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NITE Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan
Correspondence
Tomohiko Tamura
tamura-tomohiko{at}nite.go.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains TT 97-42T and TT 97-44 are AB223089 and AB223090, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) according to the classification of Lechevalier & Lechevalier (1970). However, the genera Catenuloplanes (Yokota et al., 1993) and Couchioplanes (Tamura et al., 1994), which harboured chemotype VI, were subsequently added to this family based on phylogenetic analysis. The description of this family was later emended by Koch et al. (1996) and Stackebrandt et al. (1997) on the basis of 16S rRNA gene sequence analysis. This family currently consists of 13 genera, including Micromonospora (Orskov, 1923), Actinoplanes (Couch, 1950; Stackebrandt & Kroppenstedt, 1987), Pilimelia (Kane, 1966), Dactylosporangium (Thiemann et al., 1967), Catellatospora (Asano & Kawamoto, 1986; Lee & Hah, 2002), Catenuloplanes (Kudo et al., 1999; Yokota et al., 1993), Couchioplanes (Tamura et al., 1994), Spirilliplanes (Tamura et al., 1997), Verrucosispora (Rheims et al., 1998), Virgisporangium (Tamura et al., 2001), Asanoa (Lee & Hah, 2002), Longispora (Matsumoto et al., 2003) and Salinispora (Maldonado et al., 2005).
Mangroves are formed from tropical shrubs and trees belonging to the genera Rhizophora, Bruguiera and Sonneratia. These plants grow in shore mud with tangled roots that are partly aerial. Mangroves spread from the shore into the mouths of rivers in the tropical and subtropical areas of Africa, America and Asia (Spalding et al., 1977) and they are known to be the habitat of various micro-organisms (Chapman, 1976; Odum & Heald, 1972; Robertson & Duke, 1987). During the course of taxonomic studies on the rare actinomycetes associated with mangroves, two novel strains were isolated from soil samples surrounding mangrove roots in Okinawa, Japan. These strains produced short spore chains and contained meso-diaminopimelic acid (A2pm) and glycine in their cell walls. The novel isolates belonged to the family Micromonosporaceae, but formed a lineage distinct from previously recognized genera. The data presented in this paper show that the novel isolates are members of a new genus, Polymorphospora gen. nov.
Strains TT 97-42T and TT 97-44 were isolated from soil samples collected near the roots of Bruguiera gymnorrhiza and Sonneratia alba at the mouth of the River Shiira on Iriomote Island, Okinawa, Japan. The two strains were isolated using humic acid-vitamin (HV) agar (Hayakawa & Nonomura, 1987) using the yeast extract–SDS method (Hayakawa & Nonomura, 1989). Freeze-dried cells for chemotaxonomic analyses were obtained from cultures grown in yeast extract-glucose broth (10 g yeast extract and 10 g D-glucose in 1 l distilled water, pH adjusted to 7.0) incubated at 28 °C on a rotary shaker for 4 days.
Cultures were grown on HV agar and yeast extract-starch agar (2 g yeast extract and 10 g soluble starch in 1 l distilled water, pH 7.0) by incubating at 28 °C for 3–14 days. Cultures were observed under a light microscope and under a scanning electron microscope (model JSM-5400; JEOL) by using previously described methods (Tamura et al., 1994). Scanning electron micrographs of 14-day-old cultures of strain TT 97-42T grown on HV agar are shown in Fig. 1. The strain formed short spore chains. At the periphery of the colonies, the shapes of the spores were diverse, but they showed oval to short rod morphology at the centre of the colonies. The spore sizes were 0.5–0.9 µm long x 0.8–1.3 µm wide. The cells were non-motile.
