INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,OCt. 1993, p. 715-720 Vol. 43, No. 4 0020-7713/93/040715-06$02.00/0 Copyright 0 1993, International Union of Microbiological Societies

Amycolatopsis alba sp. nov., Isolated from Soil

FREDERICK P. MERTZ* AND RAYMOND C. YAO The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

A new Amycolutopsis isolated from soil produces a new glycopeptide antibiotic related to vancomycin. Traditional taxonomic methods and contemporary fatty acid analysis techniques were used to establish the position of this species. The hyphae fragment extensively when the organism is cultured in liquid media. The organism is characterized by white aerial hyphae that bear long chains of cylindrical conidia. The reverse side is yellowish brown; a faint light brown soluble pigment is occasionally produced. The organism has a type N cell wall (meso-diaminopimelic acid), a type A whole-cell sugar pattern, and a type PI1 phospholipid pattern. Mycolic acids are not present in whole-cell hydrolysates. The major menaquinone is MK3(H4); there is also a minor amount of MKd(H4). The name proposed for this new species is Amycolutopsis &a. The type strain is strain A83850 (= NRRL 18532).

Taxonomists have long been aware that many organisms Media. Cells were grown on a rotary shaker (250 rpm) for assigned to the genus Nocardia do not have the character- 72 h at 30°C in a medium containing 30 g of Trypticase soy istic features of this genus (i.e., a type IV cell wall and the broth (BBL, Becton Dickinson, Cockeysville, Md.), 3 g of

presence of mycolic acids). Recent work by Lechevalier et yeast extract, 2 g of MgSO, a 7H20, 5 g of glucose, and 4 g al. (15) on the genus Nocardia has resulted in the reassign- of maltose in 1,000 ml of deionized water. The cells were ment of a number of its species that lack mycolic acids to harvested by centrifugation, washed twice with sterile wa- two new genera, the genera Amycolata and Amycolatopsis. ter, and then used as an inoculum. Cultures were grown on The genus Amycolatopsis is characterized by small, frag- International Streptomyces Project (ISP) media 2 to 9 (20), menting, nonmotile vegetative hyphae, a type IV cell wall actinomycete isolation agar (Difco Laboratories, Detroit, (meso-diaminopimelicacid, glutamic acid, alanine, muramic Mich.), American Type Culture Collection medium 172 (6), acid, glucosamine, galactose), and the absence of both Bennett’s agar (22), chitin agar (17), Czapek’s solution agar madurose and mycolic acids. The menaquinones are pre- (22), Emerson’s agar (22), glucose-asparagine agar (22), dominantly MK-9(H2) and MK-9(H4), and the phospholipid glycerol-glycine agar (22), nutrient agar (22), tomato paste- type is type PI1 (phosphatidylethanolamine). The guanine- oatmeal agar (22), and tap water agar (8). plus-cytosine content of the DNA ranges from 66 to 69 Cultural observations. Characteristics were recorded after mol%. 21 days of incubation at 30°C by using methods recom- Strain A83850T (T = type strain) was isolated from a mended by Shirling and Gottlieb (20). The ISCC-NBS Cen- mixture of unknown soils during the normal course of troid Color Charts (21) were used to determine color desig- isolating organisms for an antibiotic-screening program. This nations. organism has the properties of the genus Amycolatopsis; Physiological tests. Methods of Gordon et al. (8) were used however, when it was evaluated by traditional and contem- for the physiological tests. H2S production was measured porary fatty acid analysis methods, it was not similar to the with lead acetate strips (Difco). Testosterone degradation six species previously placed in this genus. On the basis of its chemical, morphological, and physiological properties, was determined with 0.1% (wtkol) testosterone in nutrient we consider strain A83850T a member of a new species, for agar. Allantoin decomposition was measured by the method which we propose the name . of Kurup and Schmitt (11). NaCl tolerance was tested at concentrations between 1 and 15%. The growth temperature range was determined by using temperatures between 5 and MATERIALS AND METHODS 50°C. Catalase, phosphatase, and urease activities were determined by methods of Blazevic and Ederer (4). Bacterial strains. Strain A83850T was isolated from soil by Resistance to antibiotics. Antibiotic disks (Difco) were using the dilution plating technique. Type strains Amyco- placed onto the surfaces of ISP medium 2 agar plates that latopsis orientalis ATCC 19795, A. orientalis subsp. lurida were seeded with 2% vegetative growth of the organism to ATCC 14930, Amycolatopsis fastidiosa ATCC 31181, and be tested. The preparations were incubated at 30°C for 1 Amycolatopsis mediterranei ATCC 13685 were obtained from the Lilly culture collection. Amycolatopsis sulphurea week. Resistance was scored as positive when no zone of ATCC 27624 and Amycolatopsis rugosa ATCC 43014 were inhibition was observed and as negative when a zone of obtained from the American Type Culture Collection, Rock- inhibition was observed. ville, Md. Data for Amycolatopsis azurea were obtained Chemotaxonomy. Whole-cell hydrolysates prepared from from reference 9. The following phages were tested against washed, lyophilized, 72-h vegetative growth were examined strain A83850T: Streptomyces phages FP4 and FP22 (6a) and by the methods of Becker et al. (3) and Lechevalier and A. orientalis phages OP1, OP2, OP3, OP4, OP5, and OP6 Lechevalier (14). Mycolic acid determinations were based (obtained from K. L. Cox, M. McHenney, and R. H. Baltz). on techniques described by Minnikin et al. (19). Phospholip- ids were extracted with CHCl,, chromatographed on Silica Gel 60 F,, thin-layer chromatography plates in CHC1,- methanol-concentrated NH40H (200: 120: 15, vol/vol) along * Corresponding author. with phospholipid standards (2), and visualized with a 10%

