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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Republic of Korea
2 Health Technology Planning and Evaluation Board, 57-1 Noryangjin-dong, Seoul, Republic of Korea
3 Department of Microbiology, College of Medicine, Chungang University, 221 Heukseok-dong, Seoul, Republic of Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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6c, C16 : 17c and/or iso-C15 : 0 2-OH and C18 : 17c as the major fatty acids. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and an unidentified phospholipid. The DNA G+C content was 65.1 mol%. Strain KSL-125T was distinguishable from the two recognized Sphingosinicella species on the basis of differential phenotypic properties, DNA–DNA relatedness data and repetitive-sequence-based PCR genomic fingerprinting patterns. The phenotypic, phylogenetic and genetic data showed that strain KSL-125T represents a novel species of the genus Sphingosinicella, for which the name Sphingosinicella soli sp. nov. is proposed. The type strain is KSL-125T (=KCTC 12482T =DSM 17328T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KSL-125T is DQ087403.
Biolog assimilation data for strain KSL-125T and the two Sphingosinicella species are presented in a supplementary table available with the online version of this paper.
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with the description of a single species, Sphingosinicella microcystinivorans, as a novel member of the family Sphingomonadaceae. Subsequently, another Sphingosinicella species, Sphingosinicella xenopeptidilytica, was described (Geueke et al., 2007). Here we report on the taxonomic characterization of a Sphingosinicella-like bacterial strain, KSL-125T, which was isolated from an alkaline soil in Korea.
An alkaline soil collected from Kwangchun, Korea, was used as the source for the isolation of bacterial strains. Strain KSL-125T was isolated with the usual dilution plating technique on 10x diluted nutrient agar (Difco) at 30 °C. The type strains of the two Sphingosinicella species were used as reference strains: S. microcystinivorans KCTC 12019T was obtained from the Korean Collection for Type Cultures (Taejon, Korea) and S. xenopeptidilytica 3-2W4T was obtained from H.-J. Busse (Geueke et al., 2007). To investigate its morphological, physiological and biochemical characteristics, strain KSL-125T was routinely cultivated at 30 °C on trypticase soy agar (TSA; Difco). The cell morphology was examined using light microscopy (E600; Nikon) and the presence of flagella was investigated using transmission electron microscopy with cells from exponentially growing cultures. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Growth in the absence of NaCl and at various NaCl concentrations (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 %, w/v) was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that NaCl was excluded. Growth at various temperatures (4–45 °C) was measured on TSA. The pH range for growth was determined in nutrient broth (Difco) adjusted to various pHs (initial pH 4.5–10.5, in increments of 0.5 pH units). The pH was adjusted to various levels prior to sterilization by the addition of HCl or Na2CO3. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on TSA and on TSA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. Catalase and oxidase activities and the hydrolysis of casein, starch, hypoxanthine, tyrosine, xanthine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and reduction of nitrate were studied as described previously (Lanyi, 1987). Assimilation of various substrates was determined by using the Biolog GN2 MicroPlate assay as recommended by the manufacturer. Assimilation results were checked after 3 days. Enzyme activity was determined by using the API ZYM system (bioMérieux). Susceptibility to antibiotics was tested on TSA plates using antibiotic discs containing the following: polymyxin B, 100 U; streptomycin, 50 µg; penicillin G, 20 U; chloramphenicol, 100 µg; ampicillin, 10 µg; cephalothin, 30 µg; gentamicin, 30 µg; novobiocin, 5 µg; tetracycline, 30 µg; kanamycin, 30 µg; lincomycin, 15 µg; oleandomycin, 15 µg; neomycin, 30 µg; carbenicillin, 100 µg. Other physiological and biochemical tests were performed with the API 20E system (bioMérieux). The occurrence of genes mlrA, mlrB, mlrC, mlrD and puf was investigated by using primers and conditions described previously (Saito et al., 2003; Geueke et al., 2007); S. microcystinivorans KCTC 12019T and S. xenopeptidilytica 3-2W4T were used as positive or negative controls.
Cell biomass for DNA extraction and for the analyses of isoprenoid quinones and polar lipids was obtained from cultivation at 30 °C in trypticase soy broth (Difco) supplemented with 1 % (v/v) Hutner's mineral base (Cohen-Bazire et al., 1957). Chromosomal DNA was isolated and purified according to the method described by Yoon et al. (1996) except that RNase T1 was used in combination with RNase A. The 16S rRNA gene was amplified by using a PCR with two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). The DNA G+C content was determined by using the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resulting nucleotides were analysed by reversed-phase HPLC. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and were analysed using reversed-phase HPLC and a YMC ODS-A (250x4.6 mm) column. Polar lipids were extracted according to the procedures described by Minnikin et al. (1984) and were identified by using two-dimensional TLC followed by spraying with the appropriate detection reagents (Minnikin et al., 1984; Komagata & Suzuki, 1987). For fatty acid methyl ester analyses, cell mass of strain KSL-125T, S. microcystinivorans KCTC 12019T and S. xenopeptidilytica 3-2W4T was harvested from TSA plates after cultivation for 7 days at 30 °C. The fatty acids were extracted and fatty acid methyl esters were prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Repetitive-sequence-based PCR (rep-PCR) genomic fingerprinting using REP, ERIC, BOX, (GTG)5 and SERE PCR primers was performed as described previously (Versalovic et al., 1994; Rademaker et al., 1998; Rajashekara et al., 1998). Computer-assisted analysis of the genomic fingerprints was performed by using GelCompar II (version 1.5) software (Applied Maths). Similarity among the patterns was calculated using Pearson's similarity coefficient and the dendrogram was constructed using the UPGMA algorithm (Sneath & Sokal, 1973).
