Selective Isolation and Numerical Classification of Novel Thermophilic

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 E1176 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1208 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1356 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1161 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey Cluster8:Streptomyces sp. A1960 Streptomyces sp. J.Lacey;Nigeria TA68 Streptomyces sp. N.fiahin;gardensoil,Yogyakarta,Indonesia Cluster9:Streptomyces sp. E1349 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1160 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey Cluster10:Streptomyces sp. E1185 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1183 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1141 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1205 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1440 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey E1446 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey

15 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes

Table1. continued.

Strain Identity Source E1430 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey E1019 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey Cluster11:Streptomyces sp. E1202 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1109 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1138 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1001 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey Cluster12:Streptomyces sp. E1008 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1122 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1016 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1364 Streptomyces sp. E.Öztürk;sheepmanuresoil,Samsun,Turkey Cluster13:Streptomyces sp. E1166 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1348 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey Cluster14:Streptomyces sp. E1124 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1173 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey E1394 Streptomyces sp. E.Öztürk;sheepmanuresoil,Samsun,Turkey E1387 Streptomyces sp. E.Öztürk,sheepmanuresoil,Samsun,Turkey E1125 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey E1415 Streptomyces sp. E.Öztürk;sheepmanuresoil,Samsun,Turkey E1391 Streptomyces sp. E.Öztürk;sheepmanuresoil,Samsun,Turkey Clustur15:S.cavourensis subsp.cavourensis,S.thermodiastaticus,S.thermoviolaceus subsp.thermoviolaceus 40300T S.cavourensis subsp. DSM40300,ISP5300,D.Giolitti829,soilItaly cavourensis NT312 Streptomyces sp. N.fiahin;aridsoil,Merida,Venezuela 40573T S.thermodiastaticus DSM40573,ISP5573,T.Cross;CUB687,J.R.Denison A1847T S.thermodiastaticus J.Lacey;RothamstedExperimentalStation,Harpenden,England,UK 40443T S.thermoviolaceus DSM40443,ISP5443,A.Henssen,R-77,mixedfreshhorseandswine subsp.thermoviolaceus manure A74 Sthermoviolaceus J.Lacey;A74;hay,Rothamsted,UK subsp.thermoviolaceus 41391 S.thermoviolaceus DSM41391,G.Vobis;MB-C18 subsp.thermoviolaceus E1119 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana E1412 Streptomyces sp. E.Öztürk;sheepmanuresoil,Samsun,Turkey TA208 Streptomyces sp. N.fiahin;gardensoil,Yogyakarta,Indonesia SingleMemberedClusters E1448 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey K45T S.megasporus C.Todd;K45,J.Lacey,A1202,Agre1869 E1100 Streptomyces sp. E.Öztürk;cottonseedcapsulecompostsoil,Adana,Turkey A1853 Streptomyces sp. J.Lacey;A600,barleygrain,Cambridge,England, K44T S.macrosporus C.Todd;K44,J.Lacey;A1201;sewagecompost,USA E1172 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey TA53 Streptomyces sp. N.fiahin;gardensoil,Yogyakarta,Indonesia E1427 Streptomyces sp. E.Öztürk;horsemanuresoil,Adana,Turkey E1201 Streptomyces sp. E.Öztürk;gardensoil,Samsun,Turkey 40574 S.thermoflavus DSM40574,ISP5574,T.Cross,CUB75,NCIB9670,N.Okafor;rotting maize,Ado-Ekiti,Nigeria

