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1 Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
2 Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
3 The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan
Correspondence
Yoko Takahashi
ytakaha{at}lisci.kitasato-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 5-10-10T and 5-38-42T are AB274966 and AB274967 respectively.
Supplementary tables detailing cultural, physiological and chemotaxonomic characteristics, cellular fatty acid contents, 16S rRNA gene sequence similarities and DNA–DNA relatedness of strains 5-10-10T and 5-38-42T and related taxa are available with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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as a member of the family Streptosporangiaceae which forms extensively branched substrate and aerial mycelia. On the basis of detailed phylogenetic analyses, the genus presently comprises 17 species and two subspecies (Gyobu & Miyadoh, 2001; Quintana et al., 2003; Stackebrandt et al., 2001). In the course of an investigation into novel actinomycetes present in soil from the southern area of Bangladesh, we isolated strains 5-10-10T and 5-38-42T that showed morphological characteristics that are typical of the genus Nonomuraea. In this paper, the characterization and classification of these strains is described and two novel species of the genus Nonomuraea are proposed.
Strains 5-10-10T and 5-38-42T were isolated from sandy soil collected at a sea beach in Cox's Bazar, Bangladesh. The strains were isolated using the standard dilution plate method and grown on humic acid-vitamin agar (Hayakawa & Nonomura, 1987) supplemented with cycloheximide (50 mg l–1), nystatin (50 mg l–1) and nalidixic acid (20 mg l–1). After 21 days of aerobic incubation at 30 °C, the strains were transferred and purified on yeast extract-malt extract agar (medium 2 of the International Streptomyces Project; ISP-2). The strains were maintained as working cultures on yeast-starch agar (pH 7.2) containing (l–1), 15.0 g soluble starch, 4.0 g yeast extract, 0.5 g K2HPO4, 0.5 g MgSO4.7H2O and 15.0 g agar. Nonomuraea fastidiosa JCM 3321T was used for comparative purposes in this study.
In order to determine cell morphology, strains 5-10-10T and 5-38-42T were grown on oatmeal (ISP-3) agar and yeast extract-malt extract (ISP-2) agar media at 30 °C for 21 days and then observed by using light (S-2400; Hitachi) and scanning electron (JSM-5600; JEOL) microscopes. Samples for scanning electron microscopy were prepared as described by Ito et al. (1989) and Ara & Kudo (2006).
The phenotypic properties of the two novel strains were examined using the following standard methods. For cultural characterization, the isolates were grown for 21 days at 30 °C on various agar media as described by Waksman (1950, 1961), Shirling & Gottlieb (1966) and Asano & Kawamoto (1986). The Colour Harmony Manual (Jacobson et al., 1958) was used to determine the names and designations of colony colours. The temperature range and NaCl tolerance for growth were determined on oatmeal agar. Utilization of carbohydrates as sole carbon sources was tested by using ISP-9 medium as a basal medium according to the modified method of Stevenson (1967). Production of melanoid pigments was examined using tyrosine agar (ISP-7).
For the analysis of chemotaxonomic characteristics, freeze-dried cells were obtained from cultures grown in yeast–glucose or trypticase soy broth on a rotary shaker at 28 °C. The isomers of diaminopimelic acid (DAP) in the cell-wall peptidoglycan were determined by TLC as described by Staneck & Roberts (1974). Reducing sugars from whole-cell hydrolysates were analysed by the paper chromatography method of Becker et al. (1965). The N-acyl group of the muramic acid in the peptidoglycan was determined according to the method of Uchida & Aida (1984). Phospholipids in cells were extracted and identified according to the method of Minnikin et al. (1984). Methyl esters of cellular fatty acids were prepared and analysed by TechnoSuruga Co., Ltd. according to the instructions of the Microbial Identification System (MIDI) using GC (HP6890; Hewlett Packard) (Sasser, 1990). Isoprenoid quinones were extracted according to the method of Collins et al. (1977, 1984) and were analysed by HPLC (802-SC; Jasco) using a CAPCELL PAK C18 column (Shiseido) (Tamaoka et al., 1983). Preparation and detection of methyl esters of mycolic acids was carried out as described by Tomiyasu (1982).
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 content (Yamasa Shoyu) was digested by bacterial alkaline phosphatase and used as the quantitative standard.
For DNA–DNA hybridization experiments, chromosomal DNA of strains 5-10-10T and 5-38-42T was isolated from biomass. Cells of the novel strains were freeze-dried and ground mechanically for preparation of chromosomal DNA as described by Raeder & Broda (1985). DNA–DNA relatedness was measured fluorometrically using the microplate hybridization method devised by Ezaki et al. (1989). Hybridization was carried out overnight at 52 °C.
