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Micromonospora Olivasterospora Sp. Nov INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1983, p. 107-112 Vol. 33. No. 1 0020-771 3/83/0101 O7-06$02.00/0 Copyright 0 1983, International Union of Microbiological Societies Micromonospora olivasterospora sp. nov. ISAO KAWAMOTO,* MITSUYOSHI YAMAMOTO, AND TAKASHI NARA Tokyo Research Laboratory, Kyowa Hukko Co., 3-6-6 Asahimachi, Machidashi, Tokyo 194, Japan A new species of bacteria is described, for which we propose the name Micromonospora olivasterospora. This organism produces new aminoglycoside antibiotics (the fortimicin complex) and is characterized by its olive black spore layer, spores with blunt spiny surfaces, an olive green soluble pigment, and its carbon utilization pattern. The type strain of M. olivasterospora is MK70 (= ATCC 21819). In the course of screening for new antibiotics, Cultural characteristics. Cultural characteristics workers isolated an actinomycete (strain MK70) were determined on ISP media, as described by Shirl- which produced the fortimicin complex (12, 13; ing and Gottlieb (14), and on several media recom- T. Nara, S. Takasawa, R. Okachi, I. Kawamoto, mended by Waksman (17). Inoculations were made by M. S. U.S. the method of Shirling and Gottlieb (14). Cultures were Yamamoto, Sato, and T. Sato, incubated at 28°C for 2 weeks. The colors of cultures patent 3,976,768, August 1976), (pseudodisac- were determined by comparing them with color chips charides consisting of fortamine and purpurosa- from the Color Harmony Manual (2). mine sugar). This organism was found to belong Physiological tests. The media used for physiological to the genus Micromonospora Orskov by mor- characterization of strains were prepared by the meth- phological and chemotaxonomic studies. In this ods of Waksman (17) and Shirling and Gottlieb (14). In report we describe this organism and discuss its addition, the medium of Gordon and Smith (3) was taxonomic position. used to determine melanoid pigment production. All cultures except those in milk and cellulose were incu- MATERIALS AND METHODS bated at 28°C for 2 weeks; cultures in milk and cultures in cellulose were incubated for 1 month. The tempera- Bacterial strains. Strain MK70 was isolated in our ture and pH for optimal growth were determined in the laboratory from a soil sample collected in Hiroshima seed medium described above after 5 days of incuba- City, Hiroshima Prefecture, Japan. The following type tion. strains were compared with our new isolate: Micro- Carbon utilization. Utilization of carbohydrates was monospora echinospora subsp. echinospora NRRL studied by the procedure of Luedemann and Brodsky 2985 (= ATCC 15837), Micromonospora echinospora (10). In addition, a chemically defined medium de- subsp. ferruginea NRRL 2995 (= ATCC 15836), Mi- scribed elsewhere (I. Kawamoto, T. Oka, and T. cromonospora echinospora subsp. pallida NRRL 2996 Nara, Agric. Biol. Chem., in press) was used as a basal (= ATCC 15838), and Micromonospora purpurea medium in this study. NRRL 2953 (= ATCC 15835). Each organism was Chemical analysis of cell components. Phospholipids inoculated into a seed medium, which consisted of 2% were examined by the method of Komura et al. (5). glucose (Nakarai Chemical Co.), 0.2% beef extract The fatty acid spectrum was determined by gas chro- (Kyokuto Seiyaku Co.), 0.2% yeast extract (Daigo matography after transesterification with diazometh- Eiyo Chemical Co.), 0.2% polypeptone (Daigo Eiyo ane. The instruments used were a Perkin-Elmer model Chemical Co.), and 0.1% CaCO, (Kanto Chemical 900 gas chromatograph equipped with a dual-flame Co.). The pH was adjusted to 7.3 before autoclaving. ionization detector and a JEOL model OISG-2 mass After 2 to 3 days of incubation at 28°C on a reciprocat- spectrograph coupled to a JEOL model 20K gas chro- ing shaker, each culture was centrifuged, washed three matograph. times with sterile saline water, and then used as an Antibiotic susceptibility. Inocula were streaked onto inoculum for various studies. the surfaces of plates containing ATCC medium 5 (1) Morphological characterization. The spore and my- and twofold dilutions of antibiotics. The minimal in- celial morphologies of cultures grown on American hibitory concentration (MIC) of each antibiotic was Type Culture Collection (ATCC) medium 5 (l),Hick- determined after 6 days of incubation at 28°C. ey-Tresner agar medium (171, and yeast extract malt extract agar medium (International Streptomyces Proj- ect [ISP] medium 2) (14) were studied by light micros- RESULTS copy and electron microscopy (model JEM-100C elec- Morphology. No true aerial mycelium was tron microscope; JEOL Ltd.). The spores and mycelia were fixed with osmium tetroxide and were embedded produced by strain MK70. The substrate myceli- in Epon 812 as described by Leudemann and Casmer um was well developed, branched, septate, and (11). Thin sections were cut with glass knives on a approximately 0.5 pm in diameter, Terminal or model JUM-7 microtome (JEOL Ltd.) and were