16 N.fiAH‹N,E.ÖZTÜRK,K.IfiIK,E.KAR‹PTAfi,R.ÖZKANCA

procedureforeachgroupof12teststrainsinorderto solutionsusing0.22mmMilliporefilters.Theexception, eliminatefalsenegativeresultsduetolossofinoculum. rifampicin,wasdissolvedindimethylformamide(0.2ml; 2.5.Degradationtests: Esculin(1.0%,w/v) BDH)andthenaddedtotheappropriateamountofsterile degradationwasdeterminedbythemethodsofWilliams distilledwater.Theantibiotictestswerereadafter3and etal. (9)andexaminedafter3days.Thedegradationof 7daysat45ºCandchemicalinhibitiontestsafter7and casein(1.0%,w/v),chitin(0.4%,w/v),pectin(0.4%, 14daysat45ºC.Organismswerescoredasresistant(+) w/v),andxylan(0.4%,w/v)wasdetectedinmodified whengrowthonthetestplateswasgreaterorequalto Bennett’sagar(24)aftereither3,7and14days; thatonpositivecontrolplateslackinginhibitors. clearingoftheareasunderandaroundthegrowthwas InoculatedBennett’sagarplateswereexaminedfor scoredaspositive.Gelatin(0.4%,w/v)andstarch(1.0%, growthafter7daysat25ºC,30ºC,37ºC,55ºCand60ºC; w/v)degradationwasreadafter3daysinmodified andatpH4,5.5,9.5,10.5and11;visiblegrowthwas Bennett’sagar(24)byfloodingplateswithtrichloroacetic scoredasapositiveresult. acid(3.0%,v/v)andiodinesolutions(25)respectively, 2.9.Codingofdata: Nearlyallofthecharacters andscoringzonesofclearingaspositive. existedinoneoftwomutuallyexclusivestatesandwere 2.6.Morphologyandpigmentation: Sporechain scoredpositive(+)ornegative(-).Qualitativemultistate morphologywasdeterminedbylightmicroscopy characters,suchassomeofthepigmentationand examinationsof14-day-oldculturesgrownoninorganic morphologicaltests,werecodedasseveralindependent salt-starchagar(ISPmedium4;Difco)at45ºC.Spore charactersandwerescoredplus(1)forthecharacter chainmorphologywasobservedusingaOptiphot stateshownandminus(0)forallalternatives.Someof binocularlightmicroscopefittedwithlongworking thetests,notablytolerancetoantibioticsandchemical distanceobjectives;sporechainswereassignedtothe inhibitors,werecodedusingtheadditivemethodof morphologicalcategoriesproposedbyPridham etal. SneathandSokal(27). (26).Severalcategoriesofaerialsporemasscolour, 2.10.Computation: Thebinarytestdataweretyped substratemyceliumcolourandtheproductionofsoluble ina+/-formatasinputtotheXprogram(28)andrun pigmentswererecognised(Table2). onanIBM-PCcomputerusingthesimplematching

2.7.Nutritionaltests: Theabilityoftheteststrains coefficient(S SM;29)whichincludesbothpositiveand touse30compoundsassolecarbonsourcesforenergy negativematches.Clusteringwasachivedusingthe andgrowthwasexaminedoncarbonutilisationagar(ISP unweightedpairgroupmethodwitharithmeticaverages medium9;Difco)for7daysat45ºC.Theteststrains algorithm(UPGMA;27).Thentheresultswereconverted werealsoinoculatedontothebasalmediumaloneasa intoadendogramusingtheNTSYS-pcstatistical negativecontrolandontothismediumsupplemented program. withglucose(1.0%,w/v)asapositivecontrol.Apositive 2.11.Testreproducibility:Sixteenrandomlyselected resultwasrecordedwhengrowthwasgreaterthanthat teststrainswereexaminedinduplicateandanestimateof inthenegativecontrol.Similarly,thecapacityofthe testvariancecalculatedandusedtoestimatetheaverage organismstouse13compoundsassolenitrogensources probability(p)ofanerroneoustestresult(30). wasdeterminedbythemethodsofWilliams etal. (9). Growthwasscoredafter7daysbycomparisonwithboth positive(thebasalmediumsupplementedwithL- 3.ResultandDiscussion Asparagine;1.0%,w/v)andnegative(basalmedium 3.1.Selectiveisolationandcolourgrouping: alone)controls;growthgreaterthanthatonthenegative Thermophilicstreptomyceteswereisolatedfromgarden controlwasscoredaspositiveandthatequaltoorless andsheepmanuresoilscollectedfromSamsunandhorse thanthatonthenegativecontrolasnegative. manureandcottenseedcapsulecompostsoilscollected 2.8.Tolerancetests: Theteststrainswereexamined fromAdana.Countsrangedfrom2.0x10 2 (sheep fortheirabilitytogrowonBennett’sagar(24) manure)to2.6x103 (horsemanure)colonyformingunits supplementedwitharangeofantibioticsandchemical pergramdryweightsample.Forty-fivestreptomycetes, inhibitorsatvariousconcentrations(Table2).Allbutone randomlychosenfrom11colourgroups,wereincluded oftheantibioticsweresterilisedbyfiltrationofaqueous inthenumericaltaxonomicstudy.

17 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes

Table2. DistributionofpositivecharacterstomajorandminorclustersdefinedintheS SM,Completeanalysis.

Cluster 1 2 5 7 10 14 15 3 4 6 8 9 11 12 13 16 Numberofstrains 5 7 8587 733 2 2 2 4 4 23 Character (w/v)

