PROPOSAL TO DESIGNATE STRAIN ATCC 3004 (IMRU 3004) AS THE NEOTYPE:STRAIN OF STREPTOMYCES ALBUS (ROSSI-DORIA)

WAKSMAN AND HENRICI1

A. J. LYONS, JR. AND T. G. PRIDHAM

Fernmentation Laboratory, Northern Regional Research Laboratory,2 Peoria, Illinois

Received for publication August 24, 1961

ABSTRACT

LYONS, A. J., JR. (Northern Regional ResearchLaboratory, Peoria, Ill.) AND T. G. PRIDHAM.Proposal to designate strain ATCC 3004 (IMRU3004) as the neotype strain of Streptomyces albus(Rossi-Doria) Waksman and Henrici. J. Bac-teriol. 83:370-380. 1962.-It is proposed thatstrain ATCC 3004 (IMRU 3004) be designatedhenceforth as the neotype strain of Streptomycesalbus (Rossi-Doria) Waksman and Henrici andas the type strain of the genus StreptomycesWaksman and Henrici. The proposal is basednot only on the fact that the holotype strain[Streptotrix (sic) alba] of Rossi-Doria is no longerextant, but also on the fact that a study of theliteiature and a taxonomic study of 55 strains

1 The title of this paper, as presented at the 61stannual meeting of the American Society for Micro-biology, was "Proposal to designate strain ATCC618 as the neotype strain of Streptomyces albus(Rossi-Doria) Waksman et Henrici" (Bacteriol.Proc., p. 74, 1961). In the original preparation ofthe manuscript we obtained information that ledus to believe that strains ATCC 618 and ATCC3004 (IMRU 3004) were both descendants of oneculture isolated about 1897. Since presentation ofthe paper we have learned this is not true and havecorrected the information. There is little doubtnow that these two strains, while remarkablysimilar, did in fact originate from two differentcultures. Because the history of strain ATCC 3004(IMRU 3004) can be traced back to about 1897,whereas strain ATCC 618 can be traced back toabout 1914, we believe the former should haveprecedence in selection of a neotype strain andhave changed the title and proposal as indicated.(ATCC refers to the American Type Culture Col-lection; IMRU refers to the culture collection ofthe Institute of Microbiology, Rutgers Univer-sity).

2 A laboratory of the Northern Utilization Re-search and Development Division, AgriculturalResearch Service, U. S. Department of Agricul-ture.

of the organism indicate the species shouldexhibit these characteristics: catenulate ovoidalspores, white aerial mycelium, coiled sporophores,.proteolytic activity, and nonchromogenicity-(inability to form brown, deep brown, or blackdiffusible pigments). Strain ATCC 3004 (IiIRU3004) exhibits these characteristics, as do 16other acquisitions. Study of the 16 additionalstrains that conform with the general definitionof the species shows differences in some physi-ological characteristics. It is suggested that thesedifferences are of subspecific significance.

Two different concepts have existed since 1916with regard to Streptomyces albus (Rossi-Doria)Waksman and Henrici. One centers aroundstrains with the following characteristics: flexuoussporolhores, colors of aerial mycelium in tints.and shades of olive-buff (yellowish gray or tan),nonchromogenicity (inability to form brown,.deep brown, or black diffusible pigments inorganic substrata), and marked abundance innature. The other concept concerns strains that.are characterized by coiled or spiralled sporo-phores with catenulate, ovoidal spores; by aerialmycelium colors generally interpreted as cre-taceous (chalk-white, often with faint tinges ofpink); by nonchromogenicity in organic sub-strata; and by their relative rareness in nature.We believe the two issues can be clarified

(Pridham and Lyons, 1961) by accepting thesecond concept, first proposed by Waksman andCurtis (1916) and stated as the concept of S.albus by Waksman and Henrici (1943) whendesignating it as the type species of the genus&Streptomyces.Waksman and Henrici additionally qualified

their description of the species to include its.ability to liquefy gelatin, to peptonize milk withproduction of an alkaline reaction, and to forman earthy or musty odor.

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We have continued our studies of 17 strains,of S. albus (Pridham and Lyons, 1961) in moredetail. Beyond the basic characteristics of thespecies, the 17 strains can now be segregated intotwo or more groups based on various physiologicalcriteria. Moreover, a careful check of the historiesof the strains has revealed that one of thesegroups contains descendants of only two strains.One was originally obtained by Berestnev(Berestneff or Berestnew), probably in the1890's. The other was isolated by Krainsky in1914. In our opinion the cultures of this groupare descendants of the earliest isolated andlabeled cultures of S. albus still extant. Becausethe Berestnev culture was isolated at a muchearlier date, we believe it should have precedencein selection of a suitable neotype strain.No type strain of the Rossi-Doria culture was

ever designated. Likewise, no neotype strainconforming to Waksman and Henrici's descrip-tion was ever designated as the type of thespecies or the type of the genus. Waksman(1961) has designated a strain "IMRU 3005" asthe "type" of the species, but this strain, origi-nally isolated and described by Wollenweber(1920) as Actinomyces albus var. cretaceus, failsto conform with the Waksman and Henrici(1943) concept and actually is a member of theS. griseus group.We intend to present a more detailed charac-

terization of the group containing descendantsof the Berestnev and Krainsky cultures (sixstrains), to propose that one of these strains,ATCC 3004 (IMRU 3004), be henceforth desig-nated as the neotype strain of Streptomyces albus(Rossi-Doria) Waksman and Henrici, and todiscuss briefly the characteristics of the remainderof the 17 strains studied.