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). Nutrient agar (Difco) was used as the medium. Physiological characteristics were analysed as described previously (Gordon et al., 1974; Yokota et al., 1993). The novel isolates formed strong red to strong vivid reddish-orange colonies on yeast extract-malt extract agar (ISP medium 2), oatmeal agar (ISP medium 3) and inorganic salts-starch agar (ISP medium 4). Soluble pigment was not produced in any of the media tested. Aerial mycelia were rarely observed to develop on HV or water agar. The novel isolates showed good to moderately good growth on ISP media 2, 3 and 4, but no growth, or very poor growth, on glycerol-asparagine agar (ISP medium 5), peptone-yeast extract iron agar (ISP medium 6) and tyrosine agar (ISP medium 7). The novel isolates utilized D-mannitol, D-melibiose, maltose, L-rhamnose, methyl 
-D-glucoside, D-galactose, D-mannose and D-glucose. Only one of the isolates, strain TT 97-42T, utilized D-raffinose. The strains were positive in tests for starch hydrolysis and NO3 reduction. However, the strains were negative in tests for aesculin hydrolysis, urea decomposition, growth in Saboraud dextrose broth, growth on MacConkey agar, utilization of sodium succinate, sodium oxalate, sodium malate and sodium citrate and resistance to 4 % NaCl, methyl violet and pyronine B.
Whole-cell sugars, cell-wall amino acids, isoprenoid quinones, cellular fatty acids, phospholipids, diaminopimelic acid isomers, acyl type of muramic acids, mycolic acids and DNA G+C content were analysed as described previously (Tamura et al., 1994). The predominant menaquinones found in the isolates were MK-10(H6) (34–35 %), MK-10(H4) (20–28 %), MK-9(H6) (20–27 %) and MK-9(H4) (17–19 %). The novel isolates contained glucose, mannose, galactose and 3-O-methylmannose as whole-cell sugars with varying amounts of xylose, but not arabinose. The cell-wall amino acids were meso-A2pm, alanine, glycine and glutamic acid (at a molar ratio of about 0.6 : 1.0 : 1.1 : 1.2). These amino acids corresponded to murein type A1
as described by Schleifer & Kandler (1972). Mycolic acids were not detected. The acyl moiety of murein contained glycolyl residues. Phosphatidylethanolamine was detected as a diagnostic phospholipid, but phosphatidylglycerol-, phosphatidylcholine- and glucosamine-containing phospholipids were not detected. The cellular fatty acids consisted of iso-branched, anteiso-branched, saturated and unsaturated fatty acids and corresponded to fatty acid pattern 2a as described by Kroppenstedt (1985). The major cellular fatty acids were iso-C16 : 0 (40–53 %), C17 : 1 (18–19 %), anteiso-C17 : 0 (11 %), anteiso-C15 : 0 (6–8 %) and C17 : 0 (5–8 %). The DNA G+C contents of strains TT 97-42T and TT 97-44 were 70.2 and 70.7 mol%, respectively.
The 16S rRNA gene was amplified by using PCR and the resulting PCR products were directly sequenced as described by Tamura & Hatano (1998) by using a DNA sequencer (ABI PRISM 3100 Genetic Analyzer; PE Applied Biosystems) according to the manufacturer's protocol. The 16S rRNA gene sequences of the novel strains and reference organisms were aligned with those of Streptomyces ambofaciens (Pernodet et al., 1989). The CLUSTAL_X 1.83 software package (Thompson et al., 1997) was used to generate evolutionary distances (Knuc value; Kimura, 1980), similarity values and a neighbour-joining phylogenetic tree (Saitou & Nei, 1987) based on the Knuc and bootstrap values (Felsenstein, 1985) for 1000 replications. PAUP 4.0 (Swofford, 2002) was used to generate a maximum-parsimony tree (Swofford & Berlocher, 1987). NJPLOT (Perrière & Gouy, 1996) was used to plot the phylogenetic trees. DNA–DNA relatedness was determined using the microplate hybridization method developed by Ezaki et al. (1988, 1989) with minor modifications (Tamura et al., 1999). Phylogenetic analysis of the 16S rRNA gene sequences revealed that the novel isolates belonged to a cluster of the family Micromonosporaceae and represented a new phyletic line that was different from each of the 13 genera in this family with validly published names (Fig. 2). This finding was supported by the topology of the maximum-parsimony tree and by the bootstrap value of 100 % obtained in the neighbour-joining analysis. Comparison of the 16S rRNA gene sequences of the novel isolates and their closest neighbours, members of the genera Dactylosporangium and Micromonospora, revealed similarity values of between 95.8 and 98.1 %. The gene sequence similarity between isolates TT 97-42T and TT 97-44 was 100 %. The DNA–DNA relatedness value between the two novel isolates was 72.7–72.9 %.
