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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 Biotechnology Research Centre, National Fisheries Research and Development Institute, Gijang-gun, Busan, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH and C16 : 0 as the major fatty acids. The DNA G+C content was 47 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain M37T within the clade comprising Photobacterium species, forming a coherent cluster with the type strains of Photobacterium profundum and Photobacterium indicum (16S rRNA gene similarity levels of 97·5–98·0 %). The mean DNA–DNA relatedness levels between strain M37T and P. profundum JCM 10084T and P. indicum DSM 5151T were in the range 12–15 %. Similarities between 16S rRNA gene sequences of strain M37T and those of the type strains of the other Photobacterium species ranged from 93·9 % (with Photobacterium fischeri) to 96·2 % (with Photobacterium phosphoreum). On the basis of phenotypic properties and phylogenetic and genomic distinctiveness, strain M37T (=KCTC 10562BPT=DSM 16190T) should be placed in the genus Photobacterium as a novel species, for which the name Photobacterium lipolyticum sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M37T is AY554009.
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MAIN TEXT |
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; at present, the genus comprises eight species with validly published names: Photobacterium phosphoreum (Reichelt & Baumann, 1973), Photobacterium leiognathi (Boisvert et al., 1967), Photobacterium angustum (Reichelt et al., 1976), Photobacterium damselae (Smith et al., 1991), Photobacterium iliopiscarium (Onarheim et al., 1994; Urakawa et al., 1999) and Photobacterium profundum (Nogi et al., 1998); Photobacterium fischeri (Beijerinck, 1889; Reichelt & Baumann, 1973) and Vibrio fischeri (Beijerinck, 1889; Lehmann & Neumann, 1896) have the same type strain and are therefore homotypic synonyms. Recently, a further species, Photobacterium indicum, was described through reclassification of Hyphomicrobium indicum (Xie & Yokota, 2004). Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Photobacterium falls within the 
-subclass of the Proteobacteria and, in particular, is closely related to the genus Vibrio (Nogi et al., 1998; Anzai et al., 2000). The genus Photobacterium is characterized chemotaxonomically by having Q-8 as the predominant respiratory lipoquinone and C16 : 1 and C16 : 0 as the major fatty acids (Nogi et al., 1998). In this study, we describe a slightly halophilic bacterial strain, M37T, with lipolytic activity, isolated from an intertidal sediment collected from the Yellow Sea of Korea. This organism was considered to be phylogenetically related to the Photobacterium and Vibrio group on the basis of 16S rRNA gene sequence comparison. Accordingly, the aim of the present work was to establish the exact taxonomic position of strain M37T by means of a polyphasic approach that combined phenotypic characterization, detailed phylogenetic analysis and genetic analysis.
Strain M37T was isolated from intertidal sediment collected in Kaehwa-do of the Yellow Sea on TCN-LB agar medium at 25 °C. TCN-LB medium for screening of lipase-producing bacterial strains was prepared as follows: tricaprylin was emulsified in 20 mM NaCl, 1 mM CaCl2 and 0·5 % (w/v) gum arabic, using a Waring blender. The tricaprylin emulsion (50 ml) was mixed with 450 ml LB agar [1 % (w/v) tryptone, 0·5 % (w/v) yeast extract, 0·5 % (w/v) NaCl and 1·5 % (w/v) agar) containing 3 % (w/v) sea salts (Sigma)]. Strain M37T, which showed an excellent ability to form a halo on the TCN-LB agar medium, indicating lipase activity, was selected and used for further study. The characterization of the lipase produced by strain M37T is described elsewhere. Cell morphology was examined by using light microscopy (Nikon E600) and transmission electron microscopy. The flagellum type was determined by using transmission electron microscopy with cells from exponentially growing cultures. The Gram reaction was determined using the bioMérieux Gram Stain kit according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber, using marine agar 2216 (MA; Difco) and MA, supplemented with nitrate, that had been prepared anaerobically. Growth in the absence of NaCl was investigated in trypticase soy broth without NaCl. Growth at various NaCl concentrations (0·5–15 %, w/v) was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth. Growth at various temperatures (4–40 °C) was measured on MA. Catalase activity was determined by bubble production in a 3 % (v/v) hydrogen peroxide solution. Oxidase activity was determined by oxidation of 1 % p-aminodimethylaniline oxalate as described by Cowan & Steel (1965). Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965). Hydrolysis of casein, starch, hypoxanthine, tyrosine and xanthine was examined on MA plates with the substrate concentrations described previously (Cowan & Steel, 1965). Hydrolysis of aesculin and gelatin and nitrate reduction were determined as described by Lanyi (1987), with the modification that artificial sea water was used. The artificial sea water contained the following (l–1 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). H2S production was tested as described previously (Bruns et al., 2001). The API ZYM system (bioMérieux) was used to determine enzyme activity. Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of substrates as sole carbon and energy sources was tested as described by Yurkov et al. (1994). The API 20E and API 20NE systems (bioMérieux) were used to examine other physiological and biochemical properties.
