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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 National Research Laboratory of Molecular Ecosystematics, Institute of Probionics, Probionic Corporation, Bio-venture Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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9c and C17 : 18c as the major fatty acids and Q-8 as the predominant ubiquinone. The DNA G+C contents of strains JG-219T and JG-220 were 43·5 and 43·0 mol%, respectively. The two strains showed no difference in their 16S rRNA gene sequences but exhibited minor differences in their phenotypic properties. Strains JG-219T and JG-220 exhibited levels of 16S rRNA gene sequence similarity of 95·2–98·7 % to the type strains of recognized Psychrobacter species. The mean level of DNA–DNA relatedness between strains JG-219T and JG-220 was 84·4 %. The two strains exhibited levels of DNA–DNA relatedness of 1·5–32·9 % to the type strains of eight phylogenetically related Psychrobacter species. On the basis of phenotypic data and phylogenetic and genetic distinctiveness, the two strains were classified as representing a novel species within the genus Psychrobacter, Psychrobacter cibarius sp. nov. The type strain is JG-219T (=KCTC 12256T=DSM 16327T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains JG-219T and JG-220 are AY639871 and AY639872, respectively.
DNA–DNA relatedness data and a neighbour-joining tree are available as supplementary material in IJSEM Online.
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-Proteobacteria (Juni, 1991
). The family also includes the genera Moraxella and Acinetobacter (Rossau et al., 1991; Bowman et al., 1996). At the time of writing, 17 species are recognized in the genus Psychrobacter, Psychrobacter immobilis (Juni & Heym, 1986), Psychrobacter frigidicola, Psychrobacter urativorans, Psychrobacter phenylpyruvicus, Psychrobacter glacincola (Bowman et al., 1996, 1997), Psychrobacter pacificensis (Maruyama et al., 2000), Psychrobacter proteolyticus (Denner et al., 2001), Psychrobacter faecalis (Kämpfer et al., 2002), Psychrobacter submarinus, Psychrobacter marincola (Romanenko et al., 2002), Psychrobacter jeotgali (Yoon et al., 2003), Psychrobacter pulmonis (Vela et al., 2003), Psychrobacter luti, Psychrobacter fozii (Bozal et al., 2003), Psychrobacter okhotskensis (Yumoto et al., 2003), Psychrobacter maritimus and Psychrobacter arenosus (Romanenko et al., 2004). The genus Psychrobacter accommodates Gram-negative, coccoid or rod-shaped, oxidase-positive, non-motile, psychrophilic or psychrotolerant and halotolerant bacteria (Juni & Heym, 1986). Psychrobacter species have been isolated from various habitats or sources, including sea water, food, various human clinical samples, poultry, sea ice, krill and Antarctic ornithogenic soil (Juni & Heym, 1986; Shaw & Latty, 1988; Juni, 1991; Bowman et al., 1996, 1997; Maruyama et al., 2000; Denner et al., 2001). In this study, we report on the taxonomic characterization of two strains, JG-219T and JG-220, which were isolated from jeotgal, a traditional Korean fermented seafood. The two strains were compared with recognized Psychrobacter species using phenotypic characteristics and phylogenetic and genetic analyses.