A.BIOCHEMICALTEST: Esculin 1.0 0 0 0 0 0 29 29 0 0 0 0 50 25 0 0 0 B.DEGRADATIONTESTS: Casein 1.0 0 86 50 0 75 71 43 100 33 0 0 100 75 50 0 0 Gelatin 0.3 100 43 100 100 100 71 100 100 0 0 100 100 100 100 100 100 Pectin 0.4 0 14 0 0 25 29 86 33 33 100 0 0 25 0 0 67 Starch 1.0 100 57 88 100 100 100 100 67 0 50 100 100 50 100 100 100 Xylan 0.4 100 100 100 170 100 100 100 100 100 50 100 100 100 100 100 100 C.NUTRITIONALTEST: Solecarbonandenergysources: D(+)Galactose 1.0 40 100 100 100 57 100 100 100 100 100 100 100 100 75 0 100 L(+)Arabinose 1.0 0 86 100 100 100 71 100 100 100 0 50 100 75 75 0 100 D(-)Fructose 1.0 20 100 100 100 100 86 100 67 100 50 100 100 100 100 100 100 D(+)Mannose 1.0 0 43 75 0 0 43 33 33 100 50 0 0 0 0 0 33 D(+)Xylose 1.0 0 86 100 100 100 57 86 100 100 50 100 100 75 75 0 67 a-L(+)Rhamnose 1.0 0 57 75 0 12 0 86 67 100 100 0 0 0 0 0 86 Turanose 1.0 0 100 25 80 100 14 29 0 67 0 50 50 75 50 0 0 D(+)Cellobiose 1.0 20 57 88 100 62 43 86 33 0 100 0 0 0 0 0 0 D(+)Lactose 1.0 20 100 100 100 75 57 86 100 100 100 100 100 100 100 100 100 Sucrose 1.0 0 71 0 60 75 0 71 67 100 50 0 0 0 100 0 0 D(+)Melezitose 1.0 0 0 12 40 88 0 0 0 0 50 0 50 0 50 50 0 D(+)Raffinose 1.0 0 29 12 60 75 57 14 0 67 50 100 0 50 100 50 0 Dextrin 1.0 40 71 100 100 100 86 86 67 100 50 100 100 100 100 100 100 Aceticacid 0.1v/v 0 57 100 0 50 0 0 67 100 100 0 0 0 0 0 0 Citricacid 1.0 0 86 100000293367500 0 0 0 00 L(+)Tartaricacid 1.0 0 0 0 0 0 14 0 0 0 100 0 0 0 0 0 0 Pyruvicacid 0.1 0 100 100 0 12 0 0 33 67 0 50 50 0 0 0 0 Sodiumacetate 0.1 0 100 62 0 25 0 0 33 33 0 0 50 0 0 0 33 Sodiumbutyrate 0.1 0 57 100 60 75 43 43 0 33 100 50 50 50 25 0 0 Sodiumcitrate 0.1 0 100 100000143333500 0 0 0 00 Sodiumpyruvate 0.1 0 100 88 60 50 29 14 67 100 50 50 50 25 0 0 0 D-Mannitol 1.0 0 100 100 60 75 51 71 100 67 100 100 0 100 50 100 67 D-Sorbitol 1.0 0 29 25 0 12 0 0 33 0 100 0 0 0 0 0 33 meso-‹nositol 1.0 20 71 100 100 100 57 71 100 100 0 100 100 50 75 50 100 Maltose 1.0 20 100 100 100 100 100 71 67 100 0 100 100 75 100 0 33 L-Sorbose 1.0 0 29 12 0 12 0 0 33 0 100 0 0 0 0 0 0 Growthofsolenitrogensources: L-Arginine 0.1 60 100 100 100 100 100 71 100 100 100 100 100 100 100 100 67 L-Cysteine 0.1 0 100 88 100 12 100 86 0 0 100 0 100 100 50 100 100 L-Histidine 0.1 100 100 100 100 100 100 100 100 100 100 100 100 75 75 100 67 L-Iso-Leucine 0.1 0 100 62 0 0 29 57 100 0 100 50 50 25 0 0 33 L-Lysin 0.1 0 100 88 100 100 100 86 100 0 100 50 100 75 100 100 100 L-Methionine 0.1 0 100 100 80 88 0 0 100 0 100 0 100 100 75 50 0 L-β-Phenylalanine 0.1 20 100 62 100 88 100 100 100 0 100 100 100 100 100 100 100 L-Proline 0.1 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 L-Serine 0.1 40 100 100 100 100 100 100 100 67 100 100 100 100 75 100 100 L-Threonine 0.1 0 100 100 20 0 100 100 100 67 100 100 100 75 0 50 100 L-Valine 0.1 0 100 50 80 25 100 100 33 0 50 100 100 75 75 100 100 Glycin 0.1 80 100 88 100 75 100 100 100 100 50 100 100 75 100 50 100 Potassiumnitrate 0.1 20 100 50 100 50 43 100 0 0 100 50 100 75 25 100 100

18 N.fiAH‹N,E.ÖZTÜRK,K.IfiIK,E.KAR‹PTAfi,R.ÖZKANCA

Table2. continued.

