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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
3 Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
4 NITE Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba 292-0818, Japan
Correspondence
Somboon Tanasupawat
Somboon.T{at}Chula.ac.th
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Published online ahead of print on 17 September 2004 as DOI 10.1099/ijs.0.63217-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains LK2-10T and LK2-5 are AB107231 and AB159778.
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for actinomycete strains that produce spores singly borne on sporophores branched from substrate hyphae. Skerman et al. (1980) listed 12 species and seven subspecies as members of the genus Micromonospora; subsequently, Micromonospora olivasterospora (Kawamoto et al., 1983), Micromonospora rosaria (Horan & Brodsky, 1986) and Micromonospora chersina (Tomita et al., 1992) have been recognized as novel species. Lee et al. (1999) transferred Catellatospora matsumotoense to the genus Micromonospora as Micromonospora matsumotoense on the basis of 16S rRNA gene sequence analysis. The genus was well defined on both a chemotaxonomic and a phylogenetic basis (Kroppenstedt, 1985; Kawamoto, 1989; Koch et al., 1996a, b; Lee et al., 1999) as well as on morphological grounds. Reclassification of Micromonospora species was carried out by Kasai et al. (2000) on the based of gyrB gene sequence analyses and DNA–DNA hybridization experiments. Consequently, the genus Micromonospora is considered to accommodate 15 species: Micromonospora aurantiaca, Micromonospora carbonacea, Micromonospora chalcea, M. chersina, Micromonospora coerulea, Micromonospora echinospora, Micromonospora gallica, Micromonospora halophytica, Micromonospora inositola, M. matsumotoense, Micromonospora nigra, M. olivasterospora, Micromonospora pallida, Micromonospora purpureochromogenes and M. rosaria. According to Kawamoto (1989), the type strain (NCTC 4582T) of M. gallica is no longer available. During investigation of novel actinomycetes from peat swamp forest soil in southern Thailand, we isolated two strains, LK2-10T and LK2-5, that showed typical morphological characteristics of the genus Micromonospora but with an unusual colony colour for this genus. Here we report on the taxonomic characterization and classification of these isolates and propose a novel species, Micromonospora eburnea sp. nov., for the strains.
Strains LK2-10T and LK2-5 were isolated from soil samples collected from peat swamp forest at Lankwai in Yala Province, Thailand. Samples were taken from the top peat layer (surface of soil). First, 0·5 g soil was suspended in 4·5 ml sterile distilled water, and the suspension was treated using a wet-heat technique (70 °C for 15 min). Next, 0·5 ml of this suspension was transferred to another 4·5 ml sterile distilled water and this step was repeated to set up a ten-fold dilution series to 10–3. At the final dilution step, aliquots of 100 µl were spread onto starch-casein nitrate agar (pH 7·2) supplemented with 37 µg novobiocin ml–1 and 223 µg mycostatin ml–1. After 21 days of aerobic incubation at 30 °C, growth of strains LK2-10T and LK2-5 was indicated by yellowish white colonies. The colonies of these strains were transferred onto yeast extract-malt extract agar (ISP2 medium).
Morphological properties of strains LK2-10T and LK2-5 grown on ISP2 medium were observed by light and scanning electron microscopy (model JSM-5410 LV; JEOL). Samples for scanning electron microscopy were prepared by cutting a block from an agar plate and then fixing it in 1 % OsO4 in 0·1 M phosphate buffer (pH 7·2) at room temperature for 4 h. The samples were dehydrated in a graded series of ethanol and then were dried with a critical-point dryer (model Samdri-780, Balzers CPD 020). The samples were placed onto a stub bearing adhesive and spatter-coated with gold under vacuum.
Phenotypic properties were examined by using several standard methods; culture characteristics were tested using 14-day cultures grown at 30 °C on various agar media (Table 1). The Jacal Colour Card L2200 (Japan Colour Research Institute) was used for determining colour designations and names. Decomposition of various compounds was examined using the basal medium recommended by Gordon et al. (1974). Temperature and NaCl tolerance were determined on ISP2 medium. Carbon source utilization was tested using ISP9 medium (Shirling & Gottlieb, 1966) supplemented with a final concentration of 1 % of the tested carbon sources and 0·05 % Casamino acid. Gelatin liquefaction, peptonization of milk, nitrite production, cellulose decomposition and starch hydrolysis were determined by cultivation on various media as described by Arai (1975) and Williams & Cross (1971). Melanin and hydrogen sulphide production were examined on slants of tyrosine agar and peptone iron agar supplemented with 0·1 % (w/v) yeast extract, respectively.
