Abstract
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences determined in this study are AB094597, AB094598, AB201475 and AB201780AB201788, as detailed in Fig. 1.
The genus Shewanella comprises Gram-negative, facultatively anaerobic gammaproteobacteria (MacDonell & Colwell, 1985) and is typical of deep-sea bacterial genera (DeLong et al., 1997). The genus includes psychrophilic and mesophilic species that are widely distributed in nature, especially in aquatic environments such as freshwater and the ocean (Semple & Westlake, 1987; Myers & Nealson, 1988; Simidu et al., 1990; Gauthier et al., 1995; Bowman et al., 1997; Makemson et al., 1997; Nogi et al., 1998; Venkateswaran et al., 1999; Ivanova et al., 2001, 2004; Satomi et al., 2003; Hirota et al., 2005). To date, at least 31 Shewanella species have been recognized. The genus Shewanella is divided into two subgenera on the basis of phylogenetic structure, growth properties in relation to pressure and polyunsaturated fatty acid production (Kato & Nogi, 2001). In this paper, we describe the results of taxonomic studies on six strains isolated from Suruga Bay, Japan. Several lines of evidence indicate that these isolates represent three novel species within the genus Shewanella.
Strains c931T, c941T, d943, c952, d954 and c959T were isolated at a depth of 24062409 m from a deep-sea sediment, using sterilized mud samplers on the unmanned submersible Kaiko positioned off Matsuzaki in Suruga Bay (34° 36.55' N 138° 34.77' E). The following reference strains were used. Shewanella benthica ATCC 43992T and Shewanella pealeana ATCC 700345T were obtained from the American Type Culture Collection (Manassas, VA, USA), Shewanella marinintestina JCM 11558T, Shewanella sairae JCM 11563T and Shewanella schlegeliana JCM 11561T were obtained from the Japan Collection of Microorganisms (Wako, Japan) and Shewanella gelidimarina ACAM 456T was obtained from the Australian Collection of Antarctic Micro-organisms (Hobart, Tasmania, Australia). Shewanella pneumatophori SCRC-2738T was obtained from Dr Kikue Hirota (Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Sapporo, Japan). Shewanella violacea DSS12T was from our laboratory stock. These bacteria were incubated on marine agar 2216 (Difco). Agar plates were incubated aerobically for 23 days at the optimum temperature. Unless indicated otherwise, physiological tests were performed with a slight modification (use of artificial seawater; 1x artificial seawater consists of 3 % NaCl, 0.07 % KCl, 1.08 % MgCl2.6H2O, 0.54 % MgSO4.7H2O, 0.1 % CaCl2.2H2O) of the general procedures described by Barrow & Feltham (1993) and Baumann et al. (1972). Optimal temperatures for growth were determined on the basis of optical density in marine broth 2216 (Difco). Acid production from sugars was assessed using modified OF medium (Hugh & Leifson 1953) containing 0.5x artificial seawater, 0.05 % (NH4)2SO4, 0.01 % yeast extract (Difco), 0.05 % Tris base, 0.5 % agar, 0.003 % bromothymol blue and 1 % test sugar (pH was adjusted to 7.1 at 20 °C), with incubation at the optimum temperature. Salt-tolerance tests were performed using medium containing 0.5 % Pepton (Difco), 0.5 % yeast extract, 0.32 % MgSO4.7H2O and 0.1 % K2HPO4, with NaCl concentrations of 015 % (w/v).
Cellular fatty acids were extracted and analysed as described by Komagata & Suzuki (1987). Isolated strains were cultured in marine broth 2216 medium at optimal temperatures. Cells were washed twice with 0.7 % NaCl at 4 °C; this was followed by centrifugation at 8000 g and freeze-drying. Dried cells (20 mg) were placed in Teflon-lined, screw-capped tubes containing 2 ml anhydrous methanolic HCl and heated to 100 °C for 3 h. After cooling, 1 ml water was added and the fatty acid methyl esters were extracted with n-hexane. Samples were analysed using a Shimadzu model GCMS-QP5050A gasliquid chromatograph/mass spectrometer with a DB-5MS column (J&W Scientific). Isoprenoid quinones were extracted with chloroform/methanol (2 : 1) from dried cells (200 mg) and purified using TLC. The purified isoprenoid quinones were analysed using reversed-phase HPLC (Komagata & Suzuki, 1987).