Cluster 1 2 5 7 10 14 15 3 4 6 8 9 11 12 13 16 Numberofstrains 5 7 8587 733 2 2 2 4 4 23 Character (w/v)

D.PHYSIOLOGICALTESTS Growthinthepresenceof: CopperIIsulphate 0.001 60 100 88 60 100 100 43 67 67 100 50 100 100 100 0 67 CopperIIsulphate 0.01 0 100 12000 0330 0 0 0 0 0 00 Ferroussulphate 0.005 100 100 100 100 100 100 100 100 100 100 0 100 100 75 100 100 Ferroussulphate 0.01 100 100 100 100 100 100 100 100 100 100 0 100 1000 75 100 100 Phenol 0.005 80 100 100 100 100 100 100 100 100 100 50 100 100 100 100 100 Phenol 0.1 80 100 100 50 100 100 100 100 100 50 50 100 100 100 100 100 Potassiumnitrate 0.005 60 100 88 80 71 100 67 100 50 50 100 100 100 100 100 100 Potassiumnitrate 0.01 60 57 88 80 75 71 86 67 100 50 50 50 75 100 100 67 Potassiumtellurite 0.001 80 71 62 20 75 100 100 100 67 100 50 100 75 100 0 100 Potassiumtellurite 0.005 80 71 38 20 50 100 100 100 67 50 0 50 50 100 0 33 Sodiumchloride 4 0 100 100 100 88 100 100 100 100 100 100 100 75 75 100 100 Sodiumchloride 7 0 100 100 40 88 51 71 100 100 100 50 100 75 75 0 67 Sodiumchloride 10 0 100 25000143333500 500 0 00 Sodiumchloride 13 0 86 0000 000 0 0 500 0 00 Sodiumcitrate 0.01 80 100 100 40 100 100 100 100 100 0 0 100 100 100 0 100 Sodiumcitrate 0.02 80 100 100 40 100 100 100 100 100 100 0 100 100 75 0 100 Zincchloride 0.001 60 100 88 100 100 86 100 100 100 100 50 100 100 100 0 100 Zincchloride 0.005 0 43 25 40 100 0 0 33 100 100 50 100 100 100 0 0 Zincchloride 0.01 0 0 0 20 100 0 0 0 0 100 50 50 75 100 0 0 Zincsulphate 0.005 20 100 62 80 88 29 43 100 100 0 100 0 75 100 50 33 Zincsulphate 0.01 0 29 25 60 75 0 43 33 33 0 100 0 75 100 50 33 Resistancetoantibiotics: (µg/ml) Ampicillin 16 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 67 Ampicillin 32 20 100 100 80 62 57 100 100 100 100 0 100 100 100 50 67 Ampicillin 64 20 100 100 0 12 0 14 100 100 100 100 0 100 50 0 0 Aztreonam 16 0 100 100 100 100 100 100 100 100 100 100 100 75 100 100 100 Aztreonam 32 0 100 100 100 100 100 100 100 100 100 50 100 75 100 100 100 Aztreonam 64 0 100 100 60 100 100 100 100 100 100 0 100 50 75 50 100 Cefepimehydrochloride 8 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Cefepimehydrochloride 16 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Cefepimehydrochloride 32 0 100 100 100 100 100 100 100 100 100 100 100 100 100 50 100 Ceftizoximesodium 8 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Ceftizoximesodium 16 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Ceftizoximesodium 32 0 100 100 80 88 86 100 100 100 100 100 100 100 100 50 33 Ceftriaxonedisodium 8 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Ceftriaxonedisodium 16 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Ceftriaxonedisodium 32 0 100 100 80 62 14 86 100 100 100 0 100 75 100 50 0 Cefuroximesodium 8 20 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Cefuroximesodium 16 0 100 100 100 100 100 100 100 100 100 100 0 75 100 100 100 Cefuroximesodium 32 0 100 100 80 50 71 86 100 100 100 100 0 75 100 50 0 Doxycyline 16 20 100 75 0 12 100 86 100 67 0 0 50 0 25 0 67 Doxycyline 32 20 71 0000571000 0 0 0 0 0 00 Doxycyline 64 0 71 00002900 0 0 0 0 0 00 Fluconazole 8 60 100 100 20 100 100 100 100 100 100 100 0 100 50 0 100 Fluconazole 16 20 100 100 0 100 100 100 100 100 100 100 0 100 25 0 100 Fluconazole 32 0 100 100 0 100 100 100 100 100 100 50 0 75 25 0 100 Fusidicacid 4 60 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

19 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes

Table2. continued.

