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Extremobiosphere Research Center of Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima, Yokosuka 237-0061, Japan
Correspondence
Atsushi Kurata
akurata{at}jamstec.go.jp
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). A. amylolytica N10T was isolated from Lake Chahannor (39° 14' N 108° 04' E) located in Inner Mongolia Autonomous Region, China. A. delamerensis 1E1T was isolated from Lake Elmenteita (0° 25' S 36° 15' E) in Kenya, East Africa. Both micro-organisms are Gram-negative, slightly halophilic, alkaliphilic and mesophilic bacteria and belong to the class Gammaproteobacteria (formerly the gamma-3 subdivision of the Proteobacteria). The first characterization of a NhaD-type Na+/H+ antiporter from a soda lake bacterium was from A. amylolytica N10T (Liu et al., 2005).
Collagenolytic enzymes are widely used in several industries (Watanabe, 2004) to hydrolyse proteins, including native collagen and gelatin. Collagen is produced in large quantities as a by-product in the livestock industry, and recently it has been shown that peptides derived from collagen possess physiological activities that are useful in food and medical products (Khare et al., 1995; Minakuchi et al., 2004; Myers et al., 1993). Over the last two decades, collagenolytic enzymes produced by various micro-organisms have been studied extensively (Bond & Van Wart, 1984; Hisano et al., 1989; Nakayama et al., 2000; Okamoto et al., 2001; Sasagawa et al., 1995; Watanabe, 2004), but there have been few reports of collagenolytic enzyme-producing micro-organisms isolated from deep-sea sediment.
In this study, we describe a psychrotolerant, obligately alkaliphilic, collagenolytic enzyme-producing bacterium that was isolated from a deep-sea sediment. Based on phylogenetic analysis of its 16S rRNA gene sequence, DNA–DNA hybridization data and phenotypic characteristics, this organism is shown to belong to the genus Alkalimonas. This study describes the first novel species of the genus Alkalimonas to be isolated from a deep-sea sediment.
Deep-sea sediment was collected off Torishima Island (30° 55.050' N 141° 48.980' E), Japan, at a depth of 4026 m in July 2005. An aliquot of the sample was spread on an alkaline plate consisting of (w/v) 1.0 % collagen from bovine Achilles tendon, type I (Sigma-Aldrich), 0.1 % bonito extract (Wako Pure Chemical), 0.5 % yeast extract (Difco), 1.0 % NaCl, 0.2 % MgSO4.7H2O, 0.02 % K2HPO4, 1.0 % NaHCO3 (sterilized separately) and 1.5 % agar (pH 9.0). After incubation at approximately 4 °C for 1 month, micro-organisms having collagenolytic activity had grown on the plate with a zone of collagen dissolution around the colonies. The isolate was subsequently purified three times on alkaline plates at 15 °C. This isolate, designated strain AC40T, was maintained on an alkaline plate without collagen at approximately 4 °C and also stored at –80 °C in alkaline broth supplemented with an equal volume of 30 % (v/v) glycerol instead of agar.
To determine phylogenetic relationships, 
1.5 kbp of the 16S rRNA gene sequence was amplified from a single colony of strain AC40T by PCR in a DNA thermal cycler (Gene Amp PCR system 9700; ABI PRISM) with the eubacterial primers 27f and 1525r and LA Taq DNA polymerase (Takara Bio). Sequencing of the gene was performed with a Dye Terminator Sequencing kit (ABI PRISM) and a DNA sequencer (model 377; ABI PRISM). Nucleotide substitution rates (Knuc; Kimura, 1980) were determined and a distance matrix tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) with the CLUSTAL_X program (Thompson et al., 1997). Alignment gaps and unidentified base positions were not taken into consideration for the calculations. The topology of the phylogenetic tree was evaluated by performing a bootstrap analysis with 1000 replications. The results of phylogenetic analysis based on 16S rRNA gene sequence data are shown in Fig. 1; strain AC40T clearly falls within the genus Alkalimonas. The nucleotide sequence of the 16S rRNA gene of strain AC40T was most closely related to those of A. amylolytica N10T and A. delamerensis 1E1T with similarities of 96.7 and 97.9 %, respectively. The generally recommended and accepted criterion for delineating bacterial species states that strains with a 16S rRNA gene sequence showing a dissimilarity of greater than 3 % are considered to belong to separate species (Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002). Thus, DNA–DNA hybridization experiments of strain AC40T with A. delamerensis 1E1T were carried out as described by Ezaki et al. (1989). Strain AC40T shared only 30 % DNA–DNA relatedness at the whole genome level with A. delamerensis 1E1T. This result suggested that strain AC40T represented a novel species of the genus Alkalimonas.
