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Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, Campus Universitario de Cartuja, University of Granada, 18071 Granada, Spain
Correspondence
Emilia Quesada
equesada{at}ugr.es
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7c (11·33 %) and 16 : 0 (11·73 %) whilst 15 : 0 iso (24·69 %), 17 : 0 iso (12·92 %) and 17 : 19c (11·03 %) are predominant in strain R22T. The DNA G+C composition is 46·0 mol% in strain FP23T and 48·7 mol% in strain R22T. Phylogenetic analyses indicate conclusively that the two strains belong to the genus Idiomarina. DNA–DNA hybridization revealed that they represent novel species. In the light of the polyphasic evidence accumulated in this study, it is proposed that they be classified as novel species of the genus Idiomarina, with the names Idiomarina fontislapidosi sp. nov. (type strain F23T=CECT 5859T=LMG 22169T) and Idiomarina ramblicola sp. nov. (type strain R22T=CECT 5858T=LMG 22170T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains F23T and R22T are AY526861 and AY526862.
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) within the 
-subclass of the Proteobacteria, was proposed by Ivanova et al. (2000) to accommodate two strains isolated from sea-water samples collected at a depth of 4000–5000 m from the north-western Pacific Ocean. These strains were described as two different species, Idiomarina abbysalis (type species) and Idiomarina zobellii. Since then two more species have been added to the genus: Idiomarina loihiensis, which was isolated from the L
‘ihi submarine volcano in Hawaii (Donachie et al., 2003), and Idiomarina baltica, which was found in the central Baltic Sea (Brettar et al., 2003). Members of Idiomarina share many phenotypic characteristics with other heterotrophic, oxidative, marine and halophilic members of the -Proteobacteria. Nevertheless, phylogenetic studies based on 16S rRNA gene sequence analysis have proved that Idiomarina represents a distinct evolutionary line. One eminent feature of the genus Idiomarina is its uniquely high content of iso-branched fatty acids, which is atypical of Proteobacteria with the sole exception of the Xanthomonas branch (Finkmann et al., 2000). They can also be distinguished from other marine bacteria by their physiological properties, being able to grow within a broad range of temperatures, pH values and NaCl concentrations. In this study we describe two strains, for which we propose the names Idiomarina fontislapidosi sp. nov. and Idiomarina ramblicola sp. nov. These two strains are the only representatives of the genus Idiomarina identified so far that have not been isolated from sea-water samples.
The strains were found during two different samplings made in 1998 (Martínez-Cánovas et al., 2004). F23T was isolated from a soil sampled at Fuente de Piedra (Málaga, S. Spain), an inland, hypersaline wetland. Strain R22T was isolated from a water sample taken in Rambla Salada (Murcia, E. Spain), a hypersaline rambla (a steep-sided river bed, normally dry but subject to flash flooding). The isolation medium was MY (Moraine & Rogovin, 1966) supplemented with 7·5 % w/v salts (Rodríguez-Valera et al., 1981); its composition is the following (g l–1): NaCl, 51·3; MgCl2.6H2O, 9; MgSO4.7H2O, 13; CaCl2.2H2O, 0·2; KCl, 1·3; NaCO3H, 0·05; NaBr, 0·15; FeCl3.6H2O, traces; glucose, 10; yeast extract, 3; malt extract, 3; proteose-peptone, 5 (pH 7). Both strains were kept and routinely grown in MH medium (Quesada et al., 1983) at 32 °C; its composition is the following (g l–1): NaCl, 51·3; MgCl2.6H2O, 9; MgSO4.7H2O, 13; CaCl2.2H2O, 0·2; KCl, 1·3; NaCO3H, 0·05; NaBr, 0·15; FeCl3.6H2O, traces; glucose, 1; yeast extract, 10; proteose-peptone, 5 (pH 7). Bacto agar (2 g l–1) was added for the preparation of solid media.
The strains were originally characterized phenotypically by Martínez-Cánovas et al. (2004) according to the methods described by Mata et al. (2002). Phenotypic data are given in the species description. Table 1 shows the main phenotypic differences between the strains F23T (I. fontislapidosi sp. nov.) and R22T (I. ramblicola sp. nov.) and the other four species of the genus Idiomarina. The same table contains the G+C contents of both strains estimated from the midpoint value (Tm) of the DNA thermal denaturation profile, as described in Martínez-Cánovas et al. (2004).
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. We determined almost the complete sequence of the 16S rRNA genes of strains F23T (1455 bp) and R-22T (1459 bp). The phylogenetic tree obtained by neighbour-joining is shown in Fig. 1. Maximum-parsimony algorithms gave a similar result. The analyses confirmed the affiliation of the novel strains to Idiomarina and their relationship with I. baltica (strain F23T) and I. loihiensis (strain R22T). Nevertheless, Stackebrandt & Pukall (1999) advise that even a 16S rRNA gene sequence similarity of over 99·5 % is insufficient evidence to affiliate an isolate to a particular species. We have demonstrated that the two new isolates can be distinguished at the species level through their phenotypic features, fatty-acid contents and by DNA–DNA hybridization. The next closest neighbours were Thalassomonas, described by Macián et al. (2001), and Colwellia, another member of the family Alteromonadaceae (Ivanova & Mikhailov, 2001).
