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1 Yunnan Institute of Microbiology and Key Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan 650091, PR China
2 College of Bio-resources and Environmental Science, Jishou University, Jishou, Hunan 416000, PR China
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b, D-38124 Braunschweig, Germany
4 Yunnan Salt & Chemical Industry Co., Ltd, Kunming, Yunnan 650091, PR China
Correspondence
Xiao-Long Cui
xlcui{at}ynu.edu.cn
or
xlcuiynu{at}yahoo.com.cn
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ABSTRACT |
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; Grant et al., 1998; Norton & Grant, 1988; Norton et al., 1993; Vreeland & Powers, 1999; Vreeland et al., 2000; McGenity et al., 2000). In a recent study of the microbial diversity of the ancient salt deposit of the Yipinglang salt mine (2 ° 18' N 10 ° 54' E) in Yunnan, south-west China, a moderately halophilic, facultatively alkaliphilic strain, YIM Y15T, was isolated from a brine sample. Polyphasic taxonomic study of the phenotypic, chemotaxonomic and genotypic characteristics and phylogenetic position of strain YIM Y15T indicated that this strain represented a novel species of the genus Salinicoccus.
Brine, saline soil and halite samples were collected from the Yipinglang salt mine at a depth of about 200 m. Serial dilutions (1 : 10) of the samples were plated on Difco marine agar 2216 (MA; pH 7.2) supplemented with 0–20 % (w/v) NaCl and ISP medium 2 agar (Shirling & Gottlieb, 1966) at 28 °C for 7–28 days. A yellow-pigmented colony, named strain YIM Y15T, was picked from a brine plate of MA. Unless otherwise indicated, morphological and physiological studies were performed with cells grown on MA supplemented with 8 % (w/v) NaCl at pH 8.0 and 37 °C and on some other media as controls, e.g. nutrient agar, tryptic soy agar (TSA; BBL) and ISP medium 2 agar. The strain was maintained both on agar slants of MA supplemented with 8 % (w/v) NaCl (pH 8.0) at 4 °C and in Difco marine broth (MB) supplemented with 20 % (v/v) glycerol at –80 °C.
Cell morphology was examined by light microscopy (model BH 2; Olympus). Gram staining was carried out using the standard Gram reaction combined with the KOH lysis test method (Gregersen, 1978). Anaerobic growth was determined using the GasPak Anaerobic System (BBL) according to the manufacturer's instructions. Motility was observed on half-strength MA, solidified with 0.3 % agar, under high-moisture conditions and in a hanging-drop preparation under a x100 objective lens with oil immersion.
Cells of strain YIM Y15T were Gram-positive cocci, approximately 0.8–1.2 µm in diameter, occurring singly and in pairs, tetrads or clumps, and strictly aerobic, non-motile and non-sporulating. Colonies were circular, convex, yellow-pigmented and non-translucent with shiny, glistening surfaces and entire margins, 2–3 mm in diameter after incubation for 3 days at 37 °C on MA supplemented with 8 % (w/v) NaCl. No diffusible pigments were produced on any media tested.
Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out as described by Cui et al. (2001). Electrophoresis of sequencing reaction products was done by using a Beckman CEQ 2000 sequencer according to the manufacturer's protocol. The resulting 16S rRNA gene sequence was compared to sequences obtained from the GenBank database to find the most closely related species. Phylogenetic analyses were performed using the software package MEGA version 2.1 (Kumar et al., 2001) after multiple alignment of sequence data by CLUSTAL_X (Thompson et al., 1997). Distances (corrected by Kimura's two-parameter model; Kimura, 1980) were calculated and clustering was performed with the neighbour-joining method (Saitou & Nei, 1987). Maximum-likelihood (Felsenstein, 1981) and parsimony (Kluge & Farris, 1969) trees (not shown) were generated using treeing algorithms contained in the PHYLIP package (Felsenstein, 1993). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by means of 1000 resamplings (Felsenstein, 1985).