715 716 MERTZ AND YAO Im. J. SYST.BACTERIOL.

FIG. 1. Scanning electron micrograph of strain A83850T grown on ISP medium 3 for 14 days at 30°C. Magnification, x 10,000. Bar = 1.0 Pm.

ethanolic molybdophosphoric acid spray (7). Menaquinones RESULTS AND DISCUSSION were extracted with chloroform-methanol (2: 1, vol/vol) and partially purified by thin-layer chromatography. Menaqui- Chemotaxonomy. Strain A83850T hydrolyzed whole cells none composition was determined by high-performance liq- contain rneso-diaminopimelic acid. The diagnostic sugars in uid chromatography by using a Bondapak C,, column and a the whole-cell extracts are arabinose and galactose. The CH,CN-(CH,),CHOH (3:1, vol/vol) eluent system. Each phospholipids present are phosphatidylethanolamine, phos- isoprenolog was detected at 254 nm and at a temperature of phatidylglycerol, and phosphatidylinositol. Thus, strain 40°C. The methods used were those of Collins (5) and A83850T contains rneso-diaminopimelic acid and has a type Kroppenstedt (10). A whole-cell sugar pattern (13) and a type PI1 phospholipid Fatty acid analysis. Fatty acid methyl esters were analyzed pattern (16). Mycolic acids were not present in whole-cell by gas-liquid chromatography with a model 5898A comput- hydrolysates. The major menaquinone detected was MK- er-controlled gas-liquid chromatography system (Hewlett 9(H,); there was also a minor amount of MK-8(H4). Packard Co., Palo Alto, Calif.). Fatty acid methyl esters Morphological characteristics. Strain A83850T fragmented were made from lyophilized whole cells grown under iden- extensively when it was grown in liquid media. The resulting tical conditions for 72 h at 30°C. The methods used were “nocardioform” units were not observed when strain those described by Miller and Berger (18). The dendrogram A83850T was grown on solid media. On ISP medium 2, strain was based on Euclidian distance and was computer gener- A83850T forms circular colonies that are 5 to 7 mm in ated. The principal-component analysis was two dimen- diameter and have raised centers. The colonies have wrin- sional and also computer generated (1). kled surfaces and are covered with fine white aerial hyphae. Scanning electron microscopy. Cultures were fixed in os- The hyphae are segmented into long strands of conidia that mium tetroxide vapors for 20 h, dehydrated, rotary shad- are arranged in a typical cobweb morphology characteristic owed with gold-palladium, and then viewed with an Etec of some nonstreptomycete actinomycetes (22). The conidia scanning electron microscope. are cylindrical and have a smooth surface, and the average VOL.43, 1993 AMYCOLATOPSIS ALBA SP. NOV. 717

