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1 Department of Oriental Medicinal Material & Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kiheung Yongin, Kyunggi 449-701, South Korea
2 Biopia Co., Ltd, 383-4 Singal-ri, Kiheung Yongin, Kyunggi-do 449-598, South Korea
3 Department of Oncology, Graduate School of East–West Medical Science, Kyung Hee University, 1 Seocheon, Kiheung Yongin, Kyunggi 449-701, South Korea
Correspondence
Deok-Chun Yang
deokchunyang{at}yahoo.co.kr
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for four strains with high G+C contents isolated from kaolin slurry, in which the strains were growing at temperatures as high as 50 °C; Thermomonas haemolytica is the type species. At the time of writing, there are four Thermomonas species with validly published names: Thermomonas haemolytica (Busse et al., 2002), Thermomonas brevis (Mergaert et al., 2003), Thermomonas fusca (Mergaert et al., 2003) and Thermomonas hydrothermalis (Alves et al., 2003). In a series of studies, we used a culture-dependent method in attempts to isolate micro-organisms from a ginseng field in order to investigate the make-up of the microbial community. In this study, one strain was isolated from soil from a ginseng field in Daejeon (South Korea) and characterized by using polyphasic approaches. These polyphasic approaches, including phylogenetic analysis based on 16S rRNA gene sequences, analysis of genomic relatedness and assessment of chemotaxonomic and phenotypic properties, were conducted to determine the precise taxonomic position of strain Ko06T. The results obtained in this study indicated that Ko06T can be assigned as a member of the genus Thermomonas but is clearly distinguishable from the Thermomonas species with validly published names.
Strain Ko06T was isolated, using direct plating onto R2A agar (Difco), from soil from a ginseng field near Daechung Lake. Single colonies on these plates were purified by transferring them onto new plates and subjecting them to an additional incubation for 3 days at 30 °C. The purified colonies were tentatively identified by means of partial 16S rRNA gene sequences.
Cell morphology and motility were observed with a Nikon light microscope (1000x magnification), with the cells being allowed to grow for 3 days at 30 °C on R2A agar. Gram reactions were conducted according to the non-staining method, as described by Buck (1982). Oxidase activity was evaluated using the oxidation of 1 % p-aminodimethylaniline oxalate. Catalase activity was determined by measuring bubble production after the application of a 3 % (v/v) hydrogen peroxide solution. Acid production from carbohydrates was assessed by using the procedures outlined by Cappuccino & Sherman (2002). Growth at various temperatures (4, 15, 25, 30, 37, 42 and 50 °C) was assessed on R2A agar, and growth at various pH values was assessed in R2A broth. Growth on nutrient agar, trypticase soy agar and MacConkey agar (Difco) was also evaluated at 30 °C. The API 20NE and API ID 32GN systems were employed in these tests, according to the recommendations of the manufacturer (bioMérieux).
Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified using TLC and subsequently analysed by HPLC, as described previously (Collins & Jones, 1981; Shin et al., 1996). For fatty acid methyl ester analyses, strain Ko06T was allowed to grow on trypticase soy agar for 48 h at 30 °C and then two loops of the well-grown cells were harvested. Fatty acid methyl esters were prepared, separated and identified with the Sherlock Microbial Identification System (MIDI) (Sasser, 1990). Total cellular lipids were extracted and analysed using TLC (Kim et al., 2005).
Genomic DNA of strain Ko06T was extracted and purified with the Qiagen Genomic-tip system 100/G and then enzymically degraded into nucleosides, as described previously (Tamaoka & Komagata, 1984; Mesbah et al., 1989). DNA–DNA hybridization was performed fluorometrically, according to the method developed by Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Five replicate hybridizations were performed for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA–DNA relatedness values.
Genomic DNA was extracted and purified with a genomic DNA isolation kit (Core Bio System). The 16S rRNA gene was amplified from the chromosomal DNA of strain Ko06T, using the universal bacterial primer set 9F and 1512R (Weisburg et al., 1991); the purified PCR products were sequenced by Genotec (Daejeon, South Korea) (Kim et al., 2005). The full sequence of the 16S rRNA gene was compiled with SeqMan software, and the 16S rRNA gene sequence of the test strain was edited using the BioEdit program (Hall, 1999). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed with the CLUSTAL X program (Thompson et al., 1997). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) in the MEGA 2 program (Kumar et al., 2001). Bootstrap analysis with 1000 replicates was also conducted to obtain confidence levels for the branches (Felsenstein, 1985). All of the species in the genus Thermomonas were included in the phylogenetic tree.
