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1 The DEEPSTAR Group, Japan Marine Science and Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
2 Marine Ecosystems Research Department, Japan Marine Science and Technology Center (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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Published online ahead of print on 12 March 2004 as DOI 10.1099/ijs.0.03049-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Y223GT is AB094412.
A 16S rRNA gene-based phylogenetic tree showing the relationship between Colwellia piezophila and piezophilic bacteria within the 
-Proteobacteria is available as supplementary material in IJSEM Online.
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; Deming & Baross, 1993; Kato et al., 1995; Yayanos et al., 1979). Piezophilic bacteria, as defined by Yayanos (2001), are bacteria that have their maximum growth rate at high pressure over all permissible temperatures. All psychropiezophilic bacteria isolated to date are members of the -Proteobacteria, according to phylogenetic classifications based on 16S rRNA gene sequence information. Nogi et al. (2002) reported that cultivated psychropiezophilic deep-sea bacteria were affiliated with one of five genera within the -Proteobacteria: Shewanella, Photobacterium, Colwellia, Moritella and Psychromonas.
In this paper, results of taxonomic studies on obligately piezophilic strains isolated from the deepest cold-seep environment in the Japan Trench are presented. Several lines of evidence indicate that two of these isolates, strains Y223GT and Y251E, represent a novel species within the genus Colwellia. In the genus Colwellia, the only deep-sea piezophilic species reported is Colwellia hadaliensis strain BNL-1T (Deming et al., 1988), although no public culture collection maintains C. hadaliensis. Species of the genus Colwellia are defined as facultative anaerobes and psychrophilic bacteria (Deming et al., 1988). Bowman et al. (1998) reported that Colwellia species produce docosahexaenoic acid (DHA; C22 : 6
3). Psychropiezophilic Shewanella and Photobacterium strains produce eicosapentaenoic acid (EPA; C20 : 53), Moritella strains produce DHA (Nogi & Kato, 1999; Kato & Nogi, 2001; Nogi et al., 1998a) and Psychromonas kaikoae produces both EPA and DHA (Nogi et al., 2002). Generally, psychropiezophilic bacteria produce either one or both of these long-chain polyunsaturated fatty acids (PUFAs). However, unlike other psychropiezophilic bacteria, our isolates did not produce either EPA or DHA in the membrane layer. Based on the taxonomic differences observed, the isolated strains appear to represent a novel obligately piezophilic Colwellia species, for which the name Colwellia piezophila sp. nov. is proposed.
Deep-sea sediment samples were collected by means of sterilized mud samplers (Ikemoto & Kyo, 1993) on the submersible Shinkai 6500 from the bottom of a small deep-sea canyon on the Japan Trench (40° 2·8' N 144° 16·6' E) at a depth of 6278 m during cruise YK 01-06, dive #6K-621. The mud samples were taken by the submersible's manipulator and put into the sample holder of the sterilized sampler. The samples were then carried to the sea surface without changing temperature, but with a change in pressure. A part of each sample was cultivated in marine broth 2216 (Difco Laboratories) at 4 °C and 60 MPa in a pressure vessel for about 2 weeks. Strains Y223GT and Y251E were isolated using the low-melting agar method (Kato et al., 1995). Colwellia maris JCM 10085T (Yumoto et al., 1998) and Colwellia psychrerythraea ATCC 27364T (Deming et al., 1988) were used as reference strains. These bacteria were maintained in marine broth 2216. C. maris was grown at 15 °C and atmospheric pressure, C. psychrerythraea was grown at 10 °C and atmospheric pressure and the isolated piezophilic strains were grown at 10 °C and 60 MPa. High-pressure cultivation utilized a liquid hydraulic system. Piezophilic bacteria were cultivated in plastic bags containing liquid medium in a pressure vessel (made of stainless steel SUS304). To supply oxygen to the cultures for the optimal growth pressure and temperature tests and the O/F test, fluorinert (FC-72; Sumitomo-3M) saturated with gas was added (20 % total volume). This culture method followed a previously reported procedure (Kato et al., 1994; Yanagibayashi et al., 1999).
Optimal growth pressure and temperature were determined by optical density. Cells were counted and cell form was confirmed microscopically in marine broth 2216 under aerobic conditions at each pressure and temperature tested.
