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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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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains KSL-2T, KSL-5 and KSL-6 are AY835924, AY835925 and AY835926, respectively.
A table detailing the fatty acid composition of the three isolates is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and, at the time of writing, comprises 12 species, Nocardioides albus (Prauser, 1976), N. luteus (Prauser, 1984), N. simplex (O'Donnell et al., 1982), N. jensenii (Suzuki & Komagata, 1983; Collins et al., 1989), N. plantarum (Collins et al., 1994), N. pyridinolyticus (Yoon et al., 1997), N. nitrophenolicus (Yoon et al., 1999), N. aquaticus (Lawson et al., 2000), N. aquiterrae (Yoon et al., 2004), N. ganghwensis (Yi & Chun, 2004a), N. aestuarii (Yi & Chun, 2004b) and N. alkalitolerans (Yoon et al., 2005). In this study, we describe the taxonomic characterization of three Nocardioides-like strains, KSL-2T, KSL-5 and KSL-6, with cell wall peptidoglycan based on LL-2,6-diaminopimelic acid (DAP), which were isolated from an alkaline soil (approximate pH 9·0–10·0) in Korea.
Strains KSL-2T, KSL-5 and KSL-6 were isolated by the usual dilution plating technique at 30 °C on 10x diluted nutrient agar (NA; Difco) with pH adjusted to 10·0. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM). Presence of flagella was examined by TEM using cells from exponentially growing cultures. Gram-reaction was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth at temperatures between 4 and 45 °C was measured on 2x diluted NA (pH 9·0). Growth at different NaCl concentrations was investigated in trypticase soy broth (Difco) lacking NaCl and in conventional trypticase soy broth. The pH range for growth was determined in 2x diluted nutrient broth (NB; Difco) that was adjusted to various pH values (initial pH 4·0–12·0 at intervals of 0·5 pH units). The pH of the 2x diluted NB was adjusted prior to sterilization by the addition of Na2CO3 (below pH 10·5) or KOH (above pH 10·5). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with 2x diluted NA (pH 9·0) that had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, gelatin, hypoxanthine, starch, Tweens 20, 40, 60 and 80, tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described previously (Lanyi, 1987). Utilization of various substrates as the sole carbon and energy sources was determined according to Shirling & Gottlieb (1966). Enzyme activity was determined using the API ZYM system (bioMérieux).
Cell biomass for cell wall and isoprenoid quinone analyses and for DNA extraction was obtained from cultivation in 2x diluted NB at 30 °C. Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. Chromosomal DNA was isolated and purified according to Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize RNA contamination. For fatty acid methyl ester analysis, cell mass was harvested from agar plates after incubation for 5 days on 2x diluted NA (pH 9·0) at 30 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by a modified method of Tamaoka & Komagata (1984) in which DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed according to Yoon et al. (2004). DNA–DNA hybridization was performed fluorometrically using photobiotin-labelled DNA probes and microdilution wells (Ezaki et al., 1989). Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strains KSL-2T, KSL-5 and KSL-6 are given in the species description (see below) and are shown in Table 1. The 16S rRNA gene sequences of strains KSL-2T, KSL-5 and KSL-6 determined in this study comprised 1476 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. The 16S rRNA gene sequences of the three isolates were identical. In a neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, the three strains fell within the cluster of the radiation comprising Nocardioides species (Fig. 1). Similar topologies were found in the tree generated with the maximum-likelihood algorithm (data not shown). Strains KSL-2T, KSL-5 and KSL-6 exhibited 16S rRNA gene sequence similarity values of 97·2 % to N. aquiterrae CJ-14T and 93·6–96·8 % to the type strains of the other Nocardioides species. Sequence similarities to all other species of the family Nocardioidaceae included in the phylogenetic analysis were lower than 94·7 % (Fig. 1).
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, and had MK-8(H4) as the predominant menaquinone. The fatty acid profiles were characterized by a common core of straight-chain, branched, unsaturated and 10-methyl fatty acids in similar amounts; the major fatty acid was iso-C16 : 0 (42·4–42·6 %) (fatty acid profiles are presented in Supplementary Table S1 in IJSEM Online). The fatty acid profiles were similar to those of Nocardioides species (Yoon et al., 1997, 2004; Lawson et al., 2000; Yi & Chun, 2004a). The DNA G+C content of strains KSL-2T, KSL-5 and KSL-6 was 73, 74 and 74 mol%, respectively.