Strains JG-219T and JG-220 were isolated by the dilution plate technique on marine agar 2216 (MA; Difco) at 30 °C. Growth at various temperatures from 4 to 37 °C was measured on MA, and growth at various pH values and tolerance to NaCl were measured in marine broth 2216 (MB; Difco). Growth in the absence of NaCl was investigated in trypticase soy broth (Difco) lacking NaCl. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on anaerobically prepared MA. Cell morphology and presence of flagella were examined by light microscopy (Nikon E600) and transmission electron microscopy of cells grown on MA. Gram reaction was determined using the bioMérieux Gram staining kit according to the manufacturer's instructions. Catalase activity was determined by bubble production in a 3 % (v/v) hydrogen peroxide solution on MA. Oxidase activity was determined by oxidation of 1 % (w/v) p-aminodimethylaniline oxalate. Nitrate reduction was investigated as described by Lanyi (1987). Hydrolysis of hypoxanthine, tyrosine and xanthine was performed on MA using the substrate concentrations recommended by Cowan & Steel (1965). Urease activity and hydrolysis of Tween 80, casein, gelatin, aesculin and starch were determined as described by Cowan & Steel (1965). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates for growth was determined as described by Yurkov et al. (1994). Requirements of yeast extract (0·005 g), biotin (10 µg), p-aminobenzoic acid (1 mg), thiamin hydrochloride (1 mg) and vitamin B12 (1 mg) were investigated in the liquid medium as used for the substrate utilization test supplemented with 0·1 % (w/v) acetate as the sole carbon and energy source. The API ZYM system (bioMérieux) was used to determine enzyme activity. Antibiotic sensitivity was tested by spreading bacterial suspension on MA and applying discs impregnated with the following antibiotics (content per disc): ampicillin (10 µg), penicillin G (10 U), lincomycin (15 µg), polymyxin B (300 U), streptomycin (30 µg), tetracycline (30 µg) and neomycin (15 µg).
Cell biomass for isoprenoid quinone analysis and for DNA extraction was obtained from cultivation for 3 days in MB at 30 °C. Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phased HPLC. For fatty acid methyl ester (FAME) analysis, cell mass of strains JG-219T and JG-220 was harvested from agar plates after cultivation for 6 days on MA at 30 °C. FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990).
Chromosomal DNA was extracted and purified by the procedure described by Yoon et al. (1996). 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 reassociation was determined by the microplate hybridization method (Ezaki et al., 1989) using photobiotin-labelled DNA probes. Type strains of P. fozii, P. okhotskensis, P. pulmonis, P. luti, P. faecalis, P. glacincola, P. proteolyticus and P. urativorans were used as reference strains for DNA–DNA hybridization. P. faecalis DSM 14664T, P. glacincola DSM 12194T, P. proteolyticus DSM 13887T and P. urativorans DSM 14009T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. P. fozii CECT 5889T, P. luti CECT 5885T and P. pulmonis CECT 5989T were obtained from the Colección Española de Cultivos Tipo (CECT), Burjassot, Spain. P. okhotskensis JCM 11840T was obtained from the Japan Collection of Microorganisms (JCM), Wako-shi, Japan.
16S rRNA gene sequence amplification was performed according to the method described by Yoon et al. (1998) using two universal primers. Sequencing of the amplified 16S rRNA gene was performed as described by Yoon et al. (2003). Alignment of sequences was carried out with the CLUSTAL W program (Thompson et al., 1994) and gaps at the 5' and 3' ends of the alignment were omitted from further analysis. Evolutionary distances were calculated using the Kimura two-parameter correction with the CLUSTAL W package (Thompson et al., 1994). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) on the basis of distance matrix data. The reliability of grouping was assessed by 1000 bootstrap resamplings of the neighbour-joining dataset using the CLUSTAL W package.
Morphological, cultural, physiological and biochemical characteristics of strains JG-219T and JG-220 are shown in Table 1 or are given under the species description below. The major respiratory lipoquinone of strains JG-219T and JG-220 was ubiquinone-8 (Q-8), at peak area ratios of approximately 83 and 86 %, respectively. The result of cellular fatty acid analysis of strains JG-219T and JG-220 is summarized in Table 2. The two strains had cellular fatty acid profiles that contained large amounts of straight-chain and unsaturated fatty acids. These fatty acid profiles were similar to those of Psychrobacter species in containing C18 : 19c and C17 : 18c as the predominant components (Bozal et al., 2003; Romanenko et al., 2002, 2004; Yoon et al., 2003; Yumoto et al., 2003). The DNA G+C contents of strains JG-219T and JG-220 were 43·5 and 43·0 mol%, respectively.
