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1 Extremophiles Laboratory, Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran
2 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
3 Pasteur Institute of Iran, Tehran, Iran
Correspondence
Mohammad Ali Amoozegar
amozegar{at}khayam.ut.ac.ir
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain QW6T is DQ767692.
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, 1992), Salinicoccus alkaliphilus (Zhang et al., 2002), Salinicoccus salsiraiae (França et al., 2006), Salinicoccus jeotgali (Aslam et al., 2007), Salinicoccus luteus (Zhang et al., 2007), Salinicoccus siamensis (Pakdeeto et al., 2007) and Salinicoccus kunmingensis (Chen et al., 2007).
The textile industry produces large quantities of wastewater containing pollutants such as azo dyes and toxic oxyanions that are of major environmental concern. Moderately halophilic bacteria can play an important role in bioremediation and transformation of these toxic compounds in such polluted saline environments. In this study, a toxic-oxyanion-tolerant, moderately halophilic, aerobic, Gram-positive coccus, designated strain QW6T, isolated from wastewaters of the textile industry in Qom, Iran, is described on the basis of a polyphasic study. The salinity of the wastewater samples from Qom region was determined to be about 3 % (w/v, as total salt) and the pH value of the sample was 7.9. Aliquots of the sample were added to 10 % MH medium (Ventosa et al., 1982) supplemented with sea salts solution as follows (%, w/v): NaCl, 8.1; MgCl2, 0.7; MgSO4, 0.96; CaCl2, 0.036; KCl, 0.2; NaHCO3, 0.006; NaBr, 0.0026; proteose-peptone no. 3, 0.5; yeast extract, 1.0, and glucose, 0.1. The pH was adjusted to 7.5 with 1 M KOH prior to autoclaving. Incubation was carried out at 35 °C under aerobic conditions on an orbital shaking incubator (orbital incubator SI 50; Stuart Scientific) at 150 r.p.m. After 2 days incubation, cultures were streaked on agar plates and gave rise to different types of colonies. Pure cultures were isolated after repeated restreaking.
A pure culture of strain QW6T was grown and maintained in 10 % MH medium solidified by adding 15 g agar (M593 agar) (http://www.dsmz.de/media/med593.htm).
Incubation was carried out on a shaker at 150 r.p.m. and 35 °C. Morphological, physiological and biochemical characteristics of the isolate were studied in 10 % MH medium at pH 7.5 and 35 °C, unless stated otherwise.
Reference strains S. roseus DSM 5351T, S. hispanicus DSM 5352T, S. alkaliphilus DSM 16010T, S. salsiraiae DSM 19496T and S. kunmingensis DSM 17847T were obtained from the DSMZ.
Cell morphology was examined by light microscopy and scanning electron microscopy (SEM) using cells from exponentially growing cultures. Strain QW6T was prepared for SEM according to the method of Bozzola & Russell (1999) and specimens were observed on a Zeiss DSM 960 EM. Gram staining was performed by the Burke method (Murray et al., 1994) and the result was confirmed by the KOH test (Baron & Finegold, 1990). Motility was analysed by the wet-mount method (Murray et al., 1994). Catalase, oxidase and urease activities, nitrate reduction, hydrolysis of aesculin and production of indole were tested as recommended by Smibert & Krieg (1994). Hydrolysis of Tween 80 was examined as described by Harrigan & McCance (1976). Methyl red and Voges–Proskauer tests were done as recommended by Smibert & Krieg (1994). Determination of acid production from carbohydrates, as well as utilization of carbon and nitrogen sources, was performed as recommended by Ventosa et al. (1982).