MATERIALS AND METHODS

Strains studied. Of 17 strains that conformto the characteristics of S. albus, 6 are of par-ticular importance. Their numbers and historiesare:

Strain 618-1 was received in 1959 from theATCC as ATCC 618. It is described in theATCC catalog as having been received by theATCC from A. J. Kluyver, Delft, Netherlands,in 1926. The culture was forwarded to the ATCCon oatmeal agar, along with a note stating thatthe culture was isolated by Krainsky and de-scribed in Centr. Bakteriol. Parasitenk., Abt. 2,

41:649, 1914. Kluyver received the strain fromthe Centraalbureau voor Schimmelcultures(CBS) on July 19, 1922. The CBS, in turn, hadreceived it from Beijerinck in March 1918. Thecorrespondence concerning this strain leaveslittle doubt that it was the original Krainskystrain.

Strain 618-2 was received in 1955 from R.Gordon, Institute of Microbiology, RutgersUniversity, as ATCC 618.

Strain 618-3 was received in 1955 from T.Yamaguchi, Institute of Applied Microbiology,University of Tokyo, Japan, as ATCC 618.

Strain 3004-1 was received in 1959 from S. A.Waksman, Institute of Microbiology, RutgersUniversity as IMRU 3004. This strain wasreceived in the 1920's by Waksman from thePribram Collection (originally the Kral Collec-tion) in Vienna, carrying the epithet label "albus"and with a note that it was named or isolatedby Berestnew. A culture designated as Actin-omyces albus Berestnew is listed in the Kralcatalogues dated 1900 and 1902, and later in1919 as a variety of A. saprophyticus (but stilldesignated as A. albus Berestnev), with a notethat it was isolated from straw in Prague.

Strain 3004-2 was received in 1956 from theATCC as ATCC 3004. It was received by theATCC from S. A. Waksman in 1944.

Strain 3004-3 was received in 1959 from theATCC as ATCC 3004. It was received by theATCC from S. A. Waksman in 1944.

Thus, the last three strains represent descend-ants of the Berestnev culture obtained by Waks-man from the Pribram Collection.

Despite their different histories, the six strainsdescribed above are remarkably similar. Theyare hereinafter referred to collectively as theBerestnev and Krainsky strains. The histories ofthe remaining 11 strains were given by Pridhamand Lyons (1961).

Procedures. Most of the general proceduresused in this study have been described elsewhere(Committee on Bacteriological Technic, 1946;Subcommittee on the Taxonomy of the Actin-omycetes, 1958; Kulster, 1958; Gordon andMihm, 1957; Gordon and Smith, 1955; Hes-seltine, Benedict, and Pridham, 1954; Pridhamand Gottlieb, 1948; Pridham, Hall, and Shekle-ton, 1951; Pridham, Hesseltine, and Benedict,1958; Pridham and Lyons, 1961, Pridham et al.,

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1956, 1957; Tresner and Danga, 1958; Waksman,1919).

Stock cultures and inocula. All cultures pre-pared for the various observations, except forpotato slants, originated from shaken-tubeliquid preparations. The liquid inocula wereprepared by transferring a loopful of spores froma 2-week-old sporulating culture, grown at 28to 30 C on tomato paste-oatmeal (TPO) agarslants or our formula for yeast-extract (YE)agar slants, into tryptone yeast extract (TYE)broth and incubated with shaking at 28 to 30 Cfor 48 hr. All media were inoculated with an equalamount of these broth cultures.

Spore morphology and nature of spore surface.Spore morphology was determined by examina-tion of Drechsler impression slides of strains thathad been cultivated on inorganic salts-starch(ST) agar for 14 days at 28 to 30 C. The detailsof the method used were outlined by Pridhamand Lyons (1961).The nature of the spore surface of descendants

of the Berestnev and Krainsky cultures has beenreported in three publications (Pridham, 1959;Pridham and Lyons, 1961; Tresner, Davies, andBackus, 1961). Electron micrographs of twoadditional strains of S. albus, other than de-scendantsof the Berestnev and Krainsky cultures,also have been made (Pridham and Lyons, 1961).

MIorphology of sporophores. The morphology ofsporophores of the strains was determined fromexamination of 14-day petri-dish cultures grownat 28 to 30 C on ST agar, Czapek's solution (CZ)agar, glycerol-asparagine (GA) agar, and nu-trient (N) agar. N agar was prepared by addingthe appropriate amount of agar (15 g/liter) tonutrient broth (Difco) and sterilizing for 20 minat 121 C.

