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1 Department of Botany and Microbiology, and Institute of Energy and the Environment, University of Oklahoma, Norman, OK 73019, USA
2 The Institute of Life Sciences and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, Jerusalem, Israel
3 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain
Correspondence
Mostafa S. Elshahed
mostafa{at}ou.edu
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain M6T is AY458601.
Scanning electron micrographs showing cells of strain M6T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Oren, 2002). A shared character of all members of the family is the absolute requirement for high concentrations of NaCl.
The genus Haloferax was first described by Torreblanca et al. (1986) and currently comprises six species with validly published names: Haloferax volcanii (Mullakhanbhai & Larsen, 1975), Haloferax denitrificans (Tomlinson et al., 1986), Haloferax gibbonsii (Juez et al., 1986), Haloferax mediterranei (Rodriguez-Valera et al., 1983), Haloferax alexandrinus (Asker & Ohta, 2002) and Haloferax lucentense (Gutierrez et al., 2002). Members of the genus Haloferax are characterized by extreme pleomorphism and a relatively low salt requirement compared with other genera of the Halobacteriaceae.
In spite of the fact that members of the Halobacteriaceae are generally considered to have an absolute requirement for at least 1·5 M NaCl for growth, a few studies have demonstrated that members of the family may be isolated from a variety of low-salt environments. Rodriguez-Valera et al. (1979) isolated Halococcus species from sea water. Also, Natrinema isolates have been recovered from low-salt saltern ponds (McGenity et al., 1998). In addition, several culture-independent analyses have detected 16S rRNA gene sequences suggestive of the presence of Halobacteriaceae species in other low-salt environments, including coastal marshes (Munson et al., 1997) and deep-sea vent black smoker chimney structures (Takai et al., 2001). In the course of our studies of microbial diversity in the sulfide- and sulfur-rich Zodletone spring (Oklahoma, USA), 16S rRNA genes were cloned from microbial mats (Elshahed et al., 2004). Clone libraries were observed to contain many clones related to the family Halobacteriaceae, even though the spring has a stream-water salinity of only 1 %. Field measurements indicated the presence of relatively high salt habitats near the shallow stream. These habitats presumably provide a suitable environment for members of the Halobacteriaceae to survive and grow (Elshahed et al., 2004). Eighteen halophilic strains were isolated from the microbial mats and mineral crusts that form near the stream using high-salt medium supplemented with antibiotics to suppress the growth of halophilic or halotolerant bacteria (Elshahed et al., 2004). Six of these strains were studied and a preliminary investigation (colony and cell morphology, salt tolerance profile, lipid pattern and partial 16S rRNA gene sequence) suggested that all six isolates belong to a single species. In this paper, one of the isolates, strain M6T, has been characterized in detail; it is proposed that this strain represents a novel species of the genus Haloferax.
The isolation procedure was described previously in detail (Elshahed et al., 2004). Characterization of strain M6T followed the guidelines outlined by Oren et al. (1997) for describing novel species of the Halobacteriaceae. Physiological, biochemical and nutritional tests were performed in halophilic medium (HM), pH 7·0, adapted from Oren (2002), containing (g l–1): NaCl (150), MgCl2.7H2O (20), K2SO4 (5), CaCl2.2H2O (0·1) and yeast extract (5). Cultures were incubated at 37 °C with shaking (200 r.p.m.) unless otherwise specified. Growth was monitored by measuring the increase in OD600. Growth was evaluated at NaCl concentrations of 0–37 % (saturation), Mg2+ concentrations of 0–200 mM, a temperature range of 4–60 °C and at pH 3–11. Suitable organic buffers were included in the pH range and pH optimum determination experiments (25 mM MES, MOPS, HEPES or TES) to prevent changes in pH due to acid production during growth. Substrate utilization was tested by lowering the yeast extract concentration to 0·1 g l–1, including the substrate at a concentration of 0·5 g l–1 and adding 25 mM MOPS as a buffer. Acid production from a variety of substrates was tested in a similar, but unbuffered HM medium, with 0·5 g substrate l–1 and phenol red (0·004 g l–1) as an indicator. Tests for the ability of strain M6T to grow utilizing S0, 
, 
, 
, DMSO and TMAO as electron acceptors, as well as its ability to ferment arginine, were performed in HM prepared anaerobically in serum tubes (Balch & Wolfe, 1976; Bryant, 1972). 