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) and Ara & Kudo (2006). The sequences were multiple-aligned with selected sequences obtained from the GenBank/EMBL/DDBJ databases using the CLUSTAL_X program (Thompson et al., 1997). The alignment was manually verified and adjusted prior to construction of a phylogenetic tree. A phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) in CLUSTAL_X based on a comparison of 1262 nucleotides present in all of the strains as a result of elimination of gaps and ambiguous nucleotides from the sequences between positions 34 and 1491 (position numbering according to Escherichia coli). Thermopolyspora flexuosa DSM 43186T was used as an outgroup. The confidence values of the branches of the phylogenetic tree were determined using bootstrap analyses based on 1000 resamplings (Felsenstein, 1985). The values for 16S rRNA gene sequence similarity (%) between Nonomuraea strains were calculated after pairwise alignment using CLUSTAL_X.
Analysis of cell morphology revealed that strains 5-10-10T and 5-38-42T produced well-developed branched substrate and aerial mycelia and did not fragment into bacillary or coccoid elements. Spiral chains of strain 5-10-10T (covered by a smooth sheath) were composed of about 8–12 non-motile spores that were borne directly, singly or in clusters, on aerial mycelia (Fig. 1a). For strain 5-38-42T, spiral to hooked chains composed of 12–17 non-motile spores with a smooth surface were observed (Fig. 1b). A spore sheath was not observed for strain 5-38-42T.
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and in Supplementary Table S2 (available in IJSEM Online).
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6c (4.5 %), C17 : 18c (3.8 %), C15 : 0 (1.8 %), summed feature 3 (C16 : 17c/iso-C15 : 0 2-OH) (1.8 %), 10-methyl C18 : 0 (1.6 %), anteiso-C17 : 0 (1.6 %), iso-C14 : 0 (1.5 %), C15 : 0 (1.2 %) and C18 : 19c (1.1 %) are present in strain 5-10-10T. For strain 5-38-42T, the major fatty acids are C16 : 0 (17.4 %), C17 : 18c (11.5 %) and 10-methyl C17 : 0 (8.4 %), with smaller amounts of C15 : 0 (7.5 %), C17 : 0 (6.7 %), summed feature 3 (C16 : 17c/iso-C15 : 0 2-OH) (5.8 %), C14 : 0 (4.6 %), C18 : 19c (4.5 %), 10-methyl C16 : 0 (3.3 %), 10-methyl C18 : 0 (2.5 %), anteiso-C17 : 0 (2.4 %), C17 : 16c (2.4 %), iso-C15 : 0 (2.2 %), anteiso-C15 : 0 (2.0 %), C18 : 0 (1.9 %), 2-OH C16 : 0 (1.9 %) and C16 : 19c (1.3 %) (Table 1
and Supplementary Table S4 in IJSEM Online). The major menaquinones observed were MK-9(H4) for strain 5-10-10T and MK-9(H6) and MK-9(H4) for strain 5-38-42T.
For the phylogenetic analysis, almost complete 16S rRNA gene sequences were determined for strains 5-10-10T and 5-38-42T consisting of 1513 and 1461 nucleotides, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strains 5-10-10T and 5-38-42T formed a coherent and monophyletic clade with N. fastidiosa that was supported by a bootstrap value of 57 % (Fig. 2). Levels of gene sequence similarities between these strains ranged from 96.7 % to 98.0 % (see Supplementary Table S5 in IJSEM Online).
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Strains 5-10-10T and 5-38-42T displayed morphological characteristics that are characteristic of other species of the genus Nonomuraea. In addition, based on their menaquinones and the predominant fatty acids in whole-cell methyl esters (Gyobu & Miyadoh, 2001; Quintana et al., 2003; Kämpfer et al., 2005), strains 5-10-10T and 5-38-42T should be placed in the genus Nonomuraea. The quinone system and fatty acid profiles also supported the affiliation of strains 5-10-10T and 5-38-42T as members of the genus Nonomuraea. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains 5-10-10T and 5-38-42T clustered with N. fastidiosa in a group under the genus Nonomuraea.
Strains 5-10-10T and 5-38-42T could be distinguished from each other by morphological and cultural characteristics (see Fig. 1, Table 1 and Supplementary Table S1). Based on their menaquinones and the predominant fatty acids in whole-cell methyl esters (see Table 1 and Supplementary Tables S3 and S4), strains 5-10-10T and 5-38-42T represent two separate species in the genus Nonomuraea. The two novel isolates appeared to be relatively distantly related to other recognized representatives of the genus Nonomuraea. DNA–DNA hybridization is the standard criterion for the delineation of species with members of the same species having values of 
70 % DNA–DNA relatedness (Wayne et al., 1987). The DNA–DNA relatedness of strains 5-10-10T and 5-38-42T to the closely related type strain of N. fastidiosa was <70 % (30–32 %) and the relatedness value between strains 5-10-10T and 5-38-42T was 58 %.
