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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 The Centre for Traditional Micro-organism Resources, Keimyung University, Shindang-Dong, Dalseo-gu, Daegu, Korea
3 National Research Laboratory of Molecular Ecosystematics, Institute of Probionic, Probionic Corporation, Bio-venture Centre, 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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7c and/or iso-C15 : 0 2-OH and C18 : 1 7c as the major fatty acids. The DNA G+C content of strain TF-22T was 46·0 mol%. Phylogenetic analyses based on 16S rDNA sequences showed that strain TF-22T falls within the 
-subclass of the Proteobacteria and forms a coherent cluster with Alteromonas macleodii and Alteromonas marina. Levels of 16S rDNA similarity between strain TF-22T and the type strains of two Alteromonas species were in the range 98·1–98·6 %. The level of DNA–DNA relatedness between strain TF-22T and the type strains of two Alteromonas species was 15·7–18·5 %. Therefore, on the basis of phenotypic properties, phylogeny and genomic distinctiveness, strain TF-22T should be placed in the genus Alteromonas as a novel species, for which the name Alteromonas litorea sp. nov. is proposed. The type strain is TF-22T (=KCCM 41775T=JCM 12188T).
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with four Alteromonas species. Previously, there were at least 21 species with validly published names within the genus Alteromonas. However, the majority of these species have been reclassified as belonging to other genera, i.e. Marinomonas, Pseudoalteromonas or Shewanella (van Landschoot & de Ley, 1983; MacDonell & Colwell, 1985; Coyne et al., 1989; Gauthier et al., 1995; Sawabe et al., 2000; Ivanova et al., 2000, 2001), and only two Alteromonas species, Alteromonas macleodii (Baumann et al., 1972) and Alteromonas fuliginea (Romanenko et al., 1994), remained in this genus. However, A. fuliginea was found to belong to Pseudoalteromonas citrea on the basis of DNA–DNA hybridization (Ivanova et al., 1998). Phylogenetic analysis based on 16S rDNA sequences showed that A. fuliginea falls within the evolutionary radiation encompassed by the genus Pseudoalteromonas, not that encompassed by the genus Alteromonas (Yoon et al., 2003). Another Alteromonas species, Alteromonas marina, has recently been described (Yoon et al., 2003). In this study, we describe a slightly halophilic bacterial strain, TF-22T, which was considered to be an Alteromonas-like organism on the basis of the results of 16S rDNA sequence comparison. Accordingly, the aim of the present work was to establish the exact taxonomic position of strain TF-22T by using a polyphasic approach involving phenotypic, genetic and chemotaxonomic analyses.
Strain TF-22T was isolated from an intertidal sediment, collected from Daepo Beach, Yellow Sea of Korea, by using the dilution plating technique on marine agar 2216 (MA; Difco). Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The flagellum type was determined by transmission electron microscopy using 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 with anaerobically prepared MA. Growth in the absence of NaCl was investigated in trypticase–soy broth lacking NaCl. Growth at various NaCl concentrations (0·5–15 %, w/v) was investigated in marine broth (MB; Difco) or trypticase–soy broth. Growth at various temperatures was measured on MA at 4–45 °C. Catalase and oxidase activities and hydrolysis of casein, starch, Tween 20, Tween 40, Tween 60 and Tween 80 were determined as described by Cowan & Steel (1965). Hydrolysis of 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 (per litre 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 the activity of some enzymes. 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 Baumann & Baumann (1981). Other physiological and biochemical tests were performed with the API 20E and API 20NE systems (bioMérieux).
Cell biomass of strain TF-22T for respiratory lipoquinone analysis and for DNA extraction was obtained from MB cultures at 30 °C. For fatty acid methyl ester analysis, cell mass of strain TF-22T was obtained from agar plates after cultivation for 2 days at 30 °C on MA. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), except that ribonuclease T1 was used with ribonuclease A. Isoprenoid quinones were extracted and analysed as described previously (Komagata & Suzuki, 1987), using reverse-phase HPLC. For quantitative analysis of cellular fatty acid compositions, a loop 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 reverse-phase HPLC.
16S rDNA was amplified by a PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rDNA and phylogenetic analysis 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 remaining three values were used to calculate similarity values. The DNA relatedness values quoted are the means of the three values.