Antibiotic susceptibility tests were performed on Mueller–Hinton agar plus 10 % (w/v) sea salts seeded with a bacterial suspension containing 1.5x106 c.f.u. ml–1 using discs (HiMedia) impregnated with various antibiotics. The plates were incubated at 35 °C for 48 h and the inhibition zone was interpreted according the manufacturer's manual. To determine the optimal temperature and pH for growth of the strain, broth cultures were incubated at temperatures of 5–55 °C at intervals of 5 °C and at pH 5–11 at intervals of 0.5 pH units. pH values below 6, pH 6–9 and pH values above 9 were obtained using sodium acetate/acetic acid, Tris/HCl and glycine/sodium hydroxide buffers, respectively. Growth at different salt contents (0, 2.5, 5, 7.5, 10, 15, 20, 25 and 30 %, w/v) was tested on MH medium at pH 7.5. Growth was monitored by turbidity at OD600 using a spectroscopic method (model UV-160 A; Shimadzu). Other physiological and biochemical tests were performed as described previously (Mata et al., 2002; Quesada et al., 1984). As the habitat of the isolate was polluted by toxic compounds such as tellurite, selenite and selenate oxyanions, the resistance of strain QW6T to these oxyanions was measured by the agar dilution method of Washington & Sutter (1980). Melted nutrient agar (20 ml) plus various concentrations of tellurite (0.1–40 mM), selenite (1 mM to 1 M) and selenate (1 mM to 1.2 M) were poured separately into 8 cm plates. Then, 10 µl bacterial suspension (1.5x108 c.f.u. ml–1) was inoculated onto each plate using a sampler followed by incubation at 35 °C for 7 days. Minimum inhibitory concentrations for tellurite, selenite and selenate oxyanions were determined. Each plate was prepared in triplicate.
Preparation and hydrolysis of the cell wall was carried out using the method of Schleifer (1985) and the interpeptide bridge in the cell-wall peptidoglycan was analysed by using the method described by Schleifer & Kandler (1972). Cell-wall hydrolysates were separated by one- or two-dimensional chromatography on cellulose thin-layer plates (Merck).
Isoprenoid quinone analysis was carried out as described by Monciardini et al. (2003). Cellular fatty acids were extracted from cells of strain QW6T, S. roseus DSM 5351T, S. hispanicus DSM 5352T, S. alkaliphilus DSM 16010T and S. salsiraiae DSM 19496T that had been cultivated on 10 % MH medium at 37 °C for 1 day according to Stead et al. (1992) and were analysed by GC (Groth et al., 1996).
For determination of DNA base composition and DNA–DNA hybridization, DNA was isolated using a French pressure cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). The DNA G+C content was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989). DNA–DNA hybridization was carried out by the Identification Service of the DSMZ, as described by De Ley et al. (1970) incorporating the modifications described by Huß et al. (1983) using a Cary 100 Bio UV/VIS spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in situ temperature probe (Varian).
Genomic DNA of the isolate was extracted with a Genelute DNA extraction kit (Sigma) by following the recommended procedure of the manufacturer.
The 16S rRNA gene of the isolate was amplified using universal primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (5'-AAGGAGGTGATCCAGCCGCA-3'). The amplification was performed by initial denaturation at 95 °C for 5 min followed by: 10 cycles of 93 °C for 1 min, 63 °C for 1 min, 71 °C for 1.5 min; 20 cycles of 93 °C for 1 min, 67 °C for 1 min, 71 °C for 2 min; and a final extension at 71 °C for 5 min. The purified PCR product was sequenced in both directions using an automated sequencer by the SeqLab Laboratory (Göttingen, Germany).
Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and MEGA version 3 (Kumar et al., 2004) after multiple alignment of data available from public databases by CLUSTAL_X (Thompson et al., 1997).
Pairwise evolutionary distances were computed using the correction of Jukes & Cantor (1969) and clustering was performed using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein, 1985).
Cells of the novel isolate were Gram-positive, non-spore-forming, non-motile cocci that were strictly aerobic and occurred singly, in pairs, in tetrads or in clumps (Fig. 1). Colonies were round, smooth, entire, opaque and approximately 2 mm in diameter forming an orangish pink, non-diffusible pigment after 2 days at 35 °C on 10 % MH medium. Strain QW6T grew at sea salts concentrations in the range 1–25 % (w/v) in MH medium. Growth did not occur at concentrations of sea salts greater than 25 % (w/v). The temperature range for growth was 5–45 °C and the pH range was 6.5–10.0. Optimal growth occurred in MH medium supplemented with 7.5–10 % (w/v) sea salts at 35 °C and pH 7.5. The novel isolate was resistant to potassium tellurite, sodium selenite and sodium selenate up to concentrations of 12, 600 and 1000 mM, respectively.
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Detailed physiological and biochemical characteristics of the strain are shown in Table 1 and in the species description.
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, 1992; Zhang et al., 2002, 2007; França et al., 2006; Aslam et al., 2007; Pakdeeto et al., 2007; Chen et al., 2007).
