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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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with the description of a single species, Microbulbifer hydrolyticus. Subsequently, two further Microbulbifer species, Microbulbifer salipaludis (Yoon et al., 2003a) and Microbulbifer maritimus (Yoon et al., 2004), were described. Pseudomonas elongata was reclassified into the genus Microbulbifer, as Microbulbifer elongatus (Yoon et al., 2003b). In this study, we report on the taxonomic characterization of a Microbulbifer-like bacterial strain (ISL-39T) that was isolated from a marine solar saltern of the Yellow Sea in Korea.
Strain ISL-39T was isolated by means of the standard dilution plating technique, at 25 °C on marine agar 2216 (MA; Difco) supplemented with 6 % (w/v) NaCl. The type strains of four Microbulbifer species were used as reference strains for DNA–DNA hybridization: M. hydrolyticus DSM 11525T and M. elongatus DSM 6810T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and M. salipaludis SM-1T and M. maritimus TF-17T were obtained from previous studies (Yoon et al., 2003a, 2004). The morphological, physiological and biochemical characteristics of strain ISL-39T were investigated using routine cultivation on MA at 37 °C. The cell morphology was examined by using light microscopy (E600; Nikon) and transmission electron microscopy. Flagellation was investigated by using a CM-20 transmission electron microscope (Philips) 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 on 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 NaCl was excluded. Growth at various NaCl concentrations was investigated in marine broth 2216 (Difco) or trypticase soy broth (Difco). Growth at various temperatures (4–50 °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), except that artificial seawater was used for the preparation of media. The artificial seawater contained the following (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). Susceptibility to various antibiotics was investigated on MA plates by using antibiotic discs with the following concentrations: 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 and 100 µg carbenicillin. Acid production from carbohydrates was tested as described by Leifson (1963). The utilization of various substrates for growth was determined as described by Yurkov et al. (1994). Enzyme activities were determined by using the API ZYM system (bioMérieux).
Cell biomass for DNA extraction and for isoprenoid quinone analysis was obtained from cultivation in marine broth 2216 at 37 °C. Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996), except that RNase T1 was used in combination with RNase A to minimize contamination from RNA. The 16S rRNA gene was amplified by using PCR with two universal primers, as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and the phylogenetic analysis were performed as described by Yoon et al. (2003a). Isoprenoid quinones were investigated as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For cellular fatty acid analysis, cell mass was harvested from MA plates after cultivation of strain ISL-39T for 2 days at 37 °C. The fatty acids were extracted and the fatty acid methyl esters prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by using the method of Tamaoka & Komagata (1984), with the modification that the DNA was hydrolysed using nuclease P1 (Sigma) 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), 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 the DNA–DNA relatedness values.
Morphological, cultural, physiological and biochemical characteristics of strain ISL-39T are given in the species description (see below) or in Table 1. The almost-complete 16S rRNA gene sequence of strain ISL-39T determined in this study comprised 1491 nt, representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence. Comparative 16S rRNA gene sequence analysis revealed that strain ISL-39T was most closely related phylogenetically to members of the genus Microbulbifer. In the phylogenetic tree based on the neighbour-joining algorithm, strain ISL-39T fell within the clade comprising the Microbulbifer species (Fig. 1). Strain ISL-39T exhibited 16S rRNA gene sequence similarity values of 97.5, 97.5, 96.2, 94.7 and 94.5 % with respect to the type strains of M. hydrolyticus, M. salipaludis, M. elongatus, M. maritimus and ‘Microbulbifer arenaceous’, respectively. The values for sequence similarity with respect to other species used in the phylogenetic analysis were below 90.8 %. The chemotaxonomic properties of ISL-39T confirm the phylogenetic results, indicating an affiliation with the genus Microbulbifer (González et al., 1997; Yoon et al., 2003a, b, 2004). The predominant isoprenoid quinone detected in strain ISL-39T was Q-8 (at a peak area ratio of approximately 92 %). The fatty acid profile of strain ISL-39T showed the presence of large amounts of branched, straight-chain, unsaturated, hydroxyl and cyclo fatty acids; the major components (>10 % of total fatty acids) were iso-C15 : 0, C16 : 0 and iso-C17 : 0 (Table 2). This fatty acid profile was similar to those of the Microbulbifer species, although there were differences in the proportions of some fatty acids, probably because of differences in cultivation conditions and extraction procedures (Table 2). The DNA G+C content of strain ISL-39T was 57.7 mol%. Strain ISL-39T exhibited DNA–DNA relatedness values of 7–16 % with respect to the type strains of four recognized Microbulbifer species, indicating that ISL-39T represents a different genomic species (Wayne et al., 1987). Strain ISL-39T is distinguishable from the recognized Microbulbifer species through differences in several phenotypic characteristics, as shown in Table 1. The phylogenetic and genetic distinctiveness of ISL-39T, together with the differential phenotypic properties, are sufficient to permit the allocation of this strain to a species that is separate from the recognized Microbulbifer species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain ISL-39T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer celer sp. nov. is proposed.