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, and the amino acid composition was analysed with an automatic amino acid analyser. The isomers of diaminopimelic acid (A2pm) in cell walls were determined by the method of Staneck & Roberts (1974). The acyl group of muramic acid in peptidoglycan was determined by the method of Uchida & Aida (1984). The reducing sugars from whole cell hydrolysates were analysed by the HPLC method of Mikami & Ishida (1983). Phospholipids in cells were extracted and analysed by the method of Minnikin et al. (1984). Fatty acid methyl ester analysis was performed by GLC according to the instructions of the Microbial Identification System (MIDI) (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Isoprenoid quinones were extracted by the method of Collins et al. (1977) and were analysed by HPLC equipped with a Cosmosil 5C18 column (4·6x150 mm; Nacalai Tesque). The elution solvent was a mixture of methanol and 2-propanol (2 : 1, v/v).
Chromosomal DNA was isolated from cells grown in ISP2 broth according to the method of Tamaoka (1994). The G+C content of the DNA was determined using the HPLC method of Tamaoka & Komagata (1984). An equimolar mixture of nucleotides for analysis of DNA base composition (Yamasa Shoyu) was used as the quantitative standard. DNA–DNA relatedness was measured fluorometrically using the microplate hybridization method devised by Ezaki et al. (1989). Hybridization was carried out at 55 °C for 2 h.
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and sequencing of the PCR products were carried out as described by Nakajima et al. (1999). The 16S rRNA gene sequence was multiply aligned with selected sequences obtained from the GenBank/EMBL/DDBJ databases by using CLUSTAL W version 1.81. Alignment was manually verified and adjusted prior to the construction of a phylogenetic tree. The phylogenetic tree was constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony methods (Kluge & Farris, 1969) in the MEGA program version 2.1. Confidence values of branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. 16S rRNA gene sequence similarities among closely related species were calculated manually after pairwise alignments obtained using the CLUSTAL X program (Thompson et al., 1997). Gaps and ambiguous nucleotides were eliminated from the calculations.
The morphological and chemical properties of strains LK2-10T and LK2-5 are consistent with their classification in the genus Micromonospora (Kawamoto, 1989). Strains LK2-10T and LK2-5 produced well-developed and branched substrate hyphae on ISP2 medium, but no aerial hyphae. Spores were borne singly on the substrate hyphae, and were 0·45 µm in diameter. Spores had a rough and nodular surface and were non-motile. The colour of the substrate mycelium was yellowish white to dull orange (Table 1
). Pale-yellow soluble pigment is produced in ISP2 and ISP3 media and in nutrient agar.
Strains LK2-10T and LK2-5 had identical chemotaxonomic characteristics, and these were similar to those of members of the genus Micromonospora. Cell wall hydrolysates of the two strains contained glutamic acid, glycine, alanine and A2pm in a molar ratio of 1·0 : 0·9 : 0·5 : 1·1 (calculated by defining the amount of glutamic acid as 1·0), and the isomer of A2pm was meso, indicating that these strains have wall chemotype II of Lechevalier & Lechevalier (1970) and peptidoglycan type A1
of Schleifer & Kandler (1972). The acyl type of cell wall muramic acid was glycolyl. The strains contained glucose, xylose, arabinose, galactose, mannose and ribose as whole-cell sugars, but rhamnose was not detected (whole-cell sugar pattern D of Lechevalier & Lechevalier, 1970). Characteristic phospholipids were diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannosides and phosphatidylethanolamine, but not phosphatidylcholine. This pattern corresponds to phospholipid type II of Lechevalier et al. (1977). The major cellular fatty acids of strains LK2-10T and LK2-5 were iso-C15 : 0, iso-C16 : 0, iso-C17 : 0, anteiso-C15 : 0, C17 : 0 and anteiso-C17 : 0. This pattern corresponds to fatty acid type 3b of Kroppenstedt (1985). Mycolic acids were absent. The predominant menaquinones were MK-9(H4), MK-10(H4) and MK-9(H6), and minor amounts of MK-9(H8), MK-10(H6), MK-10(H8) and MK-9(H2) were also present. The DNA G+C contents of strains LK2-10T and LK2-5 were 71·5 and 71·2 mol%.
