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1 Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch, Russian Academy of Sciences, 690022 Vladivostok, Prospekt 100 Let Vladivostoku, 159, Russia
2 Institute of Marine Biology, Far-Eastern Branch, Russian Academy of Sciences, 690041 Vladivostok, Russia
3 GBF – Gesellschaft für Biotechnologische Forschung, D-38124 Braunschweig, Germany
4 Institute of Microbiology, Russian Academy of Sciences, 117811 Moscow, Russia
5 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Erko Stackebrandt
Erko{at}dsmz.de
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL accession number for the 16S rDNA sequence of strain KMM 241T is AJ488501.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Members of Glaciecola have been isolated from sea-ice diatom assemblage samples collected from coastal areas of eastern Antarctica and seem to be restricted to sea-ice habitats (Bowman et al., 1998). The genus Glaciecola is phylogenetically closely related to Alteromonas macleodii within the 
-subclass of Proteobacteria. Alteromonas macleodii was originally described by Baumann et al. (1972) and the genus was later emended (Gauthier et al., 1995). Strain KMM 241T was isolated from internal liquor of a specimen of the ascidian Halocynthia aurantium, collected from coastal sea water at a depth of 5 m in Troitsa Bay, Peter the Great Bay, Sea of Japan, Russia, in May 1990. Strain KMM 642 was isolated from tissue of the sponge Plocamia sp., at a depth of 350 m near the Komandorskie Islands, east of Kamchatka, in August 1991. A homogenate of the internal tissue was diluted aseptically in sterile sea water. Aliquots of internal liquor and diluted homogenate were spread on sea-water medium (SWM) agar plates (l-1: 5·0 g peptone, 2·5 g yeast extract, 1·0 g glucose, 0·2 g K2HPO4, 0·05 g MgSO4, 500 ml seawater, 500 ml distilled water, 15·0 agar) and incubated for 7 days at 28 °C. Bacteria were stored at -80 °C in 30 % (v/v) glycerol. Strains KMM 241T and KMM 642 have been deposited in the Collection of Marine Micro-organisms (KMM), Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia. The strains were routinely grown on Marine 2216 agar (MA, Difco), Marine broth (MB, Difco) and SWM at 7–30 °C.
Gram-reaction, oxidase, catalase and production of amylase, caseinase, DNase, gelatinase and lipase (Tween 80) were tested according to the methods described by Baumann et al. (1984) and Smibert & Krieg (1994), using MA or SWM as the basal medium. Hydrolysis of 
-carrageenan was determined as described by Yaphe & Baxter (1955). Growth at different temperatures (4–40 °C) and pH values (5·0–10·0) was tested using MA and MB; sodium ion requirement and tolerance of various NaCl concentrations (0–12 % NaCl) were assessed using SWM prepared from the artificial sea-water base supplemented with the appropriate amount of NaCl. Motility was examined by the hanging-drop method. Leifson's medium was used for testing acid production from 1 % (w/v) carbohydrates (Leifson, 1963).
Additional biochemical tests using the API 20NE test kit (bioMérieux) and the Biolog GN MicroPlate method were performed as described by the manufacturers, except that strains were suspended in 3 or 2·5 % NaCl. Cell morphology was examined by transmission electron microscopy from exponential-phase cells grown in MB. The cells were fixed with 1 % (v/v) glutaraldehyde and negatively stained with 4 % (w/v) aqueous uranyl acetate and carbon-film. Samples were examined by a Zeiss TEM910 transmission electron microscope at an acceleration voltage of 80 kV at calibrated magnifications. For determination of cellular fatty acid composition, strains KMM 241T, KMM 642 and A. macleodii ATCC 27126T were grown on MA at 15 and 28 °C for 2 days. Whole-cell fatty acids and phospholipids were examined according to the procedures described by Svetashev et al. (1995) and Ivanova et al. (2000). DNA base composition was determined as described by Marmur & Doty (1962) and Owen et al. (1969). Optimal DNA–DNA relatedness was measured spectrophotometrically in 2x SSC with 10 % DMSO at 68 °C, using the initial reassociation rate kinetic procedure (De Ley et al., 1970). Extraction of genomic DNA, PCR-mediated amplification of the 16S rDNA and sequencing of PCR products were carried out as described by Rainey et al. (1996). Purified PCR products were sequenced directly using the Taq DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. An Applied Biosystems 310 DNA Genetic Analyzer was used for electrophoresis of the sequence reaction products. The 16S rDNA sequence of KMM 241T was compared with nucleotide sequences of the closest known relatives that were retrieved from the GenBank/EMBL databases. Phylogenetic dendrograms were reconstructed according to the method of De Soete (1983) and the neighbour-joining and maximum-likelihood methods (Felsenstein, 1993).
