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-glucosidase-producing bacterium in the family Sphingomonadaceae in the 
-4 subgroup of the Proteobacteria
1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, South Korea
2 Environmental and Molecular Microbiology Lab., Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Guseong-dong 373-1, Yuseong-gu, Daejeon 305-701, South Korea
3 Department of Bioresource Science, Ibaraki University College of Agriculture, Ami-machi, Ibaraki 300-0393, Japan
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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-4 subgroup of the Proteobacteria, and the highest degrees of sequence similarity determined were to Sphingomonas asaccharolytica IFO 10564T (97·5 %), Sphingomonas koreensis JSS26T (97·1 %), Sphingomonas mali IFO 15500T (96·7 %) and Sphingomonas pruni IFO 15498T (96·6 %). Chemotaxonomic data revealed that strain T5-04T possesses ubiquinone Q-10 predominantly, C18 : 1 as the predominant fatty acid and sphingoglycolipids, all of which corroborate its assignment to the genus Sphingomonas. The results of DNA–DNA hybridization and physiological and biochemical tests clearly demonstrated that strain T5-04T represents a distinct species. Based on polyphasic evidence, T5-04T (=KCTC 12210T=NBRC 100801T=IAM 15213T) should be classified as the type strain of a novel Sphingomonas species, for which the name Sphingomonas soli sp. nov. is proposed.
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in order to accommodate Gram-negative, strictly aerobic, chemoheterotrophic, yellow-pigmented and rod-shaped bacteria that harbour sphingoglycolipids as cell-envelope components. The genus contained a multitude of species consisting of strains of clinical origin, as well as from a variety of other environments. The relatively diverse species of the genus Sphingomonas have been classified into at least four clusters within the -4 subgroup of the Proteobacteria. The genus Sphingomonas has also been divided into four genera: Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis (Takeuchi et al., 2001).
Yabuuchi et al. (2002) suggested that the genus Sphingomonas should remain undivided and that the species of Novosphingobium, Sphingobium and Sphingopyxis constitute later homotypic synonyms of species of the genus Sphingomonas. Subsequently, Busse et al. (2003) suggested that sym-homospermidine, which is a characteristic marker within the family Sphingomonadaceae, had not been considered in the taxonomic considerations of Yabuuchi et al. (1990, 2002) and strongly advocated the proposal of Takeuchi et al. (2001). Nowadays, a great many articles have adopted the nomenclature of Takeuchi et al. (2001) in their reports of novel species in the genera Novosphingobium (Fujii et al., 2003; Sohn et al., 2004; Tiirola et al., 2005), Sphingobium (Ushiba et al., 2003), Sphingomonas (Busse et al., 2003; Li et al., 2004; Rivas et al., 2004) and Sphingopyxis (Kämpfer et al., 2002; Godoy et al., 2003; Yoon & Oh, 2005; Yoon et al., 2005). At the time of writing, the genus Sphingomonas sensu stricto contains 28 species with validly published names.
In this study, a strain was isolated from soil of a ginseng field in Daejeon city in South Korea and characterized by polyphasic approaches. The results obtained in this study demonstrate that strain T5-04T can be considered to be a member of the genus Sphingomonas, using the nomenclature provided by Takeuchi et al. (2001). However, it can also be clearly distinguished from Sphingomonas species with validly published names; we therefore propose that strain T5-04T should be considered the type strain of a novel Sphingomonas species.
Strain T5-04T was isolated from soil of a ginseng field via direct plating onto R2A agar (Difco). 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. One isolate, T5-04T, was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Extraction of genomic DNA was done using a commercial genomic DNA extraction kit (Core Biosystem). The 16S rRNA gene was amplified from the chromosomal DNA using the universal bacterial primer set 9F and 1512R and purified PCR products were sequenced by GenoTech Co. Ltd (Daejeon, South Korea) (Kim et al., 2005). Full 16S rRNA gene sequences were compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from the GenBank database. Multiple alignments were performed with the CLUSTAL X program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed by using a neighbour-joining method (Saitou & Nei, 1987) and the maximum-parsimony method (Fitch, 1972) using the MEGA3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
The 16S rRNA gene sequence of strain T5-04T was found to be a continuous stretch of 1422 nucleotides. Strain T5-04T was determined to belong to the -4 subgroup of the Proteobacteria, and the highest degrees of sequence similarity were found to be with Sphingomonas asaccharolytica IFO 10564T (97·5 %), Sphingomonas koreensis JSS26T (97·1 %), Sphingomonas mali IFO 15500T (96·7 %) and Sphingomonas pruni IFO 15498T (96·6 %). In the phylogenetic tree (Fig. 1), strain T5-04T clearly belonged to the Sphingomonas lineage, as confirmed by the high bootstrap value.
