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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 Department of Fashion Design, Hanseo University, Seosan, Chungnam 356-820, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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7c and C16 : 17c and/or iso-C15 : 0 2-OH as the major fatty acids. The DNA G+C contents were 50.1 and 50.5 mol%. Strains SW-353T and SW-369 exhibited no differences in their 16S rRNA gene sequences and showed a mean DNA–DNA relatedness level of 91 %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-353T and SW-369 belong to the genus Rheinheimera. Similarity values between the 16S rRNA gene sequences of the two isolates and the type strains of the recognized Rheinheimera species were in the range 96.6–97.9 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strains SW-353T and SW-369 differ from the recognized Rheinheimera species. On the basis of phenotypic, phylogenetic and genetic data, therefore, strains SW-353T and SW-369 represent a novel species of the genus Rheinheimera, for which the name Rheinheimera aquimaris sp. nov. is proposed. The type strain is SW-353T (=KCTC 12840T=JCM 14331T).
The cellular fatty acid compositions of strains SW-353T and SW-369 are shown in a supplementary table available with the online version of this paper.
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and, at the time of writing, the genus comprises three recognized species: Rheinheimera baltica (Brettar et al., 2002), Rheinheimera pacifica (Romanenko et al., 2003) and Rheinheimera perlucida (Brettar et al., 2006). Phylogenetic analyses based on 16S rRNA gene sequences showed that the genus Rheinheimera belongs to the Gammaproteobacteria (Brettar et al., 2002, 2006; Romanenko et al., 2003). Here, we report on the taxonomic characterization of two Rheinheimera-like bacterial strains, SW-353T and SW-369, which were isolated from seawater from the East Sea, Korea.
Seawater collected at Hwajinpo, on the East Sea of Korea, was used as the source for the isolation of bacterial strains. Strains SW-353T and SW-369 were isolated by means of the standard dilution plating technique with marine agar 2216 (MA; Difco) at 30 °C. The type strains of three Rheinheimera species were used as reference strains for DNA–DNA hybridization: R. baltica LMG 21511T and R. perlucida LMG 23581T were obtained from the Laboratorium voor Microbiologie Universiteit Gent (LMG), Ghent, Belgium, and R. pacifica JCM 12090T was obtained from the Japan Collection of Microorganisms (JCM), Saitama, Japan. The morphological, physiological and biochemical characteristics of strains SW-353T and SW-369 were investigated using routine cultivation on MA at 37 °C. Cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. Transmission electron microscopy was also used to determine whether flagella were present in cells from exponentially growing cultures: for this purpose, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined (after being air-dried) with a Philips CM-20 transmission electron microscope. The 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, using increments of 0.5 pH units). The pH was adjusted, prior to sterilization, to various levels by the addition of HCl or Na2CO3. Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations (0.5 %, w/v, and 1.0–10.0 %, w/v, using increments of 1.0 %) was investigated in MB and trypticase soy broth (Difco). Growth at various temperatures (4–50 °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 the hydrolysis of casein and starch were determined as described by Cowan & Steel (1965). DNase activity was examined by using DNase test agar with methyl green (Difco). Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965), with the modification that artificial seawater was used instead of distilled water. Hydrolysis of aesculin, gelatin and urea and reduction of nitrate were determined as described by Lanyi (1987), with the modification that artificial seawater was used instead of distilled water. The artificial seawater 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). Hydrolysis of hypoxanthine, tyrosine and xanthine was investigated on MA using the substrate concentrations described by 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). Utilization of substrates (each 0.2 %, w/v) as sole carbon and energy sources was tested as described by Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Susceptibility to antibiotics was tested on MA plates using antibiotic discs containing the following amounts: 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; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; carbenicillin, 100 µg. Other physiological and biochemical tests were performed with the API 20E, API 20NE, API 50 CH and API ZYM systems (bioMérieux). The cells were suspended in AUX medium, according to the manufacturer's instructions, to inoculate the API 50 CH system for assimilation tests.
Cell biomass of strains SW-353T and SW-369 for DNA extraction and for isoprenoid quinone analysis was obtained by means of cultivation for 2 days in MB at 37 °C. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. 16S rRNA genes were amplified by using a PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of each amplified 16S rRNA gene and a phylogenetic analysis were performed as described by Yoon et al. (2003). Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. For the fatty acid methyl ester analysis, cell mass of strains SW-353T and SW-369 was harvested from MA plates after incubation for 2 days at 37 °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 using the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed and the resultant nucleotides analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by 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. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as the DNA–DNA relatedness results.
Morphological, cultural, physiological and biochemical characteristics of strains SW-353T and SW-369 are given in the species description (see below) or are shown in Table 1, together with those of Rheinheimera species. The almost-complete 16S rRNA gene sequences of strains SW-353T and SW-369, each comprising 1492 nt (approx. 96 % of the Escherichia coli 16S rRNA gene sequence), were determined in this study. The 16S rRNA gene sequences of strains SW-353T and SW-369 were identical. Comparative 16S rRNA gene sequence analysis revealed that strains SW-353T and SW-369 were most closely related to members of the genus Rheinheimera. In the neighbour-joining phylogenetic tree constructed on the basis of 16S rRNA gene sequences, strains SW-353T and SW-369 fell within the radiation of the cluster comprising Rheinheimera species. Strains SW-353T and SW-369 exhibited 16S rRNA gene sequence similarity values of 97.8, 97.9 and 96.6 % with respect to the type strains of R. baltica, R. pacifica and R. perlucida, respectively. Strains SW-353T and SW-369 each exhibited 95.6 % 16S rRNA gene sequence similarity with respect to the type strain of Alishewanella fetalis and a value of less than 88.6 % with respect to other species included in the phylogenetic analysis (Fig. 1).
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7c and C16 : 17c and/or iso-C15 : 0 2-OH (see Supplementary Table S1 available in IJSEM Online). This fatty acid profile was similar to those of Rheinheimera species analysed previously, although there were differences in the proportions of some fatty acids, perhaps because of differences in cultivation conditions, e.g. temperatures and cultivation media (Brettar et al., 2002, 2006; Romanenko et al., 2003). The DNA G+C contents of strains SW-353T and SW-369 were 50.5 and 50.1 mol%, respectively.
The 16S rRNA gene sequence analysis revealed that strains SW-353T and SW-369 are most closely affiliated, phylogenetically, to the genus Rheinheimera (Fig. 1
). The results obtained from chemotaxonomic analyses are consistent with the result of the phylogenetic analysis based on 16S rRNA gene sequences (Brettar et al., 2002, 2006; Romanenko et al., 2003). Strains SW-353T and SW-369 showed a mean level of DNA–DNA relatedness of 91 % when their DNAs were used individually as labelled DNA probes for cross-hybridization, indicating that the two strains represent the same genomic species (Wayne et al., 1987). In view of the combined phenotypic, phylogenetic and genetic similarities, strains SW-353T and SW-369 could be considered as members of the same species. Strains SW-353T and SW-369 are distinguishable from the recognized Rheinheimera species through differences in phenotypic properties (Table 1). The levels of DNA–DNA relatedness between strains SW-353T and SW-369 and the type strains of R. baltica and R. pacifica were in the range 8–23 %. The genetic distinctiveness, together with the differential phenotypic properties and differences in the 16S rRNA gene sequences, are sufficient to allow the assignment of strains SW-353T and SW-369 to a species that is separate from the recognized Rheinheimera species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented here, strains SW-353T and SW-369 represent a novel species within the genus Rheinheimera, for which the name Rheinheimera aquimaris sp. nov. is proposed.
Description of Rheinheimera aquimaris sp. nov.
Rheinheimera aquimaris (a.qui.ma'ris. L. n. aqua water; L. gen. n. maris of the sea; N.L. gen. n. aquimaris of the water of the sea).
Cells are rods or cocci (0.4–0.7x0.5–2.5 µm) on MA and rods (0.3–0.5x1.0–5.0 µm) on trypticase soy agar. Cells are Gram-negative, non-spore-forming and motile by means of single polar flagella. After incubation for 2 days at 37 °C, colonies on MA are circular, raised, slightly wrinkled, yellowish-white in colour and 2.0–3.0 mm in diameter; colonies on trypticase soy agar (Difco) are circular, slightly convex, glistening, smooth, greyish-yellow in colour and 3.0–4.0 mm in diameter. Growth occurs at 4 and 43 °C, but not at 44 °C; optimal growth occurs at 30–37 °C. Growth occurs at pH 5.5, but not at pH 5; optimal growth occurs at pH 7.0–8.0. Growth occurs in the absence of NaCl and in the presence of 8 % (w/v) NaCl, but not in the presence of more than 9 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Casein, L-tyrosine, urea and Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine and xanthine are not. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase are absent. Tryptophan deaminase is present. D-Xylose, methyl 
-D-mannoside, D-trehalose, starch, glycogen, acetate, succinate and pyruvate are utilized, but glycerol, erythritol, D-arabinose, ribose, L-xylose, adonitol, methyl 
-D-xyloside, galactose, fructose, sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl -D-glucoside, amygdalin, arbutin, aesculin, salicin, cellobiose, lactose, melibiose, inulin, melezitose, raffinose, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, 5-ketogluconate, benzoate, formate and L-glutamate are not utilized. Utilization of sucrose is variable (positive for the type strain). Acid is not produced from the following substrates: L-arabinose, D-fructose, D-galactose, lactose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose, D-trehalose, D-xylose, myo-inositol, D-mannitol and D-sorbitol. Susceptible to chloramphenicol, gentamicin, kanamycin, neomycin, polymyxin B and streptomycin, but not to ampicillin, cephalothin, penicillin G, novobiocin, tetracycline, lincomycin or oleandomycin. Susceptibility to carbenicillin is variable (negative for type strain). The predominant ubiquinone is Q-8. The major fatty acids are C16 : 0, C18 : 17c and C16 : 17c and/or iso-C15 : 0 2-OH. The DNA G+C content is 50.1–50.5 mol% (HPLC). Other phenotypic properties are shown in Table 1 and Supplementary Table S1 (available in IJSEM Online).
The type strain, SW-353T (=KCTC 12840T=JCM 14331T), was isolated from seawater collected at Hwajinpo (East Sea, Korea).
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ACKNOWLEDGEMENTS |
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