Amycolatopsis Alba Sp. Nov., Isolated from Soil
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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 species 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 Amycolatopsis alba. 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.