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). The two strains exhibited 16S rRNA gene sequence similarity levels of 98·7, 98·7, 98·2, 98·1, 97·9, 97·7, 97·6 and 97·0 % to the type strains of P. fozii, P. okhotskensis, P. pulmonis, P. luti, P. faecalis, P. glacincola, P. proteolyticus and P. urativorans, respectively. The two strains exhibited 16S rRNA gene sequence similarity values of less than 95·2 % to the type strains of the other Psychrobacter species. Strains JG-219T and JG-220 exhibited a mean DNA–DNA relatedness value of 84·4 % when their DNAs were used individually as a labelled DNA probe for cross-hybridization. This value indicated that strains JG-219T and JG-220 were members of the same genomic species (Wayne et al., 1987). Levels of DNA–DNA relatedness between strains JG-219T and JG-220 and the type strains of P. fozii, P. okhotskensis, P. pulmonis, P. luti, P. faecalis, P. glacincola, P. proteolyticus and P. urativorans were in the range 1·5–32·9 % (DNA–DNA relatedness data are available as supplementary material in IJSEM Online).
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). Strains JG-219T and JG-220 are similar in most phenotypic characteristics except for properties such as utilization of some carbon sources and some enzyme activities (Table 1). The predominant respiratory lipoquinone and fatty acid profiles of strains JG-219T and JG-220 were similar to those of the type strains of the other Psychrobacter species (Bozal et al., 2003; Romanenko et al., 2002, 2004; Yoon et al., 2003; Yumoto et al., 2003). The phylogenetic distinctiveness and DNA–DNA relatedness data provide evidence that strains JG-219T and JG-220 differ from Psychrobacter species with validly published names. On the basis of the data presented, strains JG-219T and JG-220 should be placed as members of a novel species of the genus Psychrobacter, for which the name Psychrobacter cibarius sp. nov. is proposed.
Description of Psychrobacter cibarius sp. nov.
Psychrobacter cibarius (ci.ba.ri'us. L. masc. adj. cibarius relating to food).
Cells are Gram-negative, non-motile coccobacilli. Growth occurs at 4–32 °C (optimum 25–30 °C). Optimum pH for growth is 7·0–8·0; growth occurs weakly at pH 5·0, but not at 4·5. Growth occurs in the presence of 10 % (w/v) NaCl, but not in the presence of >11 % NaCl; optimum growth occurs in the presence of 2–5 % (w/v) NaCl. Growth does not occur without NaCl. Anaerobic growth does not occur on MA. Yeast extract, biotin, p-aminobenzoic acid, thiamin hydrochloride and vitamin B12 are not required for growth. Tyrosine is hydrolysed, but casein, gelatin, aesculin, hypoxanthine, xanthine and starch are not. Nitrate is reduced to nitrite. Using the API ZYM system (bioMérieux), esterase (C4), lipase (C14), cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. DL-alanine and DL-malate are utilized as sole carbon and energy source. D-Glucose, D-fructose, D-mannose, sucrose, D-cellobiose, D-mannitol, glycerol, D-gluconate, L-glutamate, aspartate, L-arginine, hexadecane, lactate, formate, methanol, ethanol and benzoate are not utilized. Utilization of butyrate and L-serine is variable (positive for type strain). Acid is not produced from D-sorbitol, myo-inositol, D-xylose, D-ribose, D-fructose, D-mannitol, melibiose, L-arabinose, D-melezitose, D-glucose, D-galactose, adonitol, L-rhamnose, D-mannose, D-cellobiose, lactose, sucrose, maltose, D-trehalose or D-raffinose. The major fatty acids are C18 : 19c and C17 : 18c. The predominant ubiquinone is Q-8. The DNA G+C content is 43·0–43·5 mol% (HPLC). Other characteristics are given in Table 1.
The type strain, JG-219T (=KCTC 12256T=DSM 16327T), was isolated from jeotgal, a traditional Korean fermented seafood.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bowman, J. P., Nichols, D. S. & McMeekin, T. A. (1997). Psychrobacter glacincola sp. nov., a halotolerant, psychrophilic bacterium isolated from Antarctic sea ice. Syst Appl Microbiol 20, 209–215.
Bozal, N., Montes, M. J., Tudela, E. & Guinea, J. (2003). Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov. Int J Syst Evol Microbiol 53, 1093–1100.
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