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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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9c as the major fatty acid. Its DNA G+C content was 47·6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SW-238T fell within the radiation of the cluster comprising Psychrobacter species. The 16S rRNA gene sequence of strain SW-238T had similarity levels of 94·8–97·9 % to sequences of the type strains of recognized Psychrobacter species. Levels of DNA–DNA relatedness between strain SW-238T and the type strains of 10 phylogenetically related Psychrobacter species were below 70 %. On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strain SW-238T (=KCTC 12313T=JCM 12601T) was placed in the genus Psychrobacter as the type strain of a novel species, for which the name Psychrobacter celer sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain SW-238T is AY842259.
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, and at the time of writing comprises 24 recognized species: Psychrobacter immobilis (Juni & Heym, 1986), P. frigidicola, P. urativorans and P. phenylpyruvicus (Bowman et al., 1996), P. glacincola (Bowman et al., 1997), P. pacificensis (Maruyama et al., 2000), P. proteolyticus (Denner et al., 2001), P. submarinus and P. marincola (Romanenko et al., 2002), P. faecalis (Kämpfer et al., 2002), P. pulmonis (Vela et al., 2003), P. jeotgali (Yoon et al., 2003), P. luti and P. fozii (Bozal et al., 2003), P. okhotskensis (Yumoto et al., 2003), P. maritimus and P. arenosus (Romanenko et al., 2004), P. vallis and P. aquaticus (Shivaji et al., 2005), P. nivimaris (Heuchert et al., 2004), P. alimentarius (Yoon et al., 2005a), P. cibarius (Jung et al., 2005) and P. aquimaris and P. namhaensis (Yoon et al., 2005b). In the study, we report on the detailed taxonomic characterization of a Psychrobacter-like bacterial strain, SW-238T, which was isolated from sea water of the South Sea in Korea.
Strain SW-238T was isolated by the standard dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. The type strains of 10 Psychrobacter species were used as reference strains for DNA–DNA hybridization: P. nivimaris DSM 16093T, P. proteolyticus DSM 13887T, P. vallis DSM 15337T, P. aquaticus DSM 15339T, P. marincola DSM 14160T and P. submarinus DSM 14161T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, P. alimentarius JG-100T, P. aquimaris SW-210T and P. namhaensis SW-242T were obtained from the studies of Yoon et al. (2005a, b) and P. pacificensis IFO 16270T was obtained from the Institute for Fermentation, Osaka (IFO), Osaka, Japan. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. Gram reaction was determined by using the bioMérieux Gram Stain kit according to the manufacturer's instructions. The pH range for growth was determined in marine broth 2216 (MB; Difco) adjusted to various pH values (initial pH 4·5–10·5 at intervals of 0·5 pH units). The pH was adjusted prior to sterilization by the addition of HCl and Na2CO3. Growth in the absence of NaCl was investigated in trypticase soy broth lacking NaCl. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth (Difco). Growth at various temperatures (4–45 °C) was measured on MA. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA and on MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and hydrolysis of casein, starch, and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were determined as described by Lanyi (1987). Hydrolysis of gelatin and urea was determined as described by Lanyi (1987) with a modification that artificial sea water was used instead of distilled water. The artificial sea water contained (per litre of distilled water) 23·6 g NaCl, 0·64 g KCl, 4·53 g MgCl2.6H2O, 5·94 g MgSO4.7H2O and 1·3 g CaCl2.2H2O (Bruns et al., 2001). Hydrolysis of hypoxanthine, tyrosine and xanthine was investigated on MA with the substrate concentrations described by Cowan & Steel (1965). H2S production was tested as described by Bruns et al. (2001). Acid production from carbohydrates was determined as described by Leifson (1963). Enzyme activity was determined by using the API ZYM system (bioMérieux). Utilization of substrates as sole carbon and energy sources was tested according to the method of Yurkov et al. (1994). Requirements for yeast extract and vitamins for growth were investigated in liquid medium (Yurkov et al., 1994), omitting yeast extract and vitamin B12 but supplementing with 0·1 % (w/v) acetate as the sole carbon and energy sources. Yeast extract and vitamins were added to the medium at the following concentrations (per litre): yeast extract (0·005 g), p-aminobenzoic acid (1 mg), biotin (10 µg), thiamine hydrochloride (1 mg) and vitamin B12 (1 mg). Susceptibility to antibiotics was tested on MA plates using antibiotic discs containing the following compounds: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; erythromycin, 15 µg; tetracycline, 30 µg. Other physiological tests were performed with the API 20E system (bioMérieux).
Cell biomass for DNA extraction and for respiratory lipoquinone analysis was obtained from cultivation in MB at 30 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the modification that RNase T1 was used together with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by PCR using two universal primers as described by Yoon et al. (1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). Respiratory lipoquinones were analysed by using the method of Komagata & Suzuki (1987), using reversed-phase HPLC. For fatty acid methyl ester analysis, cell mass was harvested from MA plates after incubation for 3 days at 30 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as DNA–DNA relatedness values.
