852NOTESTABLE1IsolationofyeastmutantsunabletoutilizearginineasthesolenitrogensourceDeterminationNoColonieson100phloxineBplates27000Smallredcoloniestested743Respiratorydef ID: 830854
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JOURNALOFBACTERIOLOGY,Dec.1976,p.851-852
JOURNALOFBACTERIOLOGY,Dec.1976,p.851-852Copyright©1976AmericanSocietyforMicrobiologyVol.128,No.3PrintedinU.S.A.NOTESDetection,withtheDyePhloxineB,ofYeastMutantsUnabletoUtilizeNitrogenousSubstancesastheSoleNitrogenSourceWOUTERJ.MIDDELHOVEN,*BERTBROEKHUIZEN,ANDJANVANEIJKLaboratoriumvoorMicrobiologie,Landbouwhogeschool,Wageningen,TheNetherlandsReceivedforpublication30July1976Yeastmutantsunabletodegradecertainnitrogencompoundsproducecharac-teristicsmallredcoloniesonanagarmediumcontainingthereddyephloxineB,galactose,thetestnitrogencompound,andasmallamountofammoniumchloride.ThedyephloxineB(tetrabromotetrachloro-fluorescein,alsonamedacidred92ormagdalared)hasbeenincorporatedbysomeauthors(2,6)intoagarmediatofacilitatedetectionofyeastmutants.Nagai(6)usedthedyeforthedetectionofrespiratory-deficientmutants.HornandWilkie(2)foundthatNagai'sphlox-ineBindicatormediumissuitableforthede-tectionofyeastmutantsunabletosynthesizepurineorpyrimidinebasesoraminoacids.Onindicatormediumrespiratory-deficientandauxotrophicyeastmutantsproducesmallcolo-niesthataccumulatethereddye,whereaswild-typecoloniesarebiggerandpinkorpale.Preliminaryexperimentsrevealedthatnitro-gen-limitedgrowthonglucoseammoniumagaralsocausesphloxineBaccumulation,optimalvisualcontrastbetweenredcoloniesandpinkmediumbeingobservedwith2.5mMammo-niumchloride.ThisobservationpromptedustodevelopaphloxineBmediumsuitableforthedetectionofyeastmutantsunabletogrowoncertainnitrogenoussubstancesasthesoleni-trogensource.Thewild-typestrainofSaccharomycescere-visiae,a-11278b,wasused,aswellastwomu-tantstrainsderivedfromit:a-HP100(a-his,ura),anauxotrophicmutantrequiringhisti-dineanduracil;anda-R79,omithinetransami-naseless,notutilizingarginineandornithineasthesolenitrogensource.Complexmedia(YEPGandYEPE)contained,perliteroftapwater:20gofpeptone,10gofOxoidyeastextract,and20gofglucose(YEPG)or20mlof96%ethanol(YEPE).Minimalmediacon-tained,perliterofdemineralizedwater:40gofglucoseor20gofgalactose,nitrogensource(40mMassimilableN),200mgofhistidine,and200mgofuracilifrequired,1gofmagnesiumsulfate,traceelersentsandvitaminsasde-scribedpreviously(4),and0.1Mpotassiumphosphate,pH5.5.Ifammoniumchloridewasthenitrogensource,40mMpotassiumsodiumtartratewasaddedasabuffersubstance.Cul-turemediaweresolidifiedbytheadditionof20gofOxoidagarperliter.Themediaweresteri-lizedat1200Cfor15min.Sugarsweresterilizedseparately.LabilenitrogenoussubstancesandphloxineB(colorindexno.45410,Koch-Light,Colnbrook,England)weresterilizedbySeitzfiltration.Ethanolwasaddedaseptically.ThephloxineBmediumwasidenticaltominimalmediumexceptitcontained,perliter:10mgofphloxineB,20gofgalactose,2.5mMammo-niumchloride,and40mMtestnitrogencom-pound.Yeastmutantswereobtainedbytreat-mentwithethylmethanesulfonate(1).Mu-tantsunabletoassimilateanitrogenoussub-stanceasthesolenitrogensourcewereen-richedbynystatintreatment(7,8).AseriesofphloxineBmediumplatesof11cmindiameterwasinoculatedwith100to300colony-producingunits/plate.After5to10daysat300CsmallredcoloniesweretransferredtoYEPGplatesand,after2daysat30°C,repli-catedonglucoseminimalmediumplateswithdifferentnitrogensourcestoscreenthepre-sumedmutantsforthepropertiesdesired.Mu-tantstrainswerepurifiedbystreakingonYEPG.Respiratory-deficientmutantswererec-ognizedbytheirinabilitytogrowonYEPEplatesandwerediscarded.Attemptstodetectmutantsunabletoutilizearginineasthe