Color. Methods for determination of colors ofsporulating aerial mycelia, reverses of cultures,and diffusible pigments have been outlined else-where (Hesseltine, Benedict, and Pridham, 1954;Pridham, Hesseltine, and Benedict, 1958; Prid-ham and Lyons, 1961).

Colors of sporulating aerial mycelium, andreverses of cultures grown on ST, CZ, GA, andN agars were keyed by reference to Ridgway(1912). The Ridgway color designations werethen converted to the Inter-Society ColorCouncil-National Bureau of Standards (ISCC-NBS) (Kelly and Judd, 1955) color number andname. Common color designations were used in

recording the characteristics of the culturesgrown on other media. NTo attempt was made tokey colors of diffusible pigments when these oc-curred, because of difficulties experienced withthis criterion in the past.

Proteolytic activity. The proteolytic activity ofeach of the strains was determined by fivemethods. These were: (i) cultivation in 12%plain gelatin (Difco) in tubes in which theinoculum (0.2 ml of a 48-hr TYE broth culture)was placed on the surface of the substratum.No stab was made. Cultures were incubated at28 to 30 C for 14 days. At 7 and 14 days, eachculture was refrigerated at 3 to 5 C for 1 hr andexamined for liquefaction and color of diffusiblepigment; (ii) the Waksman 15%c plain-gelatindish method, with incubation at 18 C (Waksman,1919); (iii) the Waksman 15% plain gelatin-1%starch dish method, with incubation at 18 C(Waksman, 1919); (iv) the skimmed-milk tubemethod, with incubation at 28 to 30 C as out-lined by the Subcommittee on the Taxonomy ofthe Actinomycetes (1958); and (v) the casein-plate method, with incubation at 28 to 30 C(Gordon and Smith, 1955). Suitable uninoculatedcontrol media were used for comparisons.

All cultures were observed at 14 to 15 days,and the degree of liquefaction of gelatin, peptoni-zation of milk, and clearing of casein agar wasrecorded.

Chromogenicity. The ability of the severalstrains to produce a brown, deep brown, or blackdiffusible pigment was determined by cultivationof each strain on international peptone (IP)agar (Pridham and Lyons, 1961) for 4 days at28 to 30 C. Each strain was also cultivated onDietz L-tyrosine water-agar slants for 14 daysat 28 to 30 C. Directions for preparation of L-tyrosine agar were given by the Subcommitteeon the Taxonomy of the Actinomycetes (1958).At the end of the incubation period, each culturewas examined for the presence or absence ofdiffusible pigment.

Ability to produce hydrogen sulfide. Each strainwas cultivated on peptone iron agar (Difco),supplemented with 0.1% yeast extract (Difco),for 10 days at 28 to 30 C. Readings for presenceor absence of the characteristic bluish blackcolor of substrata, indicative of hydrogen sulfideproduction, were made at 1 and 10 days.

Ability to utilize various carbon sources forgrowth. Each strain was cultivated in petri

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dishes, containing the Pridham and Gottlieb(1948) basal agar supplemented with individualcarbon compounds (D-xylose, L-arabinose, rham-nose, D-glucose, D-fructose, sucrose, lactose,maltose, raffinose, D-mannitol, i-inositol, orsalicin) each at a 1% level, for 10 days at 28to 30 C. An appropriate control containing nocarbon source was included. The plate method,a modification of the original tube method sug-gested, was outlined by the Subcommittee on theTaxonomy of the Actinomycetes (1958); 48-hrTYE broth cultures were centrifuged, washedtwice with sterilized 0.85% saline solution, sus-pended in saline solution, and inoculated drop-wise (ca. 0.05 ml). Three strains in duplicatewere inoculated on the surface of each medium.

Ability to produce acid from various carbonsources. The ability of the strains to produce acidfrom D-xylose, D-mannose, maltose, and lactosewas determined by Gordon and Smith's (1955)method. Observation of cultures was made after14 days of incubation at 28 to 30 C, to determinewhether acid had been produced.

Ability to reduce nitrates. The ability to reducenitrate in both an organic-based medium and ina chemically defined medium was determined byprocedures outlined by the Subcommittee on theTaxonomy of the Actinomycetes (1958) and bythe Committee on Bacteriological Technic(1946). Cultures were tested and observed forreduction of nitrate after 14 days growth at28 to 30 C.

Diastatic activity. Fourteen-day cultures ofeach strain grown on ST agar at 28 to 30 C wereexamined for presence of clear areas adjacentto growth of the organism (indicating starchbreakdown). Each dish culture was also floodedwith ca. 2 ml of Lugol's iodine solution and al-lowed to stand for several minutes. Disappear-ance of the blue color was considered evidence ofstarch breakdown.