DNA–DNA relatedness levels between strains KSL-2T, KSL-5 and KSL-6 were in the range of 88–93 %, indicating that the three strains are members of the same genomic species (Wayne et al., 1987). They were also almost identical in phenotypic properties. The three strains exhibited mean levels of DNA–DNA relatedness of 8–15 % to N. aquiterrae CJ-14T, the closest phylogenetic neighbour, when their DNAs were used individually as labelled DNA probes for cross-hybridization. The phylogenetic distinctiveness and DNA–DNA relatedness data were sufficient to categorize strains KSL-2T, KSL-5 and KSL-6 as a species that is distinct from the previously recognized Nocardioides species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). There were some differences in phenotypic characteristics between the three strains and some phylogenetically related Nocardioides species (Table 1
). On the basis of the data presented, strains KSL-2T, KSL-5 and KSL-6 should be classified in the genus Nocardioides as members of a novel species, for which the name Nocardioides kribbensis sp. nov. is proposed.
Description of Nocardioides kribbensis sp. nov.
Nocardioides kribbensis (kri.bben'sis. N.L. masc. adj. kribbensis arbitrary name formed from the acronym of the Korea Research Institute of Bioscience and Biotechnology, KRIBB, where taxonomic studies on this species were performed).
Cells are aerobic, non-spore-forming rods (0·8–1·0x1·5–2·0 µm) in the exponential phase of growth. Cells show rod-to-coccus morphogenesis from the early exponential phase to the stationary phase. Gram-positive but Gram-variable in old cultures. Colonies are circular, smooth, glistening, slightly convex, cream in colour and 1·0–1·5 mm in diameter after 6 days incubation on 2x diluted NA (pH 9·0). Neither substrate nor aerial mycelia are formed. Grows at 4 and 35 °C, but not at 36 °C. Optimal pH for growth is 9·0; growth occurs at pH 6·0 and 11·0, but not at pH 5·5. Growth occurs in the presence of 0–3 % (w/v) NaCl. Oxidase-positive. Tweens 20, 40 and 60 are hydrolysed. D-Sorbitol is utilized, but adonitol is 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. The DNA G+C content is 73–74 mol% (74 mol% for type strain). Other phenotypic characteristics are given in Table 1.
The type strain, KSL-2T (=KCTC 19038T=DSM 16314T), was isolated from an alkaline soil in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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.[CrossRef][Medline]
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[CrossRef]
Komagata, K. & Suzuki, K. (1987). Lipids and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–203.
Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1–67.
Lawson, P. A., Collins, M. D., Schumann, P., Tindall, B. J., Hirsch, P. & Labrenz, M. (2000). New LL-diaminopimelic acid-containing actinomycetes from hypersaline, heliothermal and meromictic Antarctic Ekho Lake: Nocardioides aquaticus sp. nov. and Friedmanniella lacustris sp. nov. Syst Appl Microbiol 23, 219–229.[Medline]
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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.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reverse-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
Yi, H. & Chun, J. (2004a). Nocardioides ganghwensis sp. nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 54, 1295–1299.
Yi, H. & Chun, J. (2004b). Nocardioides aestuarii sp. nov., isolated from tidal flat sediment. Int J Syst Evol Microbiol 54, 2151–2154.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.[CrossRef]
Yoon, J.-H., Rhee, S.-K., Lee, J.-S., Park, Y.-H. & Lee, S. T. (1997). Nocardioides pyridinolyticus sp. nov., a pyridine-degrading bacterium isolated from the oxic zone of an oil shale column. Int J Syst Bacteriol 47, 933–938.[CrossRef][Medline]
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rRNA gene sequences. Int J Syst Bacteriol 48, 187–194.[CrossRef][Medline]
Yoon, J.-H., Cho, Y.-G., Lee, S. T., Suzuki, K.-I., Nakase, T. & Park, Y.-H. (1999). Nocardioides nitrophenolicus sp. nov., a p-nitrophenol-degrading bacterium. Int J Syst Bacteriol 49, 675–680.[CrossRef][Medline]
Yoon, J.-H., Kim, I.-G., Kang, K. H., Oh, T.-K. & Park, Y.-H. (2004). Nocardioides aquiterrae sp. nov., isolated from groundwater in Korea. Int J Syst Evol Microbiol 54, 71–75.
Yoon, J.-H., Kim, I.-G., Lee, M.-H., Lee, C.-H. & Oh, T.-K. (2005). Nocardioides alkalitolerans sp. nov., isolated from an alkaline serpentinite soil in Korea. Int J Syst Evol Microbiol 55, 809–814.
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-glucosidase. All species or strains are negative for acid-fastness, hydrolysis of urea and hypoxanthine, 
-glucuronidase, N-acetyl-