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School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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8c. Tuberculostearic acid was absent. The G+C content of DNA was 72 mol%. Based on the morphological, physiological, biochemical and chemotaxonomical data presented in this study, strain JC2055T can be readily differentiated from other validly named Nocardioides species. The name Nocardioides ganghwensis sp. nov. is proposed for the isolate. The type strain is JC2055T (=IMSNU 14028T=KCTC 9920T=JCM 12124T).
The GenBank accession number for the 16S rDNA sequence of strain JC2055T is AY423718.
A supplementary table showing the fatty acid compositions of strain JC2055T and N. plantarum strain IMSNU 22067T is available in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In this study, the getbol isolate, designated JC2055T, together with nine Nocardioides type strains (Collins et al., 1994; Lawson et al., 2000; O'Donnell et al., 1982; Prauser, 1976, 1986; Suzuki & Komagata, 1983; Yoon et al., 1997, 1999, 2004) were the subject of a taxonomic investigation to elucidate the identity of the newly isolated strain.
Strain JC2055T was isolated from a sediment sample of the getbol of Ganghwa island in Korea (37° 35' 31·9'' N 126° 27' 24·5'' E) using MR2A [R2A (Difco) supplemented with artificial sea salts (Sigma)]. The isolate was routinely cultured on marine agar 2216 (MA; Difco) at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –80 °C. The reference strains used in this study are listed in Table 1. Test strains were cultured on nutrient agar (NA; Difco) at 30 °C except for Nocardioides aquaticus, for which MA was used.
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). The sequence of strain JC2055T was manually aligned with representative sequences of actinomycetes obtained from GenBank. Phylogenetic trees were inferred using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1972) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices for the neighbour-joining and Fitch–Margoliash methods were generated according to the model of Jukes & Cantor (1969). The resultant neighbour-joining tree topology was evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the JPHYDIT program (available at http://imsnu.snu.ac.kr/
pbblab/jphydit) and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000).
A nearly complete 16S rDNA sequence of strain JC2055T was obtained (1374 bp). Preliminary sequence comparison with 16S rRNA gene sequences held in GenBank indicated that our isolate belonged to the genus Nocardioides. The newly determined sequence was then aligned manually against representatives of the family Nocardioideaceae using bacterial 16S rRNA secondary structure information. Domains used to construct the phylogenetic trees were the regions available for all sequences (positions 38–1431; Escherichia coli numbering system), excluding positions showing ambiguous alignment (positions 76–95, 199–219, 455–478, 1025–1037 and 1135–1140). On the basis of 16S rDNA similarity, the closest cultured bacterial relatives were Nocardioides species (93·4–95·5 %), Aeromicrobium species (92·8–93·2 %) and Marmoricola aurantiacus DSM 12652T (93·1 %). The highest sequence similarity was to Nocardioides aquiterrae (95·5 %). This relationship between our isolate and other members of the family Nocardioideaceae was also evident in the phylogenetic tree (Fig. 1). Except for Nocardioides jensenii DSM 20641T, which formed a clade with Marmoricola aurantiacus DSM 12652T as reported previously (Urzi et al., 2000), all Nocardioides species formed a monophyletic clade. Although the branching position varied to some extent depending on the tree-making algorithms, the phyletic line of strain JC2055T was clearly placed within this Nocardioides clade based on all four tree-inferring methods used in this study. Our isolate formed a clade with Nocardioides plantarum NCIMB 12834T in the neighbour-joining, Fitch–Margoliash and two of the four most parsimonious trees with a relatively low bootstrap value (67 %), or a sister group to the subclade containing Nocardioides albus KCTC 9186T, Nocardioides luteus KCTC 9575T, Nocardioides nitrophenolicus NSP 41T and Nocardioides simplex KCTC 9106T in the maximum-likelihood and remaining most parsimonious trees.
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), NA and BM (basal medium; Baumann et al., 1972). For our isolate and N. aquaticus IMSNU 13113T, all media except MA were supplemented with half-strength artificial sea water [ASW (NaCl, 23·939 g; MgCl2, 5·079 g; Na2SO4, 3·994 g; CaCl2, 1·123 g; KCl, 0·667 g; NaHCO3, 0·196 g; KBr, 0·098 g; H3BO3, 0·027 g; SrCl2, 0·024 g; NaF, 0·003 g; distilled water, 1 l); Lyman & Fleming, 1940].
Growth under anaerobic conditions was checked in an anaerobic chamber (10 % CO2, 10 % H2, 80 % N2; Sheldon Manufacturing). The growth ranges of the test strain for temperature (between 5 and 50 °C at 5 °C intervals), pH (between pH 4 and 12 at intervals of 1) and NaCl concentration [between 0 and 15 % (w/v) at 1 % intervals] were determined using synthetic ZoBell medium [ZoBell, 1941; Bacto agar (Difco) 15 g; Bacto peptone (Difco), 5 g; yeast extract (Difco), 1 g; ferric citrate, 0·1 g; sea salts, 40 g; distilled water, 1 l). Sea salts were not added to the synthetic medium when the growth range for NaCl concentration was tested. 6 M KOH and 6 M HCl were used to adjust the final pH.