Ability to decompose tyrosine and xanthine.Determination of the ability of the strains todecompose tyrosine and xanthine was madefollowing methods outlined by Gordon andSmith (1955) and Gordon and Mihm (1957).Cultures were observed after 14 days incubationat 28 to 30 C for clearing of the substrata as anindication of decomposition.

Cultural characteristics on potato slants. Culturalcharacteristics of strains grown on white potatoslants were recorded after 14 days incubation

at 28 to 30 C. Peeled white potatoes were cutas plugs, sliced as slants, and soaked in distilledwater at 3 to 5 C for 24 hr. The slants weredrained, placed in test tubes, and sterilized for15 to 20 min at 121 C. After sterilization, steriledistilled water was added to each tube to coverall but Y4 in. of the butt.Each slant was inoculated with one loopful of

spores from a 2-week-old culture grown on TPOagar or YE agar slants.

Acid fastness. The vegetative mycelium from2-week-old cultures grown on GA agar wasspread over a slide by use of a pipette with arolling motion, fixed by flaming, and stainedaccording to direction for the Ziehl-Nielsenmethod outlined by the Committee on Bac-teriological Technic (1946). Prior to removal ofvegetative growth, any aerial mycelium wasremoved by wiping the surface of the growthwith a moist cotton pledget. Stained slides wereexamined under oil immersion at 1125 to 1800 x.

Optimal temperature range. Duplicate slants ofTPO agar and YE agar were inoculated with 0.2ml of a 48-hr TYE broth culture and incubatedat 18, 25, 28, 37, 44, and 55 C, respectively, for14 days. At the end of the incubation period,the amount of growth was estimated and ratedon a basis of +1 to +6. Cultures grown at 28 Cwere considered as a standard and recorded as+4.

Ability to produce antibiotic factors. The anti-biotic-producing capacity of each of the strainswas determined by the Waksman agar-streakmethod, with Pridham and Gottlieb basal agarcontaining 1% D-glucose and 0.1% yeast extract(Difco), and by cultivation of each strain inshaken-flask fermentations with the Pridhamand Gottlieb basal medium containing 1% D-glucose and 0.1% yeast extract and with amedium based on the medium A-4h of Warren,Prokop, and Grundy (1955) [soybean meal,1.5% (Staley's 4-S); commercial glucose, 1.5%(Cerelose); distillers' dried solubles, 0.5%(Curbay BG); glycerol, 0.25% wt/v; NaCl,0.5%; CaCO3, 0.1%; distilled water; pH (aftersterilization for 20 min at 121 C) 6.70 to 6.85].For the agar streak tests, the strains were culti-vated for 7 days at 28 to 30 C. Test bacteria,yeasts, molds, and streptomycetes were streakedat right angles to the streptomycete growth, andthe dishes were held for an additional 3 to 5

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days at 28 to 30 C. At that time zones of in-hibition were measured.

Shaken-flask media were inoculated with 1%(v/v) of a 48-hr TYE broth culture of thestreptomyeete. Seeded flasks were incubated ona rotary shaker at 28 to 30 C for 192 hr. At 48,96, 144, and 192 hr, samples were removedaseptically and filtered through cheesecloth.The filtrates were assayed against nine testorganisms by the paper-disc assay method. Thetest organisms used for the agar-streak determi-nations and the paper-disc assays were: Bacillussubtilis NRRL B-765; Staphylococcus aureusNRRL B-313; Escherichia coli NRRL B-766;Agrobacterium tumefaciens NRRL B-36; Erwiniacarotovora NRRL B-204; Xanthomonas phaseoliNRRL B-1460; Mycobacterium sp. NRRL B-692;Saccharomyces pastorianus NRRL Y-139; Can-dida albicans NRRL Y-477; and Mlucor raman-nianus NRRL 1839. For paper-disc assays, thetest organisms were grown in MI-7 agar (Pridhamet al., 1956), with the exception of 31. raman-nianus which was grown in MISA agar, a chemi-cally defined medium (Pridham et al., 1956).

RESULTS

Spore morphology and nature of spore surface.The six strains originating from the Berestnevand Krainsky cultures all formed spores thatwere elongated and ovoid. In unstained prepara-tions the spores measured 1.25 by 1.75 ,u. Inthe Drechsler impression preparations, theirdiameters approximated 1.5 ,u. The 11 otherstrains of S. albus that we have acquired alsoformed elongated, ovoid spores with thesedimensions. Electron micrographs of representa-tives of the 17 strains indicate that the sporesare smooth walled. The diameter of the aerialmycelium of the strains was approximately 1.5 ,

(Drechsler impression slide).Mlorphology of sporophores. All 17 strains of

S. albus were relegated to section Spira (Pridham,Hesseltine, and Benedict, 1958) after in situobservations of petri-dish cultures. The strainsform small, compact to extended, coiled chainsof spores. They occur on singly, oppositelvbranched, and sometimes verticillate branches.The verticillate aspect of these strains is quitedifferent from that of forms which occur in thegenus Streptoverticillium Baldacei, in that branch-ing from the main axial hyphae is irregular andnot equidistant. Also, the diameters of the axial

hyphae of streptoverticillia are much greaterthan those of S. albus. Sporophores typical of thesix Berestnev and Krainskv strains are illustratedin Fig. 1. A photomicrograph of another of the17 strains has been presented elsewhere (Pridhamand Lyons, 1961).

