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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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7c, C16 : 0 and C16 : 17c and/or iso-C15 : 0 2-OH as the major fatty acids. Their DNA G+C contents were 45·3–45·7 mol%. Strains DSW10-10T and DSW10-19 exhibited a 16S rRNA gene sequence similarity value of 100 % and possessed a mean DNA–DNA relatedness level of 85 %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains DSW10-10T and DSW10-19 fell within the radiation of the cluster encompassed by the genus Marinomonas. Strains DSW10-10T and DSW10-19 had 16S rRNA gene sequence similarity levels of 95·7–97·7 % with respect to the type strains of Marinomonas species with validly published names. Levels of DNA–DNA relatedness were low enough to indicate that the two strains constitute a distinct Marinomonas species. On the basis of phenotypic data and phylogenetic and genetic distinctiveness, strains DSW10-10T (=KCTC 12394T=DSM 17202T) and DSW10-19 were placed in the genus Marinomonas as members of a novel species, Marinomonas dokdonensis sp. nov.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains DSW10-10T and DSW10-19 and Marinomonas communis LMG 2864T are DQ011526, DQ011527 and DQ011528, respectively.
A table showing the cellular fatty acid composition of Marinomonas dokdonensis sp. nov. is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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), Alteromonas communis and Alteromonas vaga, as Marinomonas communis and Marinomonas vaga, respectively (Van Landschoot & De Ley, 1983). Subsequently, four more Marinomonas species, Marinomonas mediterranea (Solano & Sanchez-Amat, 1999), Marinomonas primoryensis (Romanenko et al., 2003), Marinomonas pontica (Ivanova et al., 2005) and Marinomonas ushuaiensis (Prabagaran et al., 2005), have been described. Phylogenetic analyses based on 16S rRNA gene sequences showed that the genus Marinomonas falls within the ‘Gammaproteobacteria’ (Anzai et al., 2000). The aim of the present study was to determine the exact taxonomic positions of strains DSW10-10T and DSW10-19 by a polyphasic taxonomic characterization.
Sea water collected in Dokdo, Korea, provided the source for isolation of bacterial strains. Strains DSW10-10T and DSW10-19 were isolated by the standard dilution plating technique on marine agar 2216 (MA; Difco) at 20 °C. The type strains of six Marinomonas species were used as reference strains for DNA–DNA hybridization: M. communis LMG 2864T, M. vaga LMG 2845T and M. pontica LMG 22531T were obtained from the Laboratorium voor Microbiologie Universiteit Gent (LMG), Gent, Belgium; M. primoryensis JCM 11775T and M. ushuaiensis 12170T were obtained from the Japan Collection of Microorganisms (JCM), Saitama, Japan; M. mediterranea CECT 4803T was obtained from the Colección Española de Cultivos Tipo (CECT), Valencia, Spain. To investigate their morphological and some physiological characteristics, the two strains were routinely cultivated on MA at 30 °C. Cell morphology was examined by light microscopy (Nikon E600) and transmission electron microscopy. The presence of flagella was determined by transmission electron microscopy using cells from exponentially growing cultures. For transmission electron microscopic observation, the cells were negatively stained with 1 % (w/v) phosphotungstic acid and after air-drying the grids were examined 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 prepared according to the formula of the Difco medium except that no NaCl was used. Growth at various NaCl concentrations [0·5 % (w/v) and 1·0–12·0 % (w/v) at intervals of 1·0 % units] was investigated in MB and trypticase soy broth (Difco). Growth at various temperatures (4–40 °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 nitrate reduction 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 described by Cowan & Steel (1965). H2S production was tested as described previously (Bruns et al., 2001). Acid production from carbohydrates was determined as described by 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). Enzyme activity was determined by using the API ZYM system (bioMérieux). Susceptibility to antibiotics was tested on MA plates using antibiotic discs containing the following concentrations: 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. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux).
Cell biomass of strains DSW10-10T and DSW10-19 for DNA extraction and for isoprenoid quinone analysis was obtained by cultivation for 3 days in MB at 30 °C. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was applied in combination with ribonuclease 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 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 strains DSW10-10T and DSW10-19 was harvested from agar plates after incubation for 3 days on MA at 30 °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 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 DNA–DNA relatedness values.
Almost complete 16S rRNA gene sequences of strains DSW10-10T and DSW10-19, comprising 1495 nucleotides (approx. 96 % of the Escherichia coli 16S rRNA sequence), were determined in this study. The sequences of strains DSW10-10T and DSW10-19 had no difference in the region compared, except for seven ambiguous nucleotides that were present in strain DSW10-10T. Comparative 16S rRNA gene sequence analyses revealed that strains DSW10-10T and DSW10-19 were most closely affiliated to the genus Marinomonas. In the neighbour-joining tree based on 16S rRNA gene sequences, strains DSW10-10T and DSW10-19 fell within the radiation of the cluster comprising Marinomonas species (Fig. 1). Similar tree topologies were found in the tree generated with the maximum-parsimony algorithm (data not shown). The 16S rRNA gene sequences of strains DSW10-10T and DSW10-19 exhibited highest similarity values (97·6–97·7 %) to the sequence of the type strain of M. pontica, and similarity values of 95·7–96·9 % to the sequences of the type strains of the other Marinomonas species. Sequence similarity values to other species used in the phylogenetic analysis were less than 90·3 % (Fig. 1). The DNA G+C contents of strains DSW10-10T and DSW10-19 were 45·3 and 45·7 mol%, respectively.
