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1 Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju, 660-701, Republic of Korea
2 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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8c and C17 : 0 cyclo as the major fatty acids. The G+C content of the genomic DNA was 66 mol% and the predominant ubiquinone was Q-9. Comparative 16S rRNA gene sequence analyses showed that strain BH843T formed a distinct phyletic line within the genus Halomonas, and the levels of 16S rRNA gene sequence similarity with respect to recognized Halomonas species were below 95.1 %. The levels of DNA–DNA relatedness between strain BH843T and the type strains of phylogenetically closely related Halomonas species were below 25 %. On the basis of phenotypic, chemotaxonomic and molecular data, strain BH843T represents a novel species within the genus Halomonas, for which the name Halomonas kribbensis is proposed. The type strain is BH843T (=KCTC 12584T=DSM 17892T).
A transmission electron micrograph of a negatively stained cell of strain BH843T and a table detailing the cellular fatty acid compositions of strain BH843T and related Halomonas species are available with the online version of this paper.
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to accommodate the genera Halomonas (Vreeland et al., 1980) and Deleya (Baumann et al., 1983), currently includes four genera of halophilic/halotolerant bacteria, i.e. Halomonas, Chromohalobacter, Alcanivorax and Cobetia, and two genera of non-halophilic/halotolerant bacteria, i.e. Zymobacter and Carnimonas (Arahal et al., 2001, 2002a; Dobson & Franzmann, 1996; Garriga et al., 1998; Yakimov et al., 1998). The members of the genus Halomonas comprise moderately halophilic, chemoorganotrophic, Gram-negative rods and are widely distributed in saline habitats. During the last decade, many species have been assigned to the genus Halomonas, but a number of them have since been reclassified and their nomenclature changed because they are phylogenetically heterogeneous (Arahal et al., 2001, 2002b; Mellado et al., 1995; Ventosa et al., 1998). During the course of studying saline environments, we have isolated halophilic, Gram-negative bacteria from a solar saltern (J. C. Lee et al., 2005; Lim et al., 2004). This paper describes a novel species belonging to the genus Halomonas.
Strain BH843T was isolated from a solar saltern of the Yellow Sea in Korea. For the isolation, a soil sample was serially diluted in a 10 % (w/v) saline solution and spread on marine agar 2216 (MA; Difco) supplemented with 10 % (w/v) NaCl (final NaCl concentration, 11.94 %, w/v) and incubated for 2 days at 32 °C. Except where indicated, the isolate was routinely grown aerobically for 2 days at 32 °C on MA supplemented with 3 % (w/v) NaCl. The NaCl requirement and tolerance were determined in nutrient broth (Difco) supplemented with modified artificial seawater containing the following (l–1): 0–20 % NaCl (w/v), 5.94 g MgSO4 . 7H2O, 4.53 g MgCl2 . 6H2O, 0.64 g KCl and 1.3 g CaCl2. Growth was tested at different temperatures (4–55 °C) and at different pH values (4.0–10.0) in marine broth (Difco) supplemented with 3 % (w/v) NaCl. Media with different pH values were prepared using appropriate biological buffers: Na2HPO4/NaH2PO4 buffer, Na2CO3/NaHCO3 buffer and Na2HPO4/NaOH buffer were used for pH values below 8.0, pH 8.0–10.0 and pH 11.0, respectively (Gomori, 1955). The cellular morphology of strain BH843T was examined using light microscopy and transmission electron microscopy as described by E. M. Lee et al. (2005). Motility was assessed at 12 and 36 h in wet mounts under a light microscope (E600; Nikon). Gram staining was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Oxidase activity was tested using a Bactident oxidase strip (Merck) and catalase activity was determined by assessing bubble production in a 3 % (v/v) hydrogen peroxide solution. Nitrate reduction and the hydrolysis of aesculin, casein, gelatin, L-tyrosine and urea were determined on MA supplemented with 3 % (w/v) NaCl according to the methods described by Cowan & Steel (1965), Lanyi (1987) and Smibert & Krieg (1994). Growth under anaerobic conditions was determined after 7 days incubation at 32 °C on MA supplemented with 3 % (w/v) NaCl, using an anaerobic chamber.
Strain BH843T formed yellow–cream and circular/slightly irregular colonies when grown at 32 °C for 2 days. The strain grew in nutrient broth supplemented with 1–14 % (w/v) NaCl, the optimum concentration being 5 % (w/v). The strain did not grow without added NaCl or in the presence of more than 15 % (w/v) NaCl. Growth occurred at pH 5.5–9.5 (optimum, pH 8.0–8.5) in marine broth supplemented with 3 % (w/v) NaCl. Growth was observed at temperatures between 10 and 40 °C, the optimum growth temperature being 32–35 °C. The cells were cocci measuring 1.8–2.2 µm in diameter or short rods 1.4–1.8 µm wide and 1.8–2.6 µm long after 2 days incubation at 32 °C on MA supplemented with 3 % (w/v) NaCl, and were motile by means of a single flagellum (see Supplementary Fig. S1 available in IJSEM Online). The strain showed oxidase-negative and catalase-positive reactions and was able to reduce nitrate to nitrite. Anaerobic growth was not observed during 7 days incubation under anaerobic conditions at 32 °C on MA supplemented with 3 % (w/v) NaCl. Other phenotypic features of strain BH843T are presented in Table 1 and in the description of the novel species. Some of these features are in accordance with those of known members of the genus Halomonas, whereas others serve to differentiate strain BH843T from closely related Halomonas species (Table 1).