Color of aerial mycelium. Observations made ofthe Berestnev and Krainsky strains to determinethe color of their aerial mycelia are presented inTable 1. The colors of aerial mycelium of theremaining 11 strains differed in no respect fromthose exhibited by the six strains. With theISCC-NBS method of designating colors, thecolor of aerial mycelium would be: "263. white"and "73. pale orange yellow."

Color of reverse of cultures. Observations onthe color of the reverses of the six strains arealso presented in Table 1. In addition to theRidgway designations recorded in Table 1,reverses of some of the remainder of the 17strains of S. albus were Naples Yellow XVI,Pinkish Buff XXIX, and Honey Yellow XXX.With the ISCC-NBS method of designatingcolors, the reverses of the S. albus strains wouldbe designated as: 28. light yellowish pink; 29.moderate yellowish pink; 73. pale orange yellow;86. light yellow; 87. moderate yellow; 88. darkyellow; 89. pale yellow; 101. light greenish yellow;and 102. moderate greenish yellow.

Chromogenicity. None of the 17 strains ofS. albus formed a brown, deep brown, or blackdiffusible pigment when grown on IP agar slantcultures for 4 days at 28 to 30 C. A very faintyellow diffusible pigment was noted with two ofthe strains that were not descendants of theBerestnev and Krainskv cultures. Culturesgrown on Dietz L(-)-tyrosine-water agar slantsfor 14 days at 28 to 30 C produced either nodiffusible pigment or very faint yellow to veryfaint yellowish-orange diffusible pigments in thecase of the six Berestnev and Krainsky cultures.The remainder of the 17 strains showed somedifferences in color of diffusible pigment. Colorsranged from very faint yellow to reddish brown.Likewise, growth of the 17 strains in 12% plaingelatin for 14 days at 28 to 30 C gave either noevidence of diffusible pigment production orevidence of only a faint yellow-brown diffusiblepigment. It is concluded that the 17 strains arenonchromogenic within the limits of our defini-tion of chromogenicity.

Production of hydrogen sulfide. None of the 17

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FIG. 1. Morphology of fruiting bodies of Streptomyces albus grown for 14 days at 28 to 30 C. A, Czapek'ssolution agar, in situ; B, inorganic salts-starch agar, cellophane impression slide; C, Czapek's solution agar,cellophane impression slide; D, inorganic salts-starch agar, Drechsler impression slide.

strains of S. albus produced the bluish-blackcoloration of substrata indicative of productionof hydrogen sulfide, when cultivated on slants ofpeptone iron agar supplemented with 0.1%yeast extract for 10 days at 28 to 30 C. Readingswere made at 1 and 10 days.

Proteolytic activity. When tested by the threemethods used, all the 17 strains of S. albusliquefied gelatin. Tubes of 12% plain gelatinwere completely liquefied after 14 days at 28

to 30 C. Diameters of zones of liquefaction on theWaksman 15% gelatin dish cultures ranged from18 to 27 mm; zones of the six Berestnev andKrainsky strains ranged from 18 to 24 mm.Diameters of zones of liquefaction on the Waks-man 15% gelatin-1% starch dish cultures rangedfrom 16 to 22 mm; zones of the six Berestnev andKrainsky strains also ranged from 16 to 22 mmdiameter.

After cultivation of the 17 strains in skimmed

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TABLE 1. Colors of aerial mycelium and reverses of strains of Streptomyces albus grown for 14 days at28 to 30 C

Designation Medium* Color of aerial mycelium Color of reverseused

Czapek's solution agar

Inorganic salts-starch agarGlycerol-asparagine agar

Nutrient agarCzapek's solution agar

Inorganic salts-starch agar

Glycerol-asparagine agar

Nutrient agarCzapek's solution agarInorganic salts-starch agar

Glycerol-asparagine agarNutrient agarCzapek's solution agarInorganic salts-starch agar

Glycerol-asparagine agar

Nutrient agar

Czapek's solution agarInorganic salts-starch agarGlycerol-asparagine agarNutrient agarCzapek's solution agarInorganic salts-starch agarGlycerol-asparagine agarNutrient agar

Cretaceoust

CretaceousCretaceous

CretaceousCretaceousNear Pale Pinkish BuffXXIX

Near Pale Pinkish Cinna-mon XXIX

CretaceousCretaceousNear Pale Pinkish BuffXXIX

CretaceousCretaceousNo aerial mycelium formedNear Pale Pinkish Cinna-mon XXIX

No aerial myceliumformed

No aerial myceliumformed

CretaceousCretaceousCretaceousCretaceousCretaceousCretaceousCretaceousCretaceous

Cartridge Buff:XXX

Cream Color XVPale OchraceousBuff XVStraw Yellow XVIIvory Yellow XXXCartridge Buff XXX

