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1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
2 Laboratory of Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
Correspondence
Antonio Ventosa
ventosa{at}us.es
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ABSTRACT |
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8c and C12 : 0 3-OH. Overall, the phenotypic, genotypic and phylogenetic results demonstrated that strain 43T represents a novel species within the genus Chromohalobacter. The name Chromohalobacter japonicus sp. nov. is proposed, with strain 43T (=CECT 7219T =CCM 7416T) as the type strain.
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; Garrity et al., 2005). The genus Chromohalobacter was proposed by Ventosa et al. (1989) with a single species, Chromohalobacter marismortui, in order to include a halophilic bacterium previously described as ‘Chromobacterium marismortui’ (isolated from the Dead Sea by Elazari-Volcani, 1940) and several isolates from a saltern in Spain. Currently, the genus Chromohalobacter includes the species Chromohalobacter canadensis (Arahal et al., 2001a), C. israelensis (Arahal et al., 2001a), C. salexigens (Arahal et al., 2001b), C. sarecensis (Quillaguamán et al., 2004) and C. nigrandesensis (Prado et al., 2006). Recently, Peçonek et al. (2006) proposed the reclassification of Pseudomonas beijerinckii (Hof, 1935) as Chromohalobacter beijerinckii and emended the description of this species. The genus Chromohalobacter includes Gram-negative, straight or slightly curved, motile rods that are moderately halophilic, i.e. with optimum salt concentrations for growth between 8 and 10 % NaCl. They are aerobic, catalase-positive and (in most species) oxidase-negative. They have been isolated from hypersaline environments such as the Dead Sea, salterns and saline soils (Ventosa, 2005). This genus represents a coherent phylogenetic cluster closely related to the genus Halomonas (Mellado et al., 1995; Arahal et al., 2002).
In this study, we have determined the taxonomic position of strain 43T, which was isolated from a Japanese salty food in Shiokara (Onishi et al., 1980), as a novel member of the genus Chromohalobacter.
Strain 43T was isolated in SGC medium containing 4 M NaCl (Onishi et al., 1980). The strain was cultivated in SW10 medium with 10 % (w/v) total salts (8.1 % NaCl, 0.7 % MgCl2, 0.96 % MgSO4, 0.036 % CaCl2, 0.2 % KCl, 0.006 % NaHCO3, 0.0026 % NaBr, 0.5 % yeast extract; Difco) (Ventosa et al., 1982). The pH was adjusted to 7.2 with 1 M KOH. When necessary, solid media were prepared by adding 2.0 % (w/v) Bacto agar (Difco).
Optimal conditions for growth were determined by growing strain 43T in SW medium at 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25 and 30 % (w/v) total salts at temperatures of 4, 15, 20, 25, 30, 37, 40, 42 and 45 °C. The pH range for the isolate was tested in SW10 medium adjusted to pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 using the appropriate buffers. The cells were cultivated with constant agitation (180 r.p.m.) and growth was monitored by measuring optical density at 600 nm. The cells of strain 43T were found to be motile, Gram-negative, non-spore-forming rods. The cells were 1.2–4.5 µm long and 0.4 µm wide at the exponential phase of growth in SW10 medium at 37 °C. Optimal growth occurred at 7.5–12.5 % (w/v) totals salts, 28–37 °C and pH 7.0–8.0; the ranges for growth for strain 43T are shown in Table 1.
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or included in the species description. Macroscopic properties were determined using the classical characterization of colony appearance. Biochemical tests were carried out at 10 % total salts and 37 °C, unless stated otherwise. Catalase activity was determined by adding a 1 % (w/v) H2O2 solution to colonies on SW10 agar medium. An oxidase test was performed using the Dry Slide (Difco). Hydrolysis of starch, gelatin and Tween 80 and the production of urease were determined as described by Cowan & Steel (1965) with the addition of 10 % total salts to the medium. Citrate utilization was determined on Simmons' citrate medium supplemented with SW10. Acid production from carbohydrates was determined using phenol red base supplemented with 1 % of the carbohydrate and SW10. Growth under anaerobic conditions was determined by incubating strain 43T in SW10 medium in an anaerobic chamber. Tests for sugar fermentation and enzymes were carried out using API 20NE and API ID 32E kits (bioMérieux) inoculated according to the manufacturer's instructions, using the inoculated fluid at 10 % NaCl and with incubation at 37 °C.
