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1 Departamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja s/n, CP 18071 Granada, Spain
2 Institut de Nutrition de l'Alimentation et des Technologies Agro-Alimentaires, Université Mentouri Constantine, Algeria
3 Institut des Sciences de la Nature, Faculté des Sciences, Université Mentouri Constantine, Algeria
Correspondence
Mercedes Monteoliva-Sánchez
mmonteol{at}ugr.es
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9c. Other fatty acids present were C16 : 19c, C18 : 0 10-methyl, C12 : 0 3-OH, C10 : 0 and C12 : 0. The G+C content of the genomic DNA was 56.0 mol%. 16S rRNA gene sequence analysis indicated that strain B2T was closely related to Salicola marasensis in the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain B2T and the type strain of Salicola marasensis was 99 %. DNA–DNA hybridization experiments between strain B2T and Salicola marasensis indicated a level of relatedness of 52 %. The phenotypic characteristics of strain B2T allowed its differentiation from recognized species of the genus Salicola. Strain B2T was able to hydrolyse starch but not aesculin. It was unable to use carbohydrates and could not use citrate, pyruvate or succinate as sole carbon and energy sources. On the basis of the polyphasic data presented, strain B2T is considered to represent a novel species of the genus Salicola, for which the name Salicola salis sp. nov. is proposed. The type strain is B2T (=CECT 7106T=LMG 23122T).
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, b) using specific inhibitors have shown that all or nearly all of the heterotrophic activity in the saltern crystallizer ponds at Eilat, Israel, could be attributed to members of the Archaea, which are well adapted to this environment (Oren, 1994). However, the use of molecular biological approaches based on sequencing of 16S rRNA genes recovered from the environment and fluorescence in situ hybridization have indicated that hypersaline environments also harbour significant communities of Bacteria (Antón et al., 1999, 2000; Eder et al., 1999).
Among the Bacteria, most halophiles described to date are moderately rather than extremely halophilic and fall into established genera with non-halophilic representatives within the families Halanaerobiaceae (exclusively halophiles) and Halomonadaceae (predominantly halophiles). However, there are a few halophilic taxa that resemble the haloarchaea in their salt requirement and tolerance: Halorhodospira, Halovibrio, Halospina, Salicola (Gammaproteobacteria), Actinopolyspora halophila (Actinobacteria) and Salinibacter (Bacteroidetes) (Antón et al., 2002; Oren, 2002; Sorokin et al., 2006; Maturrano et al., 2006).
Algeria has numerous natural hypersaline lakes (sabkhas) located in the north and in the south of the country. These environments can be considered as athalassohaline because their hypersalinity derives from the dissolution of salts of continental origin. Sodium and chloride ions dominate this ecosystem. Recently, in the course of screening micro-organisms present in Ezzemoul sabkha, we isolated several archaeal and bacterial strains and characterized them taxonomically. One of the bacterial isolates, designated strain B2T, an extremely halophilic, Gram-negative and rod-shaped bacterium, was subjected to further taxonomic study.
Strain B2T was isolated from brine samples collected from Ezzemoul sabkha, north-east Algeria. Samples were plated on hypersaline agar medium containing (per litre): 175 g NaCl, 20 g MgCl2.6H2O, 5 g K2SO4, 0.1 g CaCl2.2H2O and 5 g yeast extract. The pH of the medium was adjusted to 7.0 with NaOH. Incubation was at 37 °C.
Cell morphology of strain B2T and flagellum type were examined by transmission and scanning electron microscopy. Samples were negatively stained with 2 % (w/v) uranyl acetate (for 30 s) and after air-drying the grids were viewed in a model EM 902 transmission electron microscope (Zeiss). For ultrathin sections exponentially growing cells were harvested and fixed in cold 2.5 % (w/v) glutaraldehyde (0.1 % cacodylate buffer, pH 7.2). After 1 h of fixation, the cells were washed three times in cacodylate buffer and post-fixed for 1 h in 1 % (w/v) osmium tetroxide with propanol and embedded in resin. Thin sections were prepared with an ultramicrotome and post-stained with uranyl acetate and lead citrate. Finally, the sections were examined in a Zeiss EM 902 transmission electron microscope. For scanning electron microscopy (Zeiss DSM950), samples were fixed in a solution containing 2.5 % glutaraldehyde. After washing in 0.1 M sodium cacodylate for 4 h, the cells were dehydrated in a graded ethanol series (30, 50, 70, 90 and 100 %) at room temperature. Specimens were dried and coated with gold and examined. Gram staining was performed by using acetic-acid-fixed samples as described by Dussault (1955).
