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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 and C17 : 16c as the major fatty acids. The DNA G+C contents of strains SW-135T and SW-161T were 62·2 and 64·5 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that the two strains fall within the radiation of the cluster comprising Erythrobacter species. Strains SW-135T and SW-161T exhibited a 16S rRNA gene sequence similarity value of 96·9 % and a mean DNA–DNA relatedness level of 12·3 %. Sequence similarities between strains SW-135T and SW-161T and the type strains of recognized Erythrobacter species ranged from 96·7 to 98·5 %. Levels of DNA–DNA relatedness were low enough to indicate that strains SW-135T and SW-161T represent members of two species separate from all recognized Erythrobacter species. On the basis of polyphasic taxonomic data, strains SW-135T (=KCTC 12228T=DSM 16221T) and SW-161T (=KCTC 12227T=DSM 16225T) were classified as two novel Erythrobacter species, for which the names Erythrobacter seohaensis sp. nov. and Erythrobacter gaetbuli sp. nov. are proposed, respectively.
Published online ahead of print on 23 July 2004 as DOI 10.1099/ijs.0.63233-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SW-135T and SW-161T are AY562219 and AY562220, respectively.
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and at the time of writing comprised five recognized species, Erythrobacter longus (Shiba & Simidu, 1982), Erythrobacter litoralis (Yurkov et al., 1994), Erythrobacter citreus (Denner et al., 2002), Erythrobacter flavus (Yoon et al., 2003a) and Erythrobacter aquimaris (Yoon et al., 2004b). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the genus Erythrobacter belongs to the 
-Proteobacteria (Anzai et al., 2000; Denner et al., 2002; Yoon et al., 2003a). Some Erythrobacter species contain bacteriochlorophyll (BChl) a and carotenoids (Shiba & Simidu, 1982; Yurkov et al., 1994). Here we describe two slightly halophilic orange–yellow pigmented Erythrobacter-like strains, SW-135T and SW-161T, which were isolated from a tidal flat of the Yellow Sea in Korea. Tidal flat (Korean name, gaetbul) environments have recently provided several novel micro-organisms (Yi et al., 2003, 2004; Yoon et al., 2003b, c, 2004a). Accordingly, the aim of the present work was to elucidate the taxonomic positions of strains SW-135T and SW-161T using a polyphasic taxonomic approach that combined phenotypic, genetic and chemotaxonomic analyses.
Intertidal sediment provided the source for isolation of the bacterial strains. A standard dilution plating technique was used to isolate strains SW-135T and SW-161T on marine agar 2216 (MA; Difco) at 30 °C. E. longus DSM 6997T, E. litoralis DSM 8509T and E. citreus DSM 14432T were obtained from DSMZ, Germany. E. flavus KCCM 41642T and E. aquimaris KCCM 41818T were obtained from previous studies (Yoon et al., 2003a, 2004b). Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM). For TEM observation, cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air drying, grids were examined under a model CM-20 transmission electron microscope (Philips). Presence of flagella was examined by TEM using cells from exponentially growing cultures. Growth under anaerobic conditions was determined after incubation in a Forma anaerobic chamber on MA and MA supplemented with nitrate that had been prepared anaerobically using nitrogen gas. Growth in the absence of NaCl was investigated in trypticase soy broth without NaCl. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–45 °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 studied as described by Lanyi (1987) with a modification that artificial sea water was used for preparation of media. 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 (Levring, 1946). H2S production was tested as described by Bruns et al. (2001). For in vivo pigment-absorption spectrum analysis, the strains were cultivated aerobically in the dark at 30 °C in MB, peptone/yeast extract/glucose/vitamin (PYGV) medium (Fuerst et al., 1993; DSMZ medium no. 621) and Erythromicrobium/Roseococcus medium (Yurkov et al., 1994; DSMZ medium no. 767). E. longus DSM 6997T and E. litoralis DSM 8509T were used as positive controls for spectrum analysis. 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 detected on MA plates by using antibiotic discs (concentrations are given in Table 1). Acid production from carbohydrates was determined as described by Leifson (1963), and utilization of various substrates for growth was determined as described by Yurkov et al. (1994).