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, together with those of the six Marinomonas species. The predominant isoprenoid quinone found in strains DSW10-10T and DSW10-19 was ubiquinone-8 (Q-8) at peak area ratios of approximately 86–91 %. Strains DSW10-10T and DSW10-19 had cellular fatty acid profiles that contained large amounts of unsaturated, straight-chain and hydroxy fatty acids; the major components (>5 % of total fatty acids) were C18 : 17c (39·4 and 33·1 %, respectively), C16 : 0 (13·1 and 18·5 %, respectively), C16 : 17c and/or iso-C15 : 0 2-OH (15·1 and 10·2 %, respectively), C10 : 0 3-OH (9·1 and 7·3 %, respectively), C18 : 0 (4·3 and 10·8 %, respectively) and unknown fatty acid (8·4 and 6·4 %, respectively) (supplementary table in IJSEM Online). These fatty acid profiles were similar to those of Marinomonas species shown previously, particularly in that C18 : 17c was the major fatty acid (Romanenko et al., 2003; Ivanova et al., 2005; Prabagaran et al., 2005). However, there were differences in the proportions of some fatty acids, particularly C16 : 17c or C16 : 19c, C16 : 0 and iso-C16 : 0, between the two strains and Marinomonas species, which might have been caused by different cultivation conditions, e.g. temperatures and cultivation media (Romanenko et al., 2003; Ivanova et al., 2005; Prabagaran et al., 2005). The fatty acid C16 : 0, which is one of the major fatty acids in the two strains and M. primoryensis and M. pontica, was a minor component in the type strains of M. ushuaiensis, M. communis and M. primoryensis from the study of Prabagaran et al. (2005). These chemotaxonomic properties support the result of monothetic phylogenetic classification of strains DSW10-10T and DSW10-19 as members of the genus Marinomonas (Romanenko et al., 2003; Ivanova et al., 2005; Prabagaran et al., 2005).
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). In view of the combined phenotypic, phylogenetic and genetic similarities, strains DSW10-10T and DSW10-19 could be considered as members of the same species. Strains DSW10-10T and DSW10-19 were distinguishable from the recognized Marinomonas species by differences in phenotypic characteristics (Table 1
). Levels of DNA–DNA relatedness between strains DSW10-10T and DSW10-19 and the type strains of the six recognized Marinomonas species were in the range 8–23 %. The genetic distinctiveness, together with the differential phenotypic properties and differences in the 16S rRNA gene sequences, was sufficient to assign strains DSW10-10T and DSW10-19 in a species that is separate from the previously recognized Marinomonas species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strains DSW10-10T and DSW10-19 should be classified in the genus Marinomonas as members of a novel species, for which the name Marinomonas dokdonensis sp. nov. is proposed.
Description of Marinomonas dokdonensis sp. nov.
Marinomonas dokdonensis (dok.do.nen'sis. N.L. fem. adj. dokdonensis of Dokdo, a Korean island, from where the organisms were isolated).
Cells are Gram-negative, non-spore-forming and aerobic rods (0·4–0·6x1·0-2·5 µm). Motile by means of a single polar flagellum. Colonies on MA are circular to slightly irregular, smooth, glistening, flat, ivory-coloured and 2·5–3·5 mm in diameter after incubation for 3 days at 30 °C. Optimal growth occurs at 30 °C; growth occurs at 4 and 37 °C, but not at 38 °C. Optimal pH for growth is 7·0–8·0; growth occurs at pH 5·5 and is variable at pH 5·0 (negative for type strain), but not at pH 4·5. Optimal growth occurs in the presence of 2–3 % (w/v) NaCl; growth does not occur in the absence of NaCl and in the presence of greater than 10 % (w/v) NaCl. Anaerobic growth does not occur on MA and on MA supplemented with nitrate. Catalase-positive. Tweens 20, 40 and 60 are hydrolysed, but aesculin, casein, hypoxanthine, xanthine and L-tyrosine are not. Indole and H2S are not produced. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. D-Fructose, L-malate and pyruvate are utilized as sole carbon and energy sources, but L-arabinose, salicin, formate and L-glutamate are not utilized. Utilization of sucrose is variable (negative for type strain). Acid is produced from D-cellobiose, D-fructose, D-glucose, maltose, D-mannose, D-ribose, sucrose, D-trehalose, D-xylose, D-mannitol, D-sorbitol and myo-inositol, but not from L-arabinose, lactose, D-melezitose, D-raffinose and L-rhamnose. Acid production from D-galactose and melibiose is variable (negative for type strain). Susceptible to polymyxin B, streptomycin, chloramphenicol, cephalothin, gentamicin, tetracycline, kanamycin, lincomycin, oleandomycin, neomycin and carbenicillin, but not to ampicillin and novobiocin. The predominant respiratory lipoquinone is Q-8. The major fatty acids (>10 % of total fatty acids) are C18 : 17c, C16 : 0 and C16 : 17c and/or iso-C15 : 0 2-OH. The DNA G+C content is 45·3–45·7 mol% (HPLC). Other phenotypic properties are shown in Table 1 and in the supplementary table in IJSEM Online.
The type strain, DSW10-10T (=KCTC 12394T=DSM 17202T), was isolated from sea water. The reference strain is DSW10-19.
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ACKNOWLEDGEMENTS |
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