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Marine Biotechnology Research Centre, Korea Ocean Research and Development Institute, PO Box 29, Ansan 425-600, Republic of Korea
Correspondence
Sang-Jin Kim
s-jkim{at}kordi.re.kr
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3. This is consistent with corresponding data for Photobacterium profundum. The DNA G+C content of strain SL13T is 43·8 mol%. Phylogenetic analyses of 16S rRNA gene sequences place this bacterium in the ‘Gammaproteobacteria’, within the genus Photobacterium. Sequence similarity analysis indicates that the closest relatives of strain SL13T are Photobacterium indicum (99·3 %), P. profundum (98·5 %) and Photobacterium lipolyticum (98·2 %). The DNA–DNA hybridization levels between the isolate and its closest known phylogenetic relatives, P. indicum, P. profundum and P. lipolyticum, are 27·1, 52·4 and 20·2 %, respectively. Thus strain SL13T represents a novel species of the genus Photobacterium, for which the name Photobacterium frigidiphilum sp. nov. is proposed. The type strain is SL13T (=KCTC 12384T=JCM 12947T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SL13T is AY538749.
A table of physiological properties of strain SL13T and a graph showing growth of the strain under varying barometric pressure are available as supplementary material in IJSEM Online.
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-hydroxybutyrate under certain culture conditions, and do not utilize exogenous monomers of -hydroxybutyrate. Unlike the flagella of Vibrio species, which are also common in marine environments, the flagella of all Photobacterium species lack an enclosing sheath (Baumann & Baumann, 1984
). Vibrio iliopiscarius was proposed as a species of the genus Vibrio because it has sheathed flagella and because poly--hydroxybutyrate was not found in this strain (Onarheim et al., 1994). However, this strain was later reclassified as Photobacterium iliopiscarium comb. nov. on the basis of phylogenetic analysis of 16S rRNA gene sequences and restriction fragment length polymorphism patterns of 16S rRNA gene sequences digested with HhaI, which differ from those observed in other species of the genus Vibrio (Urakawa et al., 1998, 1999).
Species of the genus Photobacterium, which belongs to the family Vibrionaceae together with the genera Vibrio and Listonella, are widespread in marine environments (Baumann & Baumann, 1984; Okuzumi et al., 1994; Shieh et al., 2003). Photobacterium phosphoreum was first reported in 1889; at present, there are nine known species. P. phosphoreum, Photobacterium leiognathi and Photobacterium profundum were isolated from sea water, the intestinal contents of marine animals, and deep-sea sediment, respectively (Baumann & Baumann, 1984; Nogi et al., 1998). Typically, marine environments have low temperatures; consequently, psychrophiles, whose optimum growth temperature is lower than 20 °C, are abundant in marine environments. P. iliopiscarium, P. phosphoreum, P. profundum and several other species of the genus Photobacterium are psychrophilic. Of these, P. profundum was the first to be reported from the deep sea, after being isolated from the sediments of the Ryukyu Trench (5110 m deep; Nogi et al., 1998).
Here, we describe the morphological, phenotypic, phylogenetic and genomic characteristics of strain SL13T isolated from deep-sea sediment samples. On the basis of this polyphasic evidence, we propose that SL13T be assigned as a novel species of the genus Photobacterium, for which the name Photobacterium frigidiphilum sp. nov. is proposed.
Sediment samples were collected at Edison Seamount (cold seep area at 1500 m depth) using a video-guided grab sampler (TV-Grab; Preuss). Edison Seamount is positioned south of Lihir Island, Papua New Guinea, in the Western Pacific Ocean. Immediately after sampling, 
0·3 g chilled deep-sea sediment was ground in a mortar and inoculated into 30 ml of autoclaved marine broth 2216 (MB; Difco) containing 1 % (v/v) hexadecane in a 100 ml Erlenmeyer flask. After incubation at 10 °C for 7 days on an orbital shaker (JEIO TECH; 120 r.p.m.), 150 µl culture broth was spread onto plates of marine agar 2216 (MA; Difco) supplemented with 1 % (v/v) tributyrin. The plates were then incubated at 10 °C for 7 days. Individual colonies with halo zones were isolated from the tributyrin plates. This process was repeated on MA until pure cultures were obtained.