Morphological, physiological and biochemical characteristics are shown in Table 1 or are given in the species description (see below). Strain TF-22T had an unsaturated ubiquinone with eight isoprene units (ubiquinone-8) as the predominant respiratory lipoquinone. A. macleodii DSM 6062T and two strains of A. marina were found to contain ubiquinone-8 as the predominant respiratory lipoquinone (Yoon et al., 2003). Strain TF-22T had a cellular fatty acid profile containing large amounts of straight-chain, unsaturated and hydroxy fatty acids (Table 2). This fatty acid profile was similar to those of A. macleodii DSM 6062T and A. marina KCCM 41638T (Table 2). Strain TF-22T contained C16 : 0, C16 : 1 7c and/or iso-C15 : 0 2-OH and C18 : 1 7c as the major fatty acids (Table 2). The DNA G+C content of strain TF-22T was 46·0 mol%.
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). Similar tree topology was found in the trees generated with the maximum-likelihood and maximum-parsimony algorithms (data not shown). Strain TF-22T exhibited 16S rDNA similarity levels of 98·1 and 98·6 %, respectively, to the type strains of A. macleodii and A. marina (Fig. 1). Sequence similarities to all other species included in the phylogenetic analysis were less than 89·7 % (Fig. 1). DNA–DNA hybridization was performed to determine the genomic relatedness of strain TF-22T to the type strains of two Alteromonas species with validly published names. Strain TF-22T exhibited DNA–DNA relatedness levels of 15·7 and 18·5 %, respectively, to A. macleodii DSM 6062T and A. marina KCCM 41638T.
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-Proteobacteria, particularly to the genus Alteromonas (Fig. 1
). Chemotaxonomic analysis confirms the result of monothetic classification based on 16S rDNA sequence analysis. The predominant respiratory lipoquinone and cellular fatty acid profile of strain TF-22T were found to be most similar to those of Alteromonas species (Yoon et al., 2003). Accordingly, in view of these combined chemotaxonomic and phylogenetic analyses, strain TF-22T is considered as a member of the genus Alteromonas. Strain TF-22T is differentiated from A. macleodii and A. marina through some physiological and biochemical characteristics, as shown in Table 1. Levels of DNA–DNA relatedness, together with some differential phenotypic properties and phylogenetic distinctiveness, warrant the separation of strain TF-22T from the two Alteromonas species described previously (Wayne et al., 1987). Therefore, on the basis of the phenotypic, chemotaxonomic and phylogenetic data and genomic distinctiveness, we propose to include strain TF-22T in the genus Alteromonas as a novel species, Alteromonas litorea sp. nov.
Description of Alteromonas litorea sp. nov.
Alteromonas litorea (li.to.re'a. L. adj. litorea of the shore).
Cells are rods, 0·9–1·2x2·0–4·0 µm on MA. Motile by means of a single polar flagellum. Colonies on MA are circular, smooth, raised and cream-coloured and 2·0–3·0 mm in diameter after 2 days incubation at 30 °C. Grows well at 25–37 °C with an optimum between 30 and 37 °C. Growth occurs at 10 and 43 °C, but not at 4 °C or above 44 °C. Optimal growth pH is between 7·0 and 8·0. Growth is observed at pH 5·5, but not at pH 5·0. Optimal growth occurs in the presence of 2–5 % (w/v) NaCl. No growth occurs in the absence of NaCl or in the presence of more than 14 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA. Catalase-positive. Aesculin, casein, hypoxanthine, Tween 20, Tween 40, Tween 60 and tyrosine are hydrolysed. Xanthine is not hydrolysed. H2S is not produced. Indole is not produced. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are absent. When assayed with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), acid phosphatase and naphthol-AS-BI-phosphohydrolase are present, but cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Acid is not produced from D-raffinose. The predominant ubiquinone is ubiquinone-8. The major fatty acids are C16 : 0, C16 : 1 7c and/or iso-C15 : 0 2-OH and C18 : 1 7c. The DNA G+C content is 46·0 mol% (HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, TF-22T (=KCCM 41775T=JCM 12188T), was isolated from an intertidal sediment in the Yellow Sea of Korea.
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ACKNOWLEDGEMENTS |
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