TABLE 1. Cultural characteristics of A83850T after 21 days of incubation at 30°C Reverse Aerial Soluble Medium Growth Spore colof colof growth pigment ISP medium 2 Abundant 54. brO Abundant 263. White Light brown ISP medium 3 Fair 90. gy. Y Fair 263. White None ISP medium 4 Good 55. s. Br Good 263. White None ISP medium 5 Abundant 76. 1. yBr Abundant 263. White None Actinomycete isolation agar (Difco) Abundant 71. m. OY Abundant 263. White Light brown American Type Culture Collection medium 172 Good 89. p. Y. Good 263. White None Bennett’s agar Abundant 76. 1. yBr Abundant 263. White None Calcium malate agar Abundant 89. p. Y Abundant 263. White None Chitin agar Fair 93. y Gray Poor 263. White None Czapek’s agar Good 70. 1. OY Abundant 263. White None Emerson’s agar Abundant 72. d. OY Good 263. White Light brown Glucose-asparagine agar Abundant 70. 1. OY Poor 263. White None Glycerol-glycine agar Abundant 77. m. yBr Abundant 264. 1. Gray None Nutrient agar Good 70. 1. OY Fair 263. White Light yellow Tomato paste-oatmeal agar Abundant 77. m. yBr Abundant 263. White None Tap water agar Fair 93. yGray Poor 263. White None Yeast extract-dextrose agar Good 76. 1. yBr None None Light brown For color designations see reference 21. size is 1.3 by 0.4 pm (Fig. 1). No sporangia or motile cells other available strains belonging to the genus Amycolatop- were observed. sis. Strain A83850T is significantly different from all of the Cultural characteristics. Strain A83850T grew well on both other strains. The differences are discussed below. complex and defined -media. Aerial hyphae were produced Levels of biochemical similarity were measured by con- on most of the media. The aerial spore mass color was white. structing a table of similarity coefficients based on biochem- The reverse side was yellowish brown. No distinctive pig- ical data taken from Table 2 and from previous studies (9, mentation was observed. A faint light brown soluble pigment 15). The coefficient of Jaccard and the simple matching was produced in a few media. The cultural characteristics coefficient were used (12); this table of coefficients is shown are shown in Table 1. in Table 3. Physiological characteristics. The physiological character- Two broad-host-range Streptomyces bacteriophages and istics of strain A83850T and of other Amycolatopsis species six Amycolatopsis bacteriophages (see above) were tested are shown in Table 2 and are described below. Strain for their ability to form plaques on A83850T. The two A83850T was resistant to the following antibiotics, as indi- Streptomyces bacteriophages and one of the Am colatopsis cated by the absence of any zones of inhibition around bacteriophages did not form plaques on A83850ry . The other antibiotic disks containing the antibiotics: bacitracin (10 U), Amycolatopsis bacteriophages did not form plaques on neomycin (30 pg), oleandomycin (15 pg), rifampin (5 pg), A83850T but produced lysis from without at high multiplic- and tetracycline (30 pg). It was inhibited by cephalothin (30 ities of the bacteriophages. This killing from without shows pg), gentamicin (10 pg), lincomycin (2 pg), penicillin (10 U), that the bacteriophages were able to attach and inject their streptomycin (10 pg), tobramycin (10 pg), and vancomycin DNA into A83850T and suggests that strain A83850T is (30 pg). related to the genus Amycolatopsis. Successful bacterio- Identity of genus. The genus Amycolatopsis as described phage infections were not established, as evidenced by the by Lechevalier et al. (15) contains organisms that form lack of plaque formation; this may have been due to restric- branching vegetative hyphae with a tendency to fragment. tion expressed by A83850T. When formed, the aerial hyphae segment into sporelike A fatty acid analysis was performed with A83850T and all structures. No endospores, sheaths, synnemata, sporangia, of the available Amycolatopsis strains. A comparison of the or sclerotia are present. The organisms are gram positive, levels of fatty acids is shown in Table 4. The data reveal not acid fast, mesophilic, aerobic, catalase positive, and considerable quantitative differences between A8385OT and nonmotile and have type IV cell walls, and a type A the other species in the amounts of 14:O iso, 15:O iso, 150 whole-cell sugar pattern; no madurose or mycolates are anteiso, 16:O iso, 17:l C, and 17:O fatty acids present. The present. The menaquinones are predominantly MK- dendrogram in Fig. 2 shows the relationship of A83850T to 9(H2,H4).The phospholipid pattern is type PII. The guanine- the other Amycolatopsis species based on Euclidian dis- plus-cytosine content of the DNA ran es from 66 to 69 tances. Strains exhibiting levels of relatedness below 10.00 mol%. Soil actinomycete strain A83850g. is a fragmenting are interpreted as members of the same species. A83850T is strain that is characterized by the properties described related to a cluster containing A. sulphurea, A. orientalis, above. These chemotaxonomic characteristics plus its cul- and A. orientalis subsp. lurida at a level of 28.00, suggesting tural and morphological properties place strain A83850T in that A83850T is a distinct species. the genus Amycolatopsis. Previously, the following six spe- Principal-component analysis is a branch of multivariate cies and one subspecies of this genus have been described: statistics that deals with internal relationships of a set of A. amrea, A. fustidiosa, A. meditewanei, A. orientalis, A. variables (1). A two-dimensional principal-component plot orientalis subsp. lurida, A. rugosu, and A. sulphurea. based on fatty acid data is shown in Fig. 3. The values are Identity of species. The cultural, physiological, morpholo the degrees of separation between the strains involved. An ical, phage, and fatty acid characteristics of strain A83850g; examination of this principal-component plot shows that were determined and compared with the characteristics of A83850T lies at a considerable distance from A. sulphurea, 718 MERTZ AND YAO INT. J. SYST.BACTERIOL.

