Nocardioides salarius sp. nov., isolated from seawater enriched with zooplankton

At the time of writing, since the description of the genus Nocardioides within the class Actinobacteria (Prauser, 1976), 27 species of the genus have been isolated from various environments. Nocardioides simplex (Jensen, 1934), Nocardioides albus (Prauser, 1976), Nocardioides jensenii (Suzuki & Komagata, 1983; Collins et al., 1989), Nocardioides luteus (Prauser, 1984), Nocardioides alkalitolerans (Yoon et al., 2005a), Nocardioides kribbensis (Yoon et al., 2005b), Nocardioides dubius (Yoon et al., 2005c), Nocardioides lentus (Yoon et al., 2006a), Nocardioides kongjuensis (Yoon et al., 2006b), Nocardioides insulae (Yoon et al., 2007a), Nocardioides panacihumi (An et al., 2007), Nocardioides hankookensis (Yoon et al., 2008) and Nocardioides terrigena (Yoon et al., 2007b) were isolated from various soils. Nocardioides pyridinolyticus (Yoon et al., 1997) and Nocardioides oleivorans (Schippers et al., 2005) were isolated from the oxic zone of an oil shale column and crude oil, respectively. In addition, Nocardioides plantarum (Collins et al., 1994) was isolated from herbage, and Nocardioides nitrophenolicus (Yoon et al., 1999), Nocardioides aquiterrae (Yoon et al., 2004) and Nocardioides aromaticivorans (Kubota et al., 2005) were isolated from non-saline aquatic environments (industrial wastewater, groundwater and a river, respectively). Nocardioides daphniae was isolated from a water flea originating from lake water (Tóth et al., 2008). Furthermore, seven species were isolated from saline environments: Nocardioides aquaticus (Lawson et al., 2000) was isolated from a saline lake in Antarctica, Nocardioides aestuarii (Yi & Chun, 2004a) and Nocardioides ganghwensis (Yi & Chun, 2004b) were isolated from tidal flats, Nocardioides furvisabuli (Lee, 2007), Nocardioides marinisabuli (Lee et al., 2007) and Nocardioides hwasunensis (Lee et al., 2008) were isolated from beach sand and Nocardioides marinus (Choi et al., 2007) was isolated from seawater. In this study, strain CL-Z59^T was isolated from seawater enriched with zooplankton and was subjected to a polyphasic taxonomic analysis.

To isolate zooplankton-associated bacteria, seawater was sampled from the surface of the South Sea, Korea, filtered using a syringe filter (0.2 μm pore size) and dispensed into a 20 ml autoclaved glass vial. Then the 0.2 μm-filtered seawater was supplemented with zooplankton [collected using a zooplankton net (200 μm mesh size) and rinsed with 0.2 μm filter-sterilized seawater three times] and incubated at a temperature close to the in situ temperature (approx. 10–15 °C). After about 1 year, 50 μl aliquots were taken and spread on a plate of low-nutrient heterotrophic medium supplemented with 1× mixed carbons (Cho & Giovannoni, 2004). After 1 month, each colony was streaked again on a plate of low-nutrient heterotrophic medium plus 1× mixed carbons, and subsequently on marine agar 2216 (MA; Difco). Strain CL-Z59^T grew better on MA and was routinely cultivated on MA at 30 °C. The strain was maintained both on MA at 4 °C and in marine broth 2216 (Difco) supplemented with 30 % (v/v) glycerol at −80 °C.

Morphological, physiological and biochemical analyses were performed. Unless otherwise specified, all characteristics described hereafter were based on cultures grown on MA at 30 °C. Gram-staining was performed as described by Smibert & Krieg (1994). The cell morphology and motility of the isolate were observed by using phase-contrast microscopy (BX50; Olympus). Transmission electron microscopy (EX2; JEOL) was used to establish whether flagella were present. Anaerobic growth was checked on MA and ZOF medium (Lemos et al., 1985) supplemented with additional agar (1.5 %), using the GasPak anaerobic system (BBL) at 30 °C for 15 days. Catalase and oxidase activities and decomposition of casein, hypoxanthine, xanthine and l-tyrosine were determined according to the protocols described by Smibert & Krieg (1994). Nitrate reductase activity and degradation of starch and Tween 80 were investigated as described by Hansen & Sørheim (1991).