Cultural properties, cell morphology (Fig. 1), motility and the results of some basic physiological tests of strains KMM 241T and KMM 642 are described in the species description below. Cells were motile with a single unsheathed polar flagellum. Strains required sodium ions for growth and grew in 1–8 % NaCl; they grew at 7–35 °C but not at 4 or above 37 °C. Respective optima are indicated in the species description. Phenotypic properties of the novel strains and some reference organisms are compared in Table 1. Strains KMM 241T and KMM 642 differ from each other in acid production and utilization of some carbohydrates. Clear differences were observed between the novel isolates and the type strains of Glaciecola and Alteromonas species in the patterns of utilization of carbohydrates and amino acids.
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7c (35·4–43·0 %) and C18 : 17c (5·2–10·1 %) (Table 2). These profiles are similar to those of Alteromonas and Glaciecola strains, but the latter differ in the predominance of monounsaturated fatty acids over saturated fatty acids (Bowman et al., 1998). It should be noted that the fatty acid composition of strains KMM 241T and KMM 642 depended on the growth temperature. On decreasing the growth temperature from 28 to 15 °C, the proportions of C17 : 18c and C 18 : 17c increased from 3·2–2·8 to 6–8 % and from 5 to 13 % (for strain KMM 241T), respectively, while the relative amounts of C16 : 0 and C16 : 17c were constant or changed only slightly. A. macleodii did not show noticeable differences in the content of C17 : 18c and C18 : 17c when grown at different temperatures (Table 2).
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The DNA G+C contents of KMM 241T and KMM 642 were respectively 44·8 and 44·6 mol%. DNA–DNA hybridization experiments between the two strains showed DNA binding of 71 %.
16S rDNA sequence analysis revealed that the novel isolate KMM 241T is a phylogenetic neighbour of Glaciecola sp. strain HA02, described as decomposing Laminaria thallus (99·7 % similarity; accession number AB049729). Strain KMM 241T is phylogenetically closely related to G. punicea and G. pallidula (respectively 94·5 and 94·8 % sequence similarity) and somewhat less closely related to A. macleodii (93·4 % similarity). The 16S rRNA gene sequence similarity between G. pallidula and G. punicea was 93·3 %. Different algorithms consistently placed strain KMM 241T adjacent to the two Glaciecola type strains and the neighbour-joining dendrogram is shown in Fig. 2. The branching is supported by bootstrap values above 75 %.
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) and chemotaxonomic (Table 2) characteristics of the novel isolates are consistent with the description of Glaciecola species, including motility, requirement for sodium ions for growth, absence of growth in 10 % NaCl, the presence of oxidase, catalase and 
-galactosidase activities, 
-galactosidase production, the ability to hydrolyse aesculin, the lack of gelatinase, caseinase and urease activities, weak or delayed acid production from D-glucose, D-galactose and melibiose, C16 : 0, C16 : 17c and C18 : 17c as major fatty acids and a G+C content of 44·6–44·8 mol%.
