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1 Marine Environmental Research Department, Korea Ocean Research and Development Institute, Ansan 426-744, Republic of Korea
2 School of Earth and Environmental Sciences, Seoul National University, Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Byung Cheol Cho
bccho{at}snu.ac.kr
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ABSTRACT |
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A table detailing the cellular fatty acid content of strain CL-DD14T and other species of the genus Nocardioides is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), many species of the genus have been isolated from various environments including soil, herbage, groundwater, an oil-shale column, black sand, crude oil, a saline lake and a tidal flat (Yoon et al., 2006a, b, 2007; Lee, 2007). Most of these species were isolated from non-saline environments, although species such as Nocardioides aquaticus (Lawson et al., 2000), Nocardioides aestuarii (Yi & Chun, 2004b) and Nocardioides ganghwensis (Yi & Chun, 2004a) were isolated from marine environments. In the present study, a strain affiliated with the genus Nocardioides was isolated from seawater and subjected to a polyphasic taxonomic analysis.
Strain CL-DD14T was isolated from seawater around ‘Dokdo’ island in the East Sea, Korea, using a selective S medium (10 g Na2HPO4, 3 g KH2PO4, 1 g K2SO4, 30 g NaCl, 0.2 g MgSO4.7H2O, 0.01 g CaCl2, 0.001 g FeSO4.7H2O, 1 g Casamino acids, 1 g yeast extract, 20 g glucose and 20 g Bacto agar, per litre distilled water; Fialho et al., 1999). Subsequently, the isolate was routinely cultivated on marine agar 2216 (MA; Difco) at 30 °C and maintained both on MA at 4 °C and in marine broth (MB; Difco) supplemented with 30 % (v/v) glycerol at –80 °C.
Morphological, physiological and biochemical analyses were performed with MA as the basal medium at 30 °C. Gram staining was performed as described by Smibert & Krieg (1994). Cell morphology and motility were observed by phase-contrast microscopy. Transmission electron microscopy subsequent to negative staining was used to establish whether flagella were present. Anaerobic growth on MA was checked by using the GasPack anaerobic system (BBL). Catalase and oxidase activities and degradation of casein, hypoxanthine, xanthine and L-tyrosine were determined according to the protocols described by Smibert & Krieg (1994). Degradation of Tween 80 was examined as described by Hansen & Sørheim (1991).
The temperature range for growth was determined on the basis of formation of visible single colonies on MA incubated at 5–45 °C, at intervals of 5 °C for 1 month. The pH range (between pH 5 and 10 at intervals of one pH unit) for growth was determined by changes in OD600 with time in MB. The final pH was adjusted using 1 M NaOH and 1 N HCl solutions. Tolerance of strain CL-DD14T to NaCl was determined by using synthetic ZoBell broth (5 g Bacto peptone, 1 g yeast extract, 0.1 g ferric citrate, per litre distilled water) with various concentrations (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 %, w/v) of NaCl. Nitrate reduction, production of indole, arginine dihydrolase, urease, gelatinase and 
-galactosidase activities, acid production from glucose, and hydrolysis of aesculin were tested by using the API 20NE kit (bioMérieux) according to the manufacturer's instructions, except that cell suspension was prepared using half-strength artificial seawater (ASW; 24 g NaCl, 5.1 g MgCl2, 4 g Na2SO4, 1.1 g CaCl2, 0.7 g KCl, 0.2 g NaHCO3, 0.1 g KBr, 0.027 g H3BO3, 0.024 g SrCl2, 0.003 g NaF, per litre distilled water; Lyman & Fleming, 1940) as the suspension medium. Other enzymic activities were determined with the API ZYM kit (bioMérieux) and half-strength ASW as the suspension medium. Carbon utilization was tested by using a modified basal medium (11.8 g NaCl, 0.32 g KCl, 2.26 g MgCl2.6H2O, 2.97 g MgSO4.7H2O, 0.65 g CaCl2.2H2O, 0.2 g NaNO3, 0.2 g NH4Cl, per litre distilled water) supplemented with yeast extract (0.05 g l–1) based on changes in OD600 with time for 1 month. Concentrations of added carbon source were 0.2 % (final concentration). A test tube without any carbon source was used as a negative control. The results of morphological, physiological and biochemical tests are given in Table 1 and in the species description below.
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and analysed by HPLC as described by Collins (1985) at the Korean Culture Center of Microorganisms (KCCM, Seoul, Republic of Korea). Cell-wall diamino acids were determined as described by Staneck & Roberts (1974). The whole-cell fatty acid methyl esters of strain CL-DD14T, grown on MA plates at 30 °C for 1 day, were analysed by GC according to the instructions of the Microbial Identification System (MIDI) at KCCM. Strain CL-DD14T contained tetrahydrogenated menaquinone with eight isoprene units [MK-8(H4)] as the major menaquinone and LL-diaminopimelic acid as the cell-wall diamino acid. The cellular fatty acid composition of strain CL-DD14T was as follows: branched fatty acids iso-C16 : 0 (71.5 %), iso-C18 : 0 (2.1 %) and iso-C14 : 0 (2.0 %); 10-methyl fatty acids C17 : 0 (5.1 %), C18 : 0 (1.7 %) and C16 : 0 (1.4 %); unsaturated fatty acids C18 : 1
9c (4.4 %), C17 : 18c (3.8 %) and C18 : 17c (1.6 %); saturated fatty acid C16 : 0 (1.3 %), and summed feature 3, comprising iso-C15 : 0 2-OH and/or C16 : 17c, (1.3 %) (see Supplementary Table S1 available in IJSEM Online). Predominant fatty acids were iso-C16 : 0 and the 10-methyl fatty acid C17 : 0, which are typical characteristics of the genus Nocardioides.