Physiological tests were performed using a slight modification of the general procedures described by Barrow & Feltham (1993) and DeLong et al. (1997). All high-pressure physiological tests were performed in tandem with uninoculated blank controls according to the following procedure. Acid production from sugars was assessed using modified O/F medium (Hugh & Leifson, 1953) containing 0·5x artificial sea water (1·5 % NaCl, 0·035 % KCl, 0·54 % MgCl2.6H2O, 0·27 % MgSO4.7H2O, 0·05 % CaCl2.2H2O), 0·05 % NH4H2PO4, 0·005 % yeast extract, 0·1 % Na2CO3, 1 % sugar and 0·003 % bromothymol blue. The pH of the medium was adjusted to 7·1 at 20 °C. After capping, the tube was sealed with Parafilm and incubated at 60 MPa and 10 °C for several days (Nogi et al., 2002). Physiological tests under high-pressure conditions to examine hydrogen sulfide production from thiosulfate and the production of indole, oxidase and catalase were performed according to previously described methods (Nogi & Kato, 1999). Gelatinase, protease and amylase activities were detected in ultrasonically treated cultured cells.
Cells of strain Y223GT were Gram-negative rods, 2·0–3·0 µm long and 0·8–1·0 µm wide, motile by means of a single unsheathed polar flagellum. This strain was unable to grow at atmospheric pressure at 2–15 °C, although it grew well in pressure vessels under hydrostatic pressures of 10–80 MPa at 4 °C and 40–80 MPa at 10 °C. No growth occurred at 15 °C under any pressure examined. The most rapid growth rate (about 0·14 h–1) was observed at 60 MPa and 10 °C (Fig. 1a), which compares well to rates observed for the closely related obligate piezophile C. hadaliensis (0·12 h–1 at 90 MPa and 10 °C; Deming et al., 1988). The C. psychrerythraea reference strain was not able to grow under such high-pressure conditions (Fig. 1b) and the growth rate of C. maris showed greater pressure sensitivity.
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. Strains Y223GT and Y251E are facultatively anaerobic chemo-organotrophs, displaying both respiratory and fermentative types of metabolism. Other characteristics of Y223GT and Y251E are as follows. Acid, but not gas, is produced from D-glucose, maltose and xylose. Catalase and cytochrome oxidase test results are positive and gelatin is hydrolysed. Nitrate is reduced to nitrite, but nitrite is not reduced. Test results for protease, chitinase, amylase, H2S and indole production are negative. The following compounds are not utilized: L-arabinose, cellobiose, D-fructose, D-galactose, glycerol, myo-inositol, D-lactose, D-mannitol, D-mannose, D-raffinose, L-rhamnose, D-sorbitol, sucrose and D-trehalose. The G+C content of the DNA is 38·5–39·1 mol%. The major isoprenoid quinone is Q-8 (ubiquinone-8). The C. maris reference strain shares some physiological characteristics with the isolated strains, including the profile of carbohydrates utilized. However, unlike C. maris, the two Japan Trench strains do not hydrolyse starch, reduce nitrite or produce acid from cellobiose, and are unable to grow at atmospheric pressure. Although very few taxonomic characteristics of piezophilic C. hadaliensis are described in the literature (Deming et al., 1988), the main characteristics in which it differs from the newly isolated strains are its ability to hydrolyse chitin and its DNA G+C content of 45·7 mol%.
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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_W (Thompson et al., 1994). 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 are shown in Fig. 2 (see also the phylogenetic tree available as supplementary material in IJSEM Online). Strains Y223G T and Y251E fall into the genus Colwellia and are closely related to the psychrophilic strain C. maris. For analysis of relatedness, DNA–DNA hybridization was carried out at 40 °C for 4 h and measured fluorometrically using the method of Ezaki et al. (1989). A high level of DNA–DNA relatedness (98–100 %) was found between strains Y223GT and Y251E. The relatedness between Y223GT or Y251E and the C. maris reference strain was less than 49 %, as indicated by the hybridization values obtained. This is significantly lower than that accepted as the phylogenetic definition of a species (Wayne et al., 1987). This and other results shown in Table 1
and Fig. 2 suggest that strains Y223GT and Y251E represent a novel Colwellia species.