Cluster 1 2 5 7 10 14 15 3 4 6 8 9 11 12 13 16 Numberofstrains 5 7 8587 733 2 2 2 4 4 23 Character (w/v) Fusidicacid 8 40 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Fusidicacid 16 20 100 100 100 100 100 100 100 100 0 50 100 100 100 100 100 Kanamycin 8 0 29 0 60 88 0 14 0 0 0 100 0 0 25 100 0 Meropenem 8 20 100 100 100 75 100 100 100 100 100 0 100 100 75 100 100 Meropenem 16 20 100 100 100 75 57 86 100 100 100 0 100 100 75 100 100 Meropenem 32 0 86 75 0 0 14 0 33 33 100 0 0 0 0 0 0 Meropenem 64 0 71 0000 0033500 0 0 0 00 Nalidixicacid 4 60 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Nalidixicacid 8 0 100 100 80 100 100 86 100 100 100 100 100 100 75 50 100 Nalidixicacid 16 0 100 100 0 88 100 86 100 100 100 100 100 100 25 0 100 Nystatin 32 100 100 100 100 100 100 86 100 100 100 100 100 100 100 100 100 PenicillinG 5(i.u) 0 100 100 100 100 100 100 100 100 100 100 50 100 100 100 100 PenicillinG 10(i.u) 0 100 100 20 12 0 86 100 100 100 0 50 100 100 50 33 PenicillinG 15(i.u) 0 100 100 20 0 0 100 100 100 100 0 100 100 25 0 0 Rifampicin 16 20 100 100 0 12 71 86 100 100 100 50 100 0 50 50 33 Rifampicin 32 20 100 100 0 0 57 86 100 100 100 50 100 0 25 0 33 Rifampicin 64 20 100 100 0 0 57 86 100 100 100 0 50 0 25 0 33 Streptomycinsulphate 8 60 43 2500014033000 0 0 0 00 Teikoplanin 8 0 0 50000 067670 0 0 0 0 00 Teikoplanin 16 0 0 38000 000 0 0 0 0 0 00 Vancomycin 1 0 57 750001433100500 0120 014 Vancomycin 2 0 0 38000 033100500 0 0 0 00 Growthat: pH10.5 40 100 62 100 75 57 43 100 100 100 100 100 100 100 100 0 25°C 20 100 100 0 12 86 100 100 100 100 100 50 0 50 0 67 55°C 80 29 12 100 100 43 0 33 0 50 100 100 100 100 100 67 60°C 40 14 0 100 75 71 100 0 0 0 100 50 75 100 100 100 E.MORPHOLOG‹CALTESTS: Sporechainmorphology: Rectiflexibiles100 57 38 0 12 57 0 0 33 100 0 0 75 50 0 0 Retinaculiaperti 0 57 100 40 50 43 57 0 67 50 50 100 50 100 50 100 Spirales 100 43 67 100 88 86 100 100 67 100 100 100 100 75 100 100 Colourofaerialsporemass: White 100 86 0000 000 0 0 0 0 0 00 Grey 0 14 100 88 43 57 100 100 100 100 100 100 100 100 100 0 Black 0 0 0 0 57 71 0 0 0 0 0 0 0 0 0 100 Colourofsubstratemycelium: Nodistinctivesubstratemycelium 60 86 75 0 12 0 0 0 0 100 100 50 0 0 0 0 Grey0 14 25 60 50 14 57 100 0 0 100 50 100 100 50 0 Brown 40 0 0 0 0 43 43 0 33 0 0 0 0 0 0 100 Black 0 0 0 40 38 43 0 0 67 0 0 0 0 0 50 0 Colourofdiffusiblepigment: Grey 0 0 0000 000 0 0 0 0 0 00 Brown 0 0 12000 0331000 0 0 0 0 00 Violet 0 0 0000 000 0 0 0 0 0 00 Green 0 0 0 0 0 100 86 0 0 0 0 0 50 0 0 100 Nodiffusiblepigment 100 100 88 100 100 0 14 67 0 100 100 100 50 100 100 0

