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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Environmental Biotechnology National Core Research Center, Division of Environmental Biotechnology, Gyeongsang National University, 660-701, Republic of Korea
3 The Key Laboratory for Microbial Resources, Ministry of Education, P.R. China, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People's Republic of China
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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7c, C16 : 0 and C19 : 0 cyclo 8c. The genomic DNA G+C content was about 65 mol% and the predominant ubiquinone was Q-9. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate formed a branch of the species Cobetia marina. However, 23S and 16S rRNA gene sequence similarities revealed that strain BH539T was related more closely to the type strains of the genus Halomonas. Phylogenetic analyses based on 23S rRNA gene sequences also indicated that the strain formed a phyletic line within the genus Halomonas. Therefore, it was concluded that strain BH539T should be classified within the genus Halomonas, rather than Cobetia. On the basis of physiological and molecular properties, strain BH539T represents a novel species of the genus Halomonas, for which the name Halomonas taeanensis sp. nov. is proposed. The type strain is BH539T (=KCTC 12284T=DSM 16463T).
Published online ahead of print on 20 May 2005 as DOI 10.1099/ijs.0.63616-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S and 23S rRNA gene sequences of strain BH539T are AY671975 and AY829729, respectively.
A micrograph showing the general morphology of a cell of strain BH539T and a table detailing the cellular fatty acid composition of the strain are available as supplementary material in IJSEM Online.
These authors contributed equally to this work. 
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-Proteobacteria currently comprises four genera of halophilic/halotolerant bacteria, i.e. Halomonas, Chromohalobacter, Alcanivorax and the recently described Cobetia, and two genera of non-halophilic/halotolerant bacteria, Zymobacter and Carnimonas (Arahal et al., 2001a
, 2002a; Dobson & Franzmann, 1996; Garriga et al., 1998; Ventosa et al., 1998; Yakimov et al., 1998). More than half of the species of the genera of the family Halomonadaceae have been reclassified at least once and their nomenclature has been changed during the last few years. Some taxa might still be transferred to novel species or genera within the family because their current phylogenetic relationships are too heterogeneous and ambiguous (Arahal et al., 2001b; Dobson & Franzmann, 1996; Mellado et al., 1995). Therefore, Arahal et al. (2002a, b) proposed that comparative analyses of 16S and 23S rRNA gene sequences are needed to determine in detail the phylogenetic relationship of species of the genera within the family Halomonadaceae and to clarify the classification of this heterogeneous group. They re-evaluated the phylogeny of the species of the family Halomonadaceae on this basis and assigned the species ‘Halomonas marina’ to a novel genus and species, Cobetia marina, although C. marina has similar physiological and phenotypic properties to species of the genus Halomonas. Thus, in this study we determined the phylogenetic position of a moderately halophilic, Gram-negative bacterium (strain BH539T), isolated from a solar saltern in Korea, using comparative 23S and 16S rRNA gene sequence analyses. The strain is considered to represent a novel species, Halomonas taeanensis sp. nov.
Strain BH539T was isolated from a solar saltern at Taean in Korea. For isolation, soil samples were serially diluted with 10 % (w/v) saline solution, spread on marine agar 2216 (MA; Difco) with the addition of 8 % (w/v) NaCl [final concentration 9·94 % (w/v) NaCl] and incubated for 2 days at 35 °C. The isolate was routinely grown aerobically on MA with 10 % (w/v) NaCl for 2 days at 35 °C except where indicated otherwise. Requirement for and tolerance of NaCl were determined in nutrient broth (NB; Difco) supplemented with modified artificial sea water [ASW: 0–30 % (w/v) NaCl, 5·94 g MgSO4.7H2O, 4·53 g MgCl2.6H2O, 0·64 g KCl and 1·3 g CaCl2 l–1]. Growth was tested at different temperatures (4–55 °C) and pH values (5·0–10·0) in marine broth (MB; Difco) containing 10 % (w/v) NaCl. Cellular morphology of strain BH539T was examined by using light microscopy and transmission electron microscopy (TEM). Motility was observed at 12 and 36 h in wet mounts by using a light microscope (Nikon E600). For visualization of the flagella, cells were mounted on Formvar-coated copper grids (Electron Microscopy Science) and negatively stained with 2 % (w/v) uranyl acetate for 15 s, then viewed via TEM (JEOL JEM-1010).
