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1 Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch, Russian Academy of Sciences, 690022 Vladivostok, Prospect 100 Let Vladivostoku, 159, Russia
2 Laboratory of Food Science and Technology, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Göteborg, Sweden
4 Institute of Marine Biology, Far-Eastern Branch, Russian Academy of Sciences, 690041, Vladivostok, Russia
Correspondence
Lyudmila A. Romanenko
piboc{at}stl.ru
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ABSTRACT |
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9c, C17 : 18c and C18 : 17c. The DNA G+C content was 49·6 mol%. 16S rRNA gene sequence analysis revealed that strain KMM 1406T was related closely to Rheinheimera baltica DSM 14885T within the 
-Proteobacteria, with 96·8 % sequence similarity. On the basis of phenotypic and molecular data, a novel species, Rheinheimera pacifica sp. nov., is proposed. The type strain is KMM 1406T (=IAM 15043T=JCM 12090T=NRIC 0539T=CCUG 46544T).
-D-glucopyranosideThe GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of KMM 1406T is AB073132.
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MAIN TEXT |
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-Proteobacteria. Among them, the recently described genus Rheinheimera, which currently comprises a single species, Rheinheimera baltica DSM 14885T, was proposed for motile, blue-coloured, non-halotolerant bacteria isolated from Baltic sea-water samples (Brettar et al., 2002
). Phylogenetic relatives of R. baltica DSM 14885T were a clinical isolate of Alishewanella fetalis (Fonnesbech Vogel et al., 2000) with sequence similarity of 94·8 % and, more closely related (96·5–96·8 % sequence similarity), some deep-sea bacteria that were reported by Takami et al. (1999) (Brettar et al., 2002). During the course of studying biodiversity of deep-sea micro-organisms of the Pacific, we isolated the motile, non-pigmented, halotolerant bacterium KMM 1406T and characterized it by using polyphasic taxonomy. Several phenotypic and physiological properties of the new deep-sea isolate were consistent with those of R. baltica. 16S rRNA gene sequence analysis confirmed the affiliation of strain KMM 1406T to the genus Rheinheimera. Sequence similarity with its close phylogenetic relative, R. baltica DSM 14885T, was 96·8 %. Based on phenotypic features and genetic analysis, we concluded that strain KMM 1406T represents a novel species within the genus Rheinheimera, for which the name Rheinheimera pacifica sp. nov. is proposed.
Strain KMM 1406T was isolated from a sea-water sample that was obtained from a depth of 5000 m in the north-western part of the Pacific Ocean in July 1985. The sea-water sample was taken by a plastic hydrological bathometer. Aliquots (100 µl) of sea water were spread onto agar plates of sea-water medium (SWM) that contained (l-1): 5·0 g peptone, 2·5 g yeast extract, 1·0 g glucose, 0·2 g K2HPO4, 0·05 g MgSO4, 750 ml sea water, 250 ml distilled water and 15·0 g agar. Inoculated plates were incubated for 10 days at 28 °C. The bacterium was cultivated aerobically on marine agar 2216 (MA), marine broth (MB; Difco) or tryptic soy agar (TSA) at 25–28 °C and stored at -80 °C in liquid medium supplemented with 30 % (v/v) glycerol. Strain KMM 1406T has been deposited in the Collection of Marine Microorganisms (KMM) of the Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia. The type strains of Rheinheimera baltica (DSM 14885T) and Alishewanella fetalis (CCUG 30811T) were used in this study for comparison. Cell morphology was examined with a phase-contrast microscope. Motility was observed by the hanging-drop method. Cell morphology was examined by transmission electron microscopy on exponential-phase cells that