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1 Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, 361005, P. R. China
2 State Key Laboratory of Marine Geology, Tongji University, Shanghai, P. R. China
3 Center for Marine Biotechnology and Biomedicine, Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0202, USA
Correspondence
Fengping Wang
fengpingw{at}yahoo.com
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences of Shewanella psychrophila WP2T are AJ551089 and AM229307, and those of Shewanella piezotolerans WP3T are AJ551090 and AM229308, respectively.
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MAIN TEXT |
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; Skerratt et al., 2002; Venkateswaran et al., 1998, 1999; Satomi et al., 2003). At the time of writing the genus comprises more than 20 recognized species, among which Shewanella benthica, Shewanella violacea and Shewanella profunda have been isolated from deep-sea environments (Deming et al., 1984; MacDonell & Colwell, 1985; Nogi et al., 1998; Toffin et al., 2004). Kato & Nogi (2001) divided the genus Shewanella into two major branches, groups 1 and 2. Most Shewanella group 1 species are psychrophilic/psychrotolerant and piezophilic/piezotolerant and produce eicosapentanoic acid (EPA); by contrast, species in group 2 are mesophilic, pressure-sensitive and produce no or only trace amounts of EPA. Shewanella group 1 species include S. benthica, S. violacea, S. hanedai, S. gelidimarina and S. pealeana; Shewanella group 2 species include S. putrefaciens, S. oneidensis, S. algae, S. baltica, S. amazonensis and S. frigidimarina.
Shewanella strains are the most abundant Proteobacteria in the deep sea (Kato & Nogi, 2001; Kato, 1999). We have previously reported the isolation of two psychrotolerant/psychrophilic Shewanella strains, designated WP2T and WP3T, from a deep-sea sediment (Wang et al., 2004). In the present study, these two strains were further characterized based on combined phenotypic, phylogenetic and chemotaxonomic analyses. They were determined to represent two novel deep-sea Shewanella species.
Strains WP2T and WP3T were isolated from west Pacific deep-sea sediment (142° 30' 08'' E 8° 00' 11'' N) at a depth of 1914 m. The reference strain Shewanella fidelis LMG 20551T was obtained from the Belgian Co-ordinated Collections of Microorganisms (BCCM/LMG), S. benthica ATCC 43992T from the American Type Culture Collection (ATCC) and S. violacea JCM 10179T from the Japan Collection of Microorganisms (JCM). These bacteria were grown in marine 2216E medium (0.5 % tryptone, 0.1 % yeast extract, 3.4 % NaCl and 0.01 % FePO4, pH 7.6–7.8). Strain WP2T was grown at 15 °C, strain WP3T at 20 °C, S. benthica and S. violacea at 10 °C, and S. fidelis at 25 °C unless otherwise indicated. High-pressure cultivation was performed using a hand-operated pump and a quick-fit connector to the high-pressure vessels (Yayanos & Dietz, 1982; Li et al., 2006). Marine 2216E medium was inoculated with 1 % inoculum culture in exponential growth phase; 60 ml of inoculated medium was dispersed in sterile injection syringes. Duplicate syringes were placed into the high-pressure vessels and pressurized. Growth of the cultures was checked by decompressing the vessels and was monitored by measuring the optical absorption at OD600. Growth tests at different pressures were repeated three times.
Morphological, biochemical and physiological tests followed the general procedures described by Dong & Cai (2001). The ability of the strains to utilize a broad range of carbon sources was determined by using Biolog GN2 microplates. Cell morphology of the bacteria was observed by using light and transmission electron microscopy. After around 6–7 days incubation on marine 2216E agar plates at 10 °C, the two new strains formed pale-yellow colonies of 0.5 mm in diameter with rounded edges. After 5 days incubation in marine 2216E broth, the cell aggregates showed a pinkish colour. Cells of strains WP2T and WP3T were Gram-negative rods, 0.5–0.8 µm wide and 2–5 µm long, and motile by means of a single polar flagellum (Fig. 1). When incubated in liquid culture, strain WP2T grew over a temperature range of 0–20 °C, and optimally at 10–15 °C; strain WP3T was able to grow at 0–28 °C, with optimal growth at 15–20 °C. The strains were also cultivated in 2216E media modified with the addition of NaCl (0–12 %). The two new strains required NaCl for growth. They were able to grow at salinities of 1–7.2 %, with optimum growth at 3–4 % NaCl. The strains were tested for their ability to grow at pressures of 0.1, 10, 20, 30, 40 and 50 MPa. Strains WP2T and WP3T showed growth within the pressure range 0.1–50 MPa and displayed optimal growth at 20 MPa (Fig. 2).
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). Strain WP3T used acetate, D-glucose, maltose and N-acetyl-D-glucosamine as sole carbon sources. Nitrate was reduced to nitrite. Hydrogen sulfide was produced. Strain WP3T was positive for cytochrome oxidase, lipase, gelatinase, amylase and catalase, but negative for the production of chitinase (Table 1).