20 N.fiAH‹N,E.ÖZTÜRK,K.IfiIK,E.KAR‹PTAfi,R.ÖZKANCA

3.2.Numericalclassification: Theexperimentaltest wereassignednamesaccordingtothedistributionoftype errorwasestimatedfromdatacollectedonthe16 andreferencestrains.Thecharacteristicsofthemajor duplicatedcultures.Theaverageprobabilityofan andminorclustersaregiveninTable2. erroneoustestresult(p)wascalculatedfromthepooled i AggregategroupAencompassed6streptomycete variance(S 2 =0.040)oftheunitcharacters(30).Inthe isolatedstrainsin1majorand1singlemembered presentinvestigation,thetaxonomicstructurewasnot clusters.Mostofthetypeandreferences Streptomyces markedlyaffectedbythe3.50%testerror(p),afigure specieswereassignedtoclustergroupsBandDwiththe wellwithinthe10%guidelinerecommendedbySneath exceptionofStreptomyces sp.A1960andTA068,which andJohnson(30).Atesterrorofthisnatureis belongtocluster8andsinglememberedclusterTA053. comparabletothecorrespodingvaluesreportedin previousnumericaltaxonomicstudiessuchas AggregategroupBencompassed2major,3minor neutrophilic,mesophilicstreptomycetesp3.36%(9);p and4singlememberedclusters.Cluster2containedthe 1.54%(31),alkalitolerant,mesophilicstreptomycetesp typestrainsof S.thermovulgaris and S.albus ;the 3.11%(32)andthermotolerantstreptomycetesp remainingmajorcluster(cluster5)was S. 1.81%(19).Theduplicatedstrainsshowedamean thermolineatus.Thetypestrainsof S.megasporus (K0045T)and S.macrosporus (K0044T)wereseparated observedsimilarityof93.5%SSM.Mosttestswerehighly i assinglememberedclustersinclustergroupB. reliableandgaveS 2 valuesbelow0.1.Cophenetic correlationvaluesintherangeof0.6to0.95(27,33) ClustergroupCcontained28outofthe45 implythathierarchicalclusteringprocedureshaveyielded thermophilicisolateswiththreethermophilicreference goodrepresentationsofthetaxonomicstructureinherent streptomycetes, Streptomyces strainsTA053, insortedsimilaritymatrices.Thecopheneticcorrelation Streptomyces strainsA1960andTA068.Aggregate valuewas0.832(S SM COMPLETE)inthepresentstudy. groupCcontainedtwomajor,fiveminorandfoursingle Alloftheteststrainsgrewat35ºC,atpH4.5,5.5and memberedclusters. -1 9.5andinthepresenceofnystatine(16mgml ). WithinaggregategroupD,cluster15containedtype Incontrast,noneofthestrainswereabletodegrade andreferencestrainsof S.cavourensis , S. chitin(0.4%,w/v),usenalidixicacid(1.0%,w/v), thermodiastaticus,and S.thermoviolaceus .Cluster14 ninhydrin(1.0%,w/v),safranin(1.0%,w/v)andvanillin comprisedsevenstreptomyceteisolatedstrainsthat (1.0%,w/v)assolecarbonsources,growatpH11orin producedgreendiffusiblepigments.Theremainingtype thepresenceofcoppersulphate(0.02%,w/w),crystal strainS.thermoflavus formedasinglememberedcluster violet(0.001and0.005%,w/w),sodiumselenite(0.001 neighbouringcluster16. -1 and0.005%,w/w),amikacine(4and8mgml ), Itwasinterestingthatinthepresentnumerical -1 kanamycin(16and32mgml ),neomycinsulphate(8,16 taxonomicstudy,37strainsoutofthetotal45isolated -1 and32mgml ),streptomycinsulphate(16,32and strainsweredifferentlyclusteredfromtypeand -1 -1 64mgml ),teikoplanin(32mgml )andvancomycin(16, referencesstrains.Theseorganismswereassignedto -1 32and64mgml ). fourmajor(cluster1,7,10and14),fourminor(cluster Thefinaldatamatrixcontainedinformationon82 9,11,12and13)andfivesinglememberedclusters teststrainsand132unitcharacters. (E1448,E1100,E1172,E1427andE1201).Two isolates(E1119andE1412)outofthethreeremaining 3.3.ClusteringofstrainsusingtheS SM coefficient withthecompletealgorithm: Theclassificationbased strainswereclusteredwiththereferencestrainsTA208 incluster16,whereasstrainE1075wasclusteredwith ontheS SM completeanalysisisdescribedindetailasit gavethemostcompactaggregategroupsandclusters thetypestrains Streptomycesthermovulgaris (DSM togetherwithasuitablehighcopheneticcorrelationvalue 40444)andStreptomycesalbus (K15andK17)incluster (0.832).The82teststrainswereassignedtofourcluster 2.Ithaspreviouslybeenshownthat Streptomycesalbus groups,designatedAtoD,atthe67%similarity(S)level. strainshavemanypropertiesincommonwith Sevenmajor(5-8strains),nineminor(2-4strains)and neutrophilic,thermophilicstreptomycetes(11). tensinglememberedclusterswerecircumscribedator Anotherencouragingresultinthepresentstudywas abovethe82.5%similaritylevel(Figure).Theseclusters thatthetypestrains Streptomycesmegasporus (K45),

21 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes

Percentage Similarity Cluster Aggregate Cluster Name No Group and Strains

1 A Streptomyces sp. E1139, E1401, E1438, E1351, E1354 E1448

2 S. thermovulgaris T,S. albus E1075, 40444, 40787, K0017, K0015, NT307, NT550 K0045 S. megasporusT E1100 3 Streptomyces sp. B NAR64, NT471, NT297 4 Streptomyces sp. A1853 NT358, NT011, NT508 S. thermolineatusT 5 TA212, NT313, K0047, TA035, NT361, NT388, NT319, NT467

T K0044 S. macrosporus 6 Streptomyces sp. E1172 NT126, NT371 TA053 7 Streptomyces sp. E1145, E1176, E1208, E1356, E1161 E1427 Streptomyces sp. 8 A1960, TA068 9 Streptomyces sp. C E1349, E1160 Streptomyces sp. 10 E1185, E1183, E1141, E1205, E1440, E1446, E1430, E1019

11 Streptomyces sp. E1202, E1109, E1138,E1001

12 Streptomyces sp. E1008, E1122, E1016, E1364 E1201 13 Streptomycessp. E1166, E1348 Streptomycessp. 14 E1124, E1173, E1394, E1125,E1387,E1415,E1391

S. cavourensis S. thermodiastaticus 15 D S. thermoviolaceus 40300, NT312, 40573, A1847, 40443, A0074, 4139 Streptomyces sp. 16 E1119, E1412, TA208 40574 S. thermoflavus

Figure. DendrogramshowingrelationshipsbetweentheteststrainsbasedontheS SM coefficientandCompletealgorithm. T,TypeStrain.