TABLE 2. Differential characteristics of strain A83850T and Amycolatopsis species and subspecies A. orientalis Characteristic ::& A. orientalis subsp. lur- A. fastidiosa A. rugosa A. mediterranei A. sulphurea A. azurea ida Decomposition of: Hypoxan thine + + - Xanthine + + - Decarboxylation of Benzoate - - - Citrate + + + Production of: Nitrate reductase + + + Amylase ND Urease + + ND Phosphatase + - ND Resistance to: Lysozyme broth ND 5% NaCl + Growth at: 10°C + + + 45°C - - ND Aerial mycelium + + + Color of aerial mycelium White + + + Yellowish green - - - Pink + Blue + Soluble pigment + Color of soluble pigment Yellow - Brown - Blue + Acid produced from: Adonitol + + + L- Ar abinose + + + Cellobiose + + + Dextrin + + ND meso-Erythritol + + + D-Galactose + + + Inositol + + + Lactose + + + Maltose + + ND D-Mannitol + + + Me1 i b i o s e + - ND a-Methyl-D-glucoside + + ND Raffinose - - + L-Rhamnose + - + Salicin + + ND Sucrose + + + D-Xylose + + ND " ND, not done. V, variable results.

A. orientalis, A. orientalis subsp. lurida, A. rugosa, A. TABLE 3. Similarity coefficients of A8385OT and seven fastidiosa, and A. mediten-anei. This lack of clustering is similar taxa" supporting evidence that these organisms are distinct spe- cies. Table 2 shows characteristics that differentiate strain Strain A83850T 100 100 A83850T and otherhzycolatopsis species. Strain A83850T is A. orientalis 80 74 similar in some ways to both A. orientalis and A. orientalis A. orientalis subsp. lurida 80 73 subsp. lurida. However, it differs in nitrate reductase pro- A. meditemanei 74 63 duction, growth temperatures, and the utilization of dextrin, A. azurea 67 63 raffinose, L-rhamnose, and sucrose. A. rugosa 55 42 All of the data described above indicate that A83850T is a A. sulphurea 55 39 new species belonging to the genus Amycolatopsis. We A. fastidiosa 32 19 propose the name Arnycolatopsis alba sp. nov. for this The simple matching coefficient (Ssm)and the Jaccard coefficient (Sj) are organism. used to express levels of biochemical similarity. VOL.43, 1993 AMYCOLATOPSIS ALBA SP. NOV. 719

TABLE 4. Fatty acid compositions of strain A83850T and six Amycolatopsis species and subspecies

% Fatty acid in: Fatty acid" Strain A. orientalis orientalis subspaA. lurida A. fastidiosa A. rugosa A. mediten-anei A. sulphurea A838SOT 14:O is0 2.85 9.52 6.82 3.09 1.20 3.68 15:O is0 29.97 12.39 14.45 7.21 1.14 9.27 9.28 15:O anteiso 6.64 1.74 2.24 1.35 4.95 15:l B 1.28 0.72 1.59 1.99 15:O 6.02 4.69 5.85 0.73 1.18 3.09 3.35 16:l is0 H 11.09 17.88 2.25 16:O is0 9.27 30.14 27.87 56.70 29.91 44.86 21.01 16:O 7.84 7.60 5.29 0.56 3.39 0.97 17.12 16:l cis-9 3.07 1.34 10.42 1.25 3.85 17:l is0 G 3.06 1.50 17:O is0 2.75 1.43 1.30 1.29 2.56 2.70 2.01 17:O anteiso 3.77 1.37 1.67 0.80 3.34 4.04 5.56 17:l B 4.18 4.25 7.79 0.65 4.83 6.13 1.84 17:l C 0.90 5.06 6.16 15.81 18.07 8.95 7.03 17:O 18.58 12.84 10.95 1.10 2.86 9.34 18:l is0 F 1.28 0.85 5.60 18:l cis-9 1.43 18:O is0 1.13 18:O 1.47 1.72 1.48 3.21 B, C, F, G, and H indicate double bond positions or configurations that are not known.

