TurkJBiol 26(2002)13-24 ©TÜB‹TAK SelectiveIsolationandNumericalClassificationofNovel ThermophilicStreptomycetes NevzatfiAH‹N,ErmanÖZTÜRK,KamilIfiIK,ErginKAR‹PTAfi,ReflitÖZKANCA OndokuzMay›sUniversity,FacultyofScienceandArts,DepartmentofBiology,55139Kurupelit,Samsun-TURKEY Received:09.05.2001 Abstract: Forty-fivethermophilicstreptomyceteswereisolatedfromdifferenthabitatsandexaminedfor163unitcharacterswith 8representativethermophilictypestrains,29referencestrainsand16randomlyselectedduplicatedorganismsformanyphenotypic properties.Thedatawereevaluatedusingthesimplematchingcoefficients(S SM),andclusteringachievedusingthecomplete algorithm.Itwasclearlyseenthatthehighcopheneticcorrelation,lowtesterrorvaluesandhighsimilaritylevelagreedwit hthe definednumericaltaxonomiccriteria.Theteststrainswereassignedtofouraggregategroupsconsistingsevenmajor(5-8strai ns), nineminor(2-4strains)andtensinglememberedclusters.Itcanbeconcludedthatthenumericaltaxonomicstudiesyieldedvaluable informationonthebiodiversityofbiotechnologicallysignificantthermophilicstreptomycetesfromnaturalhabitats. KeyWords:Streptomyces,NumericalTaxonomy YeniTermofilikStreptomycetes’lerin‹zolasyonuveNümerikS›n›fland›r›lmas› Özet: Farkl›habitatlardanizoleedilen45termofilikstreptomycetes,8termofiliktipörne¤i,29referansvebunlardanrastgele seçilmifl16duplikesufl,163birimfenotipikkarakteryönündentestedildi.Verilerinanalizisimplematchingcoefficients(SSM ), kümelendirmeifllemidecompletealgorithmkullan›larakgerçeklefltirildi.Yüksekcophenetickorelasyonvedüflüktesthatas›de¤erleri ileyüksekbenzerlikseviyesikabüledilennümeriktaxonomikkriterlerileuyumiçerisindeoldu¤uaç›kçagörüldü.Testsufllar› yedi büyük(5-8sufl),dokuzküçük(2-4sufl)veonteküyelikümeleriiçerendörtagregatgrupolarakbelirlendi.Buradan,nümerik taksonomikçal›flmalar›n,biyoteknolojikönemiolantermofilikstreptomycetes’lerindo¤alhabitatlar›ndakiçeflitlili¤ihakk›nda önemli bilgilerverdi¤isonucunavar›lmaktad›r. AnahtarSözcükler:Streptomyces,Nümeriktaksonomi Introduction Bacteriology (5)isbasednotonacombinationof Thegenus Streptomyces isoneofthemost genotypicandphenotypicproperties,butontheextensive industriallyimportantandintensivelystudiedbacterial numericaltaxonomicsurveyofWilliams etal. (9).Inthis taxa.Thecapacityofstreptomycetestoproduce study,475cultures,including394 Streptomyces type antibioticsremainsunsurpassed,despitemanyeffortsto strainsfromtheInternational Streptomyces Project, obtainsecondarymetabolitesfrommembersofother wereexaminedfor139unitcharacters.Theresultant actinomycetegenera(1-4).Anotheruniquefeatureofthe datawereexaminedusingtheJaccardandsimple genus Streptomyces isthenumberofspecieswhichit matchingcoefficientsandtheunweightedpairgroup contains.Thenumberofvalidlydescribed Streptomyces methodwitharithmeticaveragesalgorithm.Thetype speciescurrentlystandsatnearly500(5,6)butwould strainswereassignedto20majorclusters(6to71 reachseveralthousandifinvalidlypublishedpatent strains)and22single–memberedclustersthatwere descriptionswereincluded(7,8). equatedwithspecies. Streptomycetessystematics,notablythedelineation Itissurprisingthatthermophilicstreptomyceteshave ofspecies,isbecomingincreasinglyobjectiveduetothe receivedsolittleattentiongiventheirpossibleimportance applicationofthepolyphasictaxonomicapproach. inmicrobialtechnology(10-12).Relativelylittleisknown However,theclassificationofthegenus Streptomyces in aboutthetaxonomyoftheseorganismsalthough thecurrenteditionof Bergey’sManualofSystematic Streptomycesthermodiastaticus Waksman(13), 13 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes Streptomycesthermonitrificans DesaiandDhala(14), dispersethebacteria.Tenfolddilutionofthesuspensions Streptomycesthermoviolaceus Henssen(15)and wereheatedinapre-warmedwaterbathat55ºCfor6 Streptomycesthermovulgaris Henssen(15)arecitedin minutes.Aliquots(0.2ml)of10 -2 to10 -5 tenfoldserial the ApprovedListsofBacterialNames (16). dilutionswerespreadoverthesurfaceofdriedstarch- Thefirstcomprehensivestudyofthermophilic caseinagarplates(pH7.2;20)supplementedwith cyclohexemide(50mgml-1)andfiltersterilisedrifampicin streptomycetestodatewascarriedoutbyGoodfellow et -1 al. (11).Theycompared50thermophilicneutrophilic (0.5mgml ).Theinoculatedplates,fourperdilution, streptomycetesfromdiversehabitatswithrepresentative wereincubatedat55ºCfor7days.Countsofthe mesophilic,neutrophilicmarkerstrainsthathadbeen streptomyceteswereexpressedasthenumberofcolony includedintheextensivenumericaltaxonomicsurveyof formingunits(c.f.u.)pergramdryweightofsample. Williams etal. (6).Theteststrainswereexaminedfor Seventy-sevenrandomlychosenstreptomycetesisolated 135unitcharactersandtheresultantdataanalysedusing from4ofthe11soilsamplesweresubculteredonto appropriateresemblancecoefficientsandclustering starch-caseinagarplates,incubatedat45ºCfor5days