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NITE Biological Resource Center, National Institute of Technology and Evaluation, Kazusakamatari 2-5-8, Kisarazu, Chiba 292-0818, Japan
Correspondence
Hideki Yamamura
yamamura-hideki{at}nite.go.jp
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ABSTRACT |
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MAIN TEXT |
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) and currently contains 63 species with validly published names, including the recently described species Nocardia jiangxiensis, Nocardia jejuensis and Nocardia harenae (Cui et al., 2005; Lee, 2006; Seo & Lee, 2006). In recent years, many species have been isolated from clinical specimens and are considered to be opportunistic pathogens in animal infections (Watanabe et al., 2006; Brown-Elliott et al., 2006). In contrast, the natural habitats of nocardiae are widely distributed throughout terrestrial and aquatic ecosystems, including, for instance, scumming activated sludge and sediments from moats, lakes and marine environments (Attwell & Colwell, 1981; Yamamura et al., 2005). Additionally, they are considered to play a significant role as saprophytic organisms in the turnover of naturally occurring organic substances (Goodfellow et al., 1999). Nocardia species have industrial uses as well, as they are known to produce bioactive agents such as nocardicin, tubelactomicin A and brasilicardin A (Aoki et al., 1976; Komaki et al., 1999; Igarashi et al., 2000).
Strain TT 00-78T was isolated from soil in a sugar-cane field on Amami Island in Japan, using an SDS/yeast extract pre-treatment method (Hayakawa & Nonomura, 1989) and HV agar (Hayakawa & Nonomura, 1987) containing nalidixic acid (20 mg l–1). The strain formed thin, flat colonies with sparse, white aerial hyphae on HV agar containing nalidixic acid. The aim of the present study was to determine the taxonomic position of strain TT 00-78T by using a polyphasic approach.
The colonial properties of strain TT 00-78T were recorded from colonies grown on a plate containing modified Bennett's agar (Jones, 1949) and incubated for 14 days at 28 °C. Spore motility was examined in hanging drops by means of light microscopy. Gram staining was examined by using Hucker's method (Gerhardt, 1981). Acid–alcohol-fastness was examined by using a modified version of the Ziehl–Neelsen method (Gordon, 1967) in which 0.5 % (v/v) sulfuric acid was used for decolorization. The hydrolysis of complex substrates and the utilization of carbon sources were examined by using well-established procedures (Gordon et al., 1974; Isik et al., 1999). The tests for aesculin and arbutin hydrolysis (Williams et al., 1983), nitrate reduction (Gordon & Mihm, 1962) and urea hydrolysis (Gordon et al., 1974) were performed using established procedures. Growth at 45 °C was recorded on GYEA medium (Gordon & Mihm, 1962).
Diaminopimelic acid isomers and sugars in whole-cell hydrolysates were analysed on the basis of the methods established by Hasegawa et al. (1983) and Schaal (1985), respectively. Standard procedures were also used for the extraction and analysis of mycolic acids (Schaal, 1985), fatty acids (Tamura et al., 1994), isoprenoid quinones and polar lipids (Minnikin et al., 1984); comparisons were made with the appropriate controls. Chromosomal DNA from strain TT 00-78T was isolated and purified by the method of Saito & Miura (1963) with a minor modification (Hatano et al., 2003). The DNA G+C content of the strain was determined by HPLC, as described by Tamura et al. (1994). DNA–DNA hybridization was carried out as described by Kusunoki et al. (1991), using biotinylated DNA.
PCR amplification of the 16S rRNA gene from strain TT 00-78T was carried out according to the procedures described by Tamura & Hatano (2001) and directly sequenced using an ABI Prism BigDye Terminator cycle sequencing kit (PE Applied Biosystems) and an automatic DNA sequencer (model 3100 Genetic Analyzer; PE Applied Biosystems). The 16S rRNA gene sequence obtained in the present study was aligned with reference sequences for the genus Nocardia (available from EMBL/GenBank/DDBJ) by using the CLUSTAL_X program (Thompson et al., 1997). Phylogenetic trees were constructed with MEGA, version 3.1 (Kumar et al., 2001) and CLUSTAL_X (Thompson et al., 1997), using the neighbour-joining (Saitou & Nei, 1987), minimum-evolution and maximum-parsimony methods (Takahashi & Nei, 2000). The topography of the resulting tree was evaluated by means of bootstrap analysis based on 1000 replicates (Felsenstein, 1985).