Gram staining was determined by using a bioMérieux Gram Stain kit according to the manufacturer's instructions. Oxidase activity was tested by using Bactident Oxidase strips (Merck) and catalase activity was determined by bubble production in 3 % (v/v) hydrogen peroxide solution. Nitrate reduction and hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine, gelatin and xanthine were determined on MA according to the methods described by Cowan & Steel (1965), Lanyi (1987) and Smibert & Krieg (1994). Acid production from carbohydrates was tested as described by Leifson (1963); all suspension media were supplemented with ASW containing 10 % (w/v) NaCl. Growth under anaerobic conditions was determined in an anaerobic chamber (5 % H2, 10 % CO2, 85 % N2; Mart Microbiology) after 5 days incubation at 35 °C on MA.
Whole-cell fatty acids of strain BH539T were analysed by using GC/MS following the instructions of the Microbial Identification System (MIDI; Microbial ID, Inc.) after cultivation either on MA or on MA supplemented with 8 % (w/v) NaCl for 2 days at 35 °C. Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987). The DNA G+C content (mol%) was determined by reversed-phase HPLC using the method of Tamaoka & Komagata (1984). DNA–DNA hybridization was carried out to evaluate genomic DNA relatedness between strain BH539T and C. marina and closely related type strains of the genus Halomonas. Strains for comparative analysis were grown aerobically on MA for 2 days at 35 °C and chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996). Random-primed DNA labelling with digoxigenin (DIG)-dUTP and detection of hybrids by enzyme immunoassay on nylon membrane were performed by using a DIG High Prime DNA Labelling and Detection starter kit II (Roche Applied Science) according to the manufacturer's instructions.
16S and 23S rRNA gene sequences of strain BH539T were analysed as described by DeLong (1992) and Arahal et al. (2002b). The sequences were aligned together with those of representative members of selected genera by using the CLUSTAL W program (Thompson et al., 1994). Sequence similarity values were computed by using Similarity Matrix version 1.1 (Ribosomal Database Project II; http://rdp.cme.msu.edu/html/analyses.html; Cole et al., 2003). Gaps at the 5' and 3' ends of the alignment were omitted from further analyses. Phylogenetic trees based on 16S and 23S rRNA gene sequences were constructed by using the neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Evolutionary distance matrices were calculated according to the algorithm of the Kimura two-parameter model (Kimura, 1980) for the NJ method. Bootstrap analyses (1000 replications) were performed to evaluate the stability of the phylogenetic tree with the NJ method in the PHYLIP package.
Strain BH539T formed creamy, smooth and circular/slightly irregular colonies following growth at 35 °C for 2 days. The strain grew at 1–25 % (w/v) NaCl, with optimum growth at 10–12 % (w/v) NaCl. The strain did not grow without NaCl or in the presence of >30 % (w/v) NaCl. Growth occurred from pH 7·0 to 10·0 (optimum 7·5–8·0) in MB containing 10 % (w/v) NaCl. Growth was observed at temperatures of 10–45 °C, with optimum growth at 35 °C. Cells of strain BH539T were Gram-negative, strictly aerobic rods, 0·6–1·0 µm wide and 2·0–3·2 µm long. Cells were motile by means of peritrichous flagella after 2 days incubation at 35 °C on MA (see Supplementary Figure in IJSEM Online). The strain showed oxidase- and catalase-positive reactions and reduced nitrate to nitrite. Strain BH539T hydrolysed L-tyrosine and urea, but hydrolysis of aesculin, casein, gelatin, starch, Tween 80, xanthine and hypoxanthine was not observed. The isolate produced acids from D-glucose, D-trehalose, L-arabinose, lactose, glycerol, D-ribose, D-mannitol, D-mannose, D-fructose and maltose, but not from L-rhamnose, adonitol, D-raffinose, D-salicin, D-melibiose, arbutin or D-xylose. The phenotypic characteristics of strain BH539T are summarized and compared with those of C. marina and the type strains of closely related Halomonas species in Table 1.