Cream Buff XXX

Straw Yellow XVICartridge Buff XXXCream Color XVI

Light Buff XVStraw Yellow XVINear Marguerite Yellow XXXCream Color XVI

Light Buff XV

Massicot Yellow XVI

Cartridge Buff XXXCream Color XVIPale Ochraceous Buff XVStraw Yellow XVICartridge Buff XXXBarium Yellow XVICream Buff XXXStraw Yellow XVI

* Formation of spores was verified on Czapek's solution agar and inorganic salts-starch agar but noton glycerol-asparagine or nutrient agars.

t Cretaceous or chalk-white indicates that no color tabs, other than "white, " were equivalent to thecolor observed.

t Ridgway color designations representing the closest approximation that could be made.

milk for 14 days at 28 to 30 C, reproducible resultswere not obtained. Three of the 17 strains failedto peptonize milk. Reproducibility with duplicatetubes for three other strains was not achieved.Of the six Berestnev and Krainsky strains, twofailed to peptonize milk. Final pH's of the pep-tonized solutions ranged from 6.91 to 8.08. ThepH of control tubes was 6.60.

All 17 of the strains of S. albus cleared theGordon and Smith casein agar to about the same

extent after cultivation for 14 days at 28 to30 C.

It is concluded that the strains of S. albusstudied exhibit good to excellent proteolyticcapacity.

Utilization of carbon compounds. Apparentlythe 17 strains of S. albus exhibit a rather uniformcarbon utilization pattern (Table 2).

Production of acid from carbohydrates. Theresults were not as uniform as those obtained inthe carbon utilization study. Lactose was theonly carbohydrate that allowed uniform acidproduction by all the strains (Table 3).

Nitrate reduction. Some differences were noted

618-1

618-2

618-3

3004-1

3004-2

3004-3

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TABLE 2. Utilization of carbon compounds by strains of Streptomyces albus*

Nega-

cotrbo D-GlU- D-Xy- L-Arab- Rham- D-Fruc- Sucrose Lactose Maltose nose nitol tol SalicinDesignation uecotl;cose lose inose nose tose ns io onocarbon

618-1 - + ) (-) (-) ) (-) + + (-) + (-) +618-2 - + ( (-) (-) f (-) + + (-) + (-) +618-3 - + ® (-) (-) ) (-) + + (-) + (-) +3004-1 - + G (-) (-) (-) (-) + + (-) + (-) +3004-2 - + i (-) (-) 3 (-) + + (-) + (-) @3004-3 - + @ (-) (-) ) (-) + + (-) + (-) +CBS - + G (-) (-) i (-) + + (-) + (-) +

F/3-2 - + i (-) (-) 3 (-) + + (-) + (-) +C13Rf-1 - + + (-) (-) ) ( + + (-) + (-) +C13R5-2 - + + (-) (-) ( ( + + (-) + (-) +F/2 - + ) (-) (-) ( (-) + + (-) + (-) +A/7 - + > ( (-) + + (-) + (-) +298x - + i) (-) (-) (-) + + (-) + (-) +299x - + ) (-) (-) ® (-) + + (-) + (-) +FAL H123 - + 3 (-) (-) @ (-) + + (-) + (-) +618-4 - + + (-) (-) l (-) + + (-) + (-) +

* Pridham and Gottlieb basal agar, 10 days at 28 to 30 C. Symbols used: -, no growth; (-), faintgrowth. probably no utilization; (3, poor to fair growth; +, good growth and positive utilization.

in the relative abilities of the 17 strains to reducenitrate, particularly with the organic-basedmedium (Table 4).

Diastatic activtity. None of the 17 strains clearedinorganic salts-starch agar after incubation for14 days at 28 to 30 C. Addition of Lugol's iodineto these cultures changed the color of the sub-strate to an intense purplish blue indicative of astarch-iodine reaction. It is concluded that the17 strains possess little, if any, diastatic activity.

Tyrosine and xanthine decomposition. All 17of the strains of S. albus cleared the Gordon andSmith L(-)-tyrosine agar about the same aftercultivation for 14 days at 28 to 30 C. Likewise,all 17 of the strains decomposed xanthine, but tomarkedly varying degrees. Some strains gaveevidence of clearing the xanthine agar of Gordonand Mihm only directly beneath the vegetativegrowth. One of the Berestnev and Krainskystrains decomposed xanthine to this extent. Theother five gave readily detectable zones ofclearing around the growth.

Cultural characteristics on potato slants. The17 strains of S. albus all formed a grayish-whiteaerial mycelium after 14 days growth on whitepotato slants at 28 to 30 C. The plugs wereinitially (after 3 days) a purplish-gray color,

changing to a light brown after 14 days. Un-inoculated control plugs also assumed the lightbrown color after 14 days. The initial purplish-gray color of the plugs is attributed to decompo-sition of the potato by the organism and is similarto the coloration noted with potatoes just begin-ning to rot.