The temperature range for growth was determined on the basis of the formation of single colonies visible on MA incubated at 5–45 °C (using increments of 5 °C). The pH range (between pH 5 and 10, using increments of 1 pH unit) for growth was determined by assessing changes in the OD₆₀₀ over time in marine broth 2216. The final pH was adjusted using 1 M NaOH and 1 M HCl solutions. The NaCl tolerance of strain CL-Z59^T was determined using synthetic ZoBell broth (containing, l⁻¹ distilled water: 5 g Bacto peptone, 1 g yeast extract, 0.1 g ferric citrate; Yi & Chun, 2004b) with various NaCl concentrations (0, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 20 %, w/v). Nitrate reduction, indole production, arginine dihydrolase, urease, gelatinase and β-galactosidase, acid production from glucose and hydrolysis of aesculin were tested using an API 20NE kit (bioMérieux) according to the manufacturer's instructions, except that the cell suspension was prepared using artificial seawater at half strength (containing, l⁻¹ distilled water: 12 g NaCl, 5.44 g MgCl₂ . 6H₂O, 2 g Na₂SO₄, 0.73 g CaCl₂ . 2H₂O, 0.35 g KCl, 0.1 g NaHCO₃, 0.05 g KBr, 0.0135 g H₃BO₃, 0.02 g SrCl₂ . 6H₂O and 0.015 g NaF; Lyman & Fleming, 1940) as the suspension medium. Other enzymic activities were determined using an API ZYM kit (bioMérieux) and half-strength artificial seawater as the suspension medium. Carbon utilization was tested using a modified basal broth medium (containing, l⁻¹ distilled water: 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 and 0.05 g yeast extract; pH 6.7±0.5) containing 0.2 % carbon source. Carbon utilization was scored as negative when growth was equal to, or less than, that in the negative control with no carbon source. Growth was measured by monitoring changes in the OD₆₀₀ after 8–12 days incubation at 30 °C.

Isoprenoid quinones were isolated according to the method of Minnikin et al. (1984) and were analysed by HPLC as described by Collins (1985) at the Korean Culture Center of Microorganisms, Seoul, Korea. The cell-wall diamino acids were determined as described by Staneck & Roberts (1974). The whole-cell fatty acid methyl esters in strain CL-Z59^T, grown on MA at 30 °C for 1 day, were analysed at the Korean Culture Center of Microorganisms, using gas chromatography according to the instructions of the Microbial Identification System (MIDI). Genomic DNA–DNA relatedness was determined by using dot-blot hybridization (Kim et al., 2007). Prehybridization, hybridization and detection were performed using a DIG labelling and detection kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. The experiment was repeated on different days.

For 16S rRNA gene amplification with PCR, DNA was extracted from a single colony by using a boiling method (Englen & Kelley, 2000). The crude extracts served as the DNA template for the PCR reactions, which involved Taq DNA polymerase (Bioneer) and primers 27F and 1492R (Lane, 1991). The PCR product was purified by using an AccuPrep PCR purification kit (Bioneer) and was cloned using the pGEM-T Easy vector (Promega). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automated sequencer (ABI3730XL) at Macrogen Corp. (Seoul, Korea). An almost-complete 16S rRNA gene sequence (1446 bp) of strain CL-Z59^T was obtained. The sequence was compared with those available in the GenBank database by using blastn searches (Altschul et al., 1990). The 16S rRNA gene sequence of strain CL-Z59^T was manually aligned with those of the type strains of other species belonging to the genus Nocardioides and with phylogenetically related genera obtained from GenBank and the Ribosomal Database Project (Cole et al., 2003) database by using known 16S rRNA gene secondary-structure information. Phylogenetic trees were obtained by using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) 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 using bootstrap analyses based on 1000 replications for the neighbour-joining and maximum-parsimony methods. Alignment analysis was carried out using the jPHYDIT program (Jeon et al., 2005) and phylogenetic analyses were carried out using mega3 (Kumar et al., 2004). The DNA G+C content was determined at the Korean Culture Center of Microorganisms by using HPLC analysis (Tamaoka & Komagata, 1984).