intercalary chlamydospore-like swellings were stained with uranyl acetate and lead citrate. sometimes observed. These swellings were glo- 107 108 KAWAMOTO, YAMAMOTO, AND NARA INT. J. SYST.BACTERIOL. of cellulose were weakly positive. Optimal growth was observed between pH 6.8 and 7.8 and between 28 and 38°C. Carbon utilization. In both Luedemann medi- um and chemically defined medium, good growth was observed with D-xylose, D-ribose, D-fructose, D-galactose, D-mannose, maltose, sucrose, a-trehalose, and starch, but no growth was obtained with D-arabinose, L-rhamnose, L- sorbose, lactose, a-melibiose, melezitose, raffi- nose, glycerol, dulcitol, inositol, sorbitol, man- nitol, and salicin. Cellobiose was utilized poorly in the defined medium and moderately in Luede- mann medium. Antibiotic susceptibility. Strain MK70 was sus- ceptible to rifampicin (MIC, 3.13 pg/ml), chlor- amphenicol (6.25 pg/ml), chlortetracycline (3.13 pg/ml), mitomycin C (0.39 pg/ml), penicillin G (6.25 pg/ml), and ristocetin (1.56 pg/ml) and moderately susceptible to spiramycin (25 pg/ml) and cycloserine (25 pghl). As shown in Table 2, strain MK70 was resistant to the aminoglycoside antibiotics fortimicin A (MIC, 250 pg/ml), kana- mycin A (500 pg/ml), seldomycins (500 pg/ml), and spectinomycin (500 pg/ml), but was suscep- tible to neomycins (0.25 pg/ml) and streptomy- cin A (0.125 pg/ml). Phospholipids and fatty acids. The phospholip- ids in whole cells of strain MK70 consisted FIG. 1. Spore-bearing substrate mycelium of strain mainly of bisphosphatidylglycerol, phosphati- MK70 on ATCC medium 5. Optical microscopy. dylethanolamine, phosphatidylinositol, and X4,OOO. phosphatidylinositol mannosides, which were detected in a ratio of 0.21:1.00:0.52:0.74 (mea- sured as phosphorous). The fatty acids in the bose or oval and were 1.0 to 5.0 pm in diameter. total cellular lipid consisted of acids having 14 to The spores were borne singly from the substrate 18 carbons, most of which were saturated and mycelium, usually on short sporophores (0.3 to branched, such as iso-, anteiso- and 10-methyl 1.0 pm long), or occasionally they were sessile. fatty acids. Isopentadecanoic acid and isohexa- Apparent branching of the sporophores was also decanoic acid were the predominant acids (Fig. observed. The mature spores were approximate- 5). ly 1.0 pm in diameter, nonmotile, and spherical to oval in shape, with a large number of surface projections (0.1 to 0.2 pm long). Sclerotium formation and sporangium formation were not observed (Fig. 1 to 4). Cultural characteristics. Table 1 lists the cul- tural characteristics of strain MK70. Growth was good on most of the organic media used, and the colors of colonies were light brown or dark yellow, turning to olive or dark green during the formation of spores. Olive green soluble pigments were produced in ISP media 2 and 3 and were not acid-alkaline indicators. Physiological characteristics. The positive physiological properties of strain MK70 includ- ed peptonization of skim milk, hydrolysis of starch, and reduction of nitrate. The negative properties included coagulation of skim milk, FIG. 2. Blunt spiny spore of strain MK70 on yeast formation of tyrosinase, and production of mela- extract malt extract agar medium. Transmission elec- nin. Liauefaction of gelatin and decomPosition tron microscow. ~40.000. VOL. 33, 1983 MICROMONOSPORA OLI VASTEROSPORA S P. N OV. 109 FIG. 3. Blunt spiny spore of strain MK70 on Hickey-Tresner agar medium. Scanning electron microscopy. X 58,000. DISCUSSION acid in the cell wall. Therefore, a further com- parison was made with the type strains of these Strain MK70 produced spores singly on sub- taxa (M. echinospora subsp. echinospora strate mycelia and did not form any true aerial NRRL 2985 [= ATCC 158371, M. echinospora mycelium. The cell walls of this strain contained subsp. ferruginea NRRL 2995 [= ATCC 1.58361, 3-hydroxy-meso-diaminopimelic acid, glycine, and M. echinospora subsp. pallida NRRL 2996 arabinose, and xylose (4) and belong to cell wall [= ATCC 158381). Strain MK70 formed olive type I1 and sugar pattern D in the classification to olive black spore layers, whereas the type scheme of Lechevalier and Lechevalier (8). The strains of the three subspecies produced pur- phospholipid composition of strain MK70 had plish black or dark brown to black spore layers. the same pattern as the phospholipid composi- Strain MK70 produced olive to olive green solu- tions of the Micromonospora strains investigat- ble pigments in ISP media 2 and 3, but the other ed by Lechevalier et al. (7) and belongs to type strains did not. The previously described type I1 in the classification scheme of Lechevalier et strains utilized L-rhamnose, but strain MK70 did al. (7). The strain MK70 fatty acid pattern, not. Strain MK70 had no P-xylosidase activity, which was positive for iso-, anteiso- and 10- but the other strains gave positive or weakly methyl acids but negative for unsaturated acids, positive test results (Kawamoto et al., in press).
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