On the basis of cultural, morphological and physiological characteristics, biochemical reactions, chemotaxonomic characteristics and 16S rRNA gene sequences similarity, it is apparent that strains 5-10-10T and 5-38-42T represent separate species. It is proposed that the two isolates represent separate novel species of the genus Nonomuraea, Nonomuraea bangladeshensis sp. nov. (type strain, 5-10-10T) and Nonomuraea coxensis sp. nov. (type strain, 5-38-42T). Characteristics that allow the differentiation of the two novel species from N. fastidiosa are listed in Table 1.
Description of Nonomuraea bangladeshensis sp. nov.
Nonomuraea bangladeshensis (ban.gla.desh.en'sis. N.L. fem. adj. bangladeshensis pertaining to Bangladesh, from where the strain was isolated).
Forms a well-developed, branched substrate mycelium. Abundant aerial mycelium is present. Spore chains are spiral with one or two turns (8–12 spores). Sporangia are not detected. Spores are spherical to cylindrical and the spore sheath surface is smooth. Good growth occurs on tyrosine agar (ISP-7), yeast extract-malt extract agar (ISP-2), oatmeal agar (ISP-3), Bennett's agar, glucose-yeast extract agar, Hickey–Tresner agar, Waksman agar, oatmeal-nitrate agar and Seino (yeast-starch) agar. Moderate growth is found on glycerol-asparagine agar, inorganic salts-starch agar (ISP-4), nutrient agar and water agar. Poor growth occurs with glucose-asparagine agar and sucrose-nitrate agar. The colour of the substrate mycelium is shell (3ca) to cinnamon brown (3lg) on various agar media. Pale brown aerial mycelium and sporulation occur on ISP-7 agar, ISP-2 agar, ISP-3 agar, water agar and oatmeal-nitrate agar media after 14 days incubation at 28 °C. A soluble pigment is not produced. Aerobic, Gram-positive and able to grow at pH 5.0–9.0. The temperature range for growth is between 15 and 45 °C. No production of melanoid pigments. No growth is observed at 5 % NaCl. The diagnostic diamino acid isomer of the cell-wall peptidoglycan is meso-DAP. Madurose, galactose, glucose, mannose and ribose are present in whole-cell hydrolysates. The N-acyl type of the muramic acid is acetyl. Mycolic acids are absent. The polar lipids include PE and OH-PE. Phosphatidylcholine is absent. The major menaquinone is MK-9(H4), with minor amounts of MK-9(H6), MK-9(H2) and MK-9(H0). Major fatty acids are iso-C16 : 0 (26.4 %), iso-C15 : 0 (14.7 %) and 10-methyl C17 : 0 (10.5 %). The G+C content of the DNA is 72.7 mol%.
The type strain, strain 5-10-10T (=MTCC 8089T=JCM 13930T), was isolated from sandy soil from Cox's Bazar, Bangladesh.
Description of Nonomuraea coxensis sp. nov.
Nonomuraea coxensis (cox.en'sis. N.L. fem. adj. coxensis pertaining to Cox's Bazar, Bangladesh, the origin of the soil from where the type strain was isolated).
Forms a well-developed, branched substrate mycelium. Abundant aerial mycelium is present. Spore chains are spiral to hooked with 12–17 spores. Sporangia are not detected. Spores are spherical to oval and the spore surface is smooth. Good growth on yeast extract-malt extract agar (ISP-2), oatmeal agar (ISP-3), Bennett's agar, glucose-yeast extract agar, Hickey–Tresner agar, Waksman agar, 1/5 yeast-starch agar, oatmeal-nitrate agar and Seino (yeast-starch) agar. Moderate growth on tyrosine agar (ISP-7), inorganic salts-starch agar (ISP-4) and nutrient agar; poor growth on water agar, glycerol-asparagine agar and glucose-asparagine agar. Substrate mycelium is light melon yellow (4ca) to apricot (orange–brown) (4ga) on various agar media. Pale pink to white aerial mycelium and sporulation occur on ISP-2 agar, ISP-3 agar, Bennett agar's, Hickey–Tresner agar and 1/5 yeast-starch agar media after 14 days incubation at 28 °C. A soluble pigment is not produced. Aerobic. Gram-positive. Growth occurs at pH 5.0–9.0. The temperature range for growth is between 15 and 45 °C. No production of melanoid pigments. No growth on 4 % NaCl. The diagnostic diamino acid isomer of the cell-wall peptidoglycan is meso-DAP. Madurose, galactose, glucose, mannose and ribose are present in whole-cell hydrolysates. The N-acyl type of muramic acid is acetyl. Mycolic acids are absent. The polar lipids include PE and OH-PE. Phosphatidylcholine is absent. The major menaquinones are MK-9(H6) and MK-9(H4). The fatty acid profile is characterized by the presence of significant amounts of C16 : 0 (17.4 %), C17 : 18c (11.5 %) and 10-methyl C17 : 0 (8.4 %). The G+C content of the DNA is 72.3 mol%.
The type strain, strain 5-38-42T (=MTCC 8090T=JCM 13931T), was isolated from sandy soil from Cox's Bazar, Bangladesh.
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ACKNOWLEDGEMENTS |
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