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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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7c as the major fatty acid. Major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The DNA G+C content was 56.9 mol%. Strain DSW-25T exhibited 16S rRNA gene sequence similarity values of 98.4 % to the type strain of Staleya guttiformis and of 96.6–97.6 % to Sulfitobacter species. There were no distinct phenotypic, particularly chemotaxonomic, properties to differentiate Staleya guttiformis and strain DSW-25T from the genus Sulfitobacter. DNA–DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain DSW-25T differs from recognized Sulfitobacter species and Staleya guttiformis. On the basis of phenotypic, chemotaxonomic, phylogenetic and genetic data, strain DSW-25T was classified in the genus Sulfitobacter as a member of a novel species, for which the name Sulfitobacter donghicola sp. nov. is proposed. The type strain is strain DSW-25T (=KCTC 12864T =JCM 14565T). It is also proposed that Staleya guttiformis be transferred to the genus Sulfitobacter as Sulfitobacter guttiformis comb. nov.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSW-25T is EF202614.
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, b, 2006a, b). One of these isolates, DSW-25T, which is phylogenetically closely related to the genera Sulfitobacter and Staleya, is the subject of this study. The genus Sulfitobacter was described by Sorokin (1995, 1996) and, at the time of writing, the genus consists of seven recognized species, Sulfitobacter pontiacus (Sorokin, 1995, 1996), Sulfitobacter mediterraneus (Pukall et al., 1999), Sulfitobacter brevis (Labrenz et al., 2000), Sulfitobacter delicatus and Sulfitobacter dubius (Ivanova et al., 2004), Sulfitobacter marinus (Yoon et al., 2007) and Sulfitobacter litoralis (Park et al., 2007). The genus Staleya was proposed by Labrenz et al. (2000) with the description of a single species, Staleya guttiformis. Phylogenetic analysis based on 16S rRNA gene sequences showed that Staleya guttiformis represents a separate genus within the phylogenetic radiation comprising Sulfitobacter species (Wagner-Döbler et al., 2004; Yoon et al., 2007). The aim of the present study was to determine the exact taxonomic position of strain DSW-25T by a polyphasic characterization that included phenotypic and chemotaxonomic properties, detailed phylogenetic analysis based on 16S rRNA gene sequences and genetic relatedness.
Seawater off Dokdo in the East Sea of Korea was used as the source for the isolation of bacterial strains. Strain DSW-25T was isolated by means of the standard dilution-plating technique at 25 °C on marine agar 2216 (MA; Difco). The type strains of five Sulfitobacter species and Staleya guttiformis were used as reference strains for DNA–DNA hybridization: Sulfitobacter mediterraneus DSM 12244T, Sulfitobacter brevis DSM 11443T and Staleya guttiformis DSM 11458T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany); Sulfitobacter delicatus KCTC 12547T and Sulfitobacter dubius KCTC 12546T were obtained from the Korean Collection for Type Cultures (Taejon, Korea); and Sulfitobacter marinus SW-265T was maintained in our laboratory (Yoon et al., 2007).
The morphological, physiological and biochemical characteristics of strain DSW-25T were investigated using routine cultivation on MA at 25 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Flagellation was determined by using a Philips CM-20 transmission electron microscope with cells from exponentially growing cultures. For this purpose, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and the grids were examined after being air-dried. 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 of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth (Difco). Growth on trypticase soy agar (TSA; Difco), nutrient agar (NA; Difco) and MacConkey agar (Difco) was tested at 25 °C. Growth at various temperatures (4–40 °C) was measured on MA. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were investigated as described previously (Lanyi, 1987) with the modification that artificial seawater was used for preparation of 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). For in vivo pigment-absorption spectrum analysis, strain DSW-25T was cultivated aerobically in the dark at 25 °C in MB. The cultures were washed twice by centrifugation using a MOPS buffer (0.01 M MOPS/NaOH, 0.1 M KCl, 0.001 M MgCl2; pH 7.5) and disrupted by sonication with a Branson Sonifier 450. After removal of cell debris by centrifugation, the absorption spectrum of the supernatant was examined on a Beckman Coulter DU800 spectrophotometer. Susceptibility to antibiotics was investigated on MA plates by using antibiotic discs with 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. Utilization of various substrates for growth was tested as described by Baumann & Baumann (1981), using supplementation with 2 % (v/v) Hutner's mineral salts solution (Cohen-Bazire et al., 1957) and 1 % (v/v) vitamin solution (Staley, 1968). Acid production from carbohydrates was determined as described by Leifson (1963). Other physiological and biochemical tests were performed with the API 20E and API ZYM systems (bioMérieux).
Cell biomass for DNA extraction and for isoprenoid quinone and polar lipid analyses was obtained from cultivation in MB at 25 °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 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). Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using reversed-phase HPLC. For cellular fatty acid analysis, cell mass of strain DSW-25T was harvested from MA plates after cultivation for 3 days at 25 °C. The 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). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). The presence of phosphatidylcholine was identified by spraying Dragendorff's reagent (Sigma). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed using nuclease P1 (Boehringer Mannheim) and 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 are quoted as DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain DSW-25T are given in the species description (see below) or are shown in Table 1. Strain DSW-25T did not produce bacteriochlorophyll a (BChl a) aerobically in the dark. Sonicated in vivo cell extracts of strain DSW-25T showed no absorption maximum. The almost complete 16S rRNA gene sequence of strain DSW-25T determined in this study comprised 1418 nucleotides, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DSW-25T is most closely related to the genera Staleya and Sulfitobacter (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, strain DSW-25T joined the type strain of Staleya guttiformis by a bootstrap resampling value of 98.8 % (Fig. 1). Strain DSW-25T exhibited 16S rRNA gene sequence similarity values of 98.4 % to Staleya guttiformis EL-38T, 96.6–97.6 % similarity to species of the genus Sulfitobacter and Oceanibulbus indolifex and less than 96.1 % similarity to other species tested in the phylogenetic analysis.