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using reversed-phase HPLC. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996) with the exception that ribonuclease T1 was used together with ribonuclease A. For fatty acid methyl ester (FAME) analysis, cell mass of strains SW-135T and SW-161T was harvested after cultivation for 5 days at 30 °C on MA. FAMEs 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 a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. The 16S rRNA gene was amplified by PCR using two universal primers as described by Yoon et al. (1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003a). 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. From the values obtained, the highest and lowest values in each sample were excluded. DNA–DNA relatedness values are the mean of the remaining three values.
Strains SW-135T and SW-161T showed similar phenotypic characteristics except for the following. Maximum growth temperatures of strains SW-135T and SW-161T were 40 and 43 °C, respectively. Strain SW-135T grew at 10 °C, whereas strain SW-161T grew at 15 °C but not at 10 °C. Malate was utilized by strain SW-161T but not by strain SW-135T. Strain SW-135T produced acid from sucrose but strain SW-161T did not. Acid was produced from D-glucose and maltose by strain SW-161T but not by strain SW-135T. Morphological, cultural, physiological and biochemical characteristics of the two isolates are summarized in Table 1
or are given in the formal species descriptions below. Phenotypic characteristics that differentiate strains SW-135T and SW-161T from Erythrobacter species are summarized in Table 1. The predominant respiratory lipoquinone detected in strains SW-135T and SW-161T was ubiquinone-10 (Q-10), at peak area ratios of approximately 91–95 %. The fatty acid profiles of the two strains were characterized by high levels of unsaturated fatty acids C18 : 17c and C17 : 16c and significant amounts of straight-chain fatty acid C16 : 0 (Table 2). Hydroxy fatty acids were present, but branched fatty acids were not detected in the two strains. These fatty acid profiles were similar to those of the type strains of recognized Erythrobacter species, particularly E. aquimaris (Table 2). However, strains SW-135T and SW-161T differed from each other and from other recognized Erythrobacter species in the relative abundance of shared components (Table 2). The DNA G+C contents of strains SW-135T and SW-161T were 62·2 and 64·5 mol%, respectively.
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). Similarity values between strain SW-135T and the type strains of recognized Erythrobacter species ranged from 98·5 % (with E. citreus) to 96·9 % (with E. flavus, E. longus and E. litoralis). Similarities between the 16S rRNA gene sequence of strain SW-161T and those of the type strains of recognized Erythrobacter species ranged from 98·1 % (with E. flavus) to 96·7 % (with E. longus). Sequence similarities to all other species of the family Sphingomonadaceae included in the phylogenetic analysis were lower than 97·2 % (Fig. 1). DNA–DNA relatedness observed between strains SW-135T and SW-161T was 12·3 %, suggesting that the two strains represent members of different genomic species (Wayne et al., 1987). Strains SW-135T and SW-161T exhibited DNA–DNA relatedness levels of 9·7–20·2 % and 8·5–18·9 %, respectively, to the type strains of recognized species of the genus Erythrobacter. This genomic evidence demonstrated that strains SW-135T and SW-161T represent two separate species within the genus Erythrobacter (Wayne et al., 1987).
