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1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon-dong, Kiheung-gu Yongin, Kyunggi-do 449-701, South Korea
2 Environmental and Molecular Microbiology Laboratory, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
3 Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence
Deok-Chun Yang
deokchunyang{at}yahoo.co.kr
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ABSTRACT |
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9c as the predominant fatty acids. The DNA G+C content is 71.5 mol%. On the basis of genotypic and phenotypic characteristics, strain Gsoil 355T represents a novel species of the genus Solirubrobacter, for which the name Solirubrobacter soli sp. nov. is proposed. The type strain is Gsoil 355T (=KCTC 12628T=LMG 23485T).
The cellular fatty acid profiles of strain Gsoil 355T and related type strains are presented in a supplementary table available with the online version of this paper.
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MAIN TEXT |
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for a member of the Actinobacteria isolated from a burrow of the earthworm Lumbricus rubellus in an agricultural soil in Georgia, USA (Furlong et al., 2002). A novel strain of this genus, Gsoil 355T, was found in soil from a ginseng field in Daejeon, South Korea.
Strain Gsoil 355T was isolated by direct plating of the serially diluted soil sample onto R2A agar (Difco). Single colonies from these plates were transferred onto new plates and incubated for 5 days at 30 °C. The purified colonies were tentatively identified using partial 16S rRNA gene sequences. Cell morphology and motility were investigated (using a Nikon light microscope at x1000 magnification) in a 5-day-old culture of Gsoil 355T grown on R2A agar at 30 °C. The Gram reaction was tested using the non-staining method, as described by Buck (1982). Oxidase activity was evaluated via the oxidation of 1 % N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. Catalase activity was determined by measuring bubble production after the application of 3 % (v/v) hydrogen peroxide solution to well-isolated colonies. Growth at a variety of temperatures (4, 15, 25, 30, 37 and 42 °C) and pH (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 and 11) was assessed on R2A agar; the pH was adjusted by using HCl and NaOH. Growth at various salt concentrations was tested by adding 0–10 % NaCl to R2A broth. The API 20NE, API ZYM and API ID 32GN microtest systems (bioMérieux) were used to analyse the physiological and biochemical characteristics, according to the recommendations of the manufacturer.
Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified by TLC and then analysed by HPLC as described previously (Collins & Jones, 1981; Shin et al., 1996). For analysis of the fatty acid methyl esters, the strain was grown on tryptic soy agar (Difco) for 48 h at 30 °C and then two loops of the well-grown cells were harvested. Fatty acid methyl esters were prepared and identified using the Sherlock Microbial Identification System (MIDI) (Sasser, 1990).
The genomic DNA of strain Gsoil 355T was extracted and purified with Genomic-tip system 100/G (Qiagen). It was then enzymically degraded into nucleosides to determine the DNA G+C content (Tamaoka & Komagata, 1984; Mesbah et al., 1989). DNA–DNA hybridization was performed according to the method developed by Ezaki et al. (1989). Hybridization was conducted using five replications for each sample. The highest and lowest values were excluded and the DNA relatedness taken from the mean of the remaining three values.
For analysis of the 16S rRNA gene sequence, genomic DNA was extracted and purified with a genomic DNA isolation kit (Core Bio System). The 16S rRNA gene was amplified using the universal bacterial primers 9F and 1512R (Weisburg et al., 1991) and the purified PCR products were sequenced by Genotec (Daejeon, Korea) and then edited (Kim et al., 2005). 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). A phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) in the MEGA2 program (Kumar et al., 2001). A bootstrap analysis (based on 1000 replicates) was also performed (Felsenstein, 1985).
Strain Gsoil 355T produced non-pigmented colonies on R2A agar at 30 °C. The strain comprised aerobic, Gram-positive, non-motile, rod-shaped bacteria. The optimal growth temperature was about 30 °C. No growth was observed within 7 days at 37 °C and only weak growth occurred after 14 days at 15 °C. The physiological characteristics are summarized in the species description.
The 16S rRNA gene sequence-based phylogenetic analysis showed (Fig. 1) that strain Gsoil 355T is a member of the family Solirubrobacteraceae (Stackebrandt, 2004, 2005). The highest sequence similarities (97.4, 94.2 and 91.8 %) were found with respect to the type strains of S. pauli (Singleton et al., 2003), Conexibacter woesei (Monciardini et al., 2003) and Patulibacter minatonensis (Takahashi et al., 2006), respectively.
