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School of Earth and Environmental Sciences and Research Institute of Oceanography, Seoul National University, San 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Byung Cheol Cho
bccho{at}snu.ac.kr
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7c and/or iso-C15 : 0 2-OH, 40.3 %), C18 : 17c (26.6 %), C16 : 0 (16.6 %) and C10 : 0 3-OH (7.1 %). The major isoprenoid quinone was Q-8. The G+C content of the genomic DNA was 61 mol%. On the basis of the data from this polyphasic study, strain CL-YJ9T belongs to the genus Marinobacterium but is distinguishable from the recognized species. Strain CL-YJ9T therefore represents a novel species, for which the name Marinobacterium rhizophilum sp. nov. is proposed. The type strain is CL-YJ9T (=KCCM 42386T =DSM 18822T).
The cellular fatty acid compositions of strain CL-YJ9T and type strains of Marinobacterium species are available as supplementary material with the online version of this paper.
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from lignin-rich waste from a pulp mill. Later, Pseudomonas stanieri (Baumann et al., 1983) and Oceanospirillum jannaschii (Bowditch et al., 1984) were reclassified to the genus Marinobacterium as Marinobacterium stanieri and Marinobacterium jannaschii on the basis of phylogenetic analysis of 16S rRNA and gyrB genes (Satomi et al., 2002). Recently, Marinobacterium halophilum was isolated from a tidal flat area of Korea (Chang et al., 2007). In this study, strain CL-YJ9T was isolated in November 2005 from sediment associated with the roots of a plant (Suaeda japonica) growing in a tidal flat of Youngjong Island, Korea; the sediment samples were diluted in sterile seawater and a 0.1 ml aliquot was spread onto marine agar 2216 (MA; Difco). The plate was incubated at 30 °C for 1 week. Strain CL-YJ9T was isolated from the plate and repeatedly purified four times on MA at 30 °C. The purified strain was maintained on MA at 4 °C and in marine broth 2216 (Difco), supplemented with 30 % (v/v) glycerol, at –80 °C.
The 16S rRNA gene was amplified from a single colony by using a PCR with Taq DNA polymerase (Bioneer) and primers 27F and 1492R (Lane, 1991). The PCR product was purified using an AccuPrep PCR purification kit (Bioneer). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automated sequencer (ABI3730XL) at Macrogen Corp. (Seoul, Korea). The almost-complete 16S rRNA gene sequence of strain CL-YJ9T (1371 nt) obtained was compared with 16S rRNA gene sequences available in GenBank, using BLASTN (Altschul et al., 1990) searches. The sequence of strain CL-YJ9T was manually aligned with the sequences of the four recognized Marinobacterium species and of the type species of related genera obtained from GenBank and the Ribosomal Database Project (Cole et al., 2003) databases, using known 16S rRNA secondary structure information. Phylogenetic trees were obtained using 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 using bootstrap analyses based on 1000 replications (for the neighbour-joining and maximum-parsimony approaches) or 100 replications (for the maximum-likelihood approach). Alignment analysis was carried out using the jPHYDIT program (Jeon et al., 2005) and phylogenetic analysis was carried out using MEGA3 (Kumar et al., 2004) and PAUP 4.0 (Swofford, 1998). Likelihood parameters were estimated using the hierarchical ratio tests in MODELTEST 3.04 (Posada & Crandall, 1998). The DNA G+C content was determined from HPLC analysis (Tamaoka & Komagata, 1984) performed at the Korean Culture Center of Microorganisms, Seoul, Korea.
For phenotypic and chemotaxonomic analyses, strain CL-YJ9T was routinely cultivated on YTSS agar (4 g yeast extract, 2.5 g tryptone, 20 g sea salts, 18 g agar and 1 l distilled water; González et al., 1997). Anaerobic growth was tested by using the GasPak anaerobic system in a GasPak anaerobic jar with an anaerobic indicator (BBL) and checking colony formation on MA at 30 °C for 1 month. Cellular morphology and motility were examined by using phase-contrast microscopy (BX50; Olympus) and transmission electron microscopy (EX2; JEOL) with cells from exponentially growing cultures. Gram-staining and catalase, oxidase and amylase activities were assayed as described by Smibert & Krieg (1994) and poly-β-hydroxybutyric acid accumulation was tested as described by Ostle & Holt (1982). Degradation of Tween 80 was tested as described by Hansen & Sørheim (1991). Other enzyme activities were also assayed using API 20NE and API ZYM (bioMérieux) kits according to the manufacturer's instructions, except that cell suspensions were prepared using a 3 % (w/v) NaCl solution. Acid production from different carbohydrates was determined by employing the API 50 CH system (bioMérieux) according to the manufacturer's instructions. All suspension media were supplemented with 3 % NaCl (final concentration). For the API 20NE, API ZYM and API 50 CH analyses, M. georgiense KW-40T was employed as a reference strain. The temperature range for growth was determined on the basis of colony formation on YTSS agar incubated at temperatures ranging from 5 to 40 °C (using increments of 5 °C). The tolerance of the isolate to NaCl was determined by using YTSS agar supplemented with various NaCl concentrations (0, 1, 3, 5, 7, 10, 13, 15, 20 and 25 %, w/v). The pH range (5–11, using increments of 0.5 pH units) for growth was determined by monitoring changes in the OD600 over time in YTSS broth (the same composition as YTSS agar but without agar). The final pH was adjusted using NaOH and HCl solutions. Carbon utilization was tested on basal agar medium [Tris/HCl, 50 mM, pH 7.5; NH4Cl, 19 mM; K2HPO4 . 3H2O, 0.33 mM; FeSO4 . 7H2O, 0.1 mM; 1.5 % Bacto agar (Difco) in artificial seawater (NaCl, 400 mM; MgSO4 . 7H2O, 100 mM; KCl, 20 mM; CaCl2 . 2H2O, 20 mM)] containing 0.2 % carbon source, as described by Baumann & Baumann (1981) but with the slight modification that artificial seawater was used instead of half-strength artificial seawater. Growth was scored as negative when it was equal to or less than that in the negative control (lacking a carbon source) after 7 days incubation at 30 °C. All of the experiments were performed under aerobic conditions.