, 
and 
were added at a concentration of 30 mM and DMSO, TMAO and arginine were added at a concentration of 5 g l–1. 
and 
reduction under anaerobic conditions was determined by quantification of the electron acceptors using a Dionex ion chromatography system. Aerobic thiosulfate reduction was determined by the formation of black sulfide precipitate in HM medium amended with 0·5 % sodium thiosulfate. Biochemical tests were performed according to the methods outlined by Gerhardt et al. (1994). Haloferax volcanii DSM 33755T was used as a control in all tests. 16S rRNA genes were amplified using the primers Arch21f and Arch1492r (Reysenbach et al., 2000), cloned using a TOPO-TA cloning kit (Invitrogen) and sequenced at the Oklahoma Medical Research Foundation Core Sequencing Facility. Sequence alignment was performed using CLUSTAL_X (Thompson et al., 1997). Phylogenetic analysis involved evaluation of the evolutionary distance with a neighbour-joining algorithm and Jukes–Cantor corrections using PAUP 4.01b10 (Sinauer Associates). Samples were fixed for EM on a poly(lysine)-coated cover-slip using glutaraldehyde, coated with gold/palladium and examined using a JSM-880 SEM. G+C content of the total cellular DNA and DNA–DNA hybridization values were determined according to methods outlined previously (Gutierrez et al., 2002).
Strain M6T formed small (0·8–1·0 mm), salmon pink, transparent and elevated punctiform colonies with undulate margins. Cells were extremely pleomorphic and stained Gram-negative. Rods, irregular cells and flattened disc shapes were observed using phase-contrast microscopy and SEM. Rod-shaped cells (single or in pairs) were observed more frequently during the exponential growth phase, whereas irregularly shaped cells were common during the stationary phase or from colonies on agar plates. Scanning electron micrographs of strain M6T are available as supplementary material in IJSEM Online. Strain M6T grew in HM medium in a wide range of NaCl concentrations (from 60 g l–1 to saturation) with an optimum between 125 and 150 g l–1. It required at least 1 mM Mg2+ and grew best with 30 mM or more. Cells lysed if suspended in distilled water or in NaCl concentrations below 30 g l–1, but they retained their viability for prolonged incubations in NaCl solutions of 40 g l–1 and above (Elshahed et al., 2004). Strain M6T did not grow anaerobically with nitrate, sulfate, thiosulfate, DMSO or TMAO as electron acceptors, nor was it able to ferment arginine. It was, however, capable of reducing elemental sulfur to sulfide (Elshahed et al., 2004). Control experiments with Haloferax volcanii DSM 33755T indicated that this species could also reduce elemental sulfur to sulfide, albeit at a much slower rate (50 µM sulfide formed after 3 months compared with 0·4 mM in 2 weeks for strain M6T). These results suggest that strain M6T may be capable of surviving under the anaerobic conditions of the Zodletone spring by reducing elemental sulfur to sulfide. It is likely that elemental sulfur reduction is a common capability within the Halobacteriaceae, as suggested by Grant & Ross (1986) and Tindall & Trüper (1986).
Detailed results of nutritional experiments, antibiotic sensitivity and the physiological description are given in the species description. In general, strain M6T was similar to other members of the genus Haloferax in being oxidase- and catalase-positive and able to grow on a single carbon source. It is also similar in that all species are unable to grow anaerobically on DMSO or TMAO and they are unable to ferment arginine or to decarboxylate lysine and ornithine. Differences between M6T and other members of the genus Haloferax are highlighted in Table 1.
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). Sequence similarity calculations using neighbour-joining analysis indicated that the sequence of strain M6T has between 96·7 % (Haloferax mediterranei) and 98·0 % (Haloferax lucentense) similarity to sequences of other members of the genus Haloferax. The closest relative to strain M6T was Haloferax sp. strain D1227, isolated from soil contaminated with highly saline oil brine (Emerson et al., 1994). The closest relative to strain M6T outside the genus Haloferax was Halogeometricum borinquense, with a sequence similarity of only 89 %. Phospholipid analysis indicated the presence of S-DGD-1 and the absence of phosphatidylglycerosulfate, a pattern characteristic of members of the genus Haloferax (Oren, 2000). The DNA G+C content of strain M6T is 60·5 mol%, a value within the designated range of the genus (Grant et al., 2001). Results of DNA–DNA hybridization experiments (Table 1
) showed hybridization values between strain M6T and other species of the genus Haloferax ranging between 1 and 24 %, thus indicating that this strain is clearly a novel species of the genus Haloferax.
