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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
2 National Research Laboratory of Molecular Ecosystematics, Institute of Probionic, Probionic Corporation, Bio-venture Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea
3 Department of Food and Life Science, Sungkyunkwan University, Chunchun-dong 300, Jangan-gu, Suwon, Korea
Correspondence
Yong-Ha Park
yhpark{at}mail.kribb.re.kr
| ABSTRACT |
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7c as the major fatty acid. Their DNA G+C content was 64·064·1 mol%. Strains SW-46T and SW-52 showed 1 bp difference in their 16S rDNA sequences and a mean DNADNA relatedness level of 94·4 %. Phylogenetic analysis based on 16S rDNA sequences showed that strains SW-46T and SW-52 fall within the
-subclass of the Proteobacteria and form a coherent cluster with Erythrobacter longus, Erythrobacter litoralis and Erythrobacter citreus. Levels of 16S rDNA similarity between strains SW-46T and SW-52 and the type strains of these three Erythrobacter species were 96·597·9 %. Levels of DNADNA relatedness between strains SW-46T and SW-52 and the type strains of E. longus, E. litoralis and E. citreus were 3·614·7 %. Therefore, on the basis of phenotypic properties, phylogeny and genomic data, strains SW-46T and SW-52 should be placed in the genus Erythrobacter as a novel species, for which the name Erythrobacter flavus sp. nov. is proposed. The type strain is SW-46T (=KCCM 41642T =JCM 11808T).
Published online ahead of print on 21 March 2003 as DOI 10.1099/ijs.0.02510-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains SW-46T and SW-52 are AF500004 and AF500005, respectively.
| MAIN TEXT |
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-subclass of the Proteobacteria and is closely related to the genera Erythromicrobium and Porphyrobacter (Yurkov et al., 1994
There are three Erythrobacter species with validly published names: Erythrobacter longus (Shiba & Simidu, 1982
), Erythrobacter litoralis (Yurkov et al., 1994
) and E. citreus (Denner et al., 2002
). The genus Erythrobacter is characterized chemotaxonomically by having C18 : 1 as the major fatty acid and by a DNA G+C content of 6067 mol% (Shiba & Simidu, 1982
; Fuerst et al., 1993
; Yurkov et al., 1994
). However, Shiba (1991)
reported that the type strain of E. longus has a DNA G+C content of 57·4 mol%.
All three Erythrobacter species have been isolated from marine environments (Shiba & Simidu, 1982
; Yurkov et al., 1994
; Denner et al., 2002
). Recently, two slightly halophilic bacterial strains, SW-46T and SW-52, were isolated from sea water of Hwajinpo Beach, East Sea, Korea. The two isolates were phylogenetically most closely related to the genus Erythrobacter, based on the result of 16S rDNA sequence comparison. Colonies of the two strains were observed to be yellow on marine agar, unlike the first two Eythrobacter species that were described. Accordingly, the aim of the present study was to establish the exact taxonomic status of the two isolates by a polyphasic taxonomic approach. In this work, we describe the morphological, phenotypic, phylogenetic and genomic characteristics of strains SW-46T and SW-52. On this basis, we propose a novel species of the genus Erythrobacter, Erythrobacter flavus sp. nov., for strains SW-46T and SW-52.
Strains SW-46T and SW-52 (=KCCM 41643 =JCM 11809) were isolated by using the dilution-plating technique on marine agar 2216 (MA; Difco). E. longus DSM 6997T, E. litoralis DSM 8509T and E. citreus DSM 14432T, which were obtained from DSMZ, Germany, were used as reference strains. Cell biomass of strains SW-46T and SW-52 and reference strains was obtained from marine broth 2216 (MB; Difco) cultures grown at 30 °C, for respiratory lipoquinone analysis and DNA extraction. All strains were cultivated on a gyratory shaker at 150 r.p.m. For fatty acid methyl ester (FAME) analysis, cell mass of strains SW-46T and SW-52, E. longus DSM 6997T and E. litoralis DSM 8509T was obtained from agar plates after 5 days cultivation at 30 °C on MA. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy (TEM); presence or absence of flagella was examined by TEM using cells from exponentially growing cultures. The cells were negatively stained with 1 % (w/v) phosphotungstic acid and, after air drying, the grids were examined by using a model CM-20 transmission electron microscope (Philips). Growth at various NaCl concentrations was investigated in MB. Growth at 455 °C was measured on MA. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with MA that had been prepared anaerobically. Presence or absence of bacteriochlorophyll a was examined by registration of the in vitro absorption spectrum of a methanol extract of cells at 768769 nm. Susceptibility to antibiotics was detected on agar plates by using antibiotic discs (concentrations shown in Table 1
). Catalase activity was determined by bubble production in 3 % (v/v) hydrogen peroxide solution. Oxidase activity was determined by oxidation of 1 % p-aminodimethylaniline oxalate. Hydrolysis of aesculin and nitrate reduction were determined as described by Lányi (1987)
. Hydrolysis of casein, starch and Tween 80 and urease activity were determined as described by Cowan & Steel (1965)
. Hydrolysis of gelatin was studied as described by Cowan & Steel (1965)
, with the modification that artificial sea water was used. The artificial sea water contained [(l distilled water)-1]: 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
). Hydrolysis of hypoxanthine, tyrosine and xanthine was examined on MA plates with substrate concentrations as described by Cowan & Steel (1965)
. H2S production was tested as described by Bruns et al. (2001)
. Acid production from carbohydrates was determined as described by Leifson (1963)
. Utilization of various substrates for growth was determined as described by Yurkov et al. (1994)
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Respiratory lipoquinones were analysed by using reversed-phase HPLC (Komagata & Suzuki, 1987
). For quantitative analysis of cellular fatty acid composition, a loop of cell mass was harvested and FAMEs were prepared and identified by following the instructions of the Microbial Identification system (MIDI). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984)
; DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.
