolblue).respectively.Thesecond dimensionwasperformedin12.5%SDSPAGEgels

    (5W/gel,30rain;60W/gel,10h).Thegelswere stainedwithamodifiedNeuhoffscolloidalCoomassie BlueG250stain[15,16.

    2.5Proteinspotsidentification

    Theproteinsampleswereingeldigestedand

    idenrifledaccordingtothestandardmethod[.The workofMALDI-TOF-TOFMSandpeptidemass

    fingerprintingwasmainlydonebyTianjinBiochip Corp.

    3Resultsanddiscussion

    3.1Proteinsexpresseddifferentlybetweenthe mutantandthewildtype

    D.radioduransisthemostradiation-resistantorganism described[18].TherecentresearchshowedthatMn ionscouldprotectproteinagainstoxidativestressinD. radiodurans[19.DR1709isapredictedMn

    transporterinD.radiodurans.AfterDRI709was disrupted,themutanthadmuchlesssurvivalratethan thewildtypewhen~eatedwithUVirradiationand H2O210.Usingtwodimensionalelectrophoresis,the differencesbetweenthewildtypeandthemutantat theproteinlevelwereanalyzed.Theresultsshowed thattheexpressionsofoneacetokinasegene(DRI120), oneheatshockprotein(DR1691),oneputativelipase gene(DR1485),oneputativec-typecytochrome (DR2095)andotherthreehypotheticalproteins (DR0124,DR0047andDR2474)inmutantcellswere

    lowerthanthoseinthewildtype.Buttheexpression ofoneputativenosXprotein(DR1794)wasareverse (Fig.1andTable1).

    3.2DR2095mightnotserveasc-typecytochromes C-typecytochromesareproteinsthatareessentialfor thelifeofvirtuallyallorganisms.Superoxideradicals arisefromtheautoxidationofrespiratory

    dehydrogenases,whereadventitioustransferof electronsfromreducedflavins(FADH2)associated withc-typecytochromes.Itwasthoughtthatthetotal TIANShuangqieta1.INuclearScienceandTechniques20(2009)331-334

    intracellulartiterofcytochromeswasamarkerforthe proclivityofcellstogenerateoxidativestress201.After

    DR1709wasdisrupted,theexpressionoftheputative ctypecytochromewasrepressed(Fig.1),whichmight 333

    causelessoxidativestressandhighersurvivalrate. However,thesurvivalrateofthemutantwasmuch lessthanthatofthewildtype.Therefore,DR2095 mightnotserveasc-typecytochromes.

    Fig.12-DEcomassstmnedproteinpatternsofD.radioduransR1andthemutantADR1709

    Table1ProteinsidentifiedbyMALDI?-TOF--TOFMSanalysisandpeptidemassfingerprin

    ting

    Proteinsplayingimportantrolesinprotecting D.radioduransfromirradiation

    Acetokinasecatalyzesthevirtuallyirreversible synthesisofadenosinetriphosphatefromacetyl phosphateandadenosinediphosphate.Whenthe bacteriumrecoveredfromirradiatiOn.energywas necessary.Inthemutantcells,theexpressionof

    acetokinase(DRll20)reduced(Fig.1),whereless adenosinetriphosphatewasproducedandmorecells died.Theseindicatedthatafterdisruptionofthe DR1709gene,theincreasedradiationsensitivityof

    themutantcellsmaynotbeattributedtoDR1709only. Besidesthegenesdescribedabove,oneputativelipase gene,onenosXproteinandotherthreehypothetical proteinsmayalsobeinvolvedintheprocessof protectingproteinsfrombeingoxidized(Fig.1).The detajls.however,shallbefoundinfurtherstudies.The proteinsexpresseddifferentlybetweenthewildtype andthemutantmightalsoplayimportantrolesin protectingD.radioduransfromirradiation. References

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