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Int J Syst Evol Microbiol 57 (2007), 136-140; DOI  10.1099/ijs.0.64612-0
© 2007 International Union of Microbiological Societies

Nocardioides insulae sp. nov., isolated from soil

Jung-Hoon Yoon, So-Jung Kang, Choong-Hwan Lee and Tae-Kwang Oh

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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A Gram-positive, rod-shaped or coccoid bacterial strain, DS-51T, was isolated from a soil in Dokdo, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain DS-51T grew optimally at pH 8.0 and 30 °C without NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain DS-51T forms a distinct line of descent within the radiation enclosed by the genus Nocardioides. The chemotaxonomic properties of strain DS-51T were consistent with those of the genus Nocardioides: the cell-wall peptidoglycan type was based on LL-2,6-diaminopimelic acid, MK-8(H4) was the predominant menaquinone and iso-C16 : 0 was the major fatty acid. The DNA G+C content was 71.1 mol%. The 16S rRNA gene sequence of strain DS-51T had similarity levels of 92.5–95.1 % with the sequences of the type strains of Nocardioides species. Strain DS-51T could be distinguished from other Nocardioides species by differences in some phenotypic characteristics. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain DS-51T represents a novel species of the genus Nocardioides, for which the name Nocardioides insulae sp. nov. is proposed. The type strain is DS-51T (=KCTC 19180T=DSM 17944T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DS-51T is DQ786794.


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The genus Nocardioides was proposed by Prauser (1976)Go and, at the time of writing, comprises 18 recognized species, including the recently described species Nocardioides lentus (Yoon et al., 2006aGo) and Nocardioides kongjuensis (Yoon et al., 2006bGo). Here, we report on the taxonomic characterization of a Nocardioides-like strain, DS-51T, which was isolated from a soil collected in Dokdo, an island of Korea.

Strain DS-51T was isolated by using the standard dilution plating technique at 30 °C on 10x diluted nutrient agar (NA; Difco). To investigate its morphological, physiological and biochemical characteristics, strain DS-51T was routinely cultivated at 30 °C on NA and in nutrient broth (NB; Difco). The morphological, physiological, cultural and biochemical properties were examined as described by Yoon et al. (2005a)Go. Growth at various NaCl concentrations (0–8 %, w/v, using increments of 1.0 %) was investigated in trypticase soy broth prepared according to the formula of Difco medium except that no NaCl was used. The pH range for growth was determined in NB that was adjusted to various pH values (pH 4.0–10.5, using increments of 0.5 pH units) by the addition of HCl and Na2CO3. The susceptibility of strain DS-51T to various antibiotics was tested on NA plates by using antibiotic discs, as follows: 100 U polymyxin B, 50 µg streptomycin, 20 U penicillin G, 100 µg chloramphenicol, 10 µg ampicillin, 30 µg cephalothin, 30 µg gentamicin, 5 µg novobiocin, 30 µg tetracycline, 30 µg kanamycin, 15 µg lincomycin, 15 µg oleandomycin, 30 µg neomycin and 100 µg carbenicillin. Other physiological and biochemical properties were tested by using the API 20E and API ZYM systems (bioMérieux). Cell biomass for DNA extraction and for analyses of cell walls and isoprenoid quinones was obtained by cultivation in NB at 30 °C for 3 days. For fatty acid methyl ester analysis, cell mass of strain DS-51T was harvested from NA plates after incubation at 30 °C for 7 days. Chemotaxonomic and molecular systematic studies were performed as described by Yoon et al. (2005a)Go. The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987)Go.

Morphological, cultural, physiological and biochemical characteristics of strain DS-51T are given in the species description (see below) or are shown in Table 1Go. Strain DS-51T was found to be susceptible to polymyxin B, streptomycin, chloramphenicol, cephalothin, gentamicin, tetracycline, carbenicillin, kanamycin, lincomycin, neomycin and oleandomycin, but not to ampicillin, novobiocin or penicillin G. The almost-complete 16S rRNA gene sequence of strain DS-51T, comprising 1484 nt (approx. 96 % of the Escherichia coli 16S rRNA gene sequence), was determined in this study. Comparative 16S rRNA gene sequence analyses showed that strain DS-51T is most closely affiliated to the genus Nocardioides (Fig. 1Go). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain DS-51T formed a distinct phylogenetic lineage with the radiation of the cluster comprising Nocardioides species (Fig. 1Go). Strain DS-51T exhibited 16S rRNA gene sequence similarity values of 92.5–95.1 % with respect to the type strains of other Nocardioides species and showed values of less than 92.1 % sequence similarity with other species used in the phylogenetic analysis.


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  		Table 1. Differential phenotypic characteristics of strain DS-51T and other Nocardioides species

Taxa: 1, strain DS-51T; 2, N. albus; 3, N. luteus; 4, N. simplex; 5, N. plantarum; 6, N. pyridinolyticus; 7, N. nitrophenolicus; 8, N. aquaticus; 9, N. aquiterrae; 10, N. ganghwensis; 11, N. aestuarii; 12, N. alkalitolerans; 13, N. kribbensis; 14, N. oleivorans; 15, N. aromaticivorans; 16, N. lentus; 17, N. kongjuensis. Data are from Collins et al. (1989Go, 1994)Go, Lawson et al. (2000)Go, Prauser (1976Go, 1984Go, 1989)Go, Suzuki & Komagata (1983)Go, Yoon et al. (1997Go, 1999Go, 2004Go, 2005aGo, bGo, 2006aGo, b)Go, Yi & Chun, (2004aGo, b)Go, Schippers et al. (2005)Go and Kubota et al. (2005)Go. +, Positive reaction; –, negative reaction; ND, not determined; W, weakly positive reaction; V, variable reaction. Data in parentheses are for the type strain. All species are positive for the Gram stain, for catalase and for esterase lipase (C8) (not determined for N. oleivorans). All species are negative for beta-glucuronidase, N-acetyl-beta-glucosaminidase and {alpha}-fucosidase activities (not determined for N. oleivorans).