algorithms.Twoaggregateclustersweredetected,one andcheckedbothforpurityandforthepresenceofspore containedmesophilicstreptomycetesandtheotherthe vesiclesusingaNikonOptiphotbinocularlight thermophilicstrains.Thelatterwererecoveredintwo microscope. major(7to19strains),fourminor(2to3strains)and 2.2.Colourgrouping: The77thermophilic twosingle-memberedclusters.Threeofthesetaxawere streptomycetesisolateswerestreakedontoplatesof equatedwith Streptomycesmegasporus (17)Agre(18), oatmeal(Difco,ISP3;21)andpeptone-ironagars(22), Streptomycesthermoviolaceus Henssen(15)and andincubatedat45ºCfor7days.Coloniesgrowingon Streptomycesthermovulgaris Henssen(15).The theoatmealagarplateswereexaminedbyeyeto remainingclusterwasraisedtospeciesstatusas determinetheaerialsporemasscolour,thepigmentation Streptomycesthermolineatus Goodfellow,Laceyand ofsubstratemyceliumandtheproductionandcolourof Todd(11). diffusiblepigments.Thepeptone-yeastextractironagar fiahin(19)isolatedlargenumbersofthermophilic plateswereexaminedtodeterminewhethertheisolates streptomycetesfromsoiladjustedtopH7.2and10.5,at producedcharacteristicdarkcolouredmelaninpigments. 55ºCfor5days.Onehundredandtwenty-nine Thethermophilicstreptomyceteswereassignedto11 representativeisolates,32thermophilictypeand colourgroups. referencesstrainswereexaminedfor339unit 2.3.Numericaltaxonomy: Thesourcesand characters.ThedatawereanalysedusingtheJaccard, taxonomichistoriesofthe45 Streptomyces isolates,37 patternandsimplematchingcoefficientsandclustering typeandreferenceculturesand16duplicatedcultures achievedusingtheunweightedpairgroupmethodwith aregiveninTable1.Theteststrainsweremaintainedas arithmeticaveragesalgorithm.Theteststrainswere suspensionsofsporesandmycelialfragmentsinglycerol assignedtosixdistinctaggregategroupsthat at–20ºC(23). encompassed12major(5-15strains),14minor(2-4 2.4.Collectionofdata: Eachstrainwasexaminedfor strains)and13singlememberedclusters. 163unitcharacters.Inoculawerepreparedfromstrains Inthepresentstudy,markerandfreshthermophilic grownoninorganicsalts-starchagar(ISP4;22)for7 isolatesofstreptomyceteswerethesubjectofextensive daysat45ºC.Growthfromheavilyinoculatedplateswas numericalpheneticstudiesdesignedtodeterminetheir scrapedoffandsuspendedinbijouxbottlescontaining broadtaxonomicaffinitieswithoneanotherandwith approximately3mlofsterile1/4strengthRinger’s representativestreptomycetes. solution.Theinoculawerethentransferredtotestmedia usingamultipointinoculator.Thisapparatusallowsthe standardised,multiplesurfaceinoculationof90mm 2.Materialsandmethods diameterPetridisheswith12differentorganisms.Allof 2.1.Selectiveisolation: Soilsamples(1g)were thetestswerecarriedoutinPetridishes,exceptaesculin, 1 whichwasperformedintesttubes.Controlplateswere asepticallyaddedto9mlsterile /4 strengthRinger’s solution(Oxoid)andshakenfor30minutesbyhandto inoculatedatthebeginningandendoftheinoculation 14 N.fiAH‹N,E.ÖZTÜRK,K.IfiIK,E.KAR‹PTAfi,R.ÖZKANCA Table1. Designation,identityandsourceofstrainsassignedtoclustersdefinedofthe82.2%similaritylevelsintheSSM,CO MPLETEanalysis. Strain Identity Source Cluster1:Streptomyces sp. E1139 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1401 Streptomyces sp. E.Öztürk,sheepmanuresoil,Samsun,Turkey E1438 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey E1351 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1354 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey Cluster2:S.thermovulgaris,S.Albus E1075 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey 40444T S.thermovulgaris DSM40444,ISP5444,A.Henssen,MBR10;freshcowmanureHenssen1957AL 40787 S.thermovulgaris DSM40787,L.Ettlinger,LBGA3072,R.Hütter,ETH24180,A.Hessen, R;freshhorseandswinemanure K17 S.Albus C.Todd;gardensoilC1,NewcastleuponTyne,England,UK K15 S.Albus C.Todd;gardensoilC1,NewcastleuponTyne,England,UK NT307 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela NT550 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela Cluster3:Streptomyces sp. NAR64 Streptomyces sp. A.T.Bull;gardensoil,Canterbury,England,UK NT471 Streptomyces sp. N.fiahin;gardensoil,Yogyakarta,Indonesia NT297 Streptomyces sp. N.fiahin;scrublandsoil,Merida,Venezuela Cluster4:Streptomyces sp. NT358 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela NT011 Streptomyces sp. N.fiahin;gardensoil,Bolu,Turkey NT508 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela Cluster5:S.thermolineatus TA212 Streptomyces sp. N.fiahin;gardensoil,Yogyakarta,Indonesia NT313 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela K47T S.thermolineatus DSM41451,C.ToddK47,J.Lacey,A1484;sewagecompost,USA Cluster6:Streptomyces sp. NT126 Streptomyces sp. N.fiahin;aridsoil,Van,Turkey NT371 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela Cluster7:Streptomyces sp. E1145 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey