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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 as the major fatty acid. The DNA G+C content was 57.8 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain SW-265T fell within the cluster comprising Sulfitobacter species. The levels of 16S rRNA gene sequence similarity between strain SW-265T and the type strains of Sulfitobacter species ranged from 97.1 to 98.7 %. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain SW-265T differs from recognized Sulfitobacter species. On the basis of the phenotypic, phylogenetic and genetic data, strain SW-265T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter marinus sp. nov. is proposed. The type strain is SW-265T (=KCTC 12738T=JCM 13602T).
Biolog assimilation data for strain SW-265T are available in a supplementary table in IJSEM Online.
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, and, at the time of writing, consists of five species with validly published names: Sulfitobacter pontiacus (Sorokin, 1995), Sulfitobacter mediterraneus (Pukall et al., 1999), Sulfitobacter brevis (Labrenz et al., 2000) and Sulfitobacter delicatus and Sulfitobacter dubius (Ivanova et al., 2004). In this study, we report on the taxonomic characterization of a Sulfitobacter-like bacterial strain, SW-265T, which was isolated from seawater from the East Sea, Korea.
Strain SW-265T was isolated, using the standard dilution plating technique, on marine agar 2216 (MA; Difco) at 25 °C. The following strains were used as reference strains: Sulfitobacter pontiacus DSM 10014T, Sulfitobacter mediterraneus DSM 12244T and Sulfitobacter brevis DSM 11443T, obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), and Sulfitobacter delicatus KCTC 12547T and Sulfitobacter dubius KCTC 12546T, obtained from the Korean Collection for Type Cultures (Taejon, Korea). The morphological, physiological and biochemical characteristics of strain SW-265T were investigated using routine cultivation on MA at 30 °C. Cell morphology was examined by light microscopy (E600; Nikon) and transmission electron microscopy. The presence of flagella was determined using transmission electron microscopy with cells from exponentially growing cultures. For transmission electron microscopic observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after air-drying with a Philips CM-20 transmission electron microscope. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and MA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Growth in the absence of NaCl was investigated using trypticase soy broth prepared according to the formula for the Difco medium, except that no NaCl was used. Growth at various NaCl concentrations was investigated in marine broth 2216 (Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–40 °C) was measured on MA. Catalase and oxidase activities and the hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). The hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA, using the substrate concentrations described by Cowan & Steel (1965). The hydrolysis of aesculin, gelatin and urea and the reduction of nitrate were investigated as described previously (Lanyi, 1987), with the modification that artificial seawater was used in the preparation of the media. The artificial seawater contained (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). Susceptibility to antibiotics was detected on MA plates by using filter-paper discs containing the following amounts of antibiotic: 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) and tetracycline (30 µg). Haemolytic activity was recorded on MA with 5 % defibrinated sheep blood. Acid production from carbohydrates was determined as described by Leifson (1963). The oxidation of various substrates was determined by using the Biolog GN2 MicroPlate assay as recommended by the manufacturer. Other physiological and biochemical tests were performed with the API 20E and API ZYM systems (bioMérieux).
Cell biomass for respiratory lipoquinone analysis and for DNA extraction was obtained from cultivation in marine broth at 30 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), with the exception that RNase T1 was used in combination with RNase A to minimize contamination with RNA. The 16S rRNA gene was amplified by a PCR with 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). Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain SW-265T was harvested from agar plates after cultivation for 3 days at 30 °C on MA. Fatty acids were extracted and fatty acid methyl esters were 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 the DNA was hydrolysed using nuclease P1 (Boehringer Mannheim); the resultant nucleotides were analysed by reversed-phase HPLC. 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 were quoted as the DNA–DNA relatedness values.
The morphological, cultural, physiological and biochemical characteristics of strain SW-265T are given in the species description (see below) or are shown in Table 1 and Supplementary Table S1 (available in IJSEM Online). The almost-complete 16S rRNA gene sequence of strain SW-265T determined in this study comprised 1420 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. In the phylogenetic tree based on the neighbour-joining algorithm, strain SW-265T fell within the clade comprising Sulfitobacter species (Fig. 1). Strain SW-265T exhibited 16S rRNA gene sequence similarity values of 97.1–98.7 % with respect to the type strains of Sulfitobacter species and showed values of 97.0–97.1 % with respect to Staleya guttiformis EL-38T and Oceanibulbus indolifex HEL45T. Sequence similarities with respect to all other species included in the phylogenetic analysis were below 96.6 %. The fatty acid profile of strain SW-265T was composed of the following (each constituting >0.5 % of total fatty acids): unsaturated fatty acids C18 : 17c (77.1 %) and C17 : 18c (0.5 %); straight-chain fatty acids C16 : 0 (8.3 %), C17 : 0 (0.7 %), C18 : 0 (0.6 %) and C15 : 0 (0.6 %); 11-methyl C18 : 17c (6.9 %); hydroxy fatty acid C10 : 0 3-OH (3.6 %); and summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH; 1.0 %). This fatty acid profile was similar to that of Sulfitobacter species (Pukall et al., 1999; Labrenz et al., 2000; Ivanova et al., 2004). The predominant respiratory lipoquinone detected in strain SW-265T was Q-10, at a peak area ratio of approximately 89 %. The DNA G+C content of strain SW-265T was 57.8 mol%. The mean levels of DNA–DNA relatedness between strain SW-265T and the type strains of five Sulfitobacter species were in the range 9–21 %, indicating that the novel strain represents a genomic species that is distinct from the five Sulfitobacter species (Wayne et al., 1987). The differential phenotypic properties, together with the phylogenetic distinctiveness and the DNA–DNA relatedness data, were sufficient to categorize strain SW-265T as a member of a species that is distinct from the recognized Sulfitobacter species (Table 1) (Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain SW-265T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter marinus sp. nov. is proposed.
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Cells are Gram-negative and rod- or oval-shaped (0.2–0.5x0.6–1.5 µm). After 3 days incubation at 30 °C on MA, colonies are circular, slightly convex, smooth, glistening, cream-coloured and 0.5–1.0 mm in diameter. Growth occurs at 4 and 35 °C, but not at 36 °C. The optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.0, but not at pH 4.5. Growth occurs in the presence of 12 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 13 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Urease-negative. Hypoxanthine, L-tyrosine and Tweens 20, 40 and 60 are hydrolysed, but aesculin and xanthine are not. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. In assays with the API ZYM system, alkaline phosphatase, esterase (C4), esterase lipase (C8) and leucine arylamidase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. Acid is produced from D-mannitol and D-sorbitol, but not from L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose, D-trehalose, D-xylose or myo-inositol. Susceptible to chloramphenicol, cephalothin, novobiocin and streptomycin, but not to lincomycin. The predominant ubiquinone is Q-10. The major fatty acid (constituting >10 % of total fatty acids) is C18 : 17c (77.1 %). The DNA G+C content is 57.8 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1
and in Supplementary Table S1 (available in IJSEM Online).
The type strain, SW-265T (=KCTC 12738T=JCM 13602T), was isolated from seawater at Hwajinpo (East Sea, Korea).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
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