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and Baumann et al. (1972) except regarding the use of alkaline broth. Cells of strain AC40T were strictly aerobic, Gram-negative rods (0.6–0.8x1.1–2.2 µm) that were motile by means of a single polar flagellum, as shown in Fig. 2. Endospores were not detected. Strain AC40T formed creamy white, smooth, circular, convex colonies on the alkaline plates. Tests for cell growth were performed by monitoring turbidity at OD660. Strain AC40T grew over the temperature range 5–37 °C with optimal growth at 33 °C. It grew at initial pH values of 7.0–10.5 with optimal growth at pH 8.5–10.0. It did not grow at below pH 7.0, suggesting the strain belongs to the obligate alkaliphiles as defined by Horikoshi (1999). The optimum NaCl concentration for growth was 1 % (w/v), although the strain was able to grow at concentrations from 0 to 10 % NaCl. Carbohydrate utilization was investigated based on acid production, which was assessed by using a modified OF basal medium (Hugh & Leifson, 1953) consisting of artificial seawater (1.5 % NaCl, 0.035 % KCl, 0.54 % MgCl2.6H2O, 0.27 % MgSO4.7H2O, 0.05 % CaCl2.2H2O), 0.05 % (NH4)2SO4, 0.01 % yeast extract, 0.05 % Tris, 1.0 % carbohydrate (sterilized separately) and 0.003 % bromothymol blue (pH 7.1) at 33 °C. Strain AC40T utilized and produced acid from cellobiose, D-glucose, maltose, D-mannose and sucrose, but did not produce acid from L-arabinose, D-fructose, D-galactose, glycerol, myo-inositol, D-lactose, D-mannitol, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose, xylose or palatinose. The strain was positive for hydrolysis of gelatin, casein, starch, tributyrin and Tweens 20, 40 and 80. It was positive for catalase and oxidase. It was negative for reduction of nitrate to nitrite and formation of indole and H2S. Antibiotic sensitivity was tested with a BD Sensi-Disc (Becton Dickinson) according to the manufacturer's instructions. Plates were incubated at 33 °C for 2 days. Strain AC40T was susceptible to ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), nalidixic acid (30 µg), penicillin (10 U) and streptomycin (10 µg) but was resistant to gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), novobiocin (30 µg) and tetracycline (30 µg).
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). The isoprenoid quinone of strain AC40T was ubiquinone-8 (Q-8). The G+C content in the total DNA of strain AC40T was 49.3 mol%, as determined by reversed-phase HPLC (Tamaoka & Komagata, 1984). The morphological and physiological characteristics of strain AC40T are given in Table 1.
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. The cellular fatty-acid profile of strain AC40T was dominated by saturated and mono-unsaturated straight-chain components (C16 : 0 and C18 : 1), as detailed in Table 2, which is one of the distinctive features of the genus Alkalimonas (Ma et al., 2004).
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Description of Alkalimonas collagenimarina sp. nov.
Alkalimonas collagenimarina (col.la.ge.ni.ma.ri'na. N.L. n. collagenum collagen; L. adj. marinus -a -um of the sea, marine; N.L. fem. adj. collagenimarina collagenolytic bacterium isolated from the sea).
Cells are Gram-negative, strictly aerobic, short rods (0.6–0.8x1.1–2.2 µm). Motile by means of a single polar flagellum. Obligately alkaliphilic, chemo-organotrophic and psychrotolerant. On alkaline plates, colonies are creamy white, smooth, circular and convex. After incubation at 33 °C for 24 h, colonies are approximately 2–4 mm in diameter. Growth occurs at 5–37 °C. Optimum growth temperature is 33 °C. The pH range for growth is 7.0–10.5. Optimum pH for growth is 8.5–10.0. Growth occurs at 0–10 % (w/v) NaCl. NaCl optimum is 1 %. Catalase- and cytochrome oxidase-positive. Neither H2S nor indole are produced. Nitrate and nitrite are not reduced. Positive for amylase, lipase, gelatinase, protease and collagenolytic enzyme, but negative for agarase and DNase. Positive for hydrolysis of tributyrin and Tweens 20, 40 and 80. Utilizes cellobiose, D-glucose, maltose, D-mannose and sucrose with acid production. L-Arabinose, D-galactose, myo-inositol, D-lactate, D-mannitol, D-raffinose, D-sorbitol, xylose, D-fructose, glycerol, L-rhamnose, D-trehalose and palatinose are not utilized. The major isoprenoid quinone is Q-8. The predominant cellular fatty acids are C16 : 0 and C18 : 1. The presence of C14 : 1 in this species is unique among members of the genus. The DNA G+C content is 49.3 mol%. Susceptible to ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), nalidixic acid (30 µg), penicillin (10 U) and streptomycin (10 µg), but resistant to gentamicin (10 µg), kanamycin (30 µg), neomycin (30 µg), novobiocin (30 µg) and tetracycline (30 µg).
The type strain, AC40T (=JCM 14267T=NCIMB 14266T), was isolated from deep-sea sediment off Torishima Island, Japan, at a depth 4026 m.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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