The growth response to NaCl was examined in solid medium containing final concentrations of 0, 7.5, 10, 15, 20, 25 and 30 % (w/v) NaCl. Tolerance to pH was tested in solid medium at pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0. The temperature range for growth was tested by incubating cultures on agar plates at 30–50 °C. Anaerobic growth in the presence of nitrate (5 g l–1) was determined and a control test without nitrate was included. Reduction of nitrate was detected by using sulfanilic acid and 
-naphthylamine reagent (Smibert & Krieg, 1981). Formation of gas from nitrate was detected by using Durham tubes. Tests for formation of indole and hydrolysis of starch and aesculin were performed following the protocols of Gonzalez et al. (1978). Hydrolysis of gelatin and Tween 80 were tested as outlined by Gutiérrez & Gonzalez (1972). Catalase production was detected with 10 % H2O2. The oxidase reaction was performed on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'- tetramethyl-p-phenylenediamine. Formation of acids from carbohydrates and alcohols was tested in liquid media in which yeast extract was reduced to 0.1 g l–1, amended with 0.5 g NH4Cl l–1 and 5 g of the substrate tested l–1. The utilization of carbohydrates, alcohols or amino acids by strain B2T was tested in medium in which yeast extract was reduced and amended with 0.5 g NH4Cl l–1, 0.05 g KH2PO4 l–1, 1 % of the respective carbohydrates, alcohols or sugars, or 0.01 % of the respective amino acids and buffer at pH 7.0. Determination of antibiotic susceptibility was performed by spreading bacterial suspensions on the standard growth agar medium plate and applying antibiotics discs.
Analysis of fatty acid methyl esters was carried out by the Analytical Service of the DSMZ (Braunschweig, Germany), using the MID/Hewlett Packard Microbial System Identification System (MIS), which relies upon high-resolution GC to obtain the fatty acid profile.
The 16S rRNA genes were amplified by PCR using two primers: 16F27 (5'-AGAGTTTGATCMTGGCTCAG-3') and 16R1525 (5'-AAGGAGGTGWTCCARCC-3'). PCR reactions were carried out under the conditions described by Saiki et al. (1988). The PCR products were purified with Microcon-100 concentrator (Amicon) and sequenced directly using primers for the sequencing reactions previously described by Lane (1991) (16SF357: 5'-CTCCTACGGGAGGCAGCA-3'; 16SR519: 5'-GWATTACCGCGGCKGCTG-3'; 16SF945: 5'-GGGCCCGCACAAGCGGTGG-3'). Sequences were determined using an Applied Biosystems PRIZM TaqDyeDeoxy Terminator Cycle Sequencing kit and model 373A automatic DNA sequencer. Multiple sequence alignments were performed using CLUSTAL W 1.8 (Thompson et al., 1994). Phylogenetic trees were constructed by the neighbour-joining method with the MEGA 3 program package (Kumar et al., 2004).
Genomic DNA was isolated by using the method of Marmur (1961). G+C content was determined from the mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962), with a Perkin Elmer UV–visible Lambda 3B spectrophotometer at 260 nm, programmed for temperature increases of 1.0 °C min–1. Tm was determined by the graphic method described by Ferragut & Leclerc (1976), and the G+C content was calculated from this temperature by using the equation of Owen & Hill (1979). DNA–DNA hybridization experiments comparing strain B2T with Salicola marasensis CECT 7107T, Halospina denitrificans DSM 15505T, Halovibrio denitrificans DSM 15503T and [Pseudomonas halophila] DSM 3050T were performed with the method described by Ziemke et al. (1998). DNA was double-labelled using DIG-11-dUTP and biotin-16-dUTP (Boehringer Mannheim). The labelling reaction was carried out using a Boehringer Mannheim Nick-Translation kit.
Cells of strain B2T were rods of approximately 0.4–0.6x0.9–1.3 µm and motile by means of polar flagella (Fig. 1). Cells stained Gram-negative. Colonies formed on standard agar medium were cream–beige, smooth, circular and convex, had entire margins, and were about 2–3 mm in diameter following 5 days cultivation at 37 °C.