Description of hycolutopsk alba sp. nov. Amycolatopsis trate. Strain A83850T utilizes allantoin, calcium malate, alba (A.my.co.la.top' sis. M.L. fem. n.; al'ba. L. fem. adj. casein, elastin, hippurate, and L-tyrosine, but is not able to alba, white, referring to the white aerial hyphae). Cells are degrade adenine and guanine. Skim milk is hydrolyzed but aerobic, nonmotile, not acid fast, gram positive, filamentous, not peptonized. Testosterone is very weakly hydrolyzed. and differentiated into substrate and aerial hyphae with a Catalase, esculinase, gelatinase, and H,S are produced. diameter of 1.0 pm. The vegetative hyphae have a tendency Strain A83850T does not produce melanoid pigments and is to fragment when the organism is grown in liquid culture. not able to survive at 50°C for 8 h. A83850T grows at The aerial hyphae are white, and the vegetative mycelium is temperatures between 15 and 37°C. Strain A83850T conzhy- yellowish brown. A faint soluble pigment is produced on tains meso-diaminopimelic acid and has a type A whole-cell some media. Cylindrical, smooth spores are formed with a sugar pattern and a type PI1 phospholipid pattern. Mycolic typical cobweb morphology. Acid is produced from the acids are not present. The major menaquinone detected is following carbohydrates: D-arabinose, D-fructose, D-glu- cose, glycerol, D-mannose, D-ribose, and trehalose. Acid is MK-9(H4); there is also a minor amount of MK-8(H4).Strain not produced from cellulose, dulcitol, ethanol, glycogen, A83850T differs from the other Amycolatopsis species in inulin, melezitose, D-sorbitol, L-sorbose, and xylitol. The fatty acid composition (Table 4) and in morphological, following carbohydrates are utilized when ISP medium 9 is cultural, and physiological characteristics (Table 2). the basal medium: D-fructose, D-galactose, D-glucose, inos- Type strain. The type strain is A83850 (= NRRL 18532). This itol, D-mannitol, raffinose, salicin, and D-xylose. L-Arabi- strain was isolated from soil. The species description is based nose, L-rhamnose, and sucrose are not utilized. Assimilates on a single strain and thus serves as the type strain description. acetate, butyrate, formate, lactate, malate, propionate, pyruvate, and succinate, but not oxalate, mucate, and tar-

1.070 A. rugosa A83850 ..... - 2.698 A. sulphurea ..... A. orientalis lurida --..--1 - 6.465 A. orientalis -----I- 10.233 A. rugosa ..... A. sulphurea A. fastidiosa ----. - 14.000 A. mediterranei ------17.768 - 21535 *A. orientalis lurida I I I 1 1 I A. orientalis @A.fastidiosa 0.00 I 8.50 1 17.01 I 25.51 I 34.0 - 25.303 4.25 12.76 21.26 29.76 - 1.929 21.922 45.773 Euclidian Distance FIG. 3. Principal-component plot based on fatty acid data from FIG. 2. Dendrogram of strain A83850T and six Amycolutopsis strain A83850T and six Amycolutopsis species and subspecies. x species and subspecies. axis, principal component 1; y axis, principal component 2. 720 MERTZ AND YAO INT. J. SYST.BACTERIOL.