The nutritional requirements of the isolate were determined using Biolog microplates. Strain 43T was grown on SW10 medium at 37 °C for 48 h and suspended in a 10 % NaCl (w/v) solution, within the density range specified by the manufacturer (model 21101 photometer; Biolog). Immediately after the cells had been suspended in the saline solution, the suspensions were transferred into sterile multichannel pipette reservoirs (Biolog) and the Biolog GN microplates were inoculated with 125 µl cell suspension per well by means of an eight-channel repeating pipette. The inoculated Biolog plates were incubated at 37 °C for 7 days and the results were read with a MicroPlate Reader using Microlog 3.59 software to perform automated readings. Antibiotic susceptibility was determined according to the conventional Kirby–Bauer method (Bauer et al., 1966). The phenotypic characteristics of strain 43T are included in the species description.
Chromosomal DNA was isolated and purified according to the method described by Marmur (1961). The 16S rRNA gene was amplified using the universal primers 16F27 and 16R1488, as described by Mellado et al. (1995). The almost-complete nucleotide sequence was determined by NBT-Newbiotechnics (Seville, Spain) using an automated DNA sequencer (model 3100; Applied Biosystems) and was compared with reference 16S rRNA gene sequences retrieved from the GenBank and EMBL databases by BLAST searching. The subsequent sequence analysis was conducted using the ARB program package (Ludwig et al., 2004). Several treeing methods (maximum parsimony, distance matrix and maximum likelihood) were performed on the basis of the recommendations of Ludwig et al. (1998). A comparison based on the 16S rRNA gene sequences from the databases revealed that the sequence (1501 bp) of strain 43T displays the highest levels of similarity with those from Chromohalobacter species. The closest relatives were C. canadensis ATCC 43984T (99.3 % sequence similarity), C. beijerinckii ATCC 19372T (99.1 %), C. sarecensis LV4T (98.3 %) and C. marismortui ATCC 17056T (97.9 %). Sequence similarities with respect to the other Chromohalobacter species were 
96.6 %. The phylogenetic tree obtained by using the maximum-parsimony method shows strain 43T within the branch constituted by the Chromohalobacter species (Fig. 1). These results were consistent when other algorithms were used to construct phylogenetic trees.
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; Miller, 1982). Cells were cultured on TSA supplemented with 10 % NaCl (w/v) at pH 7.0 and 37 °C for 24 h. The predominant fatty acids of strain 43T were C16 : 0, C19 : 0 cyclo 8c and C12 : 0 3-OH. This composition is very similar to those described for recognized Chromohalobacter species, except that the C18 : 17c content is lower than for C. beijerinckii and C. marismortui (Table 2).
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) using the equation of Owen & Hill (1979), as previously described in detail by Ventosa et al. (1999). The DNA G+C content of strain 43T was found to be 62.9 mol%, which is within the range for species belonging to the genus Chromohalobacter (Ventosa, 2005).
DNA–DNA hybridization studies were performed by using the competition procedure of the membrane method (Johnson, 1994), described in detail by Mormile et al. (1999). The hybridization temperature was 58.1 °C, which is within the limit of validity for the filter method (De Ley & Tijtgat, 1970), and the percentage of hybridization was calculated according to Johnson (1994). The experiments were carried out in triplicate. The percentage of DNA–DNA hybridization between strain 43T and C. canadensis CECT 5385T was 38 %, while those between strain 43T and C. beijerinckii DSM 7218T, C. marismortui ATCC 17056T, C. sarecensis LV4T and C. nigrandesensis LTS-4NT were 49, 33, 32 and 28 %, respectively. These levels of DNA–DNA hybridization are low enough to justify classifying strain 43T as a member of a genotypically distinct species within the genus Chromohalobacter (Stackebrandt & Goebel, 1994; Stackebrandt et al., 2002). In addition, these genotypic differences are supported by the presence of phenotypic features that can be used to differentiate strain 43T from related species of the genus Chromohalobacter (Table 1).