The novel bacteria can be distinguished from Glaciecola species by the following characteristics: the inability to produce pigments, to form filaments and to grow at 0–4 °C and the ability to degrade agar, carrageenan and DNA (weakly) and to utilize D-glucose, sucrose, D-mannitol, glycyl L-glutamic acid, cellobiose, maltose, D-galactose, D-fructose, D-mannose, L-serine, D-trehalose and -hydroxybutyric acid.
On the basis of their phenotypic, chemotaxonomic, genomic and phylogenetic characteristics, we conclude that strains KMM 241T and KMM 642 should be placed in the same species within the genus Glaciecola as Glaciecola mesophila sp. nov.
Description of Glaciecola mesophila sp. nov.
Glaciecola mesophila (me.so'phi.la. Gr. adj. mesos medium; Gr. adj. philos loving; N.L. fem. adj. mesophila medium-temperature-loving, mesophilic).
Aerobic, Gram-negative, oxidase- and catalase-positive, non-pigmented, non-spore-forming, ovoid or curved rod-shaped cells, 1·2–1·5 µm long and 0·6–0·8 µm in diameter, motile with single unsheathed polar flagella. Does not form filaments. On MA, forms smooth, convex, non-pigmented colonies, translucent or whitish with regular edges, 3–5 mm in diameter, depressed into the agar. Slightly halophilic. Requires sodium ions for growth (1–8 % NaCl), optimum between 2 and 5 %; no growth in 10 % NaCl. Growth occurs between 7 and 35 °C, but not at 4–5 or 40 °C; optimum between 25 and 28 °C. The pH range is 5·5–9·5, optimum at pH 8·0–8·5. Chemo-organoheterotroph. Positive for lipase, amylase and -galactosidase and -galactosidase, decomposition of agar and carrageenan. Produces acid weakly from carbohydrates. Other phenotypic and biochemical tests are listed in Table 1. Predominant fatty acids are C16 : 0, C16 : 17c and C18 : 17c. Phospholipids include phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and bis-phosphatidic acid. The G+C content of the DNA is 44·6–44·8 mol% (thermal denaturation method).
The type strain, strain KMM 241T (=DSM 15026T), was isolated from the ascidian Halocynthia aurantium in coastal sea water of the Sea of Japan. A reference strain, strain KMM 642, was isolated from a sponge Plocamia sp. at a depth of 350 m near the Komandorskie Islands.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Baumann, P., Gauthier, M. J. & Baumann, L. (1984). Genus Alteromonas Baumann, Baumann, Mandel and Allen 1972, 418AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 343–352. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Bowman, J. P., McCammon, S. A., Brown, J. L. & McMeekin, T. A. (1998). Glaciecola punicea gen. nov., sp. nov. and Glaciecola pallidula gen. nov., sp. nov.: psychrophilic bacteria from Antarctic sea-ice habitats. Int J Syst Bacteriol 48, 1213–1222.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
De Soete, G. (1983). A least square algorithm for fitting additive trees to proximity data. Psychometrika 48, 621–626.[CrossRef]
Felsenstein, J. (1993). PHYLIP (phylogenetic inference package) version 3.5.1. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Gauthier, G., Gauthier, M. & Christen, R. (1995). Phylogenetic analysis of the genera Alteromonas, Shewanella, and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and proposal of twelve new species combinations. Int J Syst Bacteriol 45, 755–761.
Ivanova, E. P., Zhukova, N. V., Svetashev, V. I., Gorshkova, N. M., Kurilenko, V. V., Frolova, G. M. & Mikhailov, V. V. (2000). Evaluation of phospholipid and fatty acid compositions as chemotaxonomic markers of Alteromonas-like proteobacteria. Curr Microbiol 41, 341–345.[CrossRef][Medline]
Leifson, E. (1963). Determination of carbohydrate metabolism of marine bacteria. J Bacteriol 85, 1183–1184.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
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Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–655. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Svetashev, V. I., Vysotskii, M. V., Ivanova, E. P. & Mikhailov, V. V. (1995). Cellular fatty acids of Alteromonas species. Syst Appl Microbiol 18, 37–43.
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