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). The relationship between the cluster comprising the two isolates and recognized Erythrobacter species and the cluster comprising the genera Porphyrobacter and Erythromicrobium was supported by a bootstrap confidence value of 100 % (Fig. 1). The respiratory lipoquinone and fatty acid profiles support the result of the monothetic classification based on 16S rRNA gene sequence analysis, although no clear-cut chemotaxonomic differentiation may exist between the genus Erythrobacter and the two phylogenetically related genera Porphyrobacter and Erythromicrobium (Fuerst et al., 1993; Rainey et al., 2003; Yurkov et al., 1994). Strains SW-135T and SW-161T were isolated from the same region and were similar in most morphological and phenotypic characteristics except for thermotolerance and acid production from certain substrates (Table 1). However, the two strains were phylogenetically and genetically different. There were also phenotypic, phylogenetic and genetic differences between strains SW-135T and SW-161T and other recognized Erythrobacter species (Table 1, Fig. 1). Therefore, on the basis of the data presented, strains SW-135T and SW-161T should be placed in the genus Erythrobacter as two distinct novel species, for which the names Erythrobacter seohaensis sp. nov. and Erythrobacter gaetbuli sp. nov. are proposed, respectively.
Description of Erythrobacter seohaensis sp. nov.
Erythrobacter seohaensis (seo.ha.en'sis. N.L. masc. adj. seohaensis of Seohae, the Korean name of the Yellow Sea in Korea, from where the type strain was isolated).
Cells are rod-shaped (0·6–0·8x1·5–4·0 µm) and non-spore-forming. Colonies on MA (5 days, 30 °C) are smooth, glistening, circular, convex, orange–yellow in colour and 1·0–2·0 mm in diameter. Growth occurs at 10 and 40 °C, but not at 4 °C or above 41 °C. Optimal pH for growth is 7·0–8·0; growth is observed at pH 5·5 but not at pH 5·0. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl; growth does not occur without NaCl or in the presence of >9 % NaCl. Anaerobic growth does not occur on MA or MA supplemented with nitrate. Urease-negative. Aesculin, Tweens 20, 40, 60 and 80 and tyrosine are hydrolysed. Casein, hypoxanthine and xanthine are not hydrolysed. H2S is not produced. Acid is produced from D-cellobiose, D-melezitose and sucrose. Acid is not produced from adonitol, L-arabinose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, maltose, D-mannitol, D-mannose, melibiose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, D-trehalose or D-xylose. Butyrate is utilized, but formate, methanol, ethanol and benzoate are not utilized. The predominant respiratory lipoquinone is Q-10. The fatty acid profile is shown in Table 2. The DNA G+C content is 62·2 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, SW-135T (=KCTC 12228T=DSM 16221T), was isolated from a tidal flat of the Yellow Sea in Korea.
Description of Erythrobacter gaetbuli sp. nov.
Erythrobacter gaetbuli (gaet.bu'li. N.L. gen. n. gaetbuli of gaetbul, the Korean name for a tidal flat).
Cells are rod-shaped (0·6–0·8x2·0–4·0 µm) and non-spore-forming. Colonies on MA (5 days, 30 °C) are smooth, glistening, circular, convex, orange–yellow in colour and 1·0-2·0 mm in diameter. Growth occurs at 15 and 43 °C, but not at 10 °C or above 44 °C. Optimal pH for growth is 7·0–8·0; growth is observed at pH 5·5 but not at pH 5·0. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl; growth does not occur without NaCl or in the presence of >9 % NaCl. Anaerobic growth does not occur on MA or MA supplemented with nitrate. Urease-negative. Aesculin, Tweens 20, 40, 60 and 80 and tyrosine are hydrolysed. Casein, hypoxanthine and xanthine are not hydrolysed. H2S is not produced. Acid is produced from D-cellobiose, D-glucose, maltose and D-melezitose. Acid is not produced from adonitol, L-arabinose, D-fructose, D-galactose, myo-inositol, lactose, D-mannitol, D-mannose, melibiose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, sucrose, D-trehalose or D-xylose. Butyrate is utilized, but formate, methanol, ethanol and benzoate are not utilized. The predominant respiratory lipoquinone is Q-10. The fatty acid profile is shown in Table 2. The DNA G+C content is 64·5 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, SW-161T (=KCTC 12227T=DSM 16225T), was isolated from a tidal flat of the Yellow Sea in Korea.
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ACKNOWLEDGEMENTS |
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