solenitrogensourceweresuc-cessfulifamuta
solenitrogensourceweresuc-cessfulifamutagen-treatedyeastsuspensionwasplatedonphloxineBagarcontaining10mM-arginineinadditionto2.5mMammo-niumchlorideasthenitrogensourceandgalac-851852NOTESTABLE1.IsolationofyeastmutantsunabletoutilizearginineasthesolenitrogensourceDeterminationNo.Colonieson100phloxineBplates'........27,000Smallredcoloniestested.................743Respiratory-deficientsmallredcolonies...19Strainsnotgrowingonarginineorornithine149Arginaseless..........................10Ornithinetransaminaseless............98argR(1class).........................39Unidentified...........................2aStraina-Y1278bwastreatedwithethylmeth-anesulfonate.ThemutantsdesiredwereenrichedbynystatintreatmentanddetectedonphloxineB,galactose,10mMarginine,2.5mMammoniumchloridemedium.toseasthecarbonsource(Table1).Withglu-coseasthecarbonsource,however,mostofthesmallredcolonieswererespiratorydeficient.Galactoseisknowntobenot,orveryslowly,assimilatedbyrespiratory-deficientyeastmu-tants(5).FromTable1itcanbeseenthatabout20%ofthesmallredcoloniestestedwereunabletodegradearginine.Paleorwhitecolonies,irre-spectiveofthecolonysize,yieldedarginine-degradingstrains.Thenumberofrespiratory-deficientcolonieswasverysmall.Thearginine-negativemutantsbelongedtofivedifferentge-neticclasses:arginaseless,ornithinetransami-naseless,regulatorymutantargR(3)uninduci-bleforbothoftheseenzy-mes(onlyoneofthreeknownargRclasseswasisolatedinthisexperi-ment),andtwodifferent,asyetunidentifiedmutantsunabletogrowwithargininebutgrowingnormallyonornithineminimalme-dium.Thevarietyofthemutantsobtainedissurprisinglyhigh,especiallyinviewofthefactthatallmutantshadarisenfromonemuta-genictreatment.Ourmethodhasbeenappliedsuccessfullyfortheisolationofmutantsunabletodegradeal-lantoin.Moreover,itgavegoodresultsinob-tainingmutantsofa-HP100showingimpairedgrowthonammonium,glutamate,orureaasthesolenitrogensource.Establishedenzymedefectsincludeanabolicandkatabolicglutamicdehydrogenaseandallantoinase.PhloxineBisnotgrowthinhibitingeitherinbatchorcontinuousculturebutpromotesdeathunderconditionsofnitrogenlimitation(unpub-lisheddata).Itsaccumulationbythedeadcellsconstitutesmostofthestainingofnitrogen-limitedyeastcoloniesgrowingonphloxineBindicatormedium.LITERATURECITED1.Fink,G.R.1970.Thebiochemicalgeneticsofyeast,p.59-78.InS.P.ColowickandN.0.Kaplan(ed.),Methodsinenzymology,vol.17A.AcademicPressInc.,NewYork.2.Horn,P.,andD.Wilkie.1966.UseofmagdalaredforthedetectionofauxotrophicmutantsofSaccharomy-cescerevisiae.J.Bacteriol.91:1388.3.Messenguy,F.,andJ.M.Wiame.1969.ThecontrolofornithinetranscarbamylaseactivitybyarginaseinSaccharomycescerevisiae.FEBSLett.3:47-49.4.Middelhoven,W.J.1970.Inductionandrepressionofarginaseandornithinetransaminaseinbaker'syeast.AntonievanLeeuwenhoekJ.Microbiol.Serol.36:1-19.5.Moat,A.G.,J.J.Barnes,andE.McCurley.1966.Factorsaffectingthesurvivalofauxotrophsandpro-totrophsofSaccharomycescerevisiaeinmixedpopula-tions.J.Bacteriol.92:297-301.6.Nagai,S.1963.Diagnosticcolordifferentiationplatesforhereditaryrespirationdeficiencyinyeast.J.Bac-teriol.86:299-302.7.Snow,R.1966.Anenrichmentmethodforauxotrophicyeastmutantsusingtheantibioticnystatin.Nature(London)211:206-207.8.Thouvenot,D.R.,andC.M.Bourgeois.1971.Optimi-sationdelasalectiondemutantsdeSaccharomycescerevisiaeparlanystatine.Ann.Inst.PasteurParis120:617-625.J.BACTERIOL.