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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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Published online ahead of print on 10 June 2005 as DOI 10.1099/ijs.0.63748-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Nocardioides dubius sp. nov. KSL-104T is AY928902.
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with a single species, Nocardioides albus, and, at the time of writing, the genus comprises 14 species with validly published names (Schippers et al., 2005; Yoon et al., 2005b). Recently, a bacterial strain, KSL-104T, containing LL-2,6-diaminopimelic acid (DAP) was isolated from an alkaline soil (approximate pH 9·0–10·0) in Korea. 16S rRNA gene sequence analyses indicated that strain KSL-104T is phylogenetically related to Nocardioides jensenii and Marmoricola aurantiacus. N. jensenii was proposed by the reclassification of a species that had been previously assigned to the genus Pimelobacter (Suzuki & Komagata, 1983; Collins et al., 1989). 16S rRNA gene sequence analyses showed that N. jensenii forms an evolutionary lineage at the periphery of the phylogenetic radiation encompassed by the genus Nocardioides and joins the genus Marmoricola with a high bootstrap resampling value (Yoon et al., 1998, 2005a, b; Urzì et al., 2000; Yi & Chun, 2004a, b). The genus Marmoricola was described by Urzì et al. (2000) with a single species, Marmoricola aurantiacus, which was differentiated from the genus Nocardioides and from N. jensenii by the absence of iso- and anteiso-branched fatty acids, the presence of phosphatidylinositol and the presence of only spherical cells, occurring singly, in pairs, tetrads and small clusters (Urzì et al., 2000). Accordingly, the aim of the present study was to establish the exact taxonomic position of strain KSL-104T by a polyphasic taxonomic approach.
An alkaline soil collected from Kwangchun, Korea, was used as the source for the isolation of bacterial strains. Strain KSL-104T was isolated by the standard dilution plating technique at 30 °C on 10x diluted nutrient agar (NA; Difco) with the pH adjusted to 10·0 using Na2CO3. To investigate its morphological, physiological and biochemical characteristics, strain KSL-104T was routinely cultivated on NA at 30 °C. N. jensenii KCTC 9134T, which was used as the reference strain for DNA–DNA hybridization, was obtained from the Korean Collection for Type Cultures, Taejon, Korea. Morphological, physiological and biochemical properties were examined as described by Yoon et al. (2005c). Growth at various NaCl concentrations [0–8 % (w/v) at intervals of 1·0 %] was investigated in trypticase soy broth (TSB; Difco) lacking NaCl and TSB supplemented with NaCl. The pH range for growth was determined in nutrient broth (NB; Difco) that was adjusted to various pH values (initial pH 4·0–10·5 at intervals of 0·5 pH units). The pH of the NB was adjusted prior to sterilization by the addition of HCl or Na2CO3. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux). Cell biomass for the analyses of cell-wall components, isoprenoid quinones, polar lipids and for DNA extraction was obtained by cultivation in NB at 30 °C. For fatty acid methyl ester analysis, cell mass of strain KSL-104T was harvested from agar plates after incubation for 6 days on NA at 30 °C. Chemotaxonomic and molecular systematic studies were performed as described by Yoon et al. (2005c). Polar lipids were extracted according to Minnikin et al. (1984) and were identified using two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987).
The morphological, cultural, physiological and biochemical characteristics of strain KSL-104T are given in the species description (see later) or are shown in Table 1. The 16S rRNA gene sequence of strain KSL-104T determined in this study comprised 1473 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analyses showed that strain KSL-104T was phylogenetically affiliated to the genera Nocardioides and Marmoricola (Fig. 1). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain KSL-104T joined the cluster comprising N. jensenii and M. aurantiacus with a bootstrap resampling value of 69·4 % (Fig. 1). In trees generated with the maximum-likelihood and the maximum-parsimony algorithms, strain KSL-104T joined the cluster comprising Nocardioides species but excluding N. jensenii (data not shown). Strain KSL-104T exhibited 16S rRNA gene sequence similarity levels of 97·1 % compared with the type strain of N. jensenii and 94·0–96·5 % with the type strains of the other Nocardioides species. The similarity value between the 16S rRNA gene sequence of strain KSL-104T and that of M. aurantiacus DSM 12652T was 95·4 %.
