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1 Departamento de Microbiologia, Facultad de Farmacia, Campus Universitario de Cartuja s/n 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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ABSTRACT |
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). The current divisions are based on 16S rRNA gene sequences and chemotaxonomic criteria, particularly polar lipid composition. To date, 20 genera of haloarchaea are recognized.
Members of the genus Halorubrum (McGenity & Grant, 1995, 2001) are widely distributed in hypersaline environments and play important roles in the carbon and nitrogen cycles in this ecosystem. Currently, the genus Halorubrum comprises 12 species with validly published names: Halorubrum sodomense (Oren, 1983), Halorubrum lacusprofundi (Franzmann et al., 1988), Halorubrum saccharovorum (Tomlinson & Hochstein, 1976), Halorubrum trapanicum (Petter, 1931; McGenity & Grant, 1995), Halorubrum tebenquichense (Lizama et al., 2002), Halorubrum tibetense (Fan et al., 2004), Halorubrum terrestre (Ventosa et al., 2004), Halorubrum xinjiangense (Feng et al., 2004) and Halorubrum alkaliphilum (Feng et al., 2005). Oren & Ventosa (1996) reclassified Halorubrobacterium coriense (Kamekura & Dyall-Smith, 1995) and Halorubrobacterium distributum (Zvyagintseva & Tarasov, 1987; Kamekura & Dyall-Smith, 1995) as Halorubrum coriense and Halorubrum distributum, respectively. Natronobacterium vacuolatum (Mwatha & Grant, 1993) was also reclassified as Halorubrum vacuolatum by Kamekura et al. (1997).
In this paper, a novel halophilic archaeon designated strain 5.1T belonging to the genus Halorubrum is described. Strain 5.1T was isolated from water samples collected from Ezzemoul sabkha, in north-east Algeria. Halophilic medium modified from the formulation of Oren (1983) was used, comprising (per litre distilled water): 125 g NaCl, 100 g MgCl2.6H2O, 5 g K2SO4, 0.1 g CaCl2.2H2O, 1 g yeast extract, 1 g Casamino acids and 2 g soluble starch. The pH was adjusted to 7.0 and incubation was at 37 °C. The following reference strains were used for comparative purposes: Hrr. sodomense DSM 3755T, Hrr. saccharovorum DSM 1137T, Hrr. lacusprofundi DSM 5036T, Hrr. coriense DSM 10284T, Hrr. tebenquichense CECT 5317T, Hrr. trapanicum NRC 34021T, Hrr. distributum JCM 9100T, Hrr. xinjiangense JCM 12388T and Hrr. terrestre JCM 10247T. The reference strains were cultured in medium HE, at a final concentration of approximately 25 % (w/v) salts (Torreblanca et al., 1986) and supplemented with 0.1 % (w/v) glucose.
Phenotypic analysis of strain 5.1T was performed according to the proposed minimal standard for the description of new taxa in the order Halobacteriales (Oren et al., 1997). Cell morphology was examined using transmission electron microscopy. Gram staining was performed by using acetic-acid-fixed samples, as described by Dussault (1955). Growth rates in various salt concentrations were determined in medium supplemented with 0, 7.5, 10, 15, 20, 25 or 30 % (w/v) NaCl and the requirement for Mg2+ was tested using agar plates containing final concentrations of 0, 0.005, 0.01, 0.02, 0.1, 0.5, 0.7 or 0.8 M MgCl2. The pH range for growth was determined using solid medium at pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0. Temperatures tested included 4, 22, 30, 37, 40, 50, 55 and 60 °C. Anaerobic growth in the presence of L-arginine and nitrate was tested as described by Hartmann et al. (1980). Controls without arginine and nitrate were included and incubation was performed in the dark. Reduction of nitrate was detected by using the sulfanilic acid and 
-naphthylamine reagent (Smibert & Krieg, 1981). Tests for formation of indole and hydrolysis of starch and aesculin were performed as described by González et al. (1978). Hydrolysis of gelatin and Tween 80 was tested as outlined by Gutiérrez & González (1972). Catalase and oxidase activities were tested according to Gerhardt et al. (1994). Utilization of carbohydrates, sugar alcohols, amino acids and organic acids were carried out as described by Oren et al. (1997). Acid production from carbohydrates and sugar alcohols was detected using a phenol red solution. Antibiotic susceptibility was determined by spreading bacterial suspensions on culture plates and applying disks impregnated with the antibiotics tested (Bonelo et al., 1984).
Lipids were extracted from the strains using a modified Bligh and Dyer extraction procedure (Kates, 1972). The lipids were separated by thin-layer chromatography with single development on silica gel in a chloroform/methanol/acetic acid/water (85 : 22.5 : 10 : 4, by vol.) solvent system. In addition, two-dimensional chromatography was performed using chloroform/methanol/water (65 : 25 : 4, by vol.) in the first dimension and chloroform/methanol/acetic acid/water (80 : 12 : 15 : 4, by vol.) in the second dimension. Glycolipid spots were detected by spraying the plates with 0.5 % -naphthol in 50 % methanol, followed by 5 % sulfuric acid in ethanol and heating at 150 °C. Phospholipids were visualized with an ammonium molybdate/sulfuric acid spray.