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. The major fatty acids in strains Y223GT and Y251E were C16 : 0 (hexadecanoic acid) and C16 : 1 (hexadecenoic acid). Only a low level of similarity was observed between this fatty acid profile and those of the type strains of C. maris and C. psychrerythraea. For example, the predominant components in the fatty acid profile of the C. maris type strain differed from those in the profile of strains Y223GT and Y251E, which contained substantial amounts of C14 : 0 (tetradecanoic acid), C17 : 0 (heptadecanoic acid) and C17 : 1 (heptadecenoic acid). The type strain of C. psychrerythraea had low levels of the long-chain PUFAs EPA and DHA, unlike strains Y223GT and Y251E, which did not contain these fatty acids. Psychropiezophilic Shewanella and Photobacterium strains produce EPA (Nogi et al., 1998b, c), Moritella strains produce DHA (Nogi & Kato, 1999) and P. kaikoae produces both EPA and DHA (Nogi et al., 2002). Generally, psychropiezophilic bacteria are believed to produce EPA and/or DHA. However, Allen et al. (1999) reported that monounsaturated fatty acids, but not PUFAs, are required for growth of the piezophilic bacterium Photobacterium profundum SS9, based on analysis of pressure-sensitive mutants. In their mutation experiment, C18 : 1 fatty acid was necessary for growth under low-temperature and/or high-pressure conditions. However, our strains did not contain any monounsaturated fatty acids longer than C16 : 1. Therefore, our isolates differ from previously known psychropiezophilic bacteria in that most of the unsaturated fatty acids of these isolates have been identified as C16 : 1 (Table 2).
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; Nogi & Kato, 1999; Nogi et al., 1998a, b, c, 2002); each is affiliated with one of five genera within the -Proteobacteria: Shewanella, Photobacterium, Colwellia, Moritella and Psychromonas (DeLong et al., 1997; Nogi et al., 2002). The first obligately piezophilic bacterium, strain MT41 isolated from the Mariana Trench, was later shown to be a member of the genus Colwellia (Yayanos et al., 1981; DeLong et al., 1997). Since then, four obligately piezophilic bacterial strains have been isolated: C. hadaliensis BNL-1T (Deming et al., 1988), Shewanella benthica DB21MT-2, Moritella yayanosii DB21MT-5T (Nogi & Kato, 1999) and P. kaikoae JCM 11054T (Nogi et al., 2002). A novel obligately piezophilic species within Colwellia, based on the results of phylogenetic analysis of 16S rRNA gene sequences and several other taxonomic properties described in this paper, is therefore proposed. On the basis of the phenotypic, genotypic and phylogenetic data, it is logical to conclude that our isolates are members of the genus Colwellia and that these deep-sea isolates represent a novel species within this genus. The name Colwellia piezophila sp. nov. is proposed, with strain Y223GT (=JCM 11831T=ATCC BAA-637T) as the type strain.
Description of Colwellia piezophila sp. nov.
Colwellia piezophila (piez.o.phi'la. Gr. v. piezo to press; Gr. adj. philos loving; N.L. fem. adj. piezophila loving pressure).
Cells are Gram-negative rods, 2·0–4·0x0·8–1·0 µm, motile by means of a single unsheathed polar flagellum. Halophilic, psychrophilic and piezophilic. Optimal growth occurs at an NaCl concentration of about 3 %. No growth occurs in the absence of NaCl. The optimal temperature and pressure for growth are 10 °C and 60 MPa, respectively. No growth occurs at atmospheric pressure and 2–15 °C or at 15 °C under any pressure. Facultatively anaerobic chemo-organotroph, having both respiratory and fermentative types of metabolism. Catalase and cytochrome oxidase test results are positive, gelatin is hydrolysed, nitrate is reduced to nitrite, but nitrite is not reduced. Amylase, protease, H2S production and indole production are negative. The DNA G+C content of the type strain, Y223GT (=JCM 11831T=ATCC BAA-637T), is about 39·1 mol%. The major isoprenoid quinone is Q-8. The predominant cellular fatty acids are C16 : 0 and C16 : 1. Other characteristics are shown in Table 1.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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) and 10 °C (
). (b) Pressure-dependent growth rate of C. psychrerythraea at 10 °C (optimal temperature) (
) and of C. maris at 15 °C (optimal temperature) (
). Growth rates were determined by optical density; td, doubling time (h).