Strain Ko06T was cultured on R2A agar at 30 °C, yielding light-brownish and circular colonies. Strain Ko06T was found to be an aerobic, Gram-negative, motile, rod-shaped bacterium. It was also determined as being able to grow at 20–37 °C, but it did not grow at 4 or 50 °C. The physiological characteristics of strain Ko06T are summarized in the species description, and a comparison of distinguishing characteristics with those of related type strains is shown in Table 1.
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. The major cellular fatty acid in strain Ko06T was iso-pentadecanoic acid (C15 : 0 iso, 53.2 %). Minor amounts of other iso-branched fatty acids, C16 : 0 10-methyl (7.0 %), C11 : 0 iso 3-OH (6.7 %), C14 : 0 (5.3 %), C15 : 0 anteiso (4.8 %) and C11 : 0 iso (4.6 %) were present. C11 : 0 iso 3-OH (6.7 %) was the only hydroxy fatty acid found in strain Ko06T. The presence of iso-branched fatty acids, with C15 : 0 iso as the major fatty acid, is characteristic of genera in the Xanthomonas branch, including Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Xylella and Luteimonas (Assih et al., 2002; Roumagnac et al., 2004; Yang et al., 2005). Significant differences in fatty acid profiles were found between different species of the genus Thermomonas.
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The 16S rRNA gene sequence of strain Ko06T was found to be a continuous stretch of 1474 nt. The 16S rRNA gene sequences of related taxa were obtained from GenBank. Strain Ko06T was determined as belonging to the Gammaproteobacteria, and the highest levels of sequence similarity were with T. brevis LMG 21746T (98.4 %), T. fusca LMG 21737T (97.7 %), T. haemolytica A50-7-3T (96.5 %) and T. hydrothermalis SGM-6T (95.8 %). In the phylogenetic tree (Fig. 1), strain Ko06T clearly belongs to the Thermomonas lineage, as evident from the high bootstrap value.
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), a value which is regarded as the threshold for delineating a genomic species. Our results therefore support the classification of strain Ko06T within a separate, previously unrecognized species. The results of our polyphasic analysis support the recognition of a novel species within the genus Thermomonas, for which the name Thermomonas koreensis sp. nov. is proposed.
Description of Thermomonas koreensis sp. nov.
Thermomonas koreensis sp. nov. (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, the location of the soil sample from which the type strain was isolated).
Cells are Gram-negative, aerobic, motile rods, 3.0–4.0x0.7 µm in size after growth on R2A agar for 3 days at 30 °C. The optimal growth temperature and pH are 37 °C and pH 8.0–9.0, and grow is observed at a salt concentration of 3 %. Catalase- and oxidase-positive. Shows 
-glucosidase and protease (gelatin hydrolysis) activity and does not show arginine dihydrolase, urease or -galactosidase (PNPG) activity. Does not produce any biopolymer-hydrolysing enzymes such as cellulase, chitinase, DNase, lipase or xylanase. Assimilates D-glucose, D-maltose, acetate, L-alanine, propionate, 3-hydroxybutyrate, L-proline and does not assimilate L-arabinose, D-mannose, D-mannitol, N-acetylglucosamine, gluconate, caprate, adipate, malate, citrate, phenylacetate, L-rhamnose, D-ribose, myo-inositol, D-sucrose, itaconate, suberate, malonate, lactate, 5-ketogluconate, glycogen, 3-hydroxybenzoate, L-serine, salicin, D-melibiose, L-fucose, D-sorbitol, valerate, L-histidine, 2-ketogluconate or 4-hydroxybenzoate. DNA G+C content is 68.3 mol% (as determined by HPLC). Q-8 is the predominant quinone. The major cellular fatty acid is C15 : 0 iso, (53.2 %). Other phenotypic characteristics, such as substrates utilized and enzymes produced, are summarized in Table 1
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The type strain, Ko06T (=KCTC 12540T=NBRC 101155T), was isolated from soil from a ginseng field near Daechung Lake in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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