The almost complete 16S rRNA gene sequence of strain SW-238T, comprising 1495 nt (approximately 96 % of the Escherichia coli 16S rRNA gene sequence), was analysed. Comparative 16S rRNA gene sequence analyses revealed that strain SW-238T was phylogenetically affiliated to the genus Psychrobacter. In the neighbour-joining tree based on 16S rRNA gene sequences, strain SW-238T fell within the radiation of the cluster comprising Psychrobacter species (Fig. 1). Levels of 16S rRNA gene sequence similarity between strain SW-238T and the type strains of recognized Psychrobacter species ranged from 94·8 to 97·9 % (Fig. 1). Sequence similarities to other species included in the phylogenetic analysis were below 92·6 % (Fig. 1).
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, together with those of some phylogenetically related Psychrobacter species. Strain SW-238T did not require yeast extract or vitamins for growth in minimal salt medium, but it grew better when yeast extract was added to the medium. The predominant respiratory lipoquinone found in strain SW-238T was ubiquinone-8 (Q-8) at a peak area ratio of approximately 85 %. The major components (>1 %) of the fatty acids detected in strain SW-238T were C18 : 19c (62·3 %), C17 : 18c (11·0 %), C18 : 26,9c and/or anteiso-C18 : 0 (3·1 %), C16 : 17c and/or iso-C15 : 0 2-OH (2·9 %), C19 : 19c and/or C19 : 111c (2·8 %), C12 : 0 3-OH (2·8 %), C12 : 0 (2·5 %), C10 : 0 (1·5 %), C13 : 0 3-OH and/or iso-C15 : 1 (1·5 %), iso-C16 : 1 and/or C14 : 0 3-OH (1·5 %), C18 : 0 (1·5 %), C16 : 0 (1·3 %), C16 : 0 2-OH (1·2 %) and C16 : 19c (1·2 %). This fatty acid profile was similar to those of other Psychrobacter species shown previously, although there were differences in the proportions of some fatty acids that might have been caused by different cultivation conditions (e.g. Maruyama et al., 2000; Romanenko et al., 2002; Bozal et al., 2003; Yoon et al., 2005a, b). The DNA G+C content of strain SW-238T was 47·6 mol%. These chemotaxonomic properties support the result of monothetic phylogenetic classification that strain SW-238T may represent a member of the genus Psychrobacter. Strain SW-238T was distinguishable from some phylogenetically related Psychrobacter species based on differences in phenotypic characteristics (Table 1). Levels of DNA–DNA relatedness between strain SW-238T and the type strains of 10 Psychrobacter species that showed 16S rRNA gene sequence similarity values of greater than 97 % to strain SW-238T were in the range 6–21 %. The genetic distinctiveness, together with differential phenotypic properties and 16S rRNA gene sequence similarity data, was sufficient to categorize strain SW-238T as representing a novel Psychrobacter species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain SW-238T should be placed in the genus Psychrobacter as a member of a novel species, for which the name Psychrobacter celer sp. nov. is proposed.
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Cells are coccobacilli (0·9–1·1x1·2–2·0 µm). Colonies are circular, smooth, glistening, raised, cream-coloured and 3·0–4·0 mm in diameter after incubation for 3 days on MA at 30 °C. Growth occurs at 4 and 40 °C, but not at 41 °C. Optimal pH for growth is 7·0–8·0; growth occurs at pH 5·0 but not at 4·5. Growth occurs in the presence of 0–16 % (w/v) NaCl (optimal at 2–3 %, w/v). Yeast extract, biotin, p-aminobenzoic acid, thiamine hydrochloride and vitamin B12 are not required for growth. Tweens 20 and 60 and tyrosine are hydrolysed, but hypoxanthine is not. Indole and H2S are not produced. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8) and leucine arylamidase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase are absent. L-Arabinose, D-cellobiose, D-fructose, D-galactose, maltose, D-mannose, sucrose, D-trehalose, D-xylose, benzoate, formate, salicin and L-glutamate are not utilized. Acid is produced from D-cellobiose, D-mannose, melibiose and D-ribose. Acid is not produced from D-melezitose, D-raffinose, sucrose, D-trehalose, myo-inositol or D-sorbitol. Sensitive to cephalothin (30 µg), chloramphenicol (100 µg) and penicillin G (20 U), but not to novobiocin (5 µg). The predominant respiratory lipoquinone is Q-8. The major fatty acid is C18 : 19c. The DNA G+C content is 47·6 mol% (HPLC). Other characteristics are shown in Table 1
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The type strain, SW-238T (=KCTC 12313T=JCM 12601T), was isolated from sea water of the South Sea in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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