Air-dried smears from MA cultures were stained using the Difco Gram-staining kit, according to the manufacturer's instructions. Cell morphology and flagellum type were examined by transmission electron microscopy (model FDL5000; JEOL). Cells grown for 3 days at 10 °C on MA were negatively stained with 1 % (w/v) phosphotungstic acid. After being air-dried, the grids were examined by transmission electron microscopy at 12 000x magnification.
Biochemical tests were performed using API 20NE (bioMérieux) according to the manufacturer's instructions, but with a modified saline solution (containing, l–1 distilled water, 8·8 g MgCl2.6H2O, 1·8 g CaCl2.2H2O and 20 g NaCl) instead of the API 20NE saline solution for bacterial suspension (Sohn et al., 2004a). The cells' ability to utilize different carbon sources were examined using a Biolog GN2 MicroPlate, for which the bacterial suspensions were prepared as described above. Catalase activity was determined by means of bubble production in a 3 % (v/v) hydrogen peroxide solution, and lipase activity was assessed by observing the formation of clear zones around colonies on MA containing 1 % (v/v) tributyrin. Growth potential under anaerobic conditions was tested as described by Sohn et al. (2004b), and the presence of oxygen was monitored using resazurin as an indicator of redox potential. Luminescence was checked, in darkness, after 2 days incubation at 10 °C in MB. Susceptibility to the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129; Sigma) was determined by the disc-diffusion method using 10 and 150 µg O/129 (Donovan & Furniss, 1984).
Growth characteristics were examined using MB as a basal medium. To determine the optimal temperature for growth, the growth rates were calculated from optical density values measured in a temperature-gradient incubator (TVS126MA; Advantec). To determine the optimal pH for growth, MB was dissolved in the following buffers (Sigma) at a concentration of 20 mM: for pH 4, 5 and 5·5, MES buffer; for pH 6 and 6·5, PIPES buffer; for pH 7 and 7·5, HEPES buffer; and for pH 8, 9 and 10, AMPSO buffer (Bae et al., 2005). The requirement for NaCl was tested using modified MB, containing (per litre distilled water) 5 g bactopeptone, 1 g yeast extract and 0·01 g FePO4.4H2O, supplemented with different NaCl concentrations. The requirements for MgCl2 and/or CaCl2 were determined using modified MB containing 2 % (w/v) NaCl along with 8·8 g MgCl2 l–1 and/or 1·8 g CaCl2 l–1.
Strain SL13T was Gram-negative, rod-shaped, motile by means of a single polar flagellum and approximately 0·8–1·5 µm in width by 2·3–3·8 µm in length (Fig. 1). Colonies on MA (after incubation for 3 days at 10 °C) were cream-coloured, opaque, smooth, circular, convex with entire margins and 2·0–3·0 mm in diameter. Luminescence was not observed. The isolate was susceptible to O/129. Strain SL13T grew optimally at 14 °C and growth was possible between 6 and 20 °C (Fig. 2). SL13T did not grow at temperatures higher than 20 °C. The strain grew at rates greater than half-optimal at 6 °C. It grew well within the pH range 5·0–8·5 and optimally at pH 6·0. Optimal growth for SL13T occurred in the presence of 1·5 % (w/v) NaCl; no growth occurred at NaCl concentrations above 4·0 % or in the absence of NaCl. In addition, SL13T required either MgCl2 or CaCl2 for growth. The isolate showed catalase, oxidase, arginine dihydrolase, -glucosidase, protease and lipase activities, but no urease activity. Nitrate was reduced to nitrite, and indole was produced. As the strain was unable to grow in AUX medium (from the API 20NE test kit), no data were obtained with respect to 12C assimilation. On the basis of Biolog tests, strain SL13T could oxidize the various carbon compounds listed in the species description. The physiological characteristics of strain SL13T are listed in a supplementary table available in IJSEM Online. On the basis of those phenotypes, SL13T can be differentiated from related Photobacterium type strains (Table 1).
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The total lipid component of cells incubated at 10 °C for 3 days was extracted using the method of Folch et al. (1957). For the analysis of fatty acid composition, total lipids were converted to fatty acid methyl esters by serial addition of 1·5 % (w/v) NaOH and 5 % (v/v) HCl in methanol; both reactions were repeated at 65 °C for 20 min. Fatty acid methyl esters were analysed with a gas chromatograph (5890II; Hewlett Packard) equipped with a flame-ionization detector and a capillary column (non-polar SPB-1; 25 mx0·25 mm). Bacterial fatty acid methyl ester standards (Supelco) and equivalent chain-length values (Stransky et al., 1992) were used for fatty acid identification. The DNA G+C content (mol%) of the isolate was determined by the melting temperature method, as described by Sohn et al. (2004b). Purified chromosomal DNA extracted from Escherichia coli K-12 (=KCTC 2443) served as a control.
