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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
Tae-Kwang Oh
otk{at}kribb.re.kr
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ABSTRACT |
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6c and C18 : 17c as the major fatty acids. Its DNA G+C content was 65.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DSW-74T was most closely related to Porphyrobacter species. Similarity values between the 16S rRNA gene sequence of strain DSW-74T and those of the type strains of recognized Porphyrobacter species and of Erythromicrobium ramosum were in the range 97.4–98.7 %. Strain DSW-74T exhibited 16S rRNA gene sequence similarity values of <97.5 % to recognized Erythrobacter species and the other species used in the phylogenetic analysis. DNA–DNA relatedness levels and differential phenotypic properties made it possible to categorize strain DSW-74T as representing a novel Porphyrobacter species. On the basis of the taxonomic data presented, it is proposed that DSW-74T (=KCTC 12395T=DSM 17193T) should be classified in the genus Porphyrobacter as the type strain of a novel species, Porphyrobacter dokdonensis sp. nov.
Published online ahead of print on 31 December 2005 as DOI 10.1099/ijs.0.63840-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DSW-74T is DQ011529.
A figure showing the absorption spectra of sonicated cell extract and acetone–methanol cell extract of strain DSW-74T is available as supplementary material in IJSEM Online.
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and, at the time of writing, the genus comprises five recognized species: Porphyrobacter neustonensis (Fuerst et al., 1993), Porphyrobacter tepidarius (Hanada et al., 1997), Porphyrobacter sanguineus (Hiraishi et al., 2002), Porphyrobacter cryptus (Rainey et al., 2003) and Porphyrobacter donghaensis (Yoon et al., 2004). Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Porphyrobacter belongs within the Alphaproteobacteria (Anzai et al., 2000; Hiraishi et al., 2002; Rainey et al., 2003; Yoon et al., 2004). Here we report on the detailed taxonomic characterization of a Porphyrobacter-like bacterial strain, designated DSW-74T.
Sea water collected from off the island of Dokdo, Korea, was used as the source for isolation of bacterial strains. Strain DSW-74T was isolated by the standard dilution plating technique on marine agar 2216 (MA; Difco) at 30 °C. The type strains of the five recognized Porphyrobacter species were used as reference strains for DNA–DNA hybridization and phenotypic characterization: P. neustonensis DSM 9434T, P. tepidarius DSM 10594T, P. sanguineus DSM 11032T and P. cryptus DSM 12079T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and P. donghaensis SW-132T was obtained from the study of Yoon et al. (2004). To investigate its morphological and some physiological characteristics, strain DSW-74T was routinely cultivated on MA at 30 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. Transmission electron microscopy was also used to determine whether flagella were present in cells from exponentially growing cultures; 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. 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) that was adjusted to various pH values (initial pH 4.5–10.5 at intervals 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 in trypticase soy broth lacking NaCl. Growth at various NaCl concentrations [0.5 % (w/v) and 1.0–10.0 % (w/v) at intervals of 1.0 % units] 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 hydrolysis of casein and starch were determined as described by Cowan & Steel (1965). Hydrolysis of Tweens 20, 40, 60 and 80 was determined as described by Cowan & Steel (1965) with a modification that artificial sea water was used instead of distilled water. Hydrolysis of aesculin, gelatin and urea and reduction of nitrate were determined as described by Lanyi (1987) with a modification that artificial sea water was used instead of distilled water. The artificial sea water contained (per litre of 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 with the substrate concentrations given by Cowan & Steel (1965). Production of H2S was tested as described by Bruns et al. (2001). Acid production from carbohydrates was determined using the method of Leifson (1963). Utilization of substrates 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). For in vivo pigment-absorption spectrum analysis, two test strains were cultivated aerobically in the dark at 37 °C in liquid Erythromicrobium/Roseococcus medium (Yurkov et al., 1994; DSMZ medium no. 767) with a modification that D-glucose was used instead of acetate. The cultures were washed twice by centrifugation using a MOPS buffer (MOPS/NaOH, 0.01 M; KCl, 0.1 M; MgCl2, 0.001 M; 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 tested on MA plates using antibiotic discs containing the following: 100 U polymyxin B, 50 µg streptomycin, 20 U penicillin G, 100 µg chloramphenicol, 10 µg ampicillin, 30 µg cephalothin, 30 µg gentamicin, 5 µg novobiocin, 30 µg tetracycline, 30 µg kanamycin, 15 µg lincomycin, 15 µg oleandomycin, 30 µg neomycin or 100 µg carbenicillin. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux).
Cell biomass of strain DSW-74T for DNA extraction and for isoprenoid quinone analysis was obtained by cultivation for 6 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. The 16S rRNA gene was amplified by PCR using two universal primers (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 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 fatty acid methyl ester analysis, cell mass of strain DSW-74T was harvested from agar plates after incubation for 7 days on MA 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 the method of Tamaoka & Komagata (1984) with a modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically according to the method of Ezaki et al. (1989) by 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 DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain DSW-74T are given in the species description (see later) or are shown in Table 1, together with those of the five recognized Porphyrobacter species. Strain DSW-74T produced bacteriochlorophyll (BChl) a aerobically in the dark. The sonicated cell extract showed absorption maxima at approximately 457, 481, 583, 800, 835 and 862 nm, which indicated the presence of carotenoids and BChl a (see Supplementary Fig. S1 in IJSEM Online). The in vivo absorption spectrum of strain DSW-74T was distinguishable from those of the other Porphyrobacter species, with the exception of P. sanguineus, in that three absorption maxima existed between 799 and 870 nm (Table 1). The acetone–methanol extract had in vitro absorption peak maxima at 454, 477 and 769 nm, confirming the presence of carotenoids and BChl a (Supplementary Fig. S1).