Almost complete 16S rRNA gene sequences (1477 nt) were determined for strains LK2-10T and LK2-5; a 1421 nt fragment was used for phylogenetic analysis and compared against 16S rRNA gene sequences of members of the family Micromonosporaceae. Phylogenetic analysis based on this large dataset revealed that strains LK2-10T and LK2-5 were placed within the clade of the genus Micromonospora (data not shown). When the sequences of strains LK2-10T and LK2-5 were compared with corresponding 16S rRNA gene sequences of all type strains of recognized Micromonospora species, selected sequences of the genus Actinoplanes and the 16S rRNA gene sequence of Streptomyces ambofaciens (as an outgroup), they formed a clade with M. nigra DSM 43818T (Fig. 1). 16S rRNA gene sequence similarity values between strain LK2-10T and all of the type strains of the genus Micromonospora ranged from 98·1 % (with M. matsumotoense IMSNU 22003T) to 99·1 % (with M. nigra DSM 43818T) and strain LK2-10T showed 99·9 % sequence similarity to strain LK2-5; these similarities represent 28, 14 and one nucleotide differences, respectively. On the basis of morphological, chemotaxonomic and phylogenetic data, the isolates should be classified in the genus Micromonospora.
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. The colour of substrate mycelium of the isolates was yellowish white, which distinguishes them from all other type strains of Micromonospora species, which generally have an orange colour. Levels of DNA–DNA relatedness between strains LK2-10T and LK2-5 ranged from 89 to 92 %, whereas relatedness between the isolates and type strains of all recognized Micromonospora species except M. gallica ranged from 21 to 58 % (Table 3).
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Description of Micromonospora eburnea sp. nov.
Micromonospora eburnea (eb.ur'ne.a. L. fem. adj. eburnea ivory, referring to colour of colonies).
Aerobic, Gram-positive, mesophilic, non-motile actinomycete that forms well-developed and branched substrate hyphae. Colonies are yellowish white and turn greyish black after sporulation in ISP2 medium. Single spores are formed on substrate hyphae. Aerial mycelium is absent. The spore surface appears rough and nodular. Spores are non-motile. Pale yellow soluble pigment is produced in ISP2 and ISP3 media and in nutrient agar. Nitrate is reduced to nitrite. Utilizes D-glucose, L-rhamnose, D-melibiose, D-raffinose, glycerol, salicin, lactose, cellobiose and D-xylose; weakly utilizes D-mannitol and D-galactose but not L-arabinose, D-fructose or D-ribose. Peptonization of milk, hydrolysis of starch and gelatin liquefaction are positive, but formation of melanin and H2S production are negative. Optimal temperature for growth is 25–30 °C. No growth occurs above 45 °C. The maximum NaCl concentration for growth is 4 %. Cell wall contains glutamic acid, glycine, alanine and meso-A2pm in the molar ratio 1 : 0·9 : 0·5 : 1·1. Acyl type of the cell wall is glycolyl. The predominant menaquinone is MK-9(H4). Characteristic whole-cell sugars are xylose and arabinose. The phospholipid profile comprises diphosphatidylglycerol, phosphatidylinositolmannosides, phosphatidylinositol and phosphatidylethanolamine, but not phosphatidylcholine. The major fatty acids of the type strain are iso-C15 : 0 (24·1 %), iso-C16 : 0 (17·9 %), iso-C17 : 0 (7·7 %), anteiso-C15 : 0 (8·5 %), C17 : 0 (7·9 %) and anteiso-C17 : 0 (10·1 %). The G+C content of the DNA is 71·5 mol%. Habitat is soil.
The type strain is LK2-10T (=JCM 12345T=PCU 238T=DSM 44814T=TISTR 1531T).
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ACKNOWLEDGEMENTS |
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