 

Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the positions of strain DS-51T and some related taxa. Bootstrap values (expressed as percentages of 1000 replications) greater than 50 % are shown at the branch points. Bar, 0.01 substitutions per nucleotide position.

 
The chemotaxonomic properties supported the result of the monothetic phylogenetic classification suggesting that strain DS-51T could be a member of the genus Nocardioides (Tamura & Yokota, 1994Go; Yoon et al., 1997Go; Lawson et al., 2000Go; Urzì et al., 2000Go; Wang et al., 2001Go). The diagnostic diamino acid present in strain DS-51T was LL-2,6-diaminopimelic acid, which is characteristic of wall chemotype I sensu Lechevalier & Lechevalier (1970)Go. The predominant isoprenoid quinone detected in strain DS-51T was a tetrahydrogenated menaquinone with eight isoprene units [MK-8(H4)]. The cellular fatty acid profile (i.e. fatty acids representing >1 % of the total) comprised the following: branched fatty acids iso-C16 : 0 (49.7 %), anteiso-C17 : 0 (4.9 %), iso-C17 : 0 (2.6 %), iso-C18 : 0 (2.3 %), iso-C15 : 0 (1.6 %) and iso-C16 : 1 (2.3 %); unsaturated fatty acids C18 : 1{omega}9c (7.4 %), C17 : 1{omega}6c (5.4 %) and C17 : 1{omega}8c (1.1 %); straight-chain fatty acids C18 : 0 (2.3 %), C17 : 0 (1.9 %) and C16 : 0 (1.7 %); 10-methyl fatty acids C18 : 0 (8.3 %), C17 : 0 (2.9 %) and C16 : 0 (1.8 %), and summed feature 3 (C16 : 1{omega}7c and/or iso-C15 : 0 2-OH) (1.6 %). This fatty acid profile is similar to those of Nocardioides species, although there are differences in the proportions of some fatty acids, perhaps because of differences in the extraction and cultivation conditions (Yoon et al., 1997Go, 1999Go, 2004Go, 2005aGo, bGo; Lawson et al., 2000Go; Yi & Chun, 2004aGo, bGo; Kubota et al., 2005Go). The DNA G+C content of strain DS-51T was 71.1 mol%. The differential phenotypic properties of DS-51T, together with its phylogenetic distinctiveness, are sufficient to categorize the strain as a novel species of the genus Nocardioides (Stackebrandt & Goebel, 1994Go; Table 1Go), for which the name Nocardioides insulae sp. nov. is proposed.

Description of Nocardioides insulae sp. nov.
Nocardioides insulae (in.su'lae. L. fem. gen. n. insulae of an island, referring to the source of isolation of the type strain).

Cells are aerobic, non-spore-forming, Gram-positive rods or cocci (0.6–1.0x1.0–6.0 µm) in the exponential phase of growth. Cells show rod-to-coccus morphogenesis from the early exponential phase to the stationary phase. Oxidase-positive. Colonies are circular, smooth, glistening, slightly convex, ivory in colour and 1.0–1.5 mm in diameter after 7 days incubation on NA at 30 °C. Neither substrate nor aerial mycelium is formed. Growth occurs at 10 and 34 °C, but not at 4 or 35 °C. The optimal pH for growth is 8.0; growth occurs at pH 6.5, but not at pH 6.0. Growth occurs in the presence of 0–3 % (w/v) NaCl, but is optimum in the absence of NaCl. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities are absent. H2S and indole are not produced. Tweens 20, 40 and 60 are hydrolysed. Acetate, L-malate, pyruvate and L-glutamate are utilized as sole carbon and energy sources and salicin is weakly utilized, but citrate, succinate, benzoate and formate are not utilized. The cell-wall peptidoglycan contains LL-2,6-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone is MK-8(H4). The major fatty acid (>10 % of total fatty acids) is iso-C16 : 0. The DNA G+C content is 71.1 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1Go.

The type strain, DS-51T (=KCTC 19180T=DSM 17944T), was isolated from soil.


   ACKNOWLEDGEMENTS
 
This work was supported by the 21C Frontier Program of Microbial Genomics and Applications (grant MG05-0401-2-0) from the Ministry of Science and Technology (MOST) of the Republic of Korea.


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Collins, M. D., Cockcroft, S. & Wallbanks, S. (1994). Phylogenetic analysis of a new LL-diaminopimelic acid-containing coryneform bacterium from herbage, Nocardioides plantarum sp. nov. Int J Syst Bacteriol 44, 523–526.[Abstract/Free Full Text]

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Lechevalier, M. P. & Lechevalier, H. A. (1970). A critical evaluation of the genera of aerobic actinomycetes. In The Actinomycetales, pp. 393–405. Edited by H. Prauser. Jena: Gustav Fischer.

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Prauser, H. (1989). Genus Nocardioides Prauser 1976Go. In Bergey's Manual of Systematic Bacteriology, vol. 4, pp. 2371–2375. Edited by S. T. Williams, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.

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Suzuki, K. & Komagata, K. (1983). Pimelobacter gen. nov., a new genus of coryneform bacteria with LL-diaminopimelic acid in the cell wall. J Gen Appl Microbiol 29, 59–71.

Tamura, T. & Yokota, A. (1994). Transfer of Nocardioides fastidiosa Collins and Stackebrandt 1989 to the genus Aeromicrobium as Aeromicrobium fastidiosum comb. nov. Int J Syst Bacteriol 44, 608–611.[Abstract/Free Full Text]

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