Standard physiological and biochemical tests were performed as described previously (Smibert & Krieg, 1994). Acid-fastness was determined by Ziehl–Neelsen staining (Hendrickson & Krenz, 1991). Hydrolysis of alginic acid (0·5 %, w/v), casein [50 % skim milk (Difco), v/v], cellulose [0·5 % carboxymethyl cellulose (Sigma), w/v], starch (0·2 %, w/v) and Tween 80 (1 %, w/v) was tested using MA or NA as the basal medium. DNase test agar (Difco) was used to assay DNase activity. Decomposition of adenine (0·5 %, w/v), hypoxanthine (0·5 %, w/v), L-tyrosine (0·5 %, w/v) and xanthine (0·4 %, w/v) was checked using MA or NA according to Gordon et al. (1974). Arginine dihydrolase, 
-galactosidase, nitrate reduction, urease, acid production from glucose and indole production tests were performed using the API 20NE kit (bioMérieux), and other enzymic activities were determined using the API ZYM kit (bioMérieux). Strips for our isolate and N. aquaticus IMSNU 13113T were inoculated with a heavy bacterial suspension in half-strength ASW and AUX medium (bioMérieux) supplemented with 2·5 % (w/v) NaCl. Sole carbon source utilization was tested in 96-well tissue culture microplates (Falcon) as described by Gosink et al. (1998). BM, supplemented with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and modified by reducing the concentration of sea salts to half-strength, was used as basal medium for all test strains. The results of morphological, biochemical and physiological tests are given in Table 1
and the species description.
Chemotaxonomic characteristics of strain JC2055T were determined using cells grown at 30 °C for 3 days on MA or in MB (marine broth 2216; Difco). Menaquinone was isolated according to Minnikin et al. (1984) and analysed by HPLC (Waters) as described by Collins (1985). DNA G+C content (mol%) was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989) using a reverse-phase column (Supelcosil LC-18-S; Supelco). Analysis of fatty acid methyl esters was performed by GLC according to the instructions of the Microbial Identification System (MIDI). Nocardioides plantarum IMSNU 22067T, grown on NA at 30 °C for 4 days, was also analysed, as its fatty acid composition has never been reported. Cell wall diamino acids were determined as described by Staneck & Roberts (1974). Our isolate contained MK-8(H4) as the major menaquinone and LL-diaminopimelic acid as the diagnostic cell wall diamino acid. The results of chemotaxonomic analyses are given in the species description and as supplementary data in IJSEM Online. Most chemotaxonomic characters of strain JC2055T were typical of the genus Nocardioides (Collins et al., 1994; Lawson et al., 2000; O'Donnell et al., 1982; Yoon et al., 1997, 1999, 2004). In general, the cellular fatty acid composition of our isolate also resembled that of Nocardioides species. However, the strain had i-16 : 0 (30·4 %) and 17 : 18c (25·7 %) as the major fatty acids, and contained no tuberculostearic acid, which is present in other Nocardioides species at 0·8–13·9 %.
On the basis of 16S rDNA phylogeny and chemotaxonomy, it is clear that our isolate belongs to the genus Nocardioides. Low 16S rDNA sequence similarity values to Nocardioides species (93·4–95·5 %) indicate that strain JC2055T represents a novel genomic species in the genus (Stackebrandt & Goebel, 1994; Rosselló-Mora & Amann, 2001). Along with the fatty acid composition, a number of morphological and physiological characteristics can be used to differentiate the strain from other Nocardioides species at the phenotypic level (Table 1). Based on the polyphasic evidence it is therefore suggested that strain JC2055T be assigned to a novel species, for which the name Nocardioides ganghwensis sp. nov. is proposed.
Description of Nocardioides ganghwensis sp. nov.
Nocardioides ganghwensis (gang.hwen'sis. N.L. masc. adj., ganghwensis named after Ganghwa island in Korea, the geographical origin of the type strain).
Gram-positive and strictly aerobic bacterium. Oxidase-negative, catalase-positive and not acid-fast. Spores are not formed. Cells are non-motile rods and approximately 0·9–4·5x0·4–0·5 µm. Substrate or aerial mycelium is not observed. Colonies on MA are ivory, circular, convex, entire, glistening, opaque and butyraceous. Colonies are approximately 1–2 mm in diameter after 3 days (on MA at 30 °C) and reach a maximum diameter of 3–4 mm after 5 days. Growth occurs in 0–8 % (w/v) NaCl with the optimum being at 0–1 %. Growth occurs at pH 6–10, with the optimum being at pH 7. Growth occurs at 10–40 °C, with the optimum being at 30 °C. Major menaquinone is MK-8(H4). Cell wall diamino acid is LL-DAP. Major fatty acids are i-16 : 0 (30·4 %) and 17 : 18c (25·7 %). In addition, 18 : 19c (5·7 %), i-16 : 1 H (4·4 %), 10Me-17 : 0 (3·9 %), i-15 : 0 (2·7 %), i-17 : 0 (2·4 %) and 17 : 0 (2·0 %) are also present. Tuberculostearic acid is absent. The DNA G+C content is 72 mol%. Results of API 20NE and API ZYM kits, decomposition of high molecular mass substrates and carbon source utilization are given in Table 1. The type and only strain is JC2055T (IMSNU 14028T=KCTC 9920T=JCM 12124T). Isolated from a sediment sample of getbol (tidal flat), Ganghwa island, Korea.
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ACKNOWLEDGEMENTS |
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-chymotrypsin, 