Chromosomal DNA was purified by using a standard method (Saito & Miura, 1963). The DNA G+C content was determined using reversed-phase HPLC (Tamaoka & Komagata, 1984). For analysis of relatedness, DNADNA hybridization was carried out at 40 °C for 4 h and measured fluorometrically using the method of Ezaki et al. (1989).
16S rRNA and gyrB gene sequences were obtained by direct sequencing of PCR-amplified DNA as described previously (Kato et al., 1998; Yamamoto & Harayama, 1995). Nucleotide substitution rates (Knuc) (Kimura, 1980) were determined and a distance matrix tree was constructed with the neighbour-joining method (Saitou & Nei, 1987), using the CLUSTAL X program (Thompson et al., 1997). Alignment gaps and unidentified base positions were not taken into consideration in the calculations. The topology of the phylogenetic tree was evaluated by performing bootstrap analysis with 1000 replicates. The GenBank/DDBJ/EMBL accession numbers for the 16S rRNA and gyrB gene sequences of the isolates are shown in Fig. 1; reference sequences were obtained from the GenBank database.
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Morphological, cultural, physiological and biochemical characteristics of the isolates are shown in Table 1 or given in the species descriptions below. The cells of the novel deep-sea strains were Gram-negative rods that were motile by means of single, unsheathed, polar flagella. These strains were facultatively anaerobic chemo-organotrophs, displaying both respiratory and fermentative types of metabolism. All of the isolates were able to grow in 15 % NaCl but not in the absence of NaCl, and the optimal concentration for growth was 23 % NaCl.
Table 1. Differential characteristics of Shewanella species Taxa: 1, Shewanella kaireitica sp. nov. (strains c931T, c952 and d954); 2, Shewanella abyssi sp. nov. (strains c941T and d943); 3, Shewanella surugensis sp. nov. (strain c959T); 4, S. marinintestina; 5, S. schlegeliana; 6, S. sairae; 7, S. pealeana; 8, S. gelidimarina; 9, Shewanella fidelis; 10, S. violacea; 11, S. benthica. Cells of all species are Gram-negative rods that are motile by means of single polar flagella. The following tests were positive for all species: catalase activity, hydrolysis of gelatin and reduction of nitrate to nitrite. The following tests were negative for all species: hydrolysis of starch, production of indole and utilization of D-lactose. V, Variable depending on strain; ND, no data available. Data for reference species are from Satomi et al. (2003), Leonardo et al. (1999), Bowman et al. (1997), Ivanova et al. (2003) and Nogi etal. (1998), except where indicated otherwise.
Phenotypic analysis segregated the strains into three groups, (i) strains c931T, c952 and c954, (ii) strains c941T and d943 and (iii) strain c959T, with the following properties. (i) Strains c931T, c952 and c954 grew at 228 °C, with an optimum at 2425 °C. Strains c931T and c952 (but not strain c954) were able to grow in 7 % NaCl. Tween 80 and chitin were hydrolysed. Acid, but not gas, was produced from cellobiose, D-fructose, D-galactose, D-glucose, maltose and D-sorbitol. The G+C content of the DNA was 43.044.3 mol%. The major isoprenoid quinones were Q-7 (7080 %) and Q-8 (2030 %). (ii) Strain c941T exhibited growth at 218 °C, with an optimum at 10 °C, and strain d943 grew at 224 °C, with an optimum at 14 °C. Strains c941T and d943 did not hydrolyse Tween 80 and strain c941T utilized cellobiose. The G+C content of the DNA was 43.543.6 mol%. The major isoprenoid quinones were Q-7 (6570 %) and Q-8 (3035 %). (iii) Strain c959T exhibited growth at 221 °C, with an optimum at 13 °C. No growth occurred at temperatures above 25 °C. Tests for cytochrome oxidase, H2S production and reduction of nitrite to nitrogen were negative. Acid, but not gas, was produced from D-fructose, D-glucose, glycerol, D-mannose and sucrose. The G+C content of the DNA was 40.0 mol%. The major isoprenoid quinones were Q-7 (5 %) and Q-8 (95 %).