Acid fastness. All 17 strains of S. albus werenonacid fast when the Ziehl-Nielsen techniquewas applied to vegetative growth.

Optimal temperature range. Our studies indi-cate that the optimal temperature range forgrowth of the 17 strains of S. albus is 25 C through44 C. Although cultures incubated at 18 C gavegood growth, it was somewhat less than thatobtained within the optimal range. Culturesincubated at 55 C gave significantly less growththan those incubated within the optimal range.Uniform results were obtained with all strainswithin the range 25 C through 37 C. At 44 C,strain differences became apparent, and readingsranged from + 3 to + 5.

Antibiotic activity. When tested for capacityto produce antibiotics by the agar-streak andshaken-flask methods, the six Berestnev andKrainsky strains gave no evidence of producingantibacterial or antifungal factors. The remaining

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1 strains of S. albus produced antibiotic ac-tivity against at least two of the ten differenttest organisms. In some cases, as many as nineof the test organisms were affected.

Preliminary paper chromatographs of culturefiltrates from these strains suggest that they allproduce at least two antibacterial factors incommon. Very few evidences of antagonismwere noted when the 17 strains were studiedsimultaneously in cross-inhibition studies. Theresults suggest the possibility that the sixBerestnev and Krainsky strains may, at onetime, have possessed the capacity to produce thesame antibiotic factors elaborated by the re-maining 11 strains. The results also strengthenthe suspicion that common factors are producedby the remaining 11 strains.From the results of these studies we have

concluded that the most practical course totake is that of proposing the designation ofstrain ATCC 3004 (IMRU 3004) as the neotypestrain of S. albus. To that end a description ofthis strain is presented here.

TABLE 3. Production of acid from carbohydrates bystrains of Streptomyces albus*

Designation used D-AMannose D-Xylose Maltose Lactose

618-1 + + + + (-)G + +618-2 + + @ @ (-)(-) + +618-3 + ) ) )(-) i) + +3004-1 + + + + +3004-2 (-)(-) - - 3 (-) + +3004-3 t) + 0 + 0 (-) + +CBS + + (-)(-)(-)(-) + +F/3-1 (( + +F/3-2 0 + i + (-)(-)+ +C13R5-1 (-)(-) i 0 0 0 + +C13R5-2 _ _ - _ (-)(-) + +F/2 0 0 (-)(-)(-)(-) + +A/7 + + (-)(-)(-)(-) + +298x + + (-)(-) (-)(-) + +299x + + (-)(-) (-)(-) + +FAL H123 0 0 - _ 0(-) + +618-4 i 0 0 0 (-)- + +

* Gordon and Smith 1955 method, 14 days at 28to 30 C. Symbols used: +, marked acid produc-tion, indicator completely changed; i), moderateacid production, indicator changed except forlower portions of slant; (-), poor acid production,indicator changed slightly; -, no acid production,indicator unchanged. Results presented for twoindividual tubes for each strain and each carbo-hydrate.

TABLE 4. Reductiont of nitrate by strains ofStrepto7inyces albus*

Designation used

618-1618-2618-33004-13004-23004-3CBSF/3-1F/3-2C13R5-1C13R5-2F/2A/7298x299xFAL H123618-4

Chemicallydefined broth

Tube 1 Tube 2

(-+++

+

±

+

++

Organic-based broth

Tube 1 Tube 2

0

0

(-)

0+

0

(-

+

* Symbols used: -, nitrate not reduced; (-),very slight pink color produced on addition ofreagents; 03, pale red color produced on additionof reagents; +, deep red color produced on addi-tion of reagents. Cultivated on organic-based andchemically defined media (14 days at 28 to 30 C,a-dimethylnaphthylamine-sulfanilic acid, Zn dustreagents).

Streptomyces albus (Rossi-Doria) Waksmanand Henrici, Strain ATCC 3004 (IMRU 3004).

Spore ornamentation, shape, and dimensions:Smooth-walled, catenulate, elongated, ovoid,ca. 1.25 by 1.75 ,u.Morphology of sporophores: Compact or

extended coiled chains of spores occurring onrelatively short branches on rather long axialhyphae. Branches occur singly, oppositelybranched, and sometimes in verticils which aredifficult to detect.

Color of sporulating aerial mycelium: Cre-taceous (chalk-white) after 14 days growth at28 to 30 C on Czapek's solution agar, inorganicsalts-starch agar, and glycerol-asparagine agar.Cultures may also form near Pale Pinkish Buff17" to Pale Pinkish Cinnamon 15" (Ridgway,Plate XX1X) colored aerial mycelium (ISCC-NBS designation: 73. pale orange yellow),giving a pink tinge to an otherwise cretaceousappearance.