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). Anaerobic growth was also evaluated using trimethylamine N-oxide (TMAO), DMSO, sodium fumarate, sodium nitrite and sodium nitrate in the same medium in which Fe(III) citrate was omitted. Strain WP3T was able to utilize nitrate, fumarate, TMAO, DMSO and insoluble Fe(III) as terminal electron acceptors for anaerobic growth, whereas strain WP2T used only nitrate, TMAO and DMSO.
Strains WP2T and WP3T were grown in 250 ml marine 2216E broth. Cultures were incubated with shaking at 150 r.p.m. for 24 h. The cell membrane was extracted via the TCA method and purified with trypsin according to Schleifer & Kandler (1972). Isoprenoid quinones were extracted and purified according to Collins et al. (1977). The purified menaquinones were analysed by HPLC-MS (LCQ MAT; Finnigan) (Nishijima et al., 1997) with an APCI (atmospheric pressure chemical ionization) ion source using a solution of 2-propanol and acetonitrile (1.25 : 1, v/v). Ubiquinones and menaquinones were found in the two new strains. The major ubiquinones in strains WP2T and WP3T were ubiquinone 7 (Q7) (59 and 60 %, respectively) and ubiquinone 8 (Q8) (41 and 40 %, respectively). Traces of menaquinone 7 (MK7) and monomethylmenaquinone 7 (MMK7) were also detected.
Cells for cellular fatty acid analysis were likewise harvested after cultivation for 24 h. Fatty acids were extracted and analysed following the instructions of the Microbial Identification System operating manual (MIDI Inc.). The fatty acid profiles of strains WP2T and WP3T were typical of members of the genus Shewanella and included iso-13 : 0, 14 : 0, iso-15 : 0, 16 : 0, 16 : 1, 18 : 1 and 20 : 5 (Table 2).
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. The G+C content of the DNA was determined using HPLC analysis of hydrolysed DNA according to Tamaoka & Komagata (1984). The obtained nucleoside mixture was then separated by reversed-phase HPLC using a C18 column (Kromasil ODS, 5 µm, 250x4.6 mm inner diameter) at room temperature. The solvent was 0.05 mol l–1 NH4H2PO4, pH 4.0, with 7 % acetonitrile (Tamaoka & Komagata, 1984). The G+C content was calculated based on determination of the G/(G+T) content according to Mesbah et al. (1989). The DNA G+C content of strains WP2T and WP3T was 51 and 49 mol%, respectively.
PCR was used to amplify a portion of the gyrB gene according to the procedure of Yamamoto & Harayama (1995). PCR products of 1200 bp of the gyrB gene fragment (positions 274–1525 according to the Escherichia coli numbering scheme) were cloned into the pGEM-T vector by using a 2x rapid ligation kit according to the instructions of the manufacturer (Promega). Ligation mixtures were used to transform competent cells of E. coli XL1 Blue according to the manufacturer's recommendations (Promega). Positive clones were picked for sequencing (Sangon). The resulting sequences were searched in the NCBI database. Related sequences were aligned using the program DNAMAN (Lynnon Biosoft, v. 5.1). A phylogenetic tree was constructed from a matrix of pairwise genetic distances by the maximum-parsimony algorithm (Fitch, 1971) and the neighbour-joining method (Saitou & Nei, 1987) using the DNAMAN program, and bootstrap analyses (1000 trials) were used to provide estimates of confidence for phylogenetic tree topologies.
The gyrB gene sequences of strains WP2T and WP3T shared 84 % similarity. Strain WP2T showed highest gyrB gene sequence similarity with the type strains of S. violacea (90 %) and S. benthica (89 %), and strain WP3T showed highest similarity with the type strain of Shewanella sairae (87 %). The phylogenetic relationship of strains WP2T and WP3T with other Shewanella species based on gyrB gene sequences is shown in Fig. 3(b). Strain WP2T clustered with S. violacea and S. benthica whereas strain WP3T clustered with S. fidelis and S. sairea. This is not consistent with the results based on 16S rRNA gene sequence analyses (Fig. 3a; Wang et al., 2004).
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. Triplicate target DNA (50–200 ng) was denatured in 0.4 M NaOH and transferred onto a nylon membrane (Amersham, Pharmacia) and hybridized with sheared probes labelled with digoxin (DIG High Prime DNA Labeling and Detection Starter KitII; Roche). Hybridization was performed at 55 °C, and high-stringency washes and signal detection were performed according to the manufacturer's instructions. Intensities of the signals were analysed using the GelBase/GelBlot-Pro software system. Signals produced by hybridization of the probe with homologous target DNA were considered to represent 100 % hybridization. Levels of DNA–DNA hybridization between strains WP2T and WP3T and recognized Shewanella species are given in Table 3. Levels ranged from 60 % (WP2T/S. benthica) to 19 % (WP3T/S. benthica) (Table 3), well below the 70 % threshold used to define a genomic species (Wayne et al., 1987).