The almost-complete 16S rRNA gene sequence (1493 bp) of strain YIM Y15T was determined. A neighbour-joining tree (Fig. 1) showed that strain YIM Y15T was closely related to the type strains of the five recognized members of the genus Salinicoccus Ventosa et al. 1990, and the six strains formed a distinct clade in the phylogenetic tree with significant bootstrap support (99 %), in which strain YIM Y15T and Salinicoccus alkaliphilus JCM 11311T (Zhang et al., 2002) formed a distinct subclade (Fig. 1). The values of 16S rRNA gene sequence similarity between strain YIM Y15T and the five closest relatives were 96.0 % (S. alkaliphilus JCM 11311T), 94.9 % (Salinicoccus roseus DSM 5351T; Ventosa et al., 1990), 94.8 % [Salinicoccus jeotgali KCTC 13030T (Aslam et al., 2007); Salinicoccus salsiraiae LMG 22840T (França et al., 2006)] and 94.7 % (Salinicoccus hispanicus DSM 5352T; Marquez et al., 1990; Ventosa et al., 1992). It has been suggested that bacterial strains with less than 97 % 16S rRNA gene sequence identity are members of different genomic species (Stackebrandt & Goebel, 1994). It is therefore evident from the phylogenetic data that strain YIM Y15T represents a previously unknown species of the genus Salinicoccus.
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-phenylenediamine (bioMérieux). Hydrolysis of polymers, urease activity, nitrate reduction and Voges–Proskauer and methyl red tests were determined as described previously (Cowan & Steel, 1965; Gerhardt et al., 1981; Ventosa et al., 1982). Substrate utilization as sole carbon and energy sources, the fermentation/oxidation profile, acid production, activities of constitutive enzymes and some other physiological characteristics were examined using API 20E, API 20NE, API 50CH (with API 50 CH B/E medium) and API ZYM strips (bioMérieux) according to the manufacturer's instructions. Additionally, Biolog GP2 MicroPlates and the MicroLog computer software (Biolog) were used. All suspension media were supplemented with 2 % (w/v) NaCl. Antibiotic resistance was determined by inoculating bacterial suspension on MA at 3 °C and applying Antibiotics-Sensitabs (Tiantan Medicine Co.). Inhibition zones were measured after 5 days incubation and interpreted according to the manufacturer's manual.
Strain YIM Y15T was catalase- and oxidase-positive, with a wide growth temperature range of 4–45 °C (optimum 37 °C). It was moderately halophilic, as the optimum NaCl concentration for growth was between 8 and 10 %, with a NaCl concentration range for growth of 0.5–25 % (w/v) (Kushner, 1993). Good growth occurred on MA (containing 19.5 g NaCl l–1) and TSA (containing 5.0 g NaCl l–1), but there was no growth on nutrient agar or ISP 2. Strain YIM Y15T was a facultatively alkaliphilic organism with a growth range of pH 6.0–10.0, and optimum growth was observed at pH 8.0. The results of other phenotypic tests are listed in the species description and in Table 1.
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. The amino acid composition of the cell-wall hydrolysate was determined by using TLC as described by Staneck & Roberts (1974). Isoprenoid quinones were analysed by HPLC as described by Groth et al. (1996). For isoprenoid quinone analyses, the organism was inoculated in MB supplemented with 8 % (w/v) NaCl in flasks on a rotary shaker at 200 r.p.m. The biomass was harvested by centrifugation, washed twice with distilled water and freeze-dried. The fatty acid composition was determined as described by Sasser (1990) using the Microbial Identification System (MIDI; Microbial ID) with cells grown on MA supplemented with 8 % (w/v) NaCl for 3 days at 37 °C. DNA was isolated according to Hopwood et al. (1985) and its G+C content was determined by the thermal denaturation method (Mandel & Marmur, 1968) with a Shimadzu UV-visible spectrophotometer (UV1601).
Chemotaxonomic data for strain YIM Y15T are compatible with its assignment to the genus Salinicoccus. The major amino acid constituents of the cell-wall hydrolysate were glycine and lysine, which is compatible with the murein type of L-Lys–Gly5 described for the genus Salinicoccus (Ventosa et al., 1990). The strain studied had menaquinone 6 (MK-6) (98.8 %) and MK-7 (1.2 %) as its respiratory quinones. The fatty acid profiles of strain YIM Y15T and related type strains are given in Table 2. The major fatty acids of the strain studied were iso- and anteiso-branched fatty acids [ai-C15 : 0 (28.4 %), i-C15 : 0 (23.1 %), ai-C17 : 0 (5.8 %), i-C16 : 0 (5.8 %) and i-C17 : 0 (5.7 %)]. The DNA G+C content of strain YIM Y15T was 46.2 mol%.
