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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 The Center for Traditional Microorganism Resources, Keimyung University, Shindang-Dong, Dalseo-gu, Daegu, Korea
Correspondence
Yong-Ha Park
yhpark{at}kribb.re.kr
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ABSTRACT |
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9c as the major fatty acid. Their DNA G+C content was 44 mol%. Strains JG-100T and JG-102 showed 1 bp difference in their 16S rRNA gene sequences and a mean DNA–DNA relatedness level of 88 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains JG-100T and JG-102 form a distinct phylogenetic lineage within the cluster comprising Psychrobacter species. The 16S rRNA gene sequences of strains JG-100T and JG-102 had similarity levels of 95·2–98·4 % to sequences of the type strains of recognized Psychrobacter species. Levels of DNA–DNA relatedness between strains JG-100T and JG-102 and the type strains of some phylogenetically related Psychrobacter species were 6–24 %. On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains JG-100T and JG-102 should be placed in the genus Psychrobacter as a novel species, for which the name Psychrobacter alimentarius sp. nov. (type strain, JG-100T=KCTC 12186T=DSM 16065T) is proposed.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains JG-100T and JG-102 are AY513645 and AY513646, respectively.
A table showing the fatty acid composition of strains JG-100T and JG-102 is available as supplementary material in IJSEM Online.
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; Juni & Heym, 1986; Shaw & Latty, 1988; Bowman et al., 1996; Vela et al., 2003). Recently, many Psychrobacter species have been isolated from marine locations and related environments/materials and innovations in the study of bacterial systematics have led to a considerable increase in the number of isolates identified as Psychrobacter species (Bowman et al., 1997; Maruyama et al., 2000; Denner et al., 2001; Romanenko et al., 2002; Bozal et al., 2003; Yoon et al., 2003; Yumoto et al., 2003). Currently, the genus Psychrobacter comprises 17 valid species: Psychrobacter immobilis (Juni & Heym, 1986); Psychrobacter frigidicola, Psychrobacter urativorans and Psychrobacter phenylpyruvicus (Bowman et al., 1996); Psychrobacter glacincola (Bowman et al., 1997); Psychrobacter pacificensis (Maruyama et al., 2000); Psychrobacter proteolyticus (Denner et al., 2001); Psychrobacter submarinus and Psychrobacter marincola (Romanenko et al., 2002); Psychrobacter faecalis (Kämpfer et al., 2002); Psychrobacter pulmonis (Vela et al., 2003); Psychrobacter jeotgali (Yoon et al., 2003); Psychrobacter luti and Psychrobacter fozii (Bozal et al., 2003); Psychrobacter okhotskensis (Yumoto et al., 2003); and Psychrobacter maritimus and Psychrobacter arenosus (Romanenko et al., 2004). Recently, two Gram-negative, moderately halophilic bacterial strains, JG-100T and JG-102, were isolated from squid jeotgal, a traditional Korean fermented seafood. 16S rRNA gene sequence analysis has shown that these organisms are phylogenetically related to members of the genus Psychrobacter. P. jeotgali has been recently isolated from shrimp jeotgal (Yoon et al., 2003). Accordingly, the aim of the present study was to determine the exact taxonomic positions of strains JG-100T and JG-102 by polyphasic taxonomic characterization.