The 16S rRNA gene sequence derived from strain TT 00-78T contained the signature nucleotides characteristic of the family Nocardiaceae (Stackebrandt et al., 1997). On the basis of the phylogenetic analysis, the strain falls within the radiation of the genus Nocardia (data not shown). The chemotaxonomic and morphological characteristics of strain TT 00-78T were consistent with its assignment to the genus Nocardia (Goodfellow, 1998; Goodfellow et al., 1999). The whole-cell hydrolysate of the test strain contained meso-diaminopimelic acid, arabinose and galactose (wall chemotype IV sensu Lechevalier & Lechevalier, 1970). The major menaquinones were MK-8(H4
-cycl.) (30.3 %) and MK-8(H2) (16.8 %). The major polar lipids found were phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol (phospholipid type PII sensu Lechevalier et al., 1977). In addition, the TLC analysis revealed that the strain contained mycolic acid with an Rf value (0.46) identical to that of the reference strain used as a control. The major cellular fatty acids were hexadecanoate (43 %), hexadecenoate (20 %), tuberculostearic acid (10-methyl octadecanoate; 17 %) and cis-9 octadecanoate (10 %). The formation of branched substrate hyphae, fragmenting into rod-shaped elements (Goodfellow & Lechevalier, 1989), and relatively short aerial hyphae with chains of arthrospores were observed by microscopy (Fig. 1).
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). The 16S rRNA gene sequence similarities with phylogenetic neighbours were in the range 97.7–98.6 %. The closest phylogenetic species was N. pneumoniae, with 98.6 % sequence similarity to the type strain. The taxonomic integrity of strain TT 00-78T was supported by the DNA relatedness data. DNA relatedness values of 6.9–20.3 % were obtained with respect to the type strains of related Nocardia species (N. pneumoniae, 20.3 %; N. beijingensis, 6.9 %; N. arthritidis, 20.3 %; N. araoensis, 18.3 %), the values being well below the 70 % cut-off point recommended for the assignment of bacterial strains to the same genomic species (Wayne et al., 1987). Strain TT 00-78T was also distinguishable from its phylogenetic neighbours in comparisons of biochemical and phenotypic characteristics (Table 1).
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Description of Nocardia amamiensis sp. nov.
Nocardia amamiensis (a.ma.mi.en'sis. N.L. fem. adj. amamiensis pertaining to Amami Island, from where the organism was first isolated).
Aerobic, Gram-positive, partially acid–alcohol-fast, non-motile actinomycete that forms moderately white aerial mycelium that fragments into rod-shaped elements. Diffusible pigments are not produced. Aesculin is hydrolysed and nitrate is reduced. Arbutin and urea are not hydrolysed. Degrades uric acid, but does not degrade adenine, casein, elastin, hypoxanthine, tyrosine or xanthine. Grows at 15, 28 and 37 °C but does not grow at 5, 10 or 45 °C. Growth occurs in the presence of 0–3 % NaCl, but not at 5 % NaCl (w/v). Assimilates fumarate, L-glutamate, sebacic acid, sodium citrate, succinate (at 0.1 %, w/v), 1,2-propanediol, 2,3-butanediol, isoamyl alcohol, D-glucose, maltose, D-mannose and D-trehalose (at 1 %, w/v) as sole carbon sources, but not adipic acid, pimelic acid, sodium acetate, m-hydroxybenzoic acid, p-hydroxybenzoic acid (at 0.1 % w/v), L-arabinose, D-cellobiose, dulcitol, meso-erythritol, D-galactose, guanine, gluconate, myo-inositol, inulin, D-lactose, D-mannitol, D-melezitose, D-melibiose, methyl D-glucoside, D-raffinose, L-rhamnose, salicin, D-sorbitol, D-xylitol and D-xylose (at 1 %, w/v). The major fatty acids are hexadecanoate, hexadecenoate, tuberculostearic acid (10-methyl octadecanoate) and cis-9 octadecanoate. The G+C content of the DNA is 67.4 mol%.
The type strain, TT 00-78T (=NBRC 102102T=DSM 45066T=KCTC 19208T), was isolated from a soil sample collected from a sugar-cane field on Amami Island in Japan.
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ACKNOWLEDGEMENTS |
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