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). On the basis of the phylogenetic, chemotaxonomic and phenotypic results presented above, we propose that strain YIM Y15T represents a novel species of the genus Salinicoccus, Salinicoccus kunmingensis sp. nov.
Description of Salinicoccus kunmingensis sp. nov.
Salinicoccus kunmingensis (kun.ming.en'sis. N.L. masc. adj. kunmingensis pertaining to Kunming, a city in south-west China, near which the sample from which the type strain was isolated was collected).
Cells are Gram-positive, non-motile, non-sporulating, catalase- and oxidase-positive, obligately aerobic cocci (0.8–1.2 µm) that occur singly or in pairs, tetrads or clumps. Colonies are circular, convex, yellow-pigmented and non-translucent with shiny, glistening surfaces and entire margins, 2–3 mm in diameter after 3 days on MA supplemented with 8 % (w/v) NaCl at pH 8.0 and 37 °C. No diffusible pigments are produced on any media tested. Growth occurs at 4–45 °C (optimum 37 °C) and pH 6.0–10.0 (optimum pH 8.0). Moderately halophilic, with growth at 0.5–25 % (w/v) NaCl [optimum 8–10 % (w/v) NaCl]. Also grows well on MA supplemented with 2–15 % (w/v) MgCl2 or KCl instead of NaCl. Positive for hydrolysis of aesculin, starch and Tween 80, but negative for hydrolysis of casein, chitin, gelatin and Tween 20. Nitrate is reduced to nitrite. Voges–Proskauer and methyl red tests are negative. H2S and indole are not produced. Cells are resistant to gentamicin (10 µg), kanamycin (30 µg), lincomycin (2 µg), polymyxin B (30 µg) and streptomycin (10 µg), but ampicillin (30 µg), chloramphenicol (30 µg), nalidixic acid (20 µg), novobiocin (30 µg), rifampicin (5 µg) and tetracycline (30 µg) inhibit growth. In API 20NE strips, a positive result is obtained for assimilation of potassium gluconate and negative results are obtained for assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, capric acid, adipic acid, malate, trisodium citrate and phenylacetic acid and fermentation of glucose. Acid is produced from amygdalin, N-acetylglucosamine, D-fructose, inositol, D-lactose, D-mannitol and sucrose in API 50CH strips. In Biolog GP2 plates, the following substrates are used as sole carbon and energy sources: N-acetyl-D-glucosamine, amygdalin, L-arabinose, D-cellobiose, D-fructose, D-glucose, glycerol, 
-hydroxybutyric acid, myo-inositol, 
-D-lactose, D-mannitol, D-mannose, D-psicose, D-raffinose, L-rhamnose, D-ribose, sucrose, trehalose, Tween 40, L-alaninamide and L-asparagine. Constitutive enzymes expressed by the type strain are catalase, cytochrome oxidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, -chymotrypsin, -mannosidase, gelatinase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and valine arylamidase. Arginine dihydrolase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, -glucuronidase, trypsin, N-acetyl--glucosaminidase, -galactosidase/o-nitrophenyl--D-galactopyranosidase (ONPG), tryptophan deaminase, lysine decarboxylase, -galactosidase/p-nitrophenyl--D-galactopyranosidase (PNPG), ornithine decarboxylase, -fucosidase and urease are not produced. The assumed cell-wall murein type is L-Lys–Gly5. The predominant respiratory quinone is MK-6, and MK-7 is present in minor amounts. The major cellular fatty acids are anteiso-C15 : 0 (28.4 %) and iso-C15 : 0 (23.1 %). The DNA G+C content of the type strain is 46.2 mol%.
The type strain, YIM Y15T (=DSM 17847T =CGMCC 1.6302T), was isolated from a brine sample collected from the Yipinglang salt mine in Kunming, south-west China.
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ACKNOWLEDGEMENTS |
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