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9c (18.3 %). Minor amounts of fatty acids C14 : 0 (0.7 %), C14 : 0 iso (0.6 %), C15 : 0 (1.9 %), C16 : 0 (2.1 %), C17 : 0 (5.3 %), C18 : 0 (3.3 %), C18 : 0 iso (4.3 %), C19 : 0 (4.5 %), C20 : 0 (1.1 %), unsaturated fatty acids C16 : 1 iso H (3.0 %), C16 : 19c (0.9 %), C17 : 16c (5.5 %), C17 : 18c (4.3 %), C18 : 36c (7.3 %) and C20 : 46,9,12,15c (0.9 %), summed feature 4 (C16 : 17c/C15 : 0 iso 2-OH; 1.4 %) and summed feature 8 (unknown ECL 18.756/C19 : 111c; 7.9 %) were also detected. No significant differences were found between the fatty acid profiles of strain Gsoil 355T and S. pauli B33D1T (Singleton et al., 2003). Strain Gsoil 355T contained a menaquinone with seven isoprene units [MK-7(H4)] as the predominant isoprenoid quinone.
The DNA G+C content of strain Gsoil 355T was 71.5 mol%. The strain exhibited 25.0 % (mean of 22, 29 and 27 %) DNA–DNA relatedness with respect to S. pauli ATCC BAA-492T. The level of relatedness was less than 70 %, which is considered to be the threshold for delineating a genomic species (Wayne et al., 1987). In addition, the strain could be readily distinguished from S. pauli at the phenotypic level (Table 1). The results of the polyphasic analysis clearly show that strain Gsoil 355T represents a novel species within the genus Solirubrobacter, for which the name Solirubrobacter soli sp. nov. is proposed.
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Cells are Gram-positive, aerobic, non-motile, non-pigmented rods. Growth occurs on R2A agar at 30 °C. Good growth is observed at 25 and 30 °C. Growth occurs at 0–1.5 % (w/v) NaCl but not at higher salt concentrations. Catalase-positive and weakly oxidase-positive. N-Acetyl-
-glucosaminidase, acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase (C8), 
-glucosidase, -glucosidase, -galactosidase, leucine arylamidase and valine arylamidase are produced. Arginine dihydrolase, -chymotrypsin, -fucosidase, -galactosidase, -glucuronidase, lipase (C14), -mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin and urease are not produced. Gelatin is hydrolysed. Adipate, gluconate, L-arabinose, L-fucose, D-glucose, D-maltose, D-melibiose, L-rhamnose, D-ribose, sucrose, myo-inositol, L-proline, N-acetyl-D-glucosamine, salicin and glycogen are utilized as sole carbon sources. 2-Ketogluconate, 3-hydroxybenzoate, 3-hydroxybutyrate, 4-hydroxybenzoate, 5-ketogluconate, acetate, caprate, citrate, itaconate, lactate, L-malate, malonate, phenyl acetate, propionate, suberate, n-valerate, D-mannitol, D-sorbitol, L-alanine and L-histidine are not utilized as sole carbon sources. No reduction of nitrate to nitrite or nitrogen gas occurs. The DNA G+C content is 71.5 mol% (HPLC). The predominant quinone is MK-7(H4). The major cellular fatty acids are C16 : 0 iso (26.7 %) and C18 : 19c (18.3 %).
The type strain, Gsoil 355T (=KCTC 12628T=LMG 23485T), was isolated from soil from a ginseng field in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Collins, M. D. & Jones, D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45, 316–354.
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Kim, M. K., Im, W.-T., Ohta, H., Lee, M. & Lee, S.-T. (2005). Sphingopyxis granuli sp. nov., a -glucosidase-producing bacterium in the family Sphingomonadaceae in -4 subclass of the Proteobacteria. J Microbiol 43, 152–157.[Medline]
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mura, S. (2006). Patulibacter minatonensis gen. nov., sp. nov., a novel actinobacterium isolated using an agar medium supplemented with superoxide dismutase, and proposal of Patulibacteraceae fam. nov. Int J Syst Evol Microbiol 56, 401–406.
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