The cellular fatty acids ranged in size from C12 to C20 and included saturated, monoenoic and iso-branched components (Table 2). The major fatty acids in the strain were C12 : 0, C14 : 0, C16 : 1, C16 : 0, C18 : 1 and C20 : 53, a range similar to that reported for other Photobacterium species (Nogi et al., 1998). Of particular note among the fatty acids of SL13T was 20 : 53, which is usually found in both psychrophilic and piezophilic marine bacteria (DeLong et al., 1997; Nogi et al., 1998). The fatty acid composition of SL13T is similar to that reported for P. profundum JCM 10084T isolated from the deep sea (Nogi et al., 1998). The DNA G+C content of strain SL13T was 43·8 mol%, which is within the range (40–44 mol%) for the genus Photobacterium (Baumann & Baumann, 1984).
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). The PCR product was sequenced using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) and the resultant 16S rRNA gene sequences were manually aligned with those from other Photobacterium species and related taxa, using known 16S rRNA secondary-structure information. Phylogenetic trees were constructed using neighbour-joining (Saitou & Nei, 1987), Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993) and maximum-parsimony (Fitch, 1972) methods. Evolutionary distance matrices (for the neighbour-joining and Fitch–Margoliash methods) were generated according to the Jukes–Cantor model (Jukes & Cantor, 1969). The trees were rooted using E. coli (GenBank accession no. X80725) as an outgroup. The PHYLIP software package (Felsenstein, 1993) was used for all phylogenetic analyses. The resultant unrooted tree topology was evaluated by bootstrap analysis (1000 replicates; Felsenstein, 1985) of the neighbour-joining tree.
The 16S rRNA gene sequence of SL13T obtained in this study consisted of 1536 nt. In terms of 16S rRNA gene sequence similarity, the closest relatives of strain SL13T were Photobacterium indicum (99·3 %), P. profundum (98·5 %), Photobacterium lipolyticum (98·2 %), P. iliopiscarium (97·4 %) and P. phosphoreum (97·2 %). Lower sequence similarities (<97·0 %) were found with all other Photobacterium species with validly published names (Fig. 3).
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. The resultant estimates of DNA relatedness between the isolate and the three type strains of the most closely related Photobacterium species (P. indicum, P. profundum and P. lipolyticum) were less than 53 % (Table 3), which is significantly lower than that accepted as the phylogenetic definition of a species (Wayne et al., 1987).
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Cells are rod-shaped, 0·8–1·5 µm in width and 2·3–3·8 µm in length, motile by means of a polar flagellum and Gram-negative. Colonies are cream-coloured, opaque, smooth, circular, convex, with entire margins, and form on MA at 10 °C after 3 days. Non-luminescent. Susceptible to O/129. Facultatively anaerobic. Psychrophilic. Growth occurs between 6 and 20 °C, with an optimum at 14 °C. The pH range for growth is 5·0–8·5, with an optimum at pH 6·0. Has an absolute requirement for NaCl for growth; growth occurs at NaCl concentrations of 1·0–3·5 % (w/v) and is optimal at 1·5 % (w/v). Catalase, oxidase, arginine dihydrolase, -glucosidase, protease, -galactosidase and lipase are present. Reduction of nitrate to nitrite occurs, and indoles are produced. Does not utilize -hydroxybutyrate. The following carbon sources are utilized: dextrin, glycogen, Tweens 40 and 80, D-fructose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fructose, D-galactose, 
-D-glucose, myo-inositol, maltose, D-mannitol, D-mannose, sucrose, D-trehalose, D-glucuronic acid, DL-lactic acid, propionic acid, succinic acid, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-serine, inosine, glycerol and D-glucose-6-phosphate. The major fatty acids are oleic acid (C16 : 1) and palmitic acid (C16 : 0); the fatty acid profiles contain eicosapentaenoic acid (C20 : 53). The DNA G+C content is 43·8 mol%.
The type strain, SL13T (=KCTC 12384T=JCM 12947T), was isolated from sediments at Edison Seamount (1500 m depth) in the western Pacific Ocean.
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ACKNOWLEDGEMENTS |
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