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. The DNA G+C content (mol%) was determined by using reversed-phase HPLC according to the method of Tamaoka & Komagata (1984). The predominant isoprenoid quinone of strain BH843T was Q-9. The major cellular fatty acids of strain BH843T grown on MA were C16 : 0 (26.30 %), C19 : 0 cyclo 8c (25.77 %) and C17 : 0 cyclo (15.20 %) (see Supplementary Table S1 available in IJSEM Online). The major fatty acid profile of strain BH843T was similar to those of members of the genus Halomonas (Bouchotroch et al., 2001; Lim et al., 2004; Yoon et al., 2002). The DNA G+C content of strain BH843T was 66 mol%, which is within the range for recognized Halomonas species (Garcia et al., 2004; Mata et al., 2002; Poli et al., 2007; Ventosa et al., 1998).
PCR amplification of the 16S rRNA gene of strain BH843T was performed using primers 27f and 1492r (Lane, 1991) and the PCR product was then cloned (using a TOPO cloning kit; Invitrogen) and sequenced. The resultant 16S rRNA gene sequence (1417 nt) of strain BH843T was compared with 16S rRNA gene sequences available from GenBank, using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/), to determine its approximate phylogenetic affiliation; alignment with sequences from closely related members was performed using CLUSTAL W software (Thompson et al., 1994). Phylogenetic trees were constructed using three different methods available with PHYLIP, version 3.6 (Felsenstein, 2002): the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms. Values for similarities between the sequence of the isolate and those of related members of the genus Halomonas were computed using Similarity Matrix, version 1.1 (Ribosomal Database Project II; http://35.8.164.52/html/; Cole et al., 2003). A bootstrap analysis was performed according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package. DNA–DNA hybridization was carried out to evaluate the genomic DNA relatedness between strain BH843T and related Halomonas type strains by using the fluorometric microplate method (Ezaki et al., 1989). Halomonas anticariensis LMG 22089T, Halomonas pantelleriensis DSM 9661T, Halomonas muralis LMG 20969T, Halomonas alimentaria KCCM 41042T and Halomonas pacifica DSM 4742T were used as reference strains for DNA–DNA hybridization. Reference strains were grown in marine broth at appropriate temperatures. Chromosomal DNA from H. pantelleriensis DSM 9661T, H. alimentaria KCCM 41042T and H. pacifica DSM 4742T was isolated and purified according to the standard method described by Yoon et al. (1996). However, genomic DNA from strain BH843T, H. muralis LMG 20969T and H. anticariensis LMG 22089T was extracted using a genomic DNA purification kit (Promega), according to the manufacturer's instructions, and amplified by means of a multiple displacement amplification method (Teles et al., 2007) because genomic DNA extractions from these organisms always failed if the standard method was used. Fluorometric data recorded after 30 min incubation were used to calculate the DNA–DNA hybridization values. The highest and lowest values in each sample were excluded and the remaining three values were used to calculate the similarity value. The DNA relatedness values are means of three values.
The tree constructed using neighbour-joining analysis clearly showed that the isolate forms a distinct phyletic line (with 57 % bootstrap support) within the genus Halomonas (Fig. 1). The topologies of phylogenetic trees constructed using the maximum-likelihood and maximum-parsimony algorithms also supported the notion that the isolate belongs to the genus Halomonas and can be differentiated from recognized species of the genus Halomonas (data not shown). Strain BH843T showed 16S rRNA gene sequence similarities of 95.1, 95.0, 94.9 and 94.8 % with respect to its closest relatives, H. muralis LMG 20969T, H. anticariensis LMG 22089T, H. pantelleriensis DSM 9661T and Halomonas desiderata DSM 9502T, respectively. The values for DNA–DNA relatedness between strain BH843T and the type strains of closely related Halomonas species were 19, 25, 18, 14 and 16 % for H. anticariensis LMG 22089T, H. pantelleriensis DSM 9661T, H. muralis LMG 20969T, H. alimentaria KCCM 41042T and H. pacifica DSM 4742T, respectively, which are clearly below the 70 % threshold generally accepted for species delineation (Christensen et al., 2001; Rosselló-Mora & Amann, 2001). On the basis of the phenotypic and phylogenetic data, it is proposed that strain BH843T represents a novel species within the genus Halomonas, for which the name Halomonas kribbensis sp. nov. is proposed.
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Cells are Gram-negative, non-spore-forming cocci or rods. Colonies are yellow–cream and circular/slightly irregular. Cells are motile, each cell having a flagellum. Grows at salinities in the range 1–14 % (w/v) NaCl. Good growth occurs at 5 % (w/v) NaCl. Growth occurs between 10 and 40 °C (optimum, 32–35 °C) and at pH 5.5–9.5 (optimum, pH 8.0–8.5). Oxidase-negative and catalase-positive. Nitrate is reduced to nitrite. Aesculin and gelatin are hydrolysed, but casein, L-tyrosine and urea are not hydrolysed. The predominant isoprenoid quinone is Q-9. The major fatty acids are C16 : 0, C19 : 0 cyclo 8c and C17 : 0 cyclo. The DNA G+C content is 66 mol%.
The type strain, BH843T (=KCTC 12584T=DSM 17892T), was isolated from a solar saltern in Korea.
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ACKNOWLEDGEMENTS |
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