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Cells are Gram-negative and rod-shaped (0.2–0.4x0.8–3.5 µm). Colonies on MA are circular to slightly irregular, slightly convex, smooth, glistening, greyish yellow in colour and 3.0–4.0 mm in diameter after 2 days incubation at 37 °C. Growth occurs at 10 and 48 °C, but not at 4 or 49 °C. The optimal pH for growth is between 7.0 and 8.0; growth occurs at pH 5.0, but not at pH 4.5. Growth occurs in the presence of 15 % (w/v) NaCl, but not in the absence of NaCl or in the presence of more than 16 % (w/v) NaCl. Anaerobic growth does not occur on MA or on MA supplemented with nitrate. Hypoxanthine and Tweens 20, 40 and 60 are hydrolysed, but urea, L-tyrosine and xanthine are not. Acetate and pyruvate are utilized, but D-glucose, D-fructose, D-galactose, D-cellobiose, D-mannose, trehalose, D-xylose, L-arabinose, sucrose, maltose, citrate, succinate, benzoate, L-malate, salicin, formate and L-glutamate are not utilized. Acid is produced from melibiose, but not from D-mannitol, D-melezitose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol or myo-inositol. Susceptible to chloramphenicol, kanamycin, neomycin and novobiocin and weakly susceptible to oleandomycin and polymyxin B, but not to ampicillin, carbenicillin, cephalothin, gentamicin, lincomycin, penicillin G, streptomycin or tetracycline. The predominant ubiquinone is Q-8. The major fatty acids (>10 % of total fatty acids) are iso-C15 : 0, C16 : 0 and iso-C17 : 0. The DNA G+C content is 57.7 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1
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The type strain, ISL-39T (=KCTC 12973T=CCUG 54356T), was isolated from a marine solar saltern of the Yellow Sea in Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
González, J. M., Mayer, F., Moran, M. A., Hodson, R. E. & Whitman, W. B. (1997). Microbulbifer hydrolyticus gen. nov., sp. nov., and Marinobacterium georgiense gen. nov., sp. nov., two marine bacteria from a lignin-rich pulp mill waste enrichment community. Int J Syst Bacteriol 47, 369–376.
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Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
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Yoon, J.-H., Kim, I.-G., Shin, D.-Y., Kang, K. H. & Park, Y.-H. (2003a). Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol 53, 53–57.
Yoon, J.-H., Kim, H., Kang, K. H., Oh, T.-K. & Park, Y.-H. (2003b). Transfer of Pseudomonas elongata Humm 1946 to the genus Microbulbifer as Microbulbifer elongatus comb. nov. Int J Syst Evol Microbiol 53, 1357–1361.
Yoon, J.-H., Kim, I.-G., Oh, T.-K. & Park, Y.-H. (2004). Microbulbifer maritimus sp. nov., isolated from an intertidal sediment from the Yellow Sea, Korea. Int J Syst Evol Microbiol 54, 1111–1116.
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