Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DW01T is EF178282.
The genus Shewanella is a member of the class Gammaproteobacteria (Anzai et al., 2000) and comprises a group of Gram-negative, motile, rod-shaped, oxidase-positive, non-fermentative and facultatively anaerobic aquatic and marine bacteria (Bowman, 2005; Gauthier et al., 1995; MacDonell & Colwell, 1985; Venkateswaran et al., 1999). At the time of writing, the genus Shewanella comprised 45 recognized species (). Strains of the genus Shewanella have been isolated from a variety of sources including marine environments (Venkateswaran et al., 1998; Nealson et al., 1991; Nogi et al., 1998; Ivanova et al., 2001, 2004a,b; Bozal et al., 2002; Skerratt et al., 2002; Yoon et al., 2004b; Gram et al., 1987; Stenstrom & Molin, 1990; Gram & Huss, 1996; Satomi et al., 2006, 2007; Lee et al., 2006; Satomi et al., 2003; Simidu et al., 1990; Yang et al., 2006), sediments (Myers & Nealson, 1988; Venkateswaran et al., 1999; Toffin et al., 2004; Yoon et al., 2004a; Gao et al., 2006; Zhao et al., 2005, 2006; Miyazaki et al., 2006; Xiao et al., 2007; Yang et al., 2007), clinical samples (Brink et al., 1995; Nozue et al., 1992; Levin, 1972; Debois et al., 1975; Holmes et al., 1975), oilfield fluids (Semple & Westlake, 1987) and activated sludge (Xu et al., 2005). They have also been implicated as opportunistic pathogens of humans and aquatic animals (Aguirre et al., 1994; Brink et al., 1995) and as the causal agents of proteinaceous food spoilage (Jorgensen & Huß, 1989).
During the course of our study on gut microflora of abalone, a rod-shaped bacterial strain, designated DW01T, was isolated and subjected to a taxonomic investigation. On the basis of the polyphasic evidence, strain DW01T represents a novel species of the genus Shewanella, for which the name Shewanella haliotis sp. nov. is proposed.
Strain DW01T was isolated from an abalone sample collected from the South Sea near Yeosu (3 ° 44' N, 12 ° 44' E) located in the Republic of Korea, during July 2006, using the standard dilution plating technique. Isolation was achieved using marine agar (MA; Difco) (Yang et al., 2006) at 30 °C for 7 days. The isolate was routinely cultured on MA and maintained as a glycerol suspension (20 %, w/v) at –80 °C.
Bacterial DNA preparation, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chun & Goodfellow, 1995). The resultant sequence of strain DW01T was aligned manually against sequences obtained from the GenBank database. Phylogenetic trees were inferred from the regions available for all sequences (positions 38–1450; Escherichia coli numbering system) using the Fitch–Margoliash (Fitch & Margoliash, 1967) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant neighbour-joining tree topology was evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the jPHYDIT program (available at ) and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000).
Preliminary sequence comparison with the 16S rRNA gene sequences held in GenBank indicated that our isolate was related closely to members of the genus Shewanella. The newly determined sequence was then aligned manually against representatives of the genus Shewanella. Strain DW01T showed the highest 16S rRNA gene sequence similarity to Shewanella algae ATCC 51192T (98.3 %), followed by Shewanella profunda DSM 15900T (94.9 %), Shewanella kaireitica DSM 17170T (94.4 %) and Shewanella schlegeliana JCM 11561T (94.1 %). To elucidate the phylogenetic relationship between the novel isolate and other species of the genus Shewanella, phylogenetic trees were constructed using two different tree-making algorithms. The neighbour-joining tree (Fig. 1) showed that strain DW01T formed a monophyletic clade with S. algae ATCC 51192T with 100 % bootstrap support.
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Growth on various standard bacteriological media was tested by using nutrient agar (NA; Difco), tryptic soy agar (TSA; Difco) and R2A agar (Difco) according to the manufacturer's instructions. Cells of strain DW01T grown on TSA at 30 °C for 3–7 days were used for the physiological and biochemical tests. Motility was examined by observing the cells grown in wet mounts using phase-contrast microscope (Nikon TMS-F). The pH range (pH 3–12), growth temperature (4–42 °C), NaCl tolerance [0, 1, 2, 3, 5, 10 % (w/v)] and growth in an anaerobic chamber (CO2/H2/N2, 10 : 10 : 80; Sheldon Manufacturing) were checked using TSA, using up to 1 week incubation periods. Catalase and oxidase activities were determined using 3 % (v/v) hydrogen peroxide and Kovac's reagent (Kovacs, 1956), respectively. Other physiological and biochemical tests were performed using API 20E, API 20NE and API 50CH (bioMérieux). Enzymic activities were tested using API ZYM kit (bioMérieux) following the manufacturer's instructions. Antibiotic resistance was determined with the disc diffusion method. The results were interpreted according to the guidelines set forth by the National Committee for Clinical Laboratory Standards.
Strain DW01T was facultatively anaerobic, Gram-negative, motile and rod-shaped. Colonies grown on TSA plates for 5 days at 30 °C were circular, convex, entire margin, smooth, opaque, pink–orange coloured and approximately 5.0 mm in diameter. The novel strain grew well on MA, TSA, plate count agar (PCA; Difco) and NA. On TSA medium, strain DW01T was able to grow at 10–42 °C. The detailed results of physiological and biochemical analyses are given in Table 1 and the species description. It is evident from Table 1 that there are several phenotypic characters that readily separate strain DW01T from phylogenetically related species, namely S. algae and Shewanella amazonesis.