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6c (30.6 %), C18 : 17c (28.5 %), C16 : 0 (8.7 %), C15 : 0 2-OH (6.4 %), C18 : 0 (5.0 %), C14 : 0 2-OH (4.5 %), C18 : 15c (3.0 %), C17 : 18c (2.8 %), C17 : 0 (2.0 %), C13 : 0 2-OH (1.9 %), C16 : 0 2-OH (1.7 %), C15 : 0 (1.7 %) and C16 : 17c and/or iso-C15 : 0 2-OH (1.2 %). Branched fatty acids were found as minor components in strain DSW-74T, which was also characteristic for the five recognized Porphyrobacter species (Hiraishi et al., 2002; Rainey et al., 2003; Yoon et al., 2004). This fatty acid profile was similar to those of Porphyrobacter species analysed previously, although there were differences in the proportions of some fatty acids, perhaps owing to differences in the cultivation conditions used, e.g. temperature and cultivation medium (Hiraishi et al., 2002; Rainey et al., 2003; Yoon et al., 2004). The DNA G+C content of strain DSW-74T was 65.8 mol%.
There were no distinct phenotypic, particularly chemotaxonomic, properties for differentiating strain DSW-74T and the genus Porphyrobacter from the genera Erythrobacter and Erythromicrobium (Rainey et al., 2003). The relationship between the clade comprising strain DSW-74T and the genera Porphyrobacter and Erythromicrobium and the clade comprising Erythrobacter species was supported by a bootstrap resampling value of 98.9 % (Fig. 1
). The genera Porphyrobacter and Erythromicrobium could not be clearly distinguished because they are not well defined chemotaxonomically and are intermixed phylogenetically (Rainey et al., 2003). Indeed, Rainey et al. (2003) considered the possibility of combining the genera Erythrobacter, Porphyrobacter and Erythromicrobium into a single genus. However, it is currently necessary to place strain DSW-74T into one of the three genera. Based on its highest 16S rRNA gene sequence similarity values to Porphyrobacter species, strain DSW-74T should be placed within the genus Porphyrobacter before taxonomic criteria regarding the above three genera are proposed. There are several differential phenotypic properties between strain DSW-74T and recognized Porphyrobacter species, including motility, the abilities to hydrolyse and to utilize some substrates, maximum and optimal temperatures for growth, and absorption spectrum pattern of in vivo cell extracts (Table 1). The genetic distinctiveness was sufficient to assign strain DSW-74T to a novel species within the genus Porphyrobacter (Wayne et al., 1987). Levels of DNA–DNA relatedness between strain DSW-74T and the type strains of the five recognized Porphyrobacter species were in the range 9–25 %. Therefore, on the basis of the data presented, strain DSW-74T should be classified in the genus Porphyrobacter as the type strain of a novel species, for which the name Porphyrobacter dokdonensis sp. nov. is proposed.
Description of Porphyrobacter dokdonensis sp. nov.
Porphyrobacter dokdonensis (dok.do.nen'sis. N.L. masc. adj. dokdonensis of Dokdo, from where the strain was isolated).
Cells are pleomorphic: cocci, ovals and rods (0.4–0.6x0.5–2.5 µm) are present. Cells are Gram-negative, non-spore-forming and non-motile. Colonies on MA are circular, smooth, slightly convex, reddish orange in colour and 0.5–1.0 mm in diameter after incubation for 7 days at 37 °C. Optimal growth occurs at 35–37 °C; growth occurs at 10 and 43 °C, but not at 4 or 44 °C. Optimal pH for growth is 7.0–8.0; growth occurs at pH 5.5, but not at pH 5.0. Optimal growth occurs in the presence of 2 % (w/v) NaCl; growth occurs weakly in the absence of NaCl and in the presence of 7 % (w/v) NaCl, but not in the presence of >8 % (w/v) NaCl. Growth does not occur under anaerobic conditions on MA or on MA supplemented with nitrate. Urease-negative. L-Tyrosine and Tweens 20, 40 and 60 are hydrolysed, but hypoxanthine and xanthine are not. H2S and indole are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Maltose is utilized as the sole carbon and energy source, but benzoate, salicin and formate are not. Acid is not produced from the following substrates: L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, D-melezitose, melibiose, D-raffinose, L-rhamnose, D-ribose, sucrose, D-trehalose, D-xylose, myo-inositol or D-sorbitol. Sensitive to penicillin G, chloramphenicol, cephalothin, novobiocin and carbenicillin, but not to polymyxin B, streptomycin, ampicillin, gentamicin, tetracycline, kanamycin, lincomycin, oleandomycin or neomycin. The predominant ubiquinone is Q-10. The major fatty acids are C17 : 16c (30.6 %) and C18 : 17c (28.5 %). The DNA G+C content is 65.8 mol% (HPLC). Other phenotypic properties are given in Table 1.
The type strain, DSW-74T (=KCTC 12395T=DSM 17193T), was isolated from sea water.
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ACKNOWLEDGEMENTS |
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