Strains JG-100T and JG-102 were isolated by the dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C from jeotgal that had been prepared with squid. The type strains of 13 Psychrobacter species were used as reference strains for DNA–DNA hybridization: P. faecalis DSM 14664T, P. submarinus DSM 14161T, P. marincola DSM 14160T, P. glacincola DSM 12194T, P. frigidicola DSM 12411T, P. urativorans DSM 14009T and P. proteolyticus DSM 13887T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. P. okhotskensis JCM 11840T was obtained from the Japan Collection of Microorganisms (JCM), Saitama, Japan. P. fozii CECT 5889T, P. luti CECT 5885T and P. pulmonis CECT 5989T were obtained from the Coleccion Espanola de Cultivos Tipo (CECT), Valencia, Spain. P. jeotgali KCCM 41559T was obtained from the study of Yoon et al. (2003). P. maritimus KMM 3646T was obtained from Dr Olga I. Nedashkovskaya, Pacific institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Science, Russia. Cell biomass for respiratory lipoquinone analysis and DNA extraction was obtained from marine broth 2216 (MB; Difco) cultures at 30 °C. All strains were cultivated on a gyratory shaker at 150 r.p.m. For fatty acid methyl ester analysis, cell mass of strains JG-100T and JG-102 was obtained from agar plates after incubation for 6 days at 30 °C on MA. Cell morphology was examined using a Nikon E600 light microscopy and a Philips CM-20 transmission electron microscope. The presence or absence of flagella was determined using transmission electron microscopy with cells from exponentially growing cultures. Gram reaction was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth at various NaCl concentrations was investigated in MB or trypticase soy broth. Growth in the absence of NaCl was investigated in trypticase soy broth. Growth at various temperatures (4–45 °C) was measured on MA. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with anaerobically prepared MA. All physiological and biochemical tests were performed aerobically at 30 °C, except for temperature range determinations. Catalase and oxidase activities were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were determined as described previously (Lanyi, 1987). Hydrolysis of casein, starch, and Tween 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965). Hydrolysis of gelatin and urea was determined as described by Lanyi (1987), with the modification that artificial sea water was used. 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 (Levring, 1946). Hydrolysis of hypoxanthine, tyrosine and xanthine was examined on MA using the substrate concentrations described previously (Cowan & Steel, 1965). H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was determined as described by Leifson (1963). Enzyme activity was determined using the API ZYM system (bioMérieux). Other physiological tests were performed with the API 20E system (bioMérieux). Utilization of various substrates for growth was determined as described by Yurkov et al. (1994). Growth requirements for yeast extract and vitamins were determined in the liquid medium used for substrate utilization tests, with yeast extract and vitamin B12 omitted and supplemented with 0·1 % (w/v) acetate as the sole carbon and energy sources. Yeast extract and vitamins were added to the medium in the following amounts (l–1): 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 detected after 2–3 days on MA plates using discs containing the following antibiotics: polymyxin B, 100 U; streptomycin, 50 µg; penicillin, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; erythromycin, 15 µg; and tetracycline, 30 µg.
Chromosomal DNA was isolated and purified according to a method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For quantitative analysis of cellular fatty acid composition, a loopful of cell mass was harvested and 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). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA–DNA hybridization was performed fluorometrically using the method of Ezaki et al. (1989) with photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. Of the values obtained, the highest and lowest values for each sample were excluded; DNA relatedness values were calculated as the means of the remaining three values.
Strains JG-100T and JG-102 were Gram-negative, non-spore-forming, non-motile cocci (1·4–2·0 µm in diameter) on MA. Neither strain required yeast extract or vitamins for their growth in minimal salt medium. However, both strains grew better in the presence of yeast extract than in the presence of vitamins. Ethanol and benzoate were utilized only by strain JG-102. Other morphological, cultural, physiological and biochemical characteristics of strains JG-100T and JG-102 are given in the species description or are shown in Table 1 together with those of some Psychrobacter species.
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9c (46·5 and 54·8 %, respectively) and C17 : 18c (16·6 and 13·4 %, respectively) (see the supplementary table available in IJSEM Online). These fatty acid profiles have been observed in other Psychrobacter species, although there are differences in the proportions of some fatty acids, which may be caused by different cultivation conditions, e.g. cultivation media (Maruyama et al., 2000; Romanenko et al., 2002; Bozal et al., 2003). The DNA G+C contents of strains JG-100T and JG-102 were the same (44 mol%).
The almost-complete 16S rRNA gene sequences of strains JG-100T and JG-102 determined in this study comprised 1494 nt, corresponding to the region between positions 28 and 1524 by comparison with the Escherichia coli 16S rRNA gene sequence. The sequences of strains JG-100T and JG-102 had a 1 bp difference in the region compared. 16S rRNA gene sequence comparison revealed that the two strains had highest similarity to members of the 
-Proteobacteria, particularly to species of the genus Psychrobacter. Phylogenetic trees based on 16S rRNA gene sequences showed that strains JG-100T and JG-102 fell within the radiation of the cluster comprising Psychrobacter species (Fig. 1). Strains JG-100T and JG-102 exhibited 16S rRNA gene sequence similarity levels of 95·2–98·5 % and 95·3–98·5 %, respectively, to the type strains of all validly published Psychrobacter species. The sequence similarities between strains JG-100T and JG-102 and all other taxa included in the phylogenetic analysis were less than 93·2 %. DNA–DNA hybridization was performed to determine the genomic relatedness between strains JG-100T and JG-102 and between the two strains and the type strains of some phylogenetically related Psychrobacter species. Strains JG-100T and JG-102 exhibited a mean level of DNA–DNA relatedness of approximately 88 % when their DNAs were used individually as labelled DNA probes for cross-hybridization. Levels of DNA–DNA relatedness between these two strains and the type strains of 13 Psychrobacter species, which had 16S rRNA gene sequence similarity values of more than 97 % to strains JG-100T and JG-102, were in the range 6–24 %.