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) display a predominance of iso-branched fatty acids, which is characteristic of Idiomarina. The taxonomic significance of the fatty-acid composition of Idiomarina species is discussed in Brettar et al. (2003). 15 : 0 iso (26·75 %), 16 : 17c (11·33 %) and 16 : 0 (11·73 %) form the main peaks of the fatty-acid pattern in strain F23T whilst 15 : 0 iso (24·69 %), 17 : 0 iso (12·92 %) and 17 : 19c (11·03 %) are the most abundant fatty acids in strain R22T. As can be seen in Table 2, the fatty-acid pattern of the new isolates differs to some extent from those of the previously described species of Idiomarina.
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, all of which is fully described in Bouchotroch et al. (2001). The results (Table 3) prove that strains F23T and R22T should be accepted as two novel species of the genus Idiomarina.
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Description of Idiomarina fontislapidosi sp. nov.
Idiomarina fontislapidosi (fon'tis.la.pi.do'si. L. sb. masc. gen. fontis of the spring; L. masc. adj. lapidosi stony; N.L. sb. masc. gen. fontislapidosi of the stony spring, i.e. from Fuente de Piedra, the site from which the type strain was isolated).
Cells are slightly curved rods, 3–4 µm long and 0·75 µm wide, appearing either singly or in pairs, and often forming amorphous aggregates. They stain Gram-negative and are motile by one polar flagellum. No spores or polyhydroxyalkanoate are observed under any conditions. Colonies are round, convex, mucoid and cream coloured. Exopolysaccharide is produced. The growth pattern is uniform in a liquid medium. The bacterium is chemo-organotrophic and strictly aerobic. Anaerobic respiration with nitrate, nitrite or fumarate is negative. It does not produce acid from any of the following sugars: adonitol, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, maltose, D-mannitol, mannose, D-melezitose, L-rhamnose, sucrose, D-salicin, D-sorbitol, sorbose or D-trehalose. It is moderately halophilic, capable of growing in NaCl concentrations of 0·5 to 25 % w/v, the optimum being 3–5 % w/v. It does not require additional magnesium or potassium salts. It grows within the temperature range of 4 to 45 °C, the optimum being 32 °C, and at pH values of between 5 and 10, the optimum being 7–8. It shows positive activity for catalase, cytochrome oxidase, phosphatase, ONPG, selenite reduction, H2S production from cysteine, growth on MacConkey and hydrolysis of aesculin, gelatin, casein, Tween 20, Tween 80 and DNA. It shows no activity for the hydrolysis of starch and tyrosine, nitrate and nitrite reduction, urease, lecithinase, phenylalanine deaminase, gluconate oxidation, growth on cetrimide agar, haemolysis, indole, methyl red and Voges–Proskauer. It does not grow in synthetic media supplemented with different sole sources of carbon and energy, or carbon, nitrogen and energy (aesculin, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, L-rhamnose, D-salicin, starch, D-trehalose, acetate, citrate, formate, fumarate, gluconate, lactate, malonate, propionate, succinate, adonitol, ethanol, glycerol, myo-inositol, sorbitol, L-alanine, L-cysteine, L-histidine, DL-isoleucine, L-lysine, L-methionine, L-serine and L-valine). It is susceptible to amoxicillin (25 µg), ampicillin (10 µg), carbenicillin (100 µg), cefotaxime (30 µg), cefoxitin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), kanamycin (30 µg), nalidixic acid (30 µg), nitrofurantoin (300 µg), polymyxin B (300 UI), rifampicin (30 µg), streptomycin (10 µg), tobramycin (10 µg) and trimetroprim/sulphametoxazol (1·25/23·75 µg). It is resistant to sulphamide (250 µg). DNA G+C content of the type strain is 46·0 mol% (Tm method).
The type strain, F23T (=CECT 5859T=LMG 22169T), was isolated from a sample of soil taken from the temporally emerged banks of a hypersaline pool in the Fuente de Piedra wetland wild-fowl reserve in the province of Málaga (S. Spain).
Description of Idiomarina ramblicola sp. nov.
Idiomarina ramblicola [ram.bli.co'la. Spanish sb. fem. rambla from Arabic sb. ramla lit. sandy ground; L. suff. [in]cola denizen of; N.L. sb. fem. nom. ramblicola denizen of a rambla. A rambla is a steep-sided watercourse, often dry but subject to flash flooding (cf. OED, 2nd edn, 1989, vol. XIII, p. 153), not synonymous with Arabic wadi].