The 16S rRNA gene was amplified by PCR with Taq DNA polymerase (Bioneer) and primers 27F and 1492R (Lane, 1991). The PCR product was purified by using the AccuPrep PCR purification kit (Bioneer) and cloned by using the pGEM Easy TA vector (Promega). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automatic sequencer (ABI3730XL) at Macrogen Corp., Seoul, Republic of Korea. The almost-complete 16S rRNA gene sequence of strain CL-DD14T (1440 bp) was obtained. The sequence was compared with those available in GenBank using BLASTN searches (Altschul et al., 1990). The 16S rRNA gene sequence of strain CL-DD14T was manually aligned with those of the type strains of other species belonging to the genus Nocardioides and phylogenetically related genera obtained from GenBank by using known 16S rRNA gene secondary structure information. Phylogenetic trees were obtained by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969). The robustness of tree topologies was assessed by bootstrap analyses based on 1000 replications for the neighbour-joining and maximum-parsimony methods and 100 replications for the maximum-likelihood method. Alignment was carried out via the jPHYDIT program (Jeon et al., 2005) and phylogenetic analyses were performed with MEGA 3 (Kumar et al., 2004) and PAUP* 4.0 (Swofford, 1998). Likelihood parameters were estimated by using the hierarchical ratio tests in MODELTEST version 3.04 (Posada & Crandall, 1998). The DNA G+C content was determined by HPLC analysis (Tamaoka & Komagata, 1984) at KCCM.
The DNA G+C content of strain CL-DD14T was 72.9 mol%. 16S rRNA gene sequence analysis indicated that the closest relatives of strain CL-DD14 T were the type strains of Nocardioides aquiterrae (97.1 % similarity), Nocardioides pyridinolyticus (96.6 %) and N. aestuarii (96.4 %). Other members of the genus Nocardioides showed 16S rRNA gene sequence similarities of 94.1–96.3 % to the novel strain. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain CL-DD14T formed an evolutionary lineage within the radiation enclosing most species of the genus Nocardioides. Similarity between strain CL-DD14T and N. aquiterrae was close to the recommended guideline (97 %) for species designation at the 16S rRNA gene level (Stackebrandt & Goebel, 1994). However, a dendrogram of 16S rRNA gene sequence relationships showed that strain CL-DD14T formed a distinct branch from N. aquiterrae and was recovered as a sister group to the subclade containing Nocardioides albus and Nocardioides luteus that exhibited low 16S rRNA gene sequence similarity values (96.0–96.1 %) (Fig. 1). Furthermore, phenotypic features (NaCl requirement, temperature and pH ranges for growth, absence of motility, negative reactions for oxidase, gelatinase, nitrate reductase and naphthol-AS-BI-phosphohydrolase, presence of alkaline phosphatase, and carbon utilization pattern) clearly differentiated strain CL-DD14T from N. aquiterrae (Table 1
). In addition, strain CL-DD14T could be differentiated from N. albus and N. luteus based on phenotypic characteristics (absence of hyphae, negative in tests for gelatin, tyrosine, hypoxanthine and starch hydrolysis, and carbon utilization patterns) (Table 1). In conclusion, phenotypic features and phylogenetic analyses based on 16S rRNA gene sequences suggest that strain CL-DD14T represents a novel species of the genus Nocardioides, for which the name Nocardioides marinus sp. nov. is proposed.
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Cells are rods (about 1.0–1.8 µm long and 0.4–0.6 µm wide) in the exponential phase and coccus forms are found in the stationary phase. Cells are non-motile and strictly aerobic. Gram-positive, oxidase-negative and weakly catalase-positive. Colonies on MA are circular, smooth, convex with an entire margin and creamy white. Substrate or aerial mycelium is not observed. Growth occurs at 10–40 °C (optimum about 25–30 °C) and at pH values of 6–9 (optimum pH 7–8). Growth occurs at NaCl concentrations of 0.5–8 % (w/v) (optimum 1–3 %). Negative in tests for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease and gelatinase, and positive for aesculin hydrolysis and -galactosidase in API 20NE tests. Casein and Tween 80 are hydrolysed, but hypoxanthine, xanthine, L-tyrosine and starch are not. Major menaquinone is MK-8(H4). Cell-wall diamino acid is LL-diaminopimelic acid. The major fatty acids are iso-C16 : 0 (71.5 %) and 10-methyl C17 : 0 (5.1 %). Results of API ZYM and carbon source utilization tests are given in Table 1. The DNA G+C content is 72.9 mol%.
The type strain, CL-DD14T (=KCCM 42321T=DSM 18248T), was isolated from seawater of the East Sea, Korea.
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ACKNOWLEDGEMENTS |
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-glucosidase, esterase lipase (C8) and leucine arylamidase, but negative for 