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). Chemotaxonomic data for the isolates, i.e. predominant respiratory lipoquinone, cellular fatty acid profile and DNA G+C content, are similar to those of the genus Psychrobacter (Yoon et al., 2003; Yumoto et al., 2003). Strains JG-100T and JG-102 show almost identical phenotypic characteristics (Table 1). The genomic DNA relatedness indicated that strains JG-100T and JG-102 are members of the same species (Wayne et al., 1987). Strains JG-100T and JG-102 can be differentiated from phylogenetically related Psychrobacter species in some physiological and biochemical characteristics, such as growth temperature, acid production from carbohydrates and the ability to utilize certain substrates (Table 1). There are widely recognized criteria for species definition in current bacteriology; strains with DNA–DNA relatedness levels of less than 70 % or with more than 3 % difference in 16S rRNA gene sequences are considered to represent different species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). The DNA–DNA relatedness values between the two strains and the type strains of 13 Psychrobacter species were lower than the threshold value (70 %) suggested for species delineation in current bacterial systematics (Wayne et al., 1987); the 16S rRNA gene sequence similarities between the two strains and the type strains of 13 Psychrobacter species were more than 97 %. The level of DNA relatedness, together with differential phenotypic properties and 16S rRNA gene sequence similarity data, justify taxonomic discrimination of strains JG-100T and JG-102 from all recognized Psychrobacter species. Therefore, on the basis of the data presented, strains JG-100T and JG-102 should be placed in the genus Psychrobacter as representatives of a novel species, for which the name Psychrobacter alimentarius sp. nov. is proposed.
Description of Psychrobacter alimentarius sp. nov.
Psychrobacter alimentarius (a.li.men.ta'ri.us. L. adj. alimentarius relating to food).
Cells are cocci, 1·4–2·0 µm in diameter. Colonies on MA are smooth, low convex, circular to slightly irregular and cream in colour. Aerobic. Growth occurs at 4 and 35 °C, but not above 36 °C. Optimal pH for growth is 6·5–7·5; growth occurs at pH 5·0, but not at pH 4·5. Growth occurs in the presence of 0–12 % (w/v) NaCl, with optimum growth at 2–3 %. Yeast extract, biotin, p-aminobenzoic acid, thiamine hydrochloride and vitamin B12 are not required for growth. Tyrosine is hydrolysed, but aesculin, starch, hypoxanthine and xanthine are not. Indole and H2S are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. When assayed with the API ZYM system, lipase (C8) and leucine arylamidase are present, but trypsin, 
-chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Butyrate is utilized. D-Glucose, D-fructose, glutamate, formate and methanol are not utilized. Utilization of benzoate and ethanol is variable (negative for type strain). Acid is produced from D-cellobiose, D-mannose, melibiose and D-ribose. Acid is not produced from adonitol, maltose, D-melezitose, myo-inositol, D-raffinose, D-sorbitol, sucrose or D-trehalose. Sensitive to cephalothin (30 µg), chloramphenicol (100 µg) and erythromycin (15 µg) and weakly sensitive to novobiocin (5 µg) and penicillin (20 U). The predominant respiratory lipoquinone is Q-8. The DNA G+C content of the type and reference strains is 44 mol% (HPLC). Other characteristics are shown in Table 1.
The type strain is JG-100T (=KCTC 12186T=DSM 16065T), isolated from squid jeotgal, a traditional Korean fermented seafood. Reference strain is JG-102 (=KCTC 12187=DSM 16066).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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