The cells are slightly curved rods, 2–3 µm long and 0·75 µm wide, appearing either singly or forming amorphous aggregates. They stain Gram-negative and are motile by one polar flagellum. No spores or polyhydroxyalkanoate are observed under any conditions. Colonies are round, convex, mucoid and cream coloured. Exopolysaccharide is produced. The growth pattern is uniform in a liquid medium. The bacterium is chemo-organotrophic and strictly aerobic. Anaerobic respiration with nitrate, nitrite or fumarate is negative. It does not produce acid from any of the following sugars: adonitol, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, myo-inositol, lactose, maltose, D-mannitol, mannose, D-melezitose, L-rhamnose, sucrose, D-salicin, D-sorbitol, sorbose or D-trehalose. It is moderately halophilic, capable of growing in NaCl concentrations of 0·5 to 15 % w/v, the optimum being 3–5 % w/v. It does not require additional magnesium or potassium salts. It grows within the temperature range of 15 to 40 °C, the optimum being 32 °C, and at pH values of between 5 and 10, the optimum being 7–8. It shows positive activity for catalase, cytochrome oxidase, phosphatase, selenite reduction, gluconate oxidation, haemolysis, H2S production from cysteine and the hydrolysis of aesculin, gelatin, casein, Tween 20, Tween 80 and DNA, and no activity for the hydrolysis of starch and tyrosine, reduction of nitrate and nitrite, oxidation of urease, lecithinase and phenylalanine and growth on cetrimide agar, haemolysis, indole, methyl red and Voges–Proskauer. It does not grow in synthetic media supplemented with different sole sources of carbon and energy, or carbon, nitrogen and energy (aesculin, L-arabinose, D-cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, L-rhamnose, D-salicin, starch, D-trehalose, acetate, citrate, formate, fumarate, gluconate, lactate, malonate, propionate, succinate, adonitol, ethanol, glycerol, myo-inositol, sorbitol, L-alanine, L-cysteine, L-histidine, DL-isoleucine, L-lysine, L-methionine, L-serine and L-valine). It is susceptible to amoxicillin (25 µg), ampicillin (10 µg), carbenicillin (100 µg), cefotaxime (30 µg), cefoxitin (30 µg), chloramphenicol (30 µg), erythromycin (15 µg), nalidixic acid (30 µg), nitrofurantoin (300 µg), polymyxin B (300 UI), rifampicin (30 µg), sulphamide (250 µg) and trimetroprim/sulphametoxazol (1·25/23·75 µg). It is resistant to kanamycin (30 µg), streptomycin (10 µg) and tobramycin (10 µg). DNA G+C content of the type strain is 48·7 mol% (Tm method).
The type strain, R22T (=CECT 5858T=LMG 22170T), was isolated from a hypersaline water sample taken from Rambla Salada (Murcia, S.E. Spain).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brettar, I., Christen, R. & Höfle, M. G. (2003). Idiomarina baltica sp. nov., a marine bacterium with a high optimum growth temperature isolated from surface water of the central Baltic Sea. Int J Syst Evol Microbiol 53, 407–413.
Donachie, S. P., Hou, S., Gregory, T. S., Malahoff, A. & Alam, M. (2003). Idiomarina loihiensis sp. nov., a halophilic -Proteobacterium from the L‘ihi submarine volcano, Hawai‘i. Int J Syst Evol Microbiol 53, 1873–1879.
Finkmann, W., Altendorf, K., Stackebrandt, E. & Lipski, A. (2000). Characterization of N2O-producing Xanthomonas-like isolates from biofilters as Stenotrophomonas nitritireducens sp. nov., Luteimonas mephitis gen. nov., sp. nov. and Pseudoxanthomonas broegbernensis gen. nov., sp. nov. Int J Syst Evol Microbiol 50, 273–282.[Abstract]
Ivanova, E. P. & Mikhailov, V. V. (2001). A new family, Alteromonadaceae fam. nov., including the marine proteobacteria of the genera Alteromonas, Pseudoalteromonas, Idiomarina and Colwellia. Mikrobiologia 70, 15–23 (in Russian).
Ivanova, E. P., Romanenko, L. A., Chun, J. & 7 other authors (2000). Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov. Int J Syst Evol Microbiol 50, 901–907.[Abstract]
Macián, M. C., Ludwig, W., Scheifer, K. H., Garay, E. & Pujalte, M. J. (2001). Thalassomonas viridans gen. nov., sp. nov., a novel marine -Proteobacterium. Int J Syst Evol Microbiol 51, 1283–1289.[Abstract]
Martínez-Cánovas, M. J., Quesada, E., Martínez-Checa, F. & Béjar, V. (2004). A taxonomic study to establish the relationship between exopolysaccharide-producing bacterial strains living in diverse hypersaline habitats. Curr Microbiol 48, 348–353.[CrossRef][Medline]
Mata, J. A., Martínez-Cánovas, M. J., Quesada, E. & Béjar, V. (2002). A detailed phenotypic characterisation of the type strains of Halomonas species. Syst Appl Microbiol 25, 360–375.[CrossRef][Medline]
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Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.
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