The results of the phylogenetic analyses using 16S rRNA and gyrB gene sequences are shown in Fig. 1. These results support the conclusions described below and further clarify the taxonomic and phylogenetic positions of the novel isolates among members of the genus Shewanella and related genera. Strains c931T, d954 and c952 were most closely related to S. schlegeliana (98.698.8 % similarity for 16S rRNA, 86.086.5 % similarity for gyrB), strains c941T and d943 were most closely related to S. gelidimarina (97.798.0 % similarity for 16S rRNA, 86.887.4 % similarity for gyrB) and strain c959T was most closely related to S. gelidimarina (97.1 % similarity for 16S rRNA), but showed less than 80 % gyrB gene sequence similarity to Shewanella species. The generally recommended and accepted criteria for delineating bacterial species state that strains with 16S rRNA gene sequence dissimilarity greater than 3 % are considered to belong to separate species (Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002); within the genus Shewanella, strains with gyrB gene sequence dissimilarity greater than 10 % are considered to belong to separate species (Venkateswaran et al., 1999). However, bacterial strains with gyrB gene sequences that differ by less than 10 % cannot be allocated to the same species without support from DNADNA relatedness studies. The generally recognized criteria for delineating bacterial species state that strains with a DNADNA relatedness of less than 70 %, as measured by hybridization, represent separate species (Wayne et al., 1987).
The results of DNADNA hybridization analysis indicated that the novel isolates fell into three groups (Table 2), the same grouping revealed by phenotypic characteristics and phylogenetic analyses. There was more than 70 % DNA relatedness among the strains in each group, and each group was clearly separate, representing distinct species (i.e. showing significantly less than 70 % relatedness) according to the recommendations of Wayne et al. (1987). This, together with the other results shown in Table 1 and Fig. 1, suggests that the isolated strains represent three novel species of the genus Shewanella.
Table 2. DNADNA reassociation between the isolated strains and closely related Shewanella species
The whole-cell fatty acid compositions of strains c931T, c941T and c959T and the reference strains are shown in Table 3. The major fatty acids were as follows: strain c931T, iso-C15 : 0 (isopentadecanoic acid), C16 : 0 (hexadecanoic acid) and C16 : 1 (hexadecenoic acid); strain c941T, iso-C13 : 0 (isotridecanoic acid) and C16 : 1; strain c959T, iso-C15 : 0 and C16 : 1. For each of the strains, the fatty acid profile showed low levels of similarity to those of the reference strains. For example, strain c931T contained relatively large amounts of iso-C15 : 0 and C16 : 0 and small amounts of C20 : 5ω3 (eicosapentaenoic acid), strain c941T contained large amounts of iso-C13 : 0 and C15 : 0 (pentadecanoic acid) and small amounts of C16 : 0 and strain c959T contained large amounts of C16 : 1 and did not contain C20 : 5ω3.
Table 3. Fatty acid compositions of the novel isolates and authentic Shewanella species Taxa: 1, strain c931T; 2, strain c941T; 3, strain c959T; 4, S. marinintestina JCM 11558T; 5, S. schlegeliana JCM 11561T; 6, S. sairae JCM 11563T; 7, S. pealeana ATCC 700345T; 8, S. gelidimarina ACAM 456T; 9, S. fidelis KMM 3582T; 10, S. violacea DSS12T; 11, S.benthica ATCC 43992T. Values are percentages of total fatty acids. , Not detected; tr, trace (<1 %). Data for reference species are from Satomi et al. (2003), Leonardo et al. (1999), Bowman etal. (1997), Ivanova et al. (2003) and Kato & Nogi (2001).
Lowering of temperature has a physical effect on the fluidity of membrane lipids. As the growth temperature is lowered, biological membranes undergo a reversible change from a fluid, disordered state to a non-fluid, ordered state (Hazel & Williams, 1990). The most common adaptive change observed among deep-sea bacteria involves the incorporation of increased proportions of unsaturated fatty acids into membrane phospholipids (Allen et al., 1999). The largely psychrophilic or piezophilic Shewanella strains produce eicosapentaenoic acid (Margesin & Nogi, 2004). However, strain c959T, adapted to a deep-sea environment, contained large amounts of C16 : 1.
On the basis of the phenotypic, genotypic and phylogenetic data, it is logical to conclude that the deep-sea isolates we studied are members of the genus Shewanella and that they represent three novel species within this genus. We propose the names Shewanella kaireitica sp. nov. (type strain c931T), Shewanella abyssi sp. nov. (type strain c941T) and Shewanella surugensis sp. nov. (type strain c959T).
Description of Shewanella kaireitica sp. nov.
Shewanella kaireitica (kai.rei'tic.a. N.L. n. Kairei a research vessel; L. fem. suff. -tica adjectival suffix meaning belonging to; N.L. fem. adj. kaireitica belonging to the research vessel Kairei).