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7c (29·1 %), C16 : 0 (27·5 %), summed feature 3 (mixture of C16 : 17c and/or iso-C15 : 0 2-OH) (13·7 %) and C19 : 0 cyclo 8c (9·4 %) when grown on MA (see Supplementary Table in IJSEM Online). The fatty acid profile on MA was almost identical to that on MA supplemented with 8 % NaCl (w/v). The major fatty acid profile of strain BH539T was similar to those of other members of the genus Halomonas (Bouchotroch et al., 2001; Lim et al., 2004; Valderrama et al., 1998; Yoon et al., 2002). The DNA G+C content of strain BH539T was 65 mol%, a value within the range of previously reported Halomonas species (García et al., 2004; Mata et al., 2002; Ventosa et al., 1998).
Almost-complete 16S and 23S rRNA gene sequences of strain BH539T were obtained and used for initial BLAST searches in GenBank and for phylogenetic analyses. The tree constructed by the NJ method based on 16S rRNA gene sequence analysis indicated that strain BH539T formed a branch with the species C. marina DSM 4741T with 63·3 % bootstrap support (Fig. 1a). The topologies of phylogenetic trees built by using the ML and MP algorithms were similar to that of the tree constructed by using the NJ algorithm (data not shown). However, 16S rRNA gene sequence analysis revealed that strain BH539T was related most closely to the type strains of species of the genus Halomonas rather than to C. marina DSM 4741T. Strain BH539T shared 16S rRNA gene sequence similarity of 94·1 % with C. marina DSM 4741T. 16S rRNA gene sequence similarity levels to other Halomonas species were higher: Halomonas pacifica DSM 4742T (96·9 %), Halomonas koreensis JCM 12237T (96·0 %), Halomonas elongata ATCC 33173T (95·7 %), Halomonas eurihalina ATCC 49336T (95·7 %), Halomonas maura CECT 5298T (95·4 %), Halomonas pantelleriensis DSM 9661T (95·7 %) and Halomonas salina DSM 5928T (95·3 %). Phylogenetic analyses using 23S rRNA gene sequences were carried out to determine the phylogenetic position of strain BH539T. The tree constructed by the NJ method based on 23S rRNA gene sequences indicated that strain BH539T formed a subclade within the genus Halomonas with 70·9 % bootstrap support (Fig. 1b). The topologies of phylogenetic trees built by using the ML and MP algorithms were also similar to that of the tree constructed by using the NJ algorithm (data not shown). 23S rRNA gene sequence analysis also indicated that strain BH539T was related most closely to the type strains of species of the genus Halomonas. 23S rRNA gene similarity values of strain BH539T to closely related recognized species were as follows: Halomonas cupida DSM 4740T (96·9 %), H. eurihalina ATCC 49336T (96·5 %), H. pacifica DSM 4742T (96·4 %), H. salina DSM 5928T (96·4 %), H. pantelleriensis DSM 9661T (96·3 %), Halomonas desiderata DSM 9502T (96·3 %) and H. elongata ATCC 33173T (96·2 %), whereas strain BH539T shared 23S rRNA gene sequence similarity of just 94·1 % with C. marina DSM 4741T.
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However, on the basis of physiological and multiple molecular analyses, it was concluded that strain BH539T should be classified within the genus Halomonas rather than Cobetia. Therefore, strain BH539T represents a novel species of the genus Halomonas, for which the name Halomonas taeanensis sp. nov. is proposed.
Description of Halomonas taeanensis sp. nov.
Halomonas taeanensis (tae.an.en'sis. N.L. fem. adj. taeanensis belonging to Taean, from where the organism was isolated).
Cells are aerobic, Gram-negative, non-spore-forming rods, measuring 0·6–1·0 µm in width and 2·0–3·2 µm in length. Colonies are creamy, smooth and circular/slightly irregular. Cells are motile, each cell having several flagella. Grows at salinities of 1–25 % (w/v) NaCl. Good growth at 10–12 % (w/v). Grows between 10 and 45 °C (optimum 35 °C) and from pH 7·0 to 10·0 (optimum 7·5–8·0). Oxidase- and catalase-positive. Nitrate is reduced to nitrite. Urea and L-tyrosine are hydrolysed. The predominant isoprenoid quinone is Q-9. The major fatty acids are C18 : 17c, C16 : 0, summed feature 3 (mixture of C16 : 17c and/or iso-C15 : 0 2-OH) and C19 : 0 cyclo 8c when grown on MA. The DNA G+C content is 65 mol%.
The type strain, BH539T (=KCTC 12284T=DSM 16463T), was isolated from a solar saltern at Taean in Korea.
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ACKNOWLEDGEMENTS |
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