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; Yokota et al., 1993). The novel isolates also contained 3-O-methylmannose as a whole-cell sugar and this sugar was also detected in members of the genera Spirilliplanes and Virgisporangium (Tamura et al., 1997, 2001). Furthermore, the presence of N-glycolyl muramic acid in the cell-wall peptidoglycan and the absence of mycolic acid are characteristics of the family Micromonosporaceae. Therefore, the novel isolates were assigned to the family Micromonosporaceae based on chemotaxonomic characteristics.
The novel isolates formed spore chains and differed in this respect from the genera Dactylosporangium and Micromonospora, the closest phylogenetic neighbours. Thus, the chemotaxonomic and morphological characteristics of the novel isolates distinguish them from any of the previously recognized members of the family Micromonosporaceae (Table 1).
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Description of Polymorphospora gen. nov.
Polymorphospora (Po.ly.mor.pho.spo'ra. Gr. adj. polymorphos multiform; N.L. fem. n. spora a spore; N.L. fem. n. Polymorphospora polymorphic spore).
Gram-positive, non-acid-fast, aerobic actinomycetes that show extensive branching and non-fragmenting substrate hyphae. Short spore chains develop on short sporophores on the substrate mycelium. Immature spores are oval or of various shapes and short rods are formed (0.6–0.9 µm long x 0.8–1.5 µm wide) on maturation. Spores are non-motile. Strictly aerobic. Optimum temperature for growth generally ranges between 20 and 30 °C. The cell walls contain glutamic acid, glycine, alanine and meso-diaminopimelic acid. The chemotype is II according to Lechevalier & Lechevalier (1970) and the peptidoglycan type is presumed to be A1 according to Schleifer & Kandler (1972). Mannose, 3-O-methylmannose, glucose and galactose are detected as whole-cell sugars. The predominant cellular fatty acid is iso-C16 : 0. The major menaquinones are MK-10(H6), MK-10(H4), MK-9(H6) and MK-9(H4). Phosphatidylethanolamine is present as the diagnostic phospholipid (phospholipid pattern type PII). The acyl type of the cell-wall polysaccharides is glycolyl. Mycolic acid is not detected. The G+C content of DNA is approximately 70–71 mol%. Unique nucleotide signatures are present at positions 1244 (U) of the 16S rRNA gene. This genus belongs to the family Micromonosporaceae. The type species is Polymorphospora rubra.
Description of Polymorphospora rubra sp. nov.
Polymorphospora rubra (ru'bra. L. fem. adj. rubra red).
In addition to the morphological, chemotaxonomic and general characteristics described for the genus, the species has the following characteristics. Colonies that develop on ISP media 2, 3 and 4 are red to reddish-orange in colour. Utilizes D-mannitol, D-melibiose, maltose, L-rhamnose, methyl -D-glucoside, D-galactose, D-mannose and D-glucose. Positive in tests for starch hydrolysis and urea decomposition. No growth in the presence of 4 % NaCl. Aesculin is not hydrolysed. The major cellular fatty acids are iso-C16 : 0 and C16 : 1. The G+C content of DNA is 70 mol%.
The type strain TT 97-42T (=NBRC 101157T=DSM 44947T), was isolated from soil surrounding mangrove roots.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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