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, as well as 16S rRNA gene sequence analysis and DNA–DNA hybridization data justify the placement of strain M6T within a novel species of the genus Haloferax, for which the name Haloferax sulfurifontis sp. nov. is proposed.
Description of Haloferax sulfurifontis sp. nov.
Haloferax sulfurifontis (sul.fu.ri.fon'tis. L. masc. n. fons, fontis spring; L. neut. n. sulfur sulfur; N.L. gen. n. sulfurifontis of a sulfurous spring).
Cells are non-motile, extremely pleomorphic, occurring mainly as irregularly shaped cells (1·0–1·5 µm in diameter), particularly during the stationary phase, and as rods (1·5–1·7x0·5–0·6 µm), especially during the exponential growth phase. Occurs mostly as single cells, sometimes in pairs and clusters. Gram-negative. Colonies on agar medium with 150 g NaCl l–1 are small (0·8–1·0 mm), salmon pink, transparent and elevated with undulate margin. Halophilic. Cells lyse immediately in distilled water, within 24 h in 10–20 g NaCl l–1 and within 72 h in 30 g NaCl l–1. Cells survive prolonged incubation in 40–50 g NaCl l–1. Grows in a wide range of NaCl concentrations (60 g l–1 to saturation). Requires at least 1 mM Mg2+ for growth and grows best at 30 mM Mg2+ and above. The optimum pH and temperature values for growth are pH 6·4–6·8 and 32–37 °C. The type strain, M6T, grows at 18–50 °C and at pH 4·5–9·0. Cannot utilize 
, 
, 
, DMSO or TMAO as electron acceptors. Reduces elemental sulfur to sulfide under strictly anaerobic conditions. Chemo-organotrophic. Grows on complex media with yeast extract, Casamino acids and peptone as carbon sources. Capable of growing in defined media. The following substrates are utilized as carbon sources: acetate, benzoate, citrate, fumarate, L-glutamate, malate, succinate, glycerol, maltose, glucose, fructose, sucrose, arabinose, galactose and xylose. Alanine, aspartate, arginine, glycine, lactose, mannitol, sorbitol, ribose and starch are not utilized as carbon sources. Acid is produced in unbuffered medium from the following compounds: glycerol, xylose, maltose, sucrose, arabinose, fructose, glucose and galactose. Acid is not produced from mannitol, arabinose, lactate or sorbitol. Catalase- and oxidase-positive. Reduces thiosulfate aerobically to sulfide. Capable of aerobic nitrate reduction. Indole is formed from tryptophan. Gelatin is hydrolysed, whereas starch, casein and urea are not. Produces polyhydroxyalkanoates. Phosphatase and 
-galactosidase tests are negative. Ornithine and lysine are not decarboxylated. Resistant to ampicillin, erythromycin, chloramphenicol, carbenicillin, gentamicin, ceftriaxone, ciprofloxacin, doxycycline, cefaclor, kanamycin, nalidixic acid, oxytetracycline, penicillin G, rifampicin and bacitracin up to 100 µg ml–1. Sensitive to aphidicolin, anisomycin and novobiocin, and to high concentrations of bacitracin. The major polar lipids in the membrane are the diphytanyl ether derivatives of phosphatidylglycerol, the methyl ester of phosphatidylglycerophosphate, and S-DGD-1 as the main glycolipid. Phosphatidylglycerosulfate is absent.
The type strain is M6T (=JCM 12327T=CCM 7217T=DSM 16227T=CIP 108334T). The DNA G+C content of M6T is 60·5 mol%. Isolated from the Zodletone spring in south-western Oklahoma, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Balch, W. E. & Wolfe, R. S. (1976). New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl Environ Microbiol 32, 781–791.
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Emerson, D., Chauhan, S., Oriel, P. & Breznak, J. A. (1994). Haloferax sp. D1227, a halophilic archaeon capable of growth on aromatic compounds. Arch Microbiol 161, 445–452.[CrossRef]
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McGenity, T. J., Gemmell, R. T. & Grant, W. D. (1998). Proposal of a new halobacterial genus Natrinema gen. nov., with two species Natrinema pellirubrum nom. nov. and Natrinema pallidum nom. nov. Int J Syst Bacteriol 48, 1187–1196.[CrossRef][Medline]
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Munson, M. A., Nedwell, D. B. & Embley, T. M. (1997). Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh. Appl Environ Microbiol 63, 4729–4733.[Abstract]
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Rodriguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1979). Isolation of extreme halophiles from seawater. Appl Environ Microbiol 38, 164–165.
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