Cells are rod-shaped; cell width ranges from 0.8 to 1.0 µm and cell length ranges from 2.5 to 3.0 µm. Cells are Gram-negative and motile by means of single, unsheathed, polar flagella. Colonies on marine agar 2216 are entire, smooth, semitranslucent and salmon-coloured; 24 mm in diameter after 12 days incubation at 20 °C. The bacterium is psychrotolerant. Exhibits optimal growth at NaCl concentrations of approximately 35 %; able to grow at 7 % NaCl (with the exception of strain d954). No growth occurs in the absence of NaCl. Optimal temperature for growth is 2425 °C. No growth at temperatures above 30 °C. Facultatively anaerobic chemo-organotroph, having both respiratory and fermentative types of metabolism. Catalase and cytochrome oxidase tests are positive. H2S is produced. Nitrate is reduced to N2. Acid is formed oxidatively from cellobiose, D-fructose, D-galactose, D-glucose, maltose and D-sorbitol. No acid is produced from L-arabinose, glycerol, myo-inositol, D-lactose, D-mannitol, D-mannose, D-raffinose, L-rhamnose, sucrose, D-trehalose or xylose. The G+C content of the DNA is approximately 43.0 mol%. The major isoprenoid quinone is Q-7. The predominant cellular fatty acids are iso-C13 : 0, iso-C15 : 0, C16 : 0 and C16 : 1.
The type strain, c931T (=JCM 11836T=DSM 17170T), and strains c952 and d954 were isolated from deep-sea sediment in Suruga Bay, Japan.
Description of Shewanella abyssi sp. nov.
Shewanella abyssi (a.bys'si. N.L. gen. n. abyssi from the abyss).
Cells are rod-shaped; cell width ranges from 0.8 to 1.0 µm and cell length ranges from 2.0 to 2.5 µm. Cells are Gram-negative and motile by means of single, unsheathed, polar flagella. Colonies on marine agar 2216 are entire, smooth, semitranslucent and salmon-coloured; 24 mm in diameter after 23 days incubation at 10 °C. The bacterium is psychrotolerant. Optimal growth occurs at a NaCl concentrations of 35 %. No growth occurs in the absence of NaCl. The optimal temperature for growth is 10 °C. No growth occurs at temperatures above 25 °C. Facultatively anaerobic chemo-organotroph, having both respiratory and fermentative types of metabolism. Catalase and cytochrome oxidase tests are positive. H2S and indole are produced. Nitrate is reduced to nitrite; nitrite is reduced to N2. Acid is formed oxidatively from cellobiose. No acid is produced from L-arabinose, D-fructose, D-galactose, D-glucose, glycerol, myo-inositol, D-lactose, maltose, D-mannitol, D-mannose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose or xylose. The G+C content of the DNA is approximately 43.5 mol%. The major isoprenoid quinones are Q-7 and Q-8. The predominant cellular fatty acids are iso-C13 : 0, iso-C15 : 0, C16 : 0 and C16 : 1.
The type strain, c941T (=JCM 13041T=DSM 17171T), and strain d943 were isolated from deep-sea sediment in Suruga Bay, Japan.
Description of Shewanella surugensis sp. nov.
Shewanella surugensis (su.ru.gen'sis. N.L. fem. adj. surugensis pertaining to Suruga Bay, where the type strain was isolated).
Cells are rod-shaped; cell width ranges from 0.4 to 0.6 µm and cell length ranges from 3.2 to 4.0 µm. Cells are Gram-negative and motile by means of single, unsheathed, polar flagella. Colonies on marine agar 2216 are entire, smooth, semitranslucent and cream-coloured; 24 mm in diameter after 23 days incubation at 10 °C. The bacterium is psychrotolerant. Optimal growth occurs at an NaCl concentration of approximately 3 %. No growth occurs in the absence of NaCl. The optimal temperature for growth is 13 °C. No growth occurs at temperatures above 25 °C. Facultatively anaerobic chemo-organotroph, having both respiratory and fermentative types of metabolism. The catalase test is positive. The cytochrome oxidase test is negative. Does not produce H2S. Nitrate is reduced to nitrite. Acid is formed oxidatively from D-fructose, D-glucose, glycerol, D-mannose and sucrose. No acid is produced from L-arabinose, cellobiose, D-galactose, myo-inositol, D-lactose, maltose, D-mannitol, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose or xylose. The G+C content of the DNA is approximately 40.0 mol%. The major isoprenoid quinone is Q-8. The predominant cellular fatty acids are iso-C13 : 0, iso-C15 : 0 and C16 : 1.
The type strain, c959T (=JCM 11835T=DSM 17177T), was isolated from deep-sea sediment in Suruga Bay, Japan.