Cells were Gram-positive, non-motile and strictly aerobic. During the exponential growth phase, cells of strain CL-Z59^T were rods (0.3–0.6 μm in width and 0.6–1.6 μm in length). Colonies on MA were circular, smooth, entire, convex and creamy white. Cells were positive for catalase activity and negative for oxidase and nitrate reductase. Cells were positive for the decomposition of casein, starch, Tween 80 and l-tyrosine but were negative for the decomposition of hypoxanthine and xanthine (Table 1⇓).

Growth of strain CL-Z59^T was observed at temperatures in the range 10–35 °C, the optimum being 25–30 °C. Growth occurred at pH 6–10, the optimum pH being 6–7. The strain required NaCl for growth (1–10 %, w/v), the optimum concentration being 3 %. The other results of morphological, physiological and biochemical tests are given in the species description and in Table 1⇑.

Strain CL-Z59^T contained a tetrahydrogenated menaquinone with eight isoprene units [MK-8(H₄)] as the major menaquinone, like most Nocardioides species (Holt et al., 1994) and ll-diaminopimelic acid was the cell-wall diamino acid, as in Nocardioides species (Prauser, 1976). The fatty acid profile for strain CL-Z59^T was dominated by iso-C_16 : 0 (65.3 %; Table 2⇓), which is typically found as a major component in members of the genus Nocardioides (Yoon et al., 1999). The DNA G+C content of strain CL-Z59^T was 73.3 mol%, being within the range (67–75 mol%; Prauser, 1976; Yoon et al., 2006a) reported for recognized species of the genus Nocardioides.

Phylogenetic trees based on 16S rRNA gene sequences showed that strain CL-Z59^T falls within the radiation of the cluster comprising Nocardioides species (Fig. 1⇓). Sequence similarity indicated that the closest relative of strain CL-Z59^T was N. marinisabuli (99.2 %); the other members of the genus Nocardioides showed sequence similarities in the range 94.2–97.1 %. In all of the phylogenetic trees, strain CL-Z59^T formed a tight clade with N. marinisabuli SBS-12^T (Fig. 1⇓). This clade was related to the nearest clade, containing N. kribbensis, N. aquaticus and N. lentus (Fig. 1⇓). The levels of DNA–DNA relatedness between strain CL-Z59^T and N. marinisabuli KCCM 42681^T and between strain CL-Z59^T and N. kribbensis KSL-2^T were 33±9 % and 18±6 %, respectively, supporting the view that strain CL-Z59^T is a distinct species (Stackebrandt & Goebel, 1994). Furthermore, strain CL-Z59^T can be clearly distinguished from N. marinisabuli on the basis of phenotypic traits: pH, temperature and NaCl range for growth, the ability to decompose Tween 80 and l-tyrosine, the ability to produce acid phosphatase, α-chymotrypsin, cystine arylamidase, esterase (C4), naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase and the utilization of cellobiose, citrate, d-fructose, d-galactose, d-glucose, d-mannitol, d-ribose, sucrose and d-xylose (Table 1⇑). In addition, phenotypic, biochemical and chemotaxonomic features, including NaCl ranges for growth, optimal pH, the absence of oxidase and nitrate reductase, the carbon-utilization pattern and the fatty acid profiles, clearly served to differentiate strain CL-Z59^T from N. kribbensis (Tables 1⇑ and 2⇑). Notably, combinations of some phenotypic traits (e.g. alkaline phosphatase, α-chymotrypsin, α-galactosidase, α-glucosidase and utilization of l-arabinose, cellobiose, d-glucose, inositol, d-mannitol, succinate and d-xylose) can serve to differentiate species of the genus Nocardioides (Table 1⇑). In conclusion, based on the evidence from the polyphasic study, strain CL-Z59^T represents a novel species of the genus Nocardioides, for which the name Nocardioides salarius sp. nov. is proposed.