The predominant respiratory lipoquinone of strains SW-46T and SW-52, E. longus DSM 6997T and E. litoralis DSM 8509T was ubiquinone-10 (Q-10), the same as that of E. citreus (Denner et al., 2002
). Cellular fatty acid profiles of strains SW-46T and SW-52 are shown in Table 2
, together with those of E. longus DSM 6997T, E. litoralis DSM 8509T and E. citreus RE35F/1T. Strains SW-46T and SW-52 had cellular fatty acid profiles that contained large amounts of saturated and unsaturated fatty acids (Table 2
). The major fatty acid found in strains SW-46T and SW-52 was C18 : 1
7c, at a peak ratio of approximately 4546 % (Table 2
). The fatty acid profiles of the two strains were similar to those of the type strains of E. longus and E. citreus. The fatty acid profile of E. longus DSM 6997T obtained in this study was similar to that reported by Fuerst et al. (1993)
. However, there was a noteworthy difference in the proportion of C17 : 1
6c between strains SW-46T and SW-52 and E. litoralis DSM 8509T. The DNA G+C contents of strains SW-46T and SW-52 were 64·0 and 64·1 mol%, respectively.
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The 16S rDNA sequences of two strains determined in this study each comprised 1442 nucleotides, which represents approximately 96 % of the Escherichia coli 16S rRNA gene sequence. There is only 1 bp difference between the 16S rDNA sequences of strains SW-46T and SW-52. The strains were found to have highest 16S rDNA similarity to members of the
-Proteobacteria. Strains SW-46T and SW-52 exhibited 16S rDNA similarity levels of 96·597·9 % with the type strains of E. longus, E. litoralis and E. citreus, but <96·5 % to other species used in the phylogenetic analysis. In the phylogenetic tree based on the neighbour-joining algorithm, strains SW-46T and SW-52 formed a coherent cluster with E. citreus, E. longus and E. litoralis (Fig. 1
). The relationship between this cluster and the clade that comprised the genera Erythromicrobium and Porphyrobacter was supported by a bootstrap confidence level of 100 %. This tree topology was also generated by the maximum-parsimony and maximum-likelihood algorithms (data not shown). When the neighbour-joining and maximum-parsimony algorithms were used, the relationship between the cluster that comprised strains SW-46T and SW-52 and Erythrobacter species and the clade that comprised the genera Porphyrobacter and Erythromicrobium was supported by high bootstrap resampling values.
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Description of Erythrobacter flavus sp. nov.
Erythrobacter flavus (fla'vus. L. masc. adj. flavus yellow, the colour of colonies or pigment).
Non-spore-forming rods, 0·70·9x1·52·5 µm on MA. Gram-staining reaction is negative. Motile by means of a single polar flagellum. Colonies are yellow, smooth, glistening, circular, convex with entire margins and 1·01·5 mm in diameter after 3 days cultivation at 30 °C on MA. Optimal temperature for growth is 3037 °C. Growth occurs at 10 and 42 °C, but not at 4 °C or above 43 °C. Optimal pH for growth is 6·57·5. Growth occurs at pH 5·0, but not at pH 4·5. Optimal growth occurs in the presence of 25 % (w/v) NaCl. No growth occurs in the absence of NaCl or in the presence of >14 % NaCl. No growth occurs under anaerobic conditions on MA. Catalase-, oxidase- and urease-positive. Starch, Tween 80 and tyrosine are hydrolysed. Aesculin, casein, gelatin, hypoxanthine and xanthine are not hydrolysed. H2S is not produced. Nitrate is not reduced to nitrite. Susceptible to chloramphenicol. Resistant to penicillin, streptomycin and polymyxin B. Acid is produced from D-cellobiose and maltose. Acid production from D-trehalose is variable. Acid is not produced from adonitol, L-arabinose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, D-mannitol, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, stachyose, sucrose or D-xylose. Acetate, butyrate and pyruvate are utilized for growth. Glucose, fructose, glutamate, citrate, malate, succinate, formate, methanol, ethanol and benzoate are not utilized. Utilization of lactate is variable. The predominant respiratory lipoquinone is ubiquinone-10. The major fatty acid is C18 : 1
7c. The DNA G+C content is 64·064·1 mol% (determined by HPLC).
The type strain (SW-46T=KCCM 41642T =JCM 11808T) and reference strain (SW-52=KCCM 41643 =JCM 11809) were isolated from sea water of Hwajinpo Beach, East Sea, Korea.
| ACKNOWLEDGEMENTS |
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