22 N.fiAH‹N,E.ÖZTÜRK,K.IfiIK,E.KAR‹PTAfi,R.ÖZKANCA

Streptomycesthermolineatus (A1853), Streptomyces Inconclusion,taxonomicclustersarerepresentations macrosporus (K44)and Streptomyces thermoflavus ofnaturalrelationshipsbetweenstrainsalthoughgroup (DSM40574)formedaseparatecluster,similartothe compositionmaybeinfluencedbythechoiceofstrains resultsobtainedinGoodfellow etal. ,(11)andﬁahin’s andtests,experimentalprocedures,testerrorand (19)previousnumericaltaxonomicstudies.Incontrast, statisticsused(30,34-37).Thismeansthatnumerical Streptomyces cavourensis subsp. cavourensis (DSM taxonomiesneedtobeevaluatedinthelightofadditional 40300), Streptomycesthermodiastaticus (DSM40573) informationderivedfromtheapplicationofindependent and Streptomycesthermoviolaceus (A1847)were taxonomicmethods,notablybytheuseof clusteredwithtogetherincluster15(9,11,19). chemotaxonomicandmolecularsystematictechniques.

References

1. Bérdy,J.Areactinomycetesexhaustedasasourceofsecondary 12. Korn-Wendisch,F.andKutzner,H.J.Thefamily metabolites.Biotechnologica7,8:3-34.1995. Streptomycetaceae.InTheProkaryotes.AHandbookonthe BiologyofBacteria:Ecophysiology,Isolation,Identification, 2. Manfio,G.P.,Zakrzewska-Czerwinska,J.,Atalan,E.and Applications,pp.921-995.EditedbyBalows,A.,Trüper,H.G., Goodfellow,M.Towardsminimalstandardsforthedescriptionof Dworkin,M.,Harder,W.andSchleifer,K.H.,2nded.,vol.1. Streptomyces species.Biotechnologia7-8:242-253,1995. Springer-Verlag,NewYork.1992. 3. Nolan,R.D.andCross,T.Isolationandscreeningof 13. Waksman,S.A.GuidetotheClassificationandIdentificationof actinomycetes.InActinomycetesinBiotechnology,pp.1-32. ActinomycetesandtheirAntibiotics.Williams&WilkinsCo., EditedbyM.Goodfellow,S.T.Williams&M.Mordarski. Baltimore.1953. AcademicPress:London.1988. 14. Desai,A.J.andDhala,S.A.Streptomycesthermonitrificans sp.n., 4. Okami,Y.andHotta,K.Searchanddiscoveryofnewantibiotics, athermophilicstreptomycete.AntonievanLeeuwenhoek33,137- pp.33-67.In:M.Goodfellow,S.T.WilliamsandM.Mordarski 147.1967. (ed.),Actinomycetesinbiotechnology.AcademicPress,SanDiego; 1988. 15. Henssen,A.BeiträgezurMorphologieundSystematikder thermophilenActinomyceten.Archiw.fürMikrobiologie.26,273- 5. Williams,S.T.,Sharpe,M.E.andHolt,J.G.Bergey'sManualof 414.1957. SystematicBacteriology,Volume4.WilliamsandWilkins Company:Baltimore,1989. 16. Skerman,V.B.D.,McGowan,V.andSneath,P.H.A.Approved ListsofBacterialNames.InternationalJournalofSystematic 6. Goodfellow,M.,Manfio,G.P.andChun,J.Towardsapractical Bacteriology30:225-420.1980. speciesconceptforcultivatablebacteria.InSpecies:theUnitsof Biodiversity.EditedbyM.F.Claridge,H.A.Dawah&M.R.Wilson, 17. Krassilkinov,N.A.,Agre,N.S.andEl-Registan,G.I.New pp.25-59,London:Chapman&Hall.1997. thermophilicspeciesofthegenusMicropolyspora.Mikrobiologiya 37:1065-1072.1968. 7. Trejo,W.H.,Anevaluationofsomeconceptsandcriteriausedin thespeciationofstreptomycetes.TransactionsoftheNewYork 18. Agre,N.S.InAGuidefortheDeterminationofActinomycetes. AcademyofScience,32,986-997;1970. GeneraStreptomyces,StreptoverticilliumandChainia,pp.52-71. EditedbyT.F.Gause,T.P.Preobrazhenskaya,M.A.Sveshnikova, 8. Williams,S.T.,Wellington,E.M.H.,Goodfellow,M.,Alderson,G., L.P.Terekhova&T.S.Maksimova.Moscow:Nauka(inRussian). Sackin,M.andSneath,P.H.A.Thegenus Streptomyces–a 1983. taxonomicenigma.In:Actinomycetes,EditedbySchaal,K.P.& Pulverer,G.GustavFischerVerlag,Stuttgart,pp.47-57,1981. 19. ﬁahin,N.SelectiveIsolation,CharacterisationandClassificationof NovelThermotolerantStreptomycetes.PhDThesis.Departmentof 9. Williams,S.T.,Goodfellow,M.,Alderson,G.,Wellington,E.M. Microbiology,UniversityofNewcastleuponTyne.U.K.1995. H.,Sneath,P.H.A.andSackin,M.J.Numericalclassificationof Streptomyces andrelatedgenera.JournalofGeneralMicrobiology, 20. Küster,E.andWilliams,S.T.Selectionofmediaforisolationof 129,1743-1813,1983 streptomycetes.Nature202:928-929.1964. 10. Kutzner,H.J.ThefamilyStreptomycetaceae,pp.2028-2090.In 21. Küster,E.Outlineofacomparativestudyofcriteriausedin TheProkaryotes.EditedbyM.P.Starr,H.Stolp,H.G.Trüper,A. characterizationoftheactinomycetes.InternationalBulletinof BalowsandH.G.Schlegel.SpringerVerlag:Berlin.1981. BacteriologicalNomenclatureandTaxonomy9:97-104.1959. 11. Goodfellow,M.,Lacey,J.andTodd,C.Numericalclassificationof 22. Shirling,E.B.andGottlieb,D.Methodsforcharacterisationof thermophilicstreptomycetes.JournalofGeneralMicrobiology Streptomyces species.InternationalJournalofSystematic 133:3135-3149.1987. Bacteriology16:313-340.1966.