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, 2004, 2005a, b; Lawson et al., 2000; Urzì et al., 2000; Yi & Chun, 2004a, b). Strain KSL-104T had a cellular fatty acid profile that contained large amounts of branched, unsaturated, straight-chain and 10-methyl fatty acids. Major components (>1 %) were branched fatty acids iso-C16 : 0 (58·5 %), iso-C18 : 0 (4·3 %), iso-C16 : 1 (3·7 %), iso-C17 : 0 (3·2 %), iso-C14 : 0 (2·5 %), anteiso-C17 : 0 (2·0 %) and iso-C15 : 0 (1·9 %), straight-chain fatty acids C18 : 0 (2·1 %) and C16 : 0 (1·2 %), unsaturated fatty acids C18 : 1
9c (1·7 %), C17 : 18c (1·3 %) and C18 : 17c (1·1 %) and 10-methyl fatty acids C17 : 0 (7·0 %), C16 : 0 (4·2 %) and C18 : 0 (2·7 %). This fatty acid profile was similar to those of Nocardioides species (Yoon et al., 1997, 2004, 2005a, b; Lawson et al., 2000; Urzì et al., 2000; Yi & Chun, 2004a). The major polar lipids detected in strain KSL-104T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The DNA G+C content of strain KSL-104T was 70·6 mol%.
Strain KSL-104T was differentiated from M. aurantiacus by having a rod/coccus life cycle and by exhibiting a different fatty acid profile (Urzì et al., 2000). Phosphatidylinositol was described as being a marker to differentiate the genera Nocardioides and Marmoricola by Urzì et al. (2000), but this polar lipid was also reported to be present in N. jensenii and Nocardioides aquaticus (Collins et al., 1989; Lawson et al., 2000). There were not sufficient phenotypic, and particularly chemotaxonomic, properties to distinguish strain KSL-104T from the genus Nocardioides (Yoon et al., 1997, 2004, 2005a, b; Lawson et al., 2000; Yi & Chun, 2004a, b). Although phosphatidylethanolamine was not found in Nocardioides species, inter-specific differences in polar lipid profiles have been found in many other genera as well as in the genus Nocardioides (Collins et al., 1989; Park et al., 1999; Lawson et al., 2000). The chemotaxonomic properties of strain KSL-104T were distinguishable from those of the other genera that belong to the family Nocardioidaceae (Miller et al., 1991; Tamura & Yokota, 1994; Park et al., 1999; Wang et al., 2001). Accordingly, in view of the combined phenotypic and phylogenetic data, it appears to be appropriate for strain KSL-104T to be placed in the genus Nocardioides. Strain KSL-104T was distinguishable from N. jensenii by some phenotypic characteristics, in particular, the presence of phosphatidylethanolamine, as listed in Table 1
, and the mean DNA–DNA relatedness value with N. jensenii KCTC 9134T was 13 %. Therefore, on the basis of the data presented, strain KSL-104T should be classified in the genus Nocardioides as a representative of a novel species, for which the name Nocardioides dubius sp. nov. is proposed.
Description of Nocardioides dubius sp. nov.
Nocardioides dubius (du'bi.us. L. masc. adj. dubius doubtful of the taxonomic position).
Cells are aerobic, non-endospore-forming rods (0·8–1·0x1·5–2·5 µm) in the exponential phase of growth. Cells show rod-to-coccus morphogenesis from the early exponential phase to the stationary phase. Gram-positive. Motile by means of a single polar flagellum. Colonies are irregular, smooth, raised with erose margins, yellowish-white in colour and 2·0–3·0 mm in diameter after 3 days incubation on NA at 30 °C. Neither substrate nor aerial mycelia are formed. Growth occurs at 10 and 37 °C, but not at 4 or 38 °C. Optimal pH for growth is 7·0–8·0; growth occurs at pH 6·0 and 10·5, but not at pH 5·5. Growth occurs in the presence of 0–5 % (w/v) NaCl, with an optimum at 0 % (w/v) NaCl. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. H2S and indole are not produced. Tweens 20, 40 and 60 are not hydrolysed. Pyruvate is utilized, but maltose, D-trehalose, formate and L-malate are not. The cell-wall peptidoglycan contains LL-DAP as the diagnostic diamino acid. The predominant menaquinone is MK-8(H4). The major fatty acid is iso-C16 : 0. Major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The DNA G+C content is 70·6 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, KSL-104T (=KCTC 9992T=JCM 13008T), was isolated from an alkaline soil in Kwangchun, Korea.
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ACKNOWLEDGEMENTS |
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Komagata, K. & Suzuki, K. (1987). Lipids and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–203.
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Yoon, J.-H., Kim, I.-G., Lee, M.-H., Lee, C.-H. & Oh, T.-K. (2005a). Nocardioides alkalitolerans sp. nov., isolated from an alkaline serpentinite soil in Korea. Int J Syst Evol Microbiol 55, 809–814.
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