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Growth was observed only with acetate. Glucose, lactose, sucrose, xylose, rhamnose, galactose, raffinose, arabinose, cellobiose, mannose, maltose, adonitol, dulcitol, mannitol, cellulose, salicin, malonate, succinate, pyruvate, citrate, lactate, fumarate, formate, benzoate and amino acids were not utilized. Acid was produced in the presence of glucose, rhamnose, galactose, arabinose and maltose. Nitrate was slightly reduced to nitrite. Strain B2T hydrolysed gelatin, Tween 80 and starch but not aesculin. No indole was produced. The isolate exhibited catalase and oxidase activity. It was resistant to kanamycin, bacitracin and anisomycin but sensitive to penicillin G, chloramphenicol, ampicillin and streptomycin.
The fatty acid profiles of strain B2T and several reference strains are given in Table 1. The major hydroxy fatty acid was C12 : 0 3-OH. Note that members of the genera Marinobacter (Nguyen et al., 1999), Alcanivorax (Yakimov et al., 1998), Saccharospirillum (Labrenz et al., 2003) and Halomonas (Yoon et al., 2002) also produce C12 : 0 3-OH. The fatty acids C10 : 0, C12 : 0 and C12 : 0 3-OH are present in Salicola marasensis (Maturrano et al., 2006), [P. halophila] DSM 3050T, Halovibrio denitrificans and Halospina denitrificans (Sorokin et al., 2006), but at lower levels than found in strain B2T. Additionally, strain B2T contained C16 : 0 N alcohol (3.80 %). It also exhibited an abundance of 9c isomers of the fatty acids C16 : 1 and C18 : 1, which are reported to predominate in Marinobacter (Gorshkova et al., 2003), [P. halophila] DSM 3050T, Halospina denitrificans, Halovibrio denitrificans (Sorokin et al., 2006) and Salicola marasensis (Maturrano et al., 2006). A greater proportion of C16 : 19c and C16 : 0 but lesser proportion of C18 : 09c and C18 : 0 10-methyl was found in the fatty acid profile of strain B2T compared with that of Salicola marasensis; a number of other differences in the fatty acid profiles were also found (Table 1). It is interesting to note that strain B2T possessed C18 : 36c (6, 9, 12), an unusual unsaturated fatty acid of bacterial species.
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. The G+C content of the genomic DNA of strain B2T was 56.0 mol% as determined by the thermal denaturation method. This is somewhat lower than the value reported for Salicola marasensis (Table 2). DNA–DNA hybridization experiments between strain B2T and Salicola marasensis CECT 7107T, Halospina denitrificans DSM 15505T, Halovibrio denitrificans DSM 15503T and [P. halophila] DSM 3050T revealed levels of relatedness of 52, 40, 43 and 51 %, respectively.
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). The fatty acid profile of strain B2T was similar to that of Salicola marasensis, based on the fact that C16 : 0 and C18 : 19c comprised the dominant components. However, detailed examination of the fatty acid profiles indicated that strain B2T could be distinguished from Salicola marasensis (see Table 1). Other differences between the two organisms were also found, including the pH range for growth, and the ability to hydrolyse some substrates and to use carbohydrates and other organic compounds. The level of DNA–DNA hybridization is low enough to classify strain B2T as representing a novel species in the genus Salicola, for which we propose the name Salicola salis sp. nov.
Description of Salicola salis sp. nov.
Salicola salis [sa'lis. L. gen. n. salis (poet.) of salt water, of brine].
Cells are Gram-negative rods (0.4–0.6x0.9–1.3 µm), and are motile. Colonies are cream–beige, smooth, circular and convex with entire margins. Oxidase- and catalase-positive. Growth occurs at NaCl concentrations of 10–25 % (w/v) with an optimum between 15 and 20 % (w/v) NaCl. Temperature range for growth is 30–45 °C (optimum 37 °C). Grows at pH 6.0–9.0 (optimum about pH 7.0–7.5). Tween 80 is hydrolysed but indole is not produced. Nitrate is weakly reduced. Sugars and amino acids are not utilized. Grows on acetate. Hydrolyses starch and gelatin. Aesculin is not hydrolysed. Cells are resistant to kanamycin, bacitracin and anisomycin but susceptible to chloramphenicol and penicillin G. The major fatty acids are C10 : 0, C12 : 0, C12 : 0 3-OH, iso-C16 : 1 and/or C14 : 0 3-OH, C16 : 0 N alcohol, C16 : 19c, C16 : 0, C18 : 36c, C18 : 19c, C18 : 0 and C18 : 0 10-methyl; other fatty acids are present in minor proportions. The G+C content of the DNA is 56.0 mol% (Tm).
The type strain, B2T (=CECT 7106T=LMG 23122T), was isolated from brines of Ezzemoul sabkha (Algeria).
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ACKNOWLEDGEMENTS |
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