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NEOTYPE STRAIN OF S. ALBUS

Color of reverse of culture: Tints and shadesof yellow, yellowish pink, orange yellow, andgreenish yellow; Pale Ochraceous Buff 15'(Ridgway, Plate XV; ISCC-NBS designation:28. light yellowish pink), after 14 days growthon glycerol-asparagine agar at 28 to 30 C.

Chromogenicity: Nonchromogenic; forms nodiffusible pigment when cultivated in tryptone-yeast extract broth with shaking for 48 hr at28 to 30 C, in 12% plain gelatin for 14 days at28 to 30 C, on 15% gelatin or 15% gelatin-1%starch for 14 days at 18 C, on white potato plugsfor 14 days at 28 to 30 C, on 0.1% L( -)-tyrosine-water agar for 14 days at 28 to 30 C, or on in-ternational peptone agar for 4 days at 28 to 30 C.

Production of hydrogen sulfide: Negative;no bluish-black coloration of substrate whencultivated for 10 days at 28 to 30 C on peptoneiron agar supplemented with 0.1% yeast extract.

Proteolysis: Positive; 12% plain gelatin lique-fied after 14 days at 28 to 30 C, 15% plaingelatin or 15% plain gelatin-1% starch liquefiedafter 15 days at 18 C, skim milk peptonized after14 days at 28 to 30 C, and casein agar clearedafter 14 days at 28 to 30 C.

Amylolysis: Negative; inorganic salts-starchagar not cleared, and negative Lugol iodinereaction after 14 days at 28 to 30 C.

Utilization of carbon compounds: On Pridhamand Gottlieb basal agar, after 10 days incubationat 28 to 30 C, good growth is shown on D-glucose,lactose, maltose, D-mannitol, and salicin. Fairgrowth on D-xylose. Poor growth on D-fructose.Very faint growth on L-arabinose, rhamnose,sucrose, raffinose, and i-inositol.

Utilization of sucrose in Czapek's solutionagar: Negative; very poor growth.

Antagonistic properties: None.Characteristic features: Catenulate, smooth-

walled, ovoidal spores; coiled sporophores;cretaceous (often with tinges of pink) sporulatingaerial mycelium; nonchromogenic; inability tohydrolyze starch; inability to form antibioticfactors.

Source or habitat: A descendant of a culture,originally isolated by Berestnev and labeled"albus"; obtained successively through theKral, Pribram, and Institute of Microbiology,Rutgers University collections. Presumablyfirst isolated from straw in Prague, Czechoslo-vak.a about 1897.

Optimal temperature range: Good vegetative

growth and formation of aerial mycellium at25 C through 44 C; fair at 18 C; very poor at55 C.

Related strains: Strains of streptomycetesthat are closely related to this strain have beenreported as isolated from or near beehives, fromcases of dental caries, and from cases of humanactinomycosis. Strain ATCC 3004 is maintainedat the American Type Culture Collection, Wash-ington, D. C., as ATCC 3004, and at the In-stitute of Microbiology, Rutgers University,New Brunswick, New Jersey, as IMRU 3004.

DISCUSSION

The 17 strains labeled "albus" that we havestudied were originally relegated in our filingsystem to a group characterized by coiled sporo-phores, white aerial mycelium, inability to pro-duce hydrogen sulfide, ability to utilize D-xylose,and inability to utilize L-arabinose and rhamnose.This filing system apparently is valid for thisparticular group, as evidenced by the results ofthis current study. Except for ability to produceantibiotic factors, all of the strains have beenshown to be essentially identical. However, ifthe criteria outlined here are used, it would bedifficult to identify any given strain within thegroup without resorting to study of antibioticproduction.We believe that the Waksman and Henrici

description of S. albus should be accepted, andthat our proposal to designate strain ATCC 3004(IMRU 3004) as the neotype strain will placestreptomycete taxonomy on a firmer foundation.There yet remains the issue of clarification ofthe status of the type species of the genus No-cardia, i.e., Nocardia farcinica Trevisan.We should like to emphasize that designation

of strain ATCC 3004 (IMRU 3004) as the neo-type strain of S. albus, or characterization of the17 strains we have studied, does not establishthe limits of the species. Indeed, we believe thatthe species limits are much broader than areexhibited by the aggregate of the 17 strains.These 17 probably represent one, or possiblytwo, subspecies within the species.

ACKNOWLEDGMENTS

Determining the histories of strains ATCC618 and ATCC 3004 (IMRU 3004) has requiredconsiderable correspondence and discussion.We are grateful to the following for their as-

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sistance in this regard: S. A. Waksman, Instituteof Microbiology, Rutgers University; W. A.Clark, Director, American Type Culture Col-lection; and G. A. de Vries, Centraalbureau voorSchimmelcultures, Baarn, Holland. We shouldlike also to thank R. E. Buchanan and E. F.Lessel, Jr., Iowa State University, Ames, Iowa,for their assistance in preparation of this man-uscript.

Supported, in part, by a grant from the Sub-committee on Taxonomy of the Actinomycetesof the Committee on Taxonomy of the AmericanSociety for Microbiology.

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