were grown in TSB. Cells were negatively stained with potassium phosphotungstate (1 %, w/v; pH 7·0). Gram-reaction, oxidase and catalase activity, nitrate reduction and production of caseinase, deoxyribonuclease, gelatinase and lipase (Tween 80) were tested according to standard methods described by Smibert & Krieg (1994). Hydrolysis of starch was determined after 2 days incubation on nutrient agar medium that contained 10 % (w/v) soluble starch by flooding plates with 1 % (w/v) iodine solution. Formation of H2S from thiosulfate was detected with lead acetate paper. Ability to grow at different temperatures was tested on MA and TSA at 4, 10, 15, 25, 30, 35, 37 and 42 °C. The pH range for growth (5·0–10·0) was tested by using MB with pH values adjusted by addition of 5 M NaOH or HCl. Sodium ion requirement and tolerance of various NaCl concentrations were examined by using SWM prepared with artificial sea water with NaCl concentrations of 0, 1, 3, 5, 8, 10, 12, 15 and 20 % (w/v). The strain was tested for its ability to ferment glucose and produce acid from carbohydrates with a supplement of 1 % (w/v) of each compound by the use of marine oxidation–fermentation medium (Leifson, 1963). The ability to utilize various compounds as sole carbon and energy sources was examined in a mineral liquid medium that contained (g l-1): NH4Cl, 1·0; K2HPO4, 0·075; CaCl2, 1·45; NaCl, 5·0; MgCl2, 6·15; KCl, 0·75; and FeSO4, 0·028, supplemented with 0·2 % (w/v) of one of the test substrates. Bacterial growth was determined spectrophotometrically after 2 days cultivation. Strain KMM 1406T was characterized additionally by using API 20NE and API ZYM identification systems (bioMérieux) at 28 °C. Antibiotic sensitivity was tested by the agar-diffusion method on TSA plates by using discs impregnated with antibiotics (content per disc): ampicillin, 10 µg; benzylpenicillin, 10 U; gentamicin, 10 µg; kanamycin, 30 µg; carbenicillin, 25 µg; lincomycin, 15 µg; oleandomycin, 15 µg; polymyxin, 300 U; streptomycin, 30 µg; tetracycline, 30 µg; and neomycin, 15 µg. DNA base composition was determined as described by Marmur & Doty (1962) and Owen et al. (1969). Whole-cell fatty acids were determined as described by Svetashev et al. (1995). 16S rRNA gene sequences were determined and compared as described by Shida et al. (1997). Previously published 16S rRNA gene sequences were obtained from GenBank/EMBL/DDBJ.
Deep-sea isolate KMM 1406T was an aerobic, Gram-negative, oxidase- and catalase-positive, chemoheterotrophic, non-pigmented, rod-shaped bacterium that measured 0·6–0·8 µm in diameter and 1·8–2·0 µm in length. Cells were motile by means of four to seven polar or bipolar and lateral flagella (Fig. 1). Strain KMM 1406T did not require sodium ions for growth and grew in 0–8 % (w/v) NaCl. Growth temperature was 4–37 °C. No growth was observed in 10 % NaCl or at 40 or 41 °C. The new bacterium formed non-pigmented, smooth, transparent, shining, convex and circular colonies with entire margins that were 3–5 mm in diameter on TSA and MA after 24 h incubation and released a slightly brown diffusible pigment into the medium. Strain KMM 1406T was unable to produce acid from carbohydrates aerobically or to ferment glucose under anaerobic conditions. Hydrolytic reactions on starch, gelatin and casein were observed after 24 h, but Tween 80 and DNA were hydrolysed after 3 days incubation. Other phenotypic characteristics of strain KMM 1406T are shown in Table 1.
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9c (25·5 %), C16 : 0 (19·1 %), C18 : 17c (15·7 %), C17 : 0 (8·1 %) and C17 : 18c (11·7 %); fatty acids C15 : 0 (2·4 %), C15 : 18c (3·3 %), i-C16 : 0 (3·7 %) and i-C18 : 0 (1·3 %) were detected as minor components. The DNA G+C content of strain KMM 1406T was 49·6 mol%.