Overall, the phenotypic, phylogenetic and genotypic results presented in this study demonstrate that strain 43T represents a novel species within the genus Chromohalobacter, for which the name Chromohalobacter japonicus sp. nov. is proposed.
Description of Chromohalobacter japonicus sp. nov.
Chromohalobacter japonicus (ja.po'ni.cus. N.L. masc. adj. japonicus Japanese).
Cells are Gram-negative, non-spore-forming, straight or sometimes slightly curved, motile rods that are 0.4x1.2–4.5 µm in size and occur singly, in pairs and in short chains. Colonies on SW10 medium after 2 days incubation at 37 °C are 0.5 mm in diameter, cream, circular, regular and convex with an entire margin. Moderately halophilic, growing at NaCl concentrations in the range 5–25 % (w/v), with an optimum between 7.5 and 12.5 (w/v) NaCl. Growth occurs at temperatures from 15 to 42 °C (optimally at 28–37 °C) and at pH 5.5–9.0 (optimally at pH 7.0–8.0). Strictly aerobic. Catalase is produced. Negative for indole production, the Voges–Proskauer test, H2S production, starch, casein, aesculin, DNA and Tween 80 hydrolysis, urease activity and oxidase activity. Gelatin is hydrolysed. Methyl red-positive. Nitrate is reduced to nitrite but nitrite is not reduced. Positive for the Simmons' citrate test. Acid is produced from D-galactose, mannose, D-glucose, maltose, xylose, galacturonate, adonitol, palatinose, sucrose, D-arabitol, L-rhamnose, inositol, D-cellobiose and D-sorbitol but not from arabinose, D-fructose, glycerol, D-mannitol, trehalose, sorbitol, L-arabitol or 5-ketogluconate. Positive for ornithine decarboxylase and L-aspartic acid arylamidase. Negative for arginine dihydrolase, 
-galactosidase, lysine decarboxylase, -glucosidase, -glucuronidase, N-acetyl--glucosaminidase, 
-glucosidase, -galactosidase and -maltosidase. The following compounds are used as sole carbon and energy sources (Biolog): L-arabinose, D-fructose, D-galactose, gentiobiose, -D-glucose, D-mannitol, D-psicose, L-rhamnose, cis-aconitic acid, citric acid, D-gluconic acid, D-glucosaminic acid, -ketoglutaric acid, DL-lactic acid, succinamic acid, D-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-proline, L-pyroglutamic acid, L-serine, L-threonine and glycerol. The following compounds are not used as sole carbon and energy sources (Biolog): -cyclodextrin, dextrin, glycogen, Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, D-arabitol, cellobiose, i-erythritol, L-fucose, myo-inositol, -D-lactose, lactulose, maltose, D-mannose, D-melibiose, methyl -D-glucoside, D-raffinose, D-sorbitol, sucrose, trehalose, turanose, xylitol, methyl pyruvate, monomethyl succinate, acetic acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, 
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, -ketobutyric acid, -ketovaleric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, glucuronamide, alaninamide, L-alanine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, D-serine, DL-carnitine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Resistant to (µg per disc, unless specified otherwise): penicillin G (10 U), bacitracin (10 U), cephalothin (30), rifampicin (30), streptomycin (10), neomycin (10), erythromycin (15), kanamycin (30), vancomycin (30), nalidixic acid (30), novobiocin (30), polymyxin B (300 U) and chloramphenicol (30). Susceptible to sulfamethoxazole/trimethoprim (23.75/1.25 µg per disc). The major fatty acids are C16 : 0, C19 : 0 cyclo 8c and C12 : 0 3-OH. The DNA G+C content is 62.9 mol%.
The type strain, 43T (=CECT 7219T =CCM 7416T), was isolated from a Japanese salty food.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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