23 SelectiveIsolationandNumericalClassificationofNovelThermophilicStreptomycetes

23. Wellington,E.M.H.andWilliams,S.T.Preservationof 31. fiahin,N.,Yalç›n,‹.,Ifl›k,K.,Kariptafl,E.andÖzkanca,R. actinomyceteinoculuminfrozenglycerol.MicrobiosLetters6: Nötrofilik-mezofilikstreptomycetes’lerinizolasyonuvenümerik 152-159.1978. taksonomisi.JournalofBiotechnology,Vol:25:67-81.2001. 24. Jones,K.L.Freshisolatesofactinomycetesinwhichthepresence 32. Saddler,G.S.SelectiveIsolationandRapidIdentificationof ofsporogenousaerialmyceliaisafluctuatingcharacteristic. Streptomycetes.Ph.D.Thesis.UniversityofNewcastleuponTyne, JournalofBacteriology57:141-145.1949 U.K.1988. 25. Covan,S.T.andSteel,K.J.Manualforidentificationofmedical 33. Sackin,M.J.andJones,D.Computer-assistedclassification.In bacteria.Cambridge:CambridgeUniversityPress,1974. HandbookofNewBacterialSystematics,282-217.EditedbyM. GoodfellowandA.G.O'Donnell.AcademicPress:London.1993. 26. Pridham,T.G.Hesseltine,C.W.andBenedict,R.G.,Aguidefor theclassificationofstreptomycetesaccordingtoselectedgroups. 34. Langham,C.D.,Williams,S.T.,Sneath,P.H.A.andMortimer,A. Placementofstrainmorphologicalsections.AppliedMicrobiology, M.Newprobabilitymatricesforidentificationof Streptomyces. 6:52-73.1958. JournalofGeneralMicrobiology135:121-133.1989. 27. Sneath,P.H.A.andSokal,R.R.NumericalTaxonomy:The 35. Logan,N.A.BacterialSystematics.Oxford:BlackwellScientific PrinciplesandPracticeofNumericalClassification.W.H.Freeman: Publications.1994. SanFransisco.1973. 36. Goodfellow,M.Numericaltaxonomy.InBiologyofthe 28. Chun,J.Computer-assistedclassificationandidentificationof Prokaryotes.EditedbyH.G.SchlegelandJ.W.Lengeler. actinomycetes.Ph.D.Thesis,DepartmentofMicrobiology, Stuttgart:GeorgThiemeVerlag.1995a. UniversityofNewcastleuponTyne,U.K.1995. 37. Goodfellow,M.Actinomycetes:Actinomyces,Actinomadura, 29. Sokal,R.R.andMichener,C.D.Astatisticalmethodforevaluating Nocardia, Streptomyces andrelatedgenera.InMackieand systematicrelationships.KansasUniversityScienceBulletin38: McCarneypracticalmedicalmicrobiology.EditedbyCollee,J.G.; 1409-1438.1958. Duguid,J.P.;Fraser,A.G.;Marmin,B.P.andSimmons,A.,14th ed.ChurchillLivingstone,Edinburgh.1995b. 30. Sneath,P.H.A.andJohnson,R.Theinfluenceonnumerical taxonomicsimilaritiesoferrorsinmicrobiologicaltests.Journalof GeneralMicrobiology72:377-392.1972.