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7c (79.1 %), straight-chain fatty acids C16 : 0 (10.1 %) and C18 : 0 (1.3 %), 11-methyl-C18 : 17c (3.7 %) and hydroxy fatty acids C10 : 0 3-OH (3.4 %) and iso-C17 : 0 3-OH (1.2 %). This fatty acid profile is similar to those of members of the genera Sulfitobacter and Staleya (Labrenz et al., 2000; Ivanova et al., 2004; Yoon et al., 2007). Major polar lipids found in strain DSW-25T were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid; a minor amount of diphosphatidylglycerol was also present. The DNA G+C content of strain DSW-25T was 56.9 mol%. Strain DSW-25T exhibited mean DNA–DNA relatedness values of 17 % to Staleya guttiformis DSM 11458T and of 8–14 % to the type strains of five Sulfitobacter species that showed 16S rRNA gene sequence similarity values of greater than 97 % to strain DSW-25T.
The genera Staleya and Sulfitobacter have not been clearly distinguished, since they were not well defined chemotaxonomically or phylogenetically (Labrenz et al., 2000; Ivanova et al., 2004; Yoon et al., 2007). Labrenz et al. (2000) reported that the genus Staleya is distinguishable from the genus Sulfitobacter by differences in polar lipid and fatty acid profiles and by the presence of BChl a. However, it seems that the differences in polar lipid and fatty acid profiles are not sufficient to differentiate the two genera clearly. The presence of BChl a may not be a very significant characteristic for taxonomic separation (Labrenz et al., 1999, 2000). Accordingly, it is more appropriate that Staleya guttiformis be transferred to the genus Sulfitobacter. Strain DSW-25T is distinguishable from Staleya guttiformis by the absence of BChl a and the presence of diphosphatidylglycerol. Strain DSW-25T differs from recognized Sulfitobacter species and Staleya guttiformis by several phenotypic characteristics as listed in Table 1
. The phylogenetic and genetic distinctiveness, together with differential phenotypic properties, are sufficient to allocate strain DSW-25T to a species that is separate from the recognized Sulfitobacter species and Staleya guttiformis (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain DSW-25T should be placed in the genus Sulfitobacter within a novel species, for which the name Sulfitobacter donghicola sp. nov. is proposed.
Description of Sulfitobacter donghicola sp. nov.
Sulfitobacter donghicola (dong.hi'co.la. N.L. n. Donghae Donghae, the Korean name of the East Sea; L. suff. -cola from L. n. incola a dweller, inhabitant; N.L. masc. n. donghicola a dweller of the East Sea).
Cells are Gram-negative and rod-, oval- or coccoid-shaped (0.2–0.5x0.6–1.5 µm), multiplying by monopolar growth, i.e. by a budding process. Colonies on MA are circular, convex, smooth, glistening, greyish yellow in colour and 1.5–2.5 mm in diameter after 7 days incubation at 25 °C. Growth does not occur on TSA, NA or MacConkey agar. Growth occurs at 10 and 31 °C, but not at 4 or 32 °C. Optimal pH for growth is 7.0–8.0; growth occurs at pH 6.0 but not at pH 5.5. Growth occurs in the presence of 5 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 6 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Urease-negative. Tweens 20, 40 and 60 are hydrolysed, but aesculin, hypoxanthine, L-tyrosine 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), leucine arylamidase and acid phosphatase are present, but lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, naphthol-AS-BI-phosphohydrolase, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are absent. L-Malate and pyruvate are utilized as sole carbon and energy sources, but D-xylose, salicin, formate and benzoate are not. Acid is not produced 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, trehalose, D-xylose, myo-inositol, D-mannitol or D-sorbitol. Susceptible to chloramphenicol, cephalothin, novobiocin and streptomycin, but not to lincomycin. The predominant ubiquinone is Q-10. The major fatty acids (>10 % of total fatty acids) are C18 : 17c and C16 : 0. Major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid; a minor amount of diphosphatidylglycerol is also present. The DNA G+C content of the type strain is 56.9 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, DSW-25T (=KCTC 12864T =JCM 14565T), was isolated from seawater off Dokdo in the East Sea of Korea.
Description of Sulfitobacter guttiformis (Labrenz et al. 2000) comb. nov.
Sulfitobacter guttiformis (gut.ti.for'mis. L. fem. n. gutta a drop; L. adj. suff. -formis in the shape of; N.L. masc. adj. guttiformis drop-shaped).
Basonym: Staleya guttiformis Labrenz et al. 2000.
The description is as given by Labrenz et al. (2000). The type strain is strain EL-38T =ATCC BAA-5T =DSM 11458T =JCM 21791T.
Emended description of the genus Sulfitobacter Sorokin 1996
The description of the genus Sulfitobacter is as given by Sorokin (1995) and amended by Pukall et al. (1999), Labrenz et al. (2000), Ivanova et al. (2004), Yoon et al. (2007) and Park et al. (2007) with the following further amendment. Bacteriochlorophyll a is produced by some species, but not all.
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