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Extremobiosphere Research Center, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
Correspondence
Yuichi Nogi
nogiy{at}jamstec.go.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Present address: Laboratory of Bioresources, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 1998; Nogi et al., 1998, 2004; Yayanos et al., 1979). All piezophilic bacteria isolated to date fall into the class Gammaproteobacteria, according to phylogenetic classifications based on 16S rRNA gene sequence information (DeLong et al., 1997; Nogi & Kato, 1999; Nogi et al., 2002). Margesin & Nogi (2004) reported that 11 cultivated psychrophilic and piezophilic deep-sea species were affiliated with genera within the Gammaproteobacteria: Shewanella, Photobacterium, Colwellia, Moritella and Psychromonas. Members of the genus Psychromonas were isolated from the polar circles and deep-sea environments. Most of these isolates are not piezophilic strains. Psychromonas kaikoae JT7304T (Nogi et al., 2002) and Psychromonas profunda 2825T (Xu et al., 2003) were reported to be piezophilic strains within this group. In this study, a novel obligately piezophilic, C22 : 6 (docosahexaenoic acid)-producing bacterium of the genus Psychromonas is proposed on the basis of polyphasic studies.
Strain K41GT was isolated from deep-sea sediment samples collected in November 2000 using sterilized mud samplers on the unmanned submersible KAIKO in the Japan Trench (38° 0.5' N 143° 59.9' E), at a depth of 7542 m. This piezophilic strain was maintained on marine agar 2216 (Difco) and was grown at 6 °C and 60 MPa. High-pressure cultivation was achieved using a liquid hydraulic system. Piezophilic bacteria were cultivated in a plastic bag containing liquid medium in a pressure vessel made of stainless steel (SUS304). If necessary, oxygen-saturated fluorinert (FC-72; Sumitomo-3M) was added to supply oxygen to the cultures (20 % of total volume). This was performed according to the procedure reported previously (Kato et al., 1994; Yanagibayashi et al., 1999). Optimal pressure and temperature for growth were measured by monitoring the optical density. These tests were performed anaerobically in marine broth 2216 under various pressure and temperature conditions.
Cells of the isolated deep-sea strain K41GT were found to be Gram-negative rods, 1.5–2 µm long and 0.8–1.0 µm wide, and motile by means of a single unsheathed polar flagellum. The strain was unable to grow at atmospheric pressure at 2–15 °C, although it grew well in pressure vessels under hydrostatic pressures of 30–90 MPa at 6 °C and 60–90 MPa at 10 °C. No growth occurred at 15 °C under the pressures examined. The most rapid growth rate (about 0.14 h–1) was observed at 60 MPa and 6 °C (Fig. 1).
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) to examine hydrogen sulfide production from thiosulfate, production of indole, fermentation and oxidization, gelatinase activity and oxidase and catalase production, according to methods described previously (Nogi & Kato, 1999; Nogi et al., 2002).
The characteristics of strain K41GT and reference strains are shown in Table 1. Strain K41GT was a facultatively anaerobic, chemo-organotroph, displaying both respiratory and fermentative types of metabolism. Strain K41GT shared many physiological characteristics with members of the genus Psychromonas. However, unlike the reference strains of Psychromonas, strain K41GT did not utilize cellobiose or sucrose and reduced nitrite, and strain K41GT and Psychromonas kaikoae were unable to grow at atmospheric pressure.
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). Nucleotide substitution rates (Knuc; Kimura, 1980) were determined and a distance matrix tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) with the program CLUSTAL_X (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 bootstrapped trials. The results of phylogenetic analyses based on 16S rRNA gene sequence information support the conclusions described below and further clarify the taxonomic and phylogenetic position of the novel isolate among members of the genus Psychromonas and related genera. The results of the phylogenetic analyses are shown in Fig. 2. Strain K41GT falls into the genus Psychromonas and is closely related to the psychrotrophic species Psychromonas arctica (96.1 %) and the piezophilic species Psychromonas profunda (97.0 %). 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). For analysis of relatedness, DNA–DNA hybridization was carried out at 40 °C for 3 h and was measured fluorometrically using the method of Ezaki et al. (1989). The DNA–DNA hybridization value between strain K41GT and the reference strain Psychromonas profunda was less than 40 %. This level of relatedness is significantly lower than that accepted as the phylogenetic definition of a species (Wayne et al., 1987). This and other results in Table 1
and Fig. 2 suggest that strain K41GT represents a novel Psychromonas species.
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. The major fatty acids of strain K41GT were C14 : 1 (tetradecenoic acid), C16 : 0 (hexadecanoic acid), C16 : 1 (hexadecenoic acid) and C22 : 6. There was a low level of similarity between this fatty acid profile and those of the reference strains. For example, the predominant components in the fatty acid profiles of the reference strains differed from those in the profile of strain K41GT, which contained substantial amounts of C14 : 1 and C22 : 6 and does not have the long-chain unsaturated fatty acid C18 : 1 (octadecenoic acid).
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Description of Psychromonas hadalis sp. nov.
Psychromonas hadalis [ha.da'lis. N.L. fem. adj. hadalis (from Greek Haid
s), hadal of or relating to the deepest regions of the ocean].
Cells are Gram-negative rods, 1.5–2 µm long and 0.8–1.0 µm wide, and motile by means of a single unsheathed polar flagellum. Halophilic, psychrophilic and piezophilic. Optimal growth occurs at a NaCl concentration of about 3 %. No growth occurs in the absence of NaCl. Optimal temperature and pressure for growth are 6 °C and 60 MPa, respectively. No growth occurs at atmospheric pressure. Facultatively anaerobic, chemo-organotroph, having both respiratory and fermentative types of metabolism. Catalase- and cytochrome oxidase-positive, nitrate is reduced to nitrite and nitrite is reduced to nitrogen. Gelatin is not hydrolysed, and amylase, H2S production and indole production are negative. The DNA G+C content of the type strain is 39.1 mol%. The major isoprenoid quinone is Q-8. Predominant cellular fatty acids are C14 : 1, C16 : 0, C16 : 1 and C22 : 6. Other characteristics are shown in Table 1.
The type strain is strain K41GT (=JCM 11830T=ATCC BAA-638T), which was isolated from sediment collected from the bottom of the Japan Trench.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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DeLong, E. F., Franks, D. G. & Yayanos, A. A. (1997). Evolutionary relationships of cultivated psychrophilic and barophilic deep-sea bacteria. Appl Environ Microbiol 63, 2105–2108.[Abstract]
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Kawasaki, K., Nogi, Y., Hishinuma, M., Nodasaka, Y., Matsuyama, H. & Yumoto, I. (2002). Psychromonas marina sp. nov., a novel halophilic, facultatively psychrophilic bacterium isolated from the coast of the Okhotsk Sea. Int J Syst Evol Microbiol 52, 1455–1459.[Abstract]
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Xu, Y., Nogi, Y., Kato, C., Liang, Z., Rüger, H.-J., Kegel, D. D. & Glansdorff, N. (2003). Psychromonas profunda sp. nov., a psychropiezophilic bacterium from deep Atlantic sediments. Int J Syst Evol Microbiol 53, 527–532.
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), 6 °C (
) and 10 °C (
). Growth rates were determined based on optical density. td, Doubling time (h).