Isoprenoid quinones were extracted according to Minnikin et al. (1984) and analysed by HPLC as described by Collins (1985). For whole-cell fatty acid analyses, the strain was grown on MA at 25 °C for 5 days. Fatty acid extraction, conversion to methyl esters, gas chromatographic analysis and identification were performed at the Korean Culture Center of Microorganisms according to the protocols of the Microbial Identification System (Microbial ID).
Cells of strain CL-YJ9T were found to be Gram-negative, rod-shaped, strictly aerobic, approximately 0.3–0.4 µm wide and 0.6–0.8 µm long and motile by means of monopolar flagella. The strain grew with 1–5 % NaCl (optimum, 3 %) and at 5–30 °C (optimum, approx. 25 °C) and pH 6.0–9.0 (optimum, pH 7.0). The results of the other biochemical and physiological tests are given in Table 1 and in the species description. The major isoprenoid quinone in strain CL-YJ9T was Q-8. The major fatty acids were summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH, 40.3 %), C18 : 17c (26.6 %), C16 : 0 (16.6 %) and C10 : 0 3-OH (7.1 %) (see Supplementary Table S1, available in IJSEM Online). Strain CL-YJ9T was most closely related to M. halophilum mano11T (94.1 % sequence similarity) followed by other members of the genus Marinobacterium (92.5–93.7 % sequence similarity). Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain CL-YJ9T formed a robust cluster with the members of the genus Marinobacterium (Fig. 1): thus, it is clear that our isolate belongs to this genus. However, the low levels of sequence similarity (<94.1 %) between strain CL-YJ9T and the recognized species of the genus Marinobacterium indicated that strain CL-YJ9T represents a novel species of the genus (Stackebrandt & Goebel, 1994; Rosselló-Mora & Amann, 2001). There were differences in DNA G+C content between strain CL-YJ9T (61 mol%) and existing species of the genus Marinobacterium (55–57 mol%; no data are available for M. halophilum) (Table 1). Furthermore, strain CL-YJ9T can be differentiated from species of the genus Marinobacterium from the utilization patterns for some carbon sources (citrate and N-acetyl-D-glucosamine; Table 1). In conclusion, on the basis of the data from this polyphasic study, strain CL-YJ9T represents a novel species of the genus Marinobacterium, for which the name Marinobacterium rhizophilum sp. nov. is proposed.
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Cells are Gram-negative, strictly aerobic, straight rods, approximately 0.3–0.4 µm wide and 0.6–0.8 µm long, and motile by means of monopolar flagella. Colonies on MA are circular, smooth, entire, convex and creamy white. After 3 days on MA at 30 °C, colonies are approximately 1.0–3.0 mm in diameter. Growth occurs at temperatures in the range 5–30 °C (optimally at approx. 25 °C) and at pH 6.0–9.0 (optimum, pH 7.0). Growth occurs at NaCl concentrations of 1–5 % (optimally at 3 %, w/v). Catalase, oxidase, amylase and Tween 80 hydrolysis are present. In the API 20NE system, nitrate reductase, gelatinase, β-galactosidase (PNPG), urease, aesculin hydrolysis and indole production are absent. In the API ZYM system, alkaline and acid phosphatases, esterase (C4), leucine arylamidase, naphthol phosphohydrolase and 
-glucosidase activities are present, whereas esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, - and β-galactosidases, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, -mannosidase and -fucosidase activities are absent. In the API 50CH system, acids are produced from L-arabinose, D-adonitol, D-galactose, D-glucose, D-fructose, inositol, D-mannitol, D-sorbitol, maltose, sucrose, raffinose, turanose, D-fucose, L-fucose, D- and L-arabitol and potassium 5-ketogluconate, are produced weakly from glycerol, methyl β-D-xylopyranoside, L-rhamnose, methyl -D-glucopyranoside, trehalose, inulin, melezitose and xylitol and are not produced from erythritol, D-arabinose, D-ribose, D- or L-xylose, D-mannose, L-sorbose, dulcitol, methyl -D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, D-lactose, melibiose, starch, glycogen, gentiobiose, D-lyxose, D-tagatose, potassium gluconate or potassium 2-ketogluconate. Utilizes L-arginine, L-asparagine, betaine, D-galactose, L- and D-leucine, glucose, glutamic acid, glycine, mannose, N-acetyl-D-glucosamine, L-arabinose, D-fructose, glycerol, mannitol, trehalose and sucrose as sole carbon sources, but does not utilize citrate, dimethyl sulfide, lactose, L-cysteine, maleic acid, p-hydroxyphenylacetate, succinate or urea. Major isoprenoid quinone is Q-8. Fatty acids comprising >1 % of the total are summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH, 40.3 %), C18 : 17c (26.6 %), C16 : 0 (16.6 %), C10 : 0 3-OH (7.1 %), C12 : 0 (4.0 %) and C10 : 0 (3.9 %). The DNA G+C content of the type strain is 61 mol%.
The type strain, CL-YJ9T (=KCCM 42386T =DSM 18822T), was isolated from the rhizosphere of a coastal plant (Suaeda japonica) inhabiting a tidal flat of Youngjong Island, Korea.
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ACKNOWLEDGEMENTS |
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