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1 