Genomic DNA was extracted and purified by using the method of Lind & Ursing (1986). Purity was assessed from A260/A280 and A230/A260 ratios. The G+C content was determined from the mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962), with a Perkin Elmer UV-Vis Lambda 3B spectrophotometer at 260 nm, programmed for temperature increases of 1.0 °C min–1. The Tm was determined by using 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 studies were performed as 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.
16S rRNA genes were amplified by PCR with a primer set designed to complement the highly conserved regions of the Halobacterium salinarum and Halorubrum species 16S rRNA genes: forward primer F8 (5'-TTGATCCTGCCGGAGGCCATTG-3') and reverse primer R1462 (5'-ATCCAGCGCAGATTCCCCTAC-3'), corresponding to positions 8–30 and 1462–1441, respectively. PCR and sequencing were performed as described previously (Lizama et al., 2002). Multiple sequence alignments were performed using CLUSTAL W 1.8 (Thompson et al., 1994). A phylogenetic tree was constructed by using the neighbour-joining method with the MEGA 3 program package (Kumar et al., 2004).
On the basis of pigmentation, NaCl-dependent growth, antibiotic susceptibility and 16S rRNA gene sequences, strain 5.1T was identified as representing a member of the family Halobacteriaceae. Colonies on standard growth agar medium were circular, convex and with entire margins. Light-red-coloured colonies, about 0.5–1.0 mm in diameter, were formed after 7 days of incubation at 37 °C. Cells of strain 5.1T were motile, single, small rods. Pleomorphic forms (irregular rods and coccoid and triangular forms) were also observed in liquid culture. Mean cell dimensions of the rod forms were 0.6x1.5–3.0 µm (Fig. 1). Cells stained Gram-negative and lysed in distilled water. Strain 5.1T grew in media containing 15–25 % (w/v) NaCl, with optimum growth at 20 % (w/v) NaCl. The strain grew at pH 6.5–9.0, with optimum growth at pH 7.0–7.5. Strain 5.1T grew at temperatures between 22 and 50 °C. Mg2+ was required for growth and optimum growth occurred at 37–40 °C.
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Strain 5.1T contained C20C20 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate and a single glycolipid, chromatographically identical to S-DGD-3 of Hrr. coriense DSM 10284T.
The 16S rRNA gene sequence of strain 5.1T was determined and comparison with the 16S rRNA gene sequences of members of the family Halobacteriaceae placed strain 5.1T in the genus Halorubrum (Fig. 2). Strain 5.1T was related to Hrr. coriense (97 % 16S rRNA gene sequence similarity), Hrr. sodomense (97 % similarity), Hrr. trapanicum (96 %), Hrr. distributum (96 %), Hrr. xinjiangense (96 %), Hrr. terrestre (95 %), Hrr. tebenquichense (94 %), Hrr. saccharovorum (93 %) and Hrr. lacusprofundi (93 %).
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Description of Halorubrum ezzemoulense sp. nov.
Halorubrum ezzemoulense (ez.ze.mou.len'se. N.L. neut. adj. ezzemoulense pertaining to Ezzemoul sabkha, where the type strain was isolated).
Cells stain Gram-negative, are motile, lyse in distilled water and are pleomorphic. Cells occur mostly as single rods and exhibit a range of morphological types, including irregular rod-shaped, cocci and triangular forms. Rods are 0.6x1.5–3.0 µm. Colonies on standard agar growth medium are 0.5–1.0 mm in diameter, circular, convex, entire and red. Chemo-organotrophic, aerobic growth occurs at 15–25 % (w/v) NaCl, pH 6.5–9.0 and 22–50 °C. Optimum NaCl concentration, pH and temperature for growth are 20 % (w/v), pH 7.0–7.5 and 37–40 °C, respectively. Mg2+ is required for growth. Catalase- and oxidase-positive. Anaerobic growth with nitrate or L-arginine does not occur. Nitrate is reduced to nitrite. Indole is not produced. Gelatin is not liquefied. Starch, aesculin and Tween 80 are not hydrolysed. Lactose, xylose, rhamnose, galactose, raffinose, cellobiose, mannose, fructose, starch, inositol, sorbitol, dulcitol, adonitol, salicin, cellulose, malonate, fumarate, formate and amino acids are not utilized. Glucose, sucrose, maltose, arabinose, mannitol, glycerol, acetate, oxalate, citrate and malate are used as carbon sources. Acids are produced from glucose, lactose, xylose, galactose and rhamnose. Sensitive to novobiocin and bacitracin, but resistant to penicillin, chloramphenicol, ampicillin, rifampicin, streptomycin and neomycin. Polar lipids are C20C20 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Also contains phosphatidylglycerol sulfate and a sulfated diglycosyl diether. The G+C content of genomic DNA of the type strain is 61.9 mol% (Tm).
The type strain, 5.1T (=CECT 7099T=DSM 17463T), was isolated from Ezzemoul sabkha in Algeria.
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ACKNOWLEDGEMENTS |
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