Cell biomass of strain M37T for respiratory lipoquinone analysis and for DNA extraction was obtained from cultivation in MB at 25 °C. Isoprenoid quinones were extracted and analysed as described by Komagata & Suzuki (1987) using reverse-phase HPLC. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. For fatty acid methyl ester analysis, a loop of cell mass of strain M37T was obtained from agar plates after cultivation for 2 days at 25 °C on MA. 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 the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reverse-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 analyses were performed as described by Yoon et al. (2003). 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 are quoted as DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain M37T are shown in Table 1 or are given in the species description (see below). Strain M37T had an unsaturated ubiquinone with eight isoprene units (Q-8), at a peak area ratio of approx. 86 %, as the predominant respiratory lipoquinone, which is in agreement with previous findings that Photobacterium species contain Q-8 as the predominant respiratory lipoquinone (Nogi et al., 1998). Strain M37T had a cellular fatty acid profile containing large amounts of straight-chain and unsaturated fatty acids: the major components were C16 : 17c and/or iso-C15 : 0 2-OH (51·3 %), C16 : 0 (25·9 %) and C18 : 17c (5·9 %), C12 : 0 3-OH (4·0 %) and C14 : 0 (3·8 %). This fatty acid profile was similar to those of Photobacterium species (Nogi et al., 1998). The DNA G+C content of strain M37T was 47 mol%, which is higher than those of other Photobacterium species (Table 1).
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). Strain M37T exhibited 16S rRNA gene similarity levels of 97·5–98·0 % with respect to P. indicum NBRC 14233T and P. profundum JCM 10084T, and 93·9–96·2 % with respect to the type strains of the other Photobacterium species (Fig. 1). Sequence similarities to all other species included in the phylogenetic analysis were lower than 94·9 % (Fig. 1). Mean DNA–DNA relatedness levels between strain M37T and P. profundum JCM 10084T and between strain M37T and P. indicum DSM 5151T were 12 and 15 %, respectively, when their DNAs were used individually as labelled DNA probes for cross-hybridization. These DNA relatedness values indicate that strain M37T is a member of a genomic species that is distinct from P. profundum and P. indicum (Wayne et al., 1987).
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). The phylogenetic distinctiveness and DNA–DNA relatedness data show that strain M37T is distinct from previously described Photobacterium species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Physiological and biochemical characteristics that differentiate strain M37T from existing Photobacterium species are given in Table 1
. Therefore, on the basis of the data presented, there is sufficient evidence to classify strain M37T in the genus Photobacterium as a novel species, for which the name Photobacterium lipolyticum sp. nov. is proposed.
Description of Photobacterium lipolyticum sp. nov.
Photobacterium lipolyticum (li.po.ly'ti.cum. Gr. n. lipos fat; Gr. adj. lytikos dissolving; N.L. neut. adj. lipolyticum dissolving fat or lipid).
Cells are pleomorphic. Motile by means of a single polar flagellum. Colonies on MA are circular, smooth, glistening, low-convex, cream-coloured and 1·5–2·0 mm in diameter after 2 days incubation at 25 °C. Optimal growth occurs between 25 and 28 °C; growth occurs at 4 and 34 °C but not at 35 °C. Optimal growth pH is between 7·0 and 8·0; growth is observed at pH 5·0 but not at pH 4·5. Optimal growth occurs in the presence of 1–2 % NaCl; no growth occurs without NaCl or in the presence of more than 6 % NaCl. Growth occurs under anaerobic conditions on MA and MA with nitrate. Aesculin, starch and Tweens 20, 40, 60 and 80 are hydrolysed. Casein is weakly hydrolysed. Hypoxanthine, tyrosine, urea and xanthine are not hydrolysed. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Malate and succinate are utilized for growth. D-Xylose, benzoate, butyrate, citrate, glutamate, lactate, formate, ethanol and methanol are not utilized for growth. Acid is produced from D-cellobiose, D-glucose, D-fructose, maltose, D-mannose, D-ribose, sucrose and D-trehalose. Acid is not produced from adonitol, myo-inositol, D-mannitol, D-sorbitol, L-arabinose, D-galactose, lactose, D-melezitose, melibiose, D-raffinose or L-rhamnose. The predominant respiratory lipoquinone is Q-8. The major fatty acids are C16 : 17c and/or iso-C15 : 0 2-OH and C16 : 0. The DNA G+C content is 47 mol% (HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, M37T (=KCTC 10562BPT=DSM 16190T), was isolated from an intertidal sediment from the Yellow Sea of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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