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. Catalase activity was determined by bubble production in 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. Acid production from carbohydrates was assessed by the procedures outlined by Cappuccino & Sherman (2002). Growth at a variety of temperatures (4, 15, 25, 30, 37 and 42 °C) was assessed on R2A agar and growth at a variety of pH values was assessed in R2A broth. Substrate utilization as sole carbon source and some physiological characteristics were determined with API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux).
When strain T5-04T was cultured on R2A agar (Difco) at 30 °C, it produced yellowish coloured, circular and non-glossy colonies. Cells were aerobic, Gram-negative, non-motile rods. The strain was able to grow at 15–37 °C, but did not grow at 4 or 42 °C. Results regarding the physiological characteristics of strain T5-04T are summarized in the species description and a comparison of selective characteristics with related type strains is shown in Table 1.
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; Shin et al., 1996). Cellular fatty acids were analysed in organisms grown on trypticase soy agar (TSA; Difco) for 2 days. Cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI, 1999). The fatty acids analysed by GC (Hewlett Packard 6890) were identified by the Microbial Identification software package. Total cellular lipids were extracted three times from 50 mg dried cells with 5 ml chloroform/methanol (2 : 1, v/v) and a portion of the extracted lipids was subjected to mild alkaline hydrolysis with 0·5 M KOH in chloroform/methanol (2 : 1, v/v) for 1 h at 40 °C as described by Yabuuchi et al. (1990). Both total extracted lipids and their alkaline hydrolysates were analysed by TLC (HPTLC plate, Silica gel 60, 10x10 cm; Merck) with a solvent system composed of chloroform, methanol and water (70 : 30 : 5, by vol.). To detect glycolipid spots, orcine-sulfuric acid reagent (0·2 % orcinol in 2 M sulfuric acid) was sprayed and charred at 120 °C until the maximum purple colour developed.
The genomic DNA of strain T5-04T was extracted and purified with the Qiagen Genomic-tip system 100/G and was then enzymically degraded into nucleosides, as described previously (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. Hybridization was conducted in five replications for each sample. The highest and lowest values obtained for each sample were excluded and DNA relatedness values are expressed as the means of the remaining three values.
The cellular fatty acid profiles of strain T5-04T and related Sphingomonas type strains are shown in Table 2. The major cellular fatty acids in strain T5-04T included summed feature 7, which includes isomers of octadecenoic acid (C18 : 1, 65·5 %), and hexadecanoic acid (C16 : 0, 14·3 %). Minor amounts of the 2-hydroxy fatty acids C14 : 0 2-OH (3·4 %) and C15 : 0 2-OH (0·9 %) were also determined to be present. The presence of 2-OH fatty acids and absence of 3-OH fatty acids and the presence of octadecenoic acid isomers as major fatty acids are characteristic features of members of the genus Sphingomonas, as has been discussed previously (Godoy et al., 2003). Significant differences in fatty acid profiles were found in Sphingomonas asaccharolytica, Sphingomonas koreensis, Sphingomonas mali and Sphingomonas pruni. Q-10 was the predominant ubiquinone of strain T5-04T. The quinone system supported our assignment of strain T5-04T to the Alphaproteobacteria, in which the majority of species (including Sphingomonas species) exhibit Q-10 as the predominant quinone (Collins & Jones, 1981). Strain T5-04T was also determined to harbour sphingolipids.
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), which is the suggested threshold which delineates a genomic species. Our results therefore support the assignment of strain T5-04T to a separate, previously unrecognized species. On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequence comparisons, strain T5-04T should be placed into a novel species, for which we propose the name Sphingomonas soli sp. nov.
Description of Sphingomonas soli sp. nov.
Sphingomonas soli (so'li. L. neut. gen. n. soli of soil, the source of the type strain).
Cells are Gram-negative, non-motile, non-spore-forming rods (approx. 1 µm in length). They are oxidase-positive, exhibiting oxidative metabolism (obligately aerobic), and catalase-positive. Favourable growth occurs aerobically at 30 °C on R2A agar, nutrient agar and TSA; optimal growth occurs at pH 6·8–7·5. Forms yellowish, convex, circular colonies with whole edges on R2A agar within 3 days, approximately 1·5–3·0 mm in diameter. Q-10 is the predominant quinone. The major cellular fatty acids include octadecenoic acid (C18 : 1) and hexadecanoic acid (C16 : 0). Sphingoglycolipids are also present. The G+C content of the genomic DNA is 63·9 mol% (determined by HPLC). Carbon and nitrogen source utilization and enzyme activities are shown in Table 1
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The type strain, T5-04T (=KCTC 12210T=NBRC 100801T=IAM 15213T), was isolated from soil of a ginseng field in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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) end of the carbon chain. cis and trans isomersare indicated by the suffixes c and t, respectively.