Morphological, cultural, physiological and biochemical properties of strain KSL-125T are shown in Table 1 and Supplementary Table S1 (available in IJSEM Online) or are given in the species description. The almost-complete 16S rRNA gene sequence of strain KSL-125T determined in this study comprised 1409 nt (representing approximately 96 % of the Escherichia coli 16S rRNA gene sequence). Comparative 16S rRNA gene sequence analyses showed that strain KSL-125T is phylogenetically closely related to the family Sphingomonadaceae of the Alphaproteobacteria (Fig. 1). In the phylogenetic tree based on the neighbour-joining algorithm, strain KSL-125T joined the cluster comprising the two Sphingosinicella species with 100 % bootstrap support (Fig. 1). The 16S rRNA gene sequence of strain KSL-125T shared the same signature nucleotides as those defined for the genus Sphingosinicella, as described by Geueke et al. (2007). Strain KSL-125T exhibited 16S rRNA gene sequence similarity values of 98.9 and 99.0 % with respect to the type strains of S. microcystinivorans and S. xenopeptidilytica, respectively, and showed less than 94.2 % sequence similarity with respect to other species used in the phylogenetic analysis. Chemotaxonomic properties confirm the phylogenetic affiliation of strain KSL-125T as a member of the genus Sphingosinicella. The predominant isoprenoid quinone detected in strain KSL-125T was Q-10 (at a peak area ratio of approximately 90 %) and minor amounts of Q-8 and Q-9 were also present. Strain KSL-125T had a cellular fatty acid profile comprising large amounts of unsaturated, hydroxy and straight-chain fatty acids; the major fatty acids (>10 % total fatty acids) were C17 : 16c (27.7 %), C16 : 17c and/or iso-C15 : 0 2-OH (23.3 %) and C18 : 17c (19.0 %). This cellular fatty acid profile was generally similar to those of the two Sphingosinicella species, but was distinguishable from them in terms of the contents of some fatty acids, particularly C17 : 16c, which was present as a major component in strain KSL-125T but was only a minor component in the two Sphingosinicella species (Table 2). The major polar lipids detected in strain KSL-125T were sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and an unidentified phospholipid; a minor amount of an unidentified glycolipid was also present. The DNA G+C content of strain KSL-125T was 65.1 mol%.
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). Strain KSL-125T exhibited mean DNA–DNA relatedness values of 27 and 25 % with respect to the type strains of S. microcystinivorans and S. xenopeptidilytica, respectively. These data indicate that strain KSL-125T differs genetically from the two recognized Sphingosinicella species (Wayne et al., 1987). Strain KSL-125T is also distinguishable from the two recognized Sphingosinicella species by differences in several phenotypic characteristics (Table 1
). The phylogenetic and genetic distinctiveness and the differential phenotypic properties are sufficient to allocate strain KSL-125T to a species that is separate from the recognized Sphingosinicella species (Wayne et al., 1987; Table 1). Therefore, on the basis of the data presented, strain KSL-125T represents a novel species of the genus Sphingosinicella, for which the name Sphingosinicella soli sp. nov. is proposed.
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Cells are Gram-negative, non-spore-forming, short rods or rods, 0.3–0.5x0.7–4.0 µm. Motile by means of single polar flagella. Colonies are circular, convex, smooth, glistening, yellow in colour and 0.5–1.0 mm in diameter after 7 days cultivation at 30 °C on TSA. The optimal growth temperature is 30 °C; growth occurs at 4 and 37 °C, but not at 38 °C. The optimal pH for growth is 7.5–8.0; growth occurs at pH 6.0 and 9.5, but not at pH 5.5 or 10.0. Optimal growth occurs in the presence of 0.5 % (w/v) NaCl; growth occurs in the absence of NaCl and in the presence of 1 % (w/v) NaCl but not in the presence of >2 % (w/v) NaCl. Growth does not occur under anaerobic conditions on TSA or on TSA supplemented with nitrate. Tweens 20, 40 and 60 are hydrolysed. Casein, starch, hypoxanthine, L-tyrosine and xanthine are not hydrolysed. Lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are absent. Susceptible to polymyxin B, streptomycin, chloramphenicol, gentamicin, tetracycline, kanamycin and neomycin, but not to lincomycin or oleandomycin. Acetone/methanol extracts of freeze-dried cells show absorption maxima at 448–449 and 476 nm. The predominant ubiquinone is Q-10. The major fatty acids (>10 % of total fatty acids) are C17 : 16c, C16 : 17c and/or iso-C15 : 0 2-OH and C18 : 17c. The major polar lipids are sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine and an unidentified phospholipid. The DNA G+C content is 65.1 mol% (determined by HPLC). Other phenotypic characteristics are shown in Table 1 and Supplementary Table S1.
The type strain, KSL-125T (=KCTC 12482T =DSM 17328T), was isolated from an alkaline soil from Kwangchun, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Geueke, B., Busse, H.-J., Fleischmann, T., Kämpfer, P. & Kohler, H.-P. E. (2007). Description of Sphingosinicella xenopeptidilytica sp. nov., a β-peptide-degrading species, and emended descriptions of the genus Sphingosinicella and Sphingosinicella microcystinivorans. Int J Syst Evol Microbiol 57, 107–113.
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Minnikin, D. E., O'Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.[CrossRef]
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Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rRNA gene sequences. Int J Syst Bacteriol 48, 187–194.
Yoon, J.-H., Kim, H., Kim, I.-G., Kang, K. H. & Park, Y.-H. (2003). Erythrobacter flavus sp. nov., a slight halophile from the East Sea in Korea. Int J Syst Evol Microbiol 53, 1169–1174.
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-galactosidase, β-galactosidase, β-glucuronidase, 
50 % are shown at nodes. Rhodospirillum rubrum ATCC 11170T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.