Table 1. Phenotypic characteristics that differentiate strain DW01T from its phylogenetic neighbours of Shewanella species Strains: 1, DW01T; 2, S. algae ATCC 51192T; 3, S. amazonensis ATCC 700329T. Data from Khashe & Janda (1998), Nozue et al. (1992), Venkateswaran et al. (1998) and this study. +, Positive; –, negative; W, weakly positive; ND, no data available. All strains are Gram-negative, rod-shaped, motile and positive for catalase, oxidase, H2S production, gelatinase and reduction of nitrates to nitrites. All strains are negative for the utilization of D-glucose, adipate, glycerol, mannitol, sorbitol, maltose, lactose and sucrose as carbon sources. Two species (1 and 2) are resistant to vancomycin (30 µg per disc), ampicillin (10 µg per disc) and penicillin (10 U per disc).
Cellular fatty acids of strain DW01T were analysed as methyl esters by GLC according to the instructions of the Microbial Identification System (MIDI). The cellular fatty acid profile of strain DW01T is described in Table 2. The G+C content of the DNA was determined by using the thermal denaturation method of Marmur & Doty (1962). The DNA G+C content of DW01T was 53.7 mol%.
Table 2. Cellular fatty acid composition (%) of strain DW01T and its phylogenetic neighbours of Shewanella species Strains: 1, DW01T; 2, S. algae ATCC 51192T; 3, S. amazonensis ATCC 700329T. Data from Venkateswaran et al. (1998) and this study. Values are percentages of total fatty acids. –, Fatty acid not present; tr, trace amount (<1 %); ND, no data available.
The taxonomic relationship between strain DW01T and S. algae ATCC 51192T was further examined using DNA–DNA hybridization. Genomic relatedness was determined using a membrane filter technique (Seldin & Dubnau, 1985) with a DIG High Prime DNA Labelling and Detection Starter kit II (Roche). When DNAs were used individually as labelled DNA probes for reciprocal hybridization experiments and conducted in duplicate, the DNA–DNA relatedness value between strain DW01T and S. algae ATCC 51192T was 35.8 %. It is clear from the 16S rRNA gene sequence and DNA–DNA hybridization data that strain DW01T represents a novel species of the genus Shewanella (Wayne et al., 1987).
In addition, a number of physiological and chemotaxonomic characters clearly distinguished our isolate from other phylogenetically related species (Tables 1 and 2). Therefore, strain DW01T should be classified as a novel species of the genus Shewanella, for which the name Shewanella haliotis sp. nov. is proposed.
Description of Shewanella haliotis sp. nov.
Shewanella haliotis (ha.li.o'tis. N.L. gen. n. haliotis of Haliotis, the scientific name of abalones).
Cells are rod-shaped, Gram-negative and facultatively anaerobic bacterium. Cells grow best on media such as MA, TSA, PCA and NA, but weakly on R2A. Colonies on TSA are circular, low-convex, entire margin, smooth, opaque, pink–orange coloured and approximately 5.0 mm in diameter after 5 days at 30 °C (pH 7). Cells are motile rods and 0.5–0.7x2.0–4.3 µm in size. Growth occurs in 0–10 % (w/v) NaCl (optimum 4 %). Growth occurs in pH 5–11 (optimum pH 7) and at 10–42 °C (optimum 37 °C). Oxidase-positive, catalase-positive. Reduces nitrate to nitrite. Negative for glucose fermentation. Does not produce arginine dihydrolase, urease, lysine decarboxylase, β-galactosidase or tryptophan deaminase. Produces H2S but not acetoin or indole. Produces gelatinase, α-chymotrypsin, alkaline phosphatase and ornithine decarboxylase, but not lipase (C14). Utilizes the following substrates as sole carbon and energy sources: N-acetylglucosamine, caprate and malate. Does not utilize the following substrates: D-glucose, galactose, fructose, mannose, melibiose, D-arabinose, L-arabinose, D-xylose, aesculin, salicin, glycerol, ribose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol or inulin. Cells are sensitive to (µg per disc, unless otherwise indicated): amikacin (30), gentamicin (10), streptomycin (10), tetracycline (30), erythromycin (15), chloramphenicol (30), kanamycin (30) and nalidixic acid (30) but resistant to ampicillin (10), vancomycin (30), polymyxin B (300) and penicillin (10 U). Other physiological and biochemical characteristics are given in Table 1. Major fatty acids are iso-C15 : 0 (17.7 %), C16 : 0 (13.4 %), iso-C15 : 0 2-OH and/or C16 : 1ω7c (12.5 %) and C17 : 1ω8c (10.7 %), and complete fatty acid composition is given in Table 2. The DNA G+C content is 53.7 mol%.
The type strain, DW01T (=KCTC 12896T=JCM 14758T), was isolated from the gut microflora of abalone collected from the South Sea, Republic of Korea.
Acknowledgements
This research was supported by the Programme for the Training of Graduate Students in Regional Innovation, which was conducted by the Korean Government's Ministry of Commerce Industry and Energy and this work was also supported by the BK21 (the Ministry of Education and Human Resources Development), Republic of Korea. Special thanks are extended to Dr J. P. Euzéby for his recommendation concerning the Latin etymology for strain DW01T.References
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