The almost complete 16S rRNA gene sequence (1499 bp) of strain QW6T was determined. Alignments of this sequence with sequences belonging to type strains of the halophilic cocci available from GenBank showed that the closest relative of strain QW6T was S. roseus DSM 5351T, with a sequence similarity of 96.4 %. The sequence similarities of strain QW6T to S. hispanicus DSM 5352T, S. luteus YIM 70202T, S. jeotgali KCTC 13030T, S. salsiraiae LMG 22840T, S. siamensis PN1-2T, S. alkaliphilus DSM 16010T and S. kunmingensis YIM Y15T were 95.0, 94.9, 94.5, 94.5, 94.1, 94.0 and 92.9 %, respectively. The level of 16S rRNA gene sequence similarity between strain QW6T and other Gram-positive cocci was less than 91 %. The phylogenetic tree constructed by the neighbour-joining method is shown in Fig. 2.
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together with the data for S. jeotgali (Aslam et al., 2007). The fatty acid content of S. jeotgali was very similar to those of the other type strains, even though the cells were grown under different conditions. Strain QW6T had iso-C15 : 0 and anteiso-C15 : 0 as major fatty acids; most minor fatty acids were similar to those of the type strains of members of the genus Salinicoccus, except that strain QW6T had a remarkably higher C20 : 0 content.
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).
DNA–DNA hybridization experiments revealed low levels of relatedness between strain QW6T and S. roseus DSM 5351T (40.3 %), S. hispanicus DSM 5352T (1.3 %) and S. alkaliphilus DSM 16010T (19.6 %). According to Wayne et al. (1987), less than 70 % DNA–DNA relatedness is considered to be the threshold value for the delineation of genospecies, so the values are low enough to separate strain QW6T from these three species.
The phylogenetic distinctiveness and low DNA–DNA relatedness values, together with several phenotypic differences (physiological and biochemical, as well as DNA G+C content; Table 1
), indicate that strain QW6T represents a novel species of the genus Salinicoccus, for which the name Salinicoccus iranensis sp. nov. is proposed.
Description of Salinicoccus iranensis sp. nov.
Salinicoccus iranensis (i.ran.en'sis. N.L. masc. adj. iranensis from Iran, where the organism was isolated).
Cells are Gram-positive cocci, 0.8–1.0 µm in diameter, occurring singly, in pairs, in tetrads or in clumps. Non-motile and strictly aerobic. Endospores are not formed. Colonies are round, smooth, slightly convex and orangish pink in colour; no diffusible pigment is produced. Growth occurs in media containing 1.0–25.0 % (w/v) NaCl; 10 % (w/v) is optimal for growth. No growth occurs in the absence of NaCl. The pH range for growth is 6.5–10.0 (optimum at pH 7.5). Growth occurs at 5.0–45.0 °C (optimum at 35 °C). Catalase, oxidase, and Simmons' citrate and indole tests are positive. H2S production and methyl red, Voges–Proskauer and urease tests are negative. Tween 20 is weakly hydrolysed, whereas casein, starch, aesculin, gelatin and Tween 80 are not. Utilizes sucrose, glucose, starch, aesculin, D-mannitol, D-mannose, lactate, dextrin, salicin, inositol and fructose as sole carbon and energy sources. Acid is produced from (+)-D-glucose and fructose, but not from arabinose, maltose, galactose, glycerol, raffinose, D-melibiose, L-rhamnose, lactose or sucrose. Nitrate is reduced. L-Cysteine, L-proline and L-glycine are utilized as sole nitrogen sources, whereas urea, yeast extract, L-arginine, L-ornithine, L-asparagine and L-phenylalanine are not. Susceptible to carbenicillin (100 µg), cephalothin (30 µg), cefoxitin (30 µg), chloramphenicol (30 µg), cotrimoxazole (25 µg), erythromycin (15 µg), neomycin (30 µg), nitrofurantoin (300 µg) and tetracycline (30 µg); resistant to ampicillin (10 µg), bacitracin (10 U), clindamycin (2 µg), cloxacillin (5 µg), gentamicin (30 µg), novobiocin (5 µg), penicillin G (10 U), polymyxin B (100 U), streptomycin (10 µg) and tobramycin (10 µg); intermediately susceptible to amikacin (30 µg) and kanamycin (30 µg). Other phenotypic features of this strain are shown in Table 1. The cell wall contains peptidoglycan of the L-Lys-Gly5 type. The major respiratory lipoquinone is MK-6. Predominant fatty acids are iso-C15 : 0 and anteiso-C15 : 0. The DNA G+C content of the type strain is 54.4 mol%.
The type strain is QW6T (=DSM 18903T=CCM 7448T), isolated from wastewaters of the textile industry in Qom, Iran.
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ACKNOWLEDGEMENTS |
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