Phylogenetic relationships among strain KMM 1406T and some related taxa are shown in Fig. 2. Comparative 16S rRNA gene sequence analysis showed that the new isolate was phylogenetically most closely related to R. baltica DSM 14885T and deep-sea isolates HBT 019, HTB 010 and HTB 021 (Takami et al., 1999), with 96·8–97·4 % sequence similarity, and less closely related to A. fetalis CCUG 30811T (Fonnesbech Vogel et al., 2000), with 95·4 % sequence similarity. This 16S rDNA sequence similarity value with R. baltica DSM 14885T is appropriate for determining intrageneric relationships for species definition (Stackebrandt & Goebel, 1994), indicating that strain KMM 1406T probably represents a novel species within the genus Rheinheimera. The novel deep-sea isolate shared main physiological characteristics, assimilation pattern and spectrum of enzyme activities with R. baltica DSM 14885T. Its DNA G+C content was close to those of R. baltica strains (47·8–48·9 mol%). The fatty acid profiles of KMM 1406T and R. baltica (Brettar et al., 2002) were found to be similar to each other, but strain KMM 1406T contained a higher proportion of C17 : 0 and C17 : 18c. Together with the findings mentioned above, significant characteristics for clear differentiation of KMM 1406T from Rheinheimera baltica include its lack of blue-coloured pigmentation, presence of bipolar and lateral flagella, growth at 37 °C and in 6–8 % NaCl, positive reactions for aesculin hydrolysis, PNPG (p-nitrophenyl -D-glucopyranoside) test, arabinose and citrate utilization and inability to utilize D-glucose. It has been reported that R. baltica strains do not require NaCl for growth and that sodium ions stimulate their growth (Brettar et al., 2002). In the present study, we failed to grow R. baltica DSM 14885T without NaCl, whilst strain KMM 1406T gave good growth on the same media. In addition, NaCl was not needed to support growth of KMM 1406T. Major phenotypic differences of the new strain from the phylogenetically less related species A. fetalis were as follows: motility, inability to reduce nitrate or thiosulfate, lack of growth at 40 or 42 °C or without NaCl, hydrolysis of some compounds and assimilation pattern (Table 1
). On the basis of phenotypic characterization and phylogenetic analysis, we propose that strain KMM 1406T should be classified as a novel species, Rheinheimera pacifica sp. nov., with the type strain KMM 1406T (=JCM 12090T=NRIC 0539T=CCUG 46544T).
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Strictly aerobic, Gram-negative, chemoheterotrophic, oxidase- and catalase-positive, rod-shaped, 1·8–2·0 µm long and 0·6–0·8 µm wide and motile with four to seven polar or bipolar and lateral flagella. Sodium ions are not required for growth. Growth is observed in 0–8 % (w/v) NaCl, but not in 10 % NaCl. Grows at 4–37 °C, but not at 40–41 °C. Non-pigmented, smooth, transparent and circular colonies with entire margins are formed on MA. Gelatin, starch, casein, Tween 80 and DNA are hydrolysed. Glucose fermentation and H2S production are not detected. Glycerol, acetate, L-arginine, valine, asparagine and DL-lysine are utilized, but methionine and L-glutamic acid are not. According to the API 20NE test (bioMérieux), aesculin and gelatin hydrolysis, PNPG test, arabinose, N-acetylglucosamine, maltose and citrate assimilation are positive and the following tests are negative: nitrate reduction, indole production, arginine dihydrolase, urease and assimilation of glucose, mannose, mannitol, gluconate, L-malate, caprate, adipate and phenylacetate. Assimilation of trehalose and sucrose is positive. In API ZYM analysis, positive reactions are exhibited for alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, trypsin, -chymotrypsin, naphthol-AS-BI-phosphohydrolase and N-acetyl-
-glucosaminidase; negative reactions are exhibited for lipase C14, valine arylamidase, cystine arylamidase, acid phosphatase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, -mannosidase and -fucosidase. Susceptible to gentamicin (10 µg); weakly susceptible to polymyxin (300 U), streptomycin (30 µg), kanamycin (30 µg) and neomycin (15 µg) and resistant to carbenicillin (25 µg), lincomycin (15 µg), oleandomycin (15 µg), tetracycline (30 µg), ampicillin (10 µg) and benzylpenicillin (10 U). DNA G+C content of the type strain is 49·6 mol% (determined by the thermal denaturation method). Predominant cellular fatty acids are C16 : 0, C17 : 0, C16 : 19c, C17 : 18c and C18 : 17c. Fatty acids C15 : 0, C15 : 18c, i-C16 : 0 and i-C18 : 0 are minor components.
The type strain, KMM 1406T (=IAM 15043T=JCM 12090T=NRIC 0539T=CCUG 46544T), was isolated from deep sea water of the Pacific Ocean.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Fonnesbech Vogel, B., Venkateswaran, K., Christensen, H., Falsen, E., Christiansen, G. & Gram, L. (2000). Polyphasic taxonomic approach in the description of Alishewanella fetalis gen. nov., sp. nov., isolated from a human foetus. Int J Syst Evol Microbiol 50, 1133–1142.[Abstract]
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]
Owen, J., Hill, L. R. & Lapage, S. P. (1969). Determination of DNA base compositions from melting profiles in dilute buffers. Biopolymers 7, 503–516.[CrossRef][Medline]
Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997). Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47, 289–298.
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.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
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.
Takami, H., Kobata, K., Nagahama, T., Kobayashi, H., Inoue, A. & Horikoshi, K. (1999). Biodiversity in deep-sea sites located near the south part of Japan. Extremophiles 3, 97–102.[CrossRef][Medline]
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