ACKNOWLEDGMENTS tions. Int. J. Syst. Bacteriol. 32292-295. 10. Kroppenstedt, R. M. 1985. Fatty acid and menaquinone analysis We thank Sandy White, Eli Lilly & Co., Indianapolis, Ind., for of actinomycetes and related organisms, p. 173-199. In M. her skillful technical assistance in obtaining the scanning electron Goodfellow and D. E. Minnikin (ed.), Chemical methods in micrographs and Patti Treadway, Eli Lilly & Co., for performing the bacterial systematics. Academic Press, New York. phage plating experiments. Menaquinone determinations were per- 11. Kurup, P. Y., and J. A. Schmitt. 1973. Numerical of formed by J. S. Ruan, Academia Sinica, Beijing, People’s Republic Nocardia . Can. J. Microbiol. 19:1035-1048. of China. 12. Kurylowicz, W., A. Paszkiewicz, W. Woznicka, W. Kurzat- kowski, and T. Szulga. 1975. Numerical taxonomy of Strepto- REFERENCES mycetes, p. 37. Polish Medical Publishers, Warsaw. 1. Alderson, G. 1985. The application and relevance of nonhierar- 13. Lechevalier, M. P., and H. Lechevalier. 1970. Chemical compo- chic methods in bacterial taxonomy, p. 227-263. In M. Good- sition as a criterion in the classification of aerobic actino- fellow, D. Jones, and F. G. Priest (ed.), Computer-assisted mycetes. Int. J. Syst. Bacteriol. 20:435443. bacterial systematics. Academic Press, New York. 14. Lechevalier, M. P., and H. Lechevalier. 1980. A university 2. Ames, G. F. 1968. Lipids of Salmonella typhimurium and laboratory approach, p. 227-233. In A. Dietz and D. W. Thayer Escherichia coli: structure and metabolism. J. Bacteriol. 95: (ed.), The chemotaxonomy of actinomycetes. Society for Indus- 833-842. trial Microbiology Special Publication no. 6. Society for Indus- 3. Becker, B., M. P. Lechevalier, R. E. Gordon, and H. E. trial Microbiology, Arlington, Va. Lechevalier. 1964. Rapid differentiation between Nocardia and 15. Lechevalier, M. P., H. Prauser, D. P. Labeda, and J. S. Ruan. Streptomyces by paper chromatography of whole-cell hydroly- 1986. Two new genera of nocardioform actinomycetes: Amyco- sates. Appl. Microbiol. 12:421423. lata gen. nov. and Amycolatopsis gen. nov. Int. J. Syst. 4. Blazevic, D. J., and G. M. Ederer. 1975. Principles of biochem- Bacteriol. 36:29-37. ical tests in diagnostic microbiology. John Wiley & Sons, Inc., 16. Lechevalier, M. P., A. E. Stern, and H. A. Lechevalier. 1981. New York. Phospholipids in the taxonomy of actinomycetes, p. 111-116. In 5. Collins, M. D. 1985. Isoprenoid quinone analysis in bacterial K. P. Schaal and G. Pulverer (ed.), Actinomycetes. Gustav classification and identification, p. 267-287. In M. Goodfellow Fischer Verlag, New York. and D. E. Minnikin (ed.), Chemical methods in bacterial sys- 17. Makkar, N. S., and T. Cross. 1982. Actinoplanetes in soil and on tematics. Academic Press, New York. plant litter from freshwater habitats. J. Appl. Bacteriol. 52:209- 6. Cote, R., P.-M. Daggett, M. J. Gantt, R. Hay, S.-C. Hay, and P. 218. Pienta. 1984. ATCC media handbook, 1st ed. American Type 18. Miller, L., and T. Berger. 1985. Bacterial identification by gas Culture Collection, Rockville, Md. chromatography of whole cell fatty acids. Hewlett-Packard 6a.Cox. K. L.. and R. H. Baltz. 1984. Restriction of bacterioDhage Application Note 228-41. Hewlett-Packard Co., Avondale, Pa. plaque formation in “Streptomyces” species. J. Bacterioi. 16: 19. Minnikin, D. E., I. G. Hutchinson, and A. B. Caldicott. 1980. 499-504. Thin-layer chromatography of methanolysates of mycolic acid- Dittmer, J. C., and R. L. Lester. 1968, A simple, specific spray containing . J. Chromatogr. 188221-233. for the detection of phospholipids on thin-layer chromatograms. 20. Shirling, E. B., and D. Gottlieb. 1966. Methods for character- J. Lipid Res. 5126-127. ization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313- Gordon, R. E., D. A. Barnett, J. E. Handerhan, and C. Pang. 340. 1974. Nocardia coeliaca, Nocardia autotrophica, and the no- 21. U. S. Department of Commerce National Bureau of Standards. cardin strain. Int. J. Syst. Bacteriol. 24:54-63. 1958. ISCC-NBS centroid color charts, standard sample no. Henssen, A., H. W. Kothe, and R. M. Kroppenstedt. 1987. 2106. U. S. Department of Commerce, Washington, D.C. Transfer of Pseudonocardia azurea and Pseudonocardiafastid- 22. Waksman, S. A. 1961. The actinomycetes, vol. 2, p. 328-334. iosa to the genus Amycolatopsis, with emended species descrip- The Williams & Wilkins Co., Baltimore.