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; Venkateswaran et al., 1999). The more rapid evolution of the gyrB gene compared with that of the 16S rRNA gene makes it a more appropriate choice for differentiating between closely related strains. The 16S rRNA gene sequence of strain WP2T had highest similarity (97 %) with S. fidelis KMM 3589, and strain WP3T highest similarity (97 %) with S. benthica ATCC 43992T. By contrast, the gyrB gene sequence of strain WP2T exhibited highest similarity with those of the type strains of S. violacea and S. benthica (90 and 89 %, respectively), and the gyrB gene sequence of strain WP3T exhibited highest similarity with that of the type strain of S. sairea (87 %). Discrepancies between gyrB and 16S rRNA gene sequences were previously evident among the Shewanella species examined by Venkateswaran et al. (1999). DNA–DNA hybridization results indicated that strain WP2T is most closely related to S. benthica. DNA–DNA hybridization and gyrB sequence analysis both placed strain WP2T, S. violacea and S. benthica within a single phylogenetic clade. The results of the polyphasic taxonomic investigations performed in this study suggest that strains WP2T and WP3T represent two novel Shewanella species. The names Shewanella psychrophila sp. nov. and Shewanella piezotolerans sp. nov. are proposed for strains WP2T and WP3T, respectively.
Description of Shewanella psychrophila sp. nov.
Shewanella psychrophila (psy.chro'phi.la. Gr. adj. psychros cold; Gr. adj. philos loving; N.L. fem. adj. psychrophila cold loving).
Cells are Gram-negative rods that are 0.5–0.8 µm wide and 2–5 µm long when grown at 0.1 MPa. Motile by means of a single polar flagellum. Facultatively anaerobic, with nitrate, TMAO and DMSO as electron acceptors. Colonies on agar plates are circular with a round edge and pale-yellow in colour. Growth occurs within the temperature range 0–20 °C, with optimal growth at 10–15 °C. Growth occurs within the pressure range 0.1–50 MPa, with optimal growth at 20 MPa. Growth occurs at NaCl concentrations between 1 and 7.2 % (optimum at 3–4 %), and at pH 6–8 (optimum at pH 7). Positive for oxidase and catalase. Hydrogen sulfide is not produced. The following compounds are utilized for respiration (Biolog): Tweens 40 and 80, N-acetyl-D-glucosamine, myo-inositol, maltose, sucrose, D-trehalose, cis-aconitic acid, 
-ketovaleric acid, bromosuccinic acid, succinamic acid, L-alaninamide, D-alanine, L-glutamic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, D-serine L-serine, L-threonine, DL-carnitine, thymidine and DL--glycerol phosphate. Produces EPA. Major quinones are Q7 and Q8; MK7 and MMK7 are also present. The G+C content of the DNA is 51 mol%.
The type strain, WP2T (=JCM 13876T=CGMCC 1.6159T), was isolated from west Pacific deep-sea sediment.
Description of Shewanella piezotolerans sp. nov.
Shewanella piezotolerans (pie.zo'to.le.rans. Gr. v. piezo to press; L. part. adj. tolerans tolerating; N.L. fem. part. adj. piezotolerans pressure tolerating).
Cells are Gram-negative rods, 0.5–0.8 µm wide and 2–5 µm long, and motile by means of a single polar flagellum. Facultatively anaerobic, with nitrate, fumarate, TMAO, DMSO and insoluble Fe(III) as terminal electron acceptors. Grows at temperatures of 0–28 °C, with optimal growth occurring at 15–20 °C. Growth occurs within the pressure range 0.1–50 MPa, with optimal growth at 20 MPa. Growth occurs at NaCl concentrations between 1 and 7.2 % (optimum at 3–4 %), and at pH 6–8 (optimum at pH 7). Lipase, oxidase, catalase, gelatinase and amylase are produced. Hydrogen sulfide is produced from thiosulfate. Cells are able to reduce nitrate to nitrite. The following carbon sources are used: -cyclodextrin, dextrin, Tweens 40 and 80, N-acetyl-D-glucosamine, gentiobiose, -D-glucose, maltose, acetic acid, DL-lactic acid, propionic acid, L-alanine, L-alanyl glycine, L-asparagine, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-leucine, L-serine, L-threonine, inosine, uridine and thymidine. Produces EPA. Major quinones are Q7 and Q8; MK7 and MMK7 are also present. The G+C content of the DNA is 49 mol%.
The type strain, WP3T (=JCM 13877T=CGMCC 1.6160T), was isolated from west Pacific deep-sea sediment.
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ACKNOWLEDGEMENTS |
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) and WP3T (
) at different pressures (at 15 °C). For strain WP2T, 100 % growth rate: 1/td=0.405; for strain WP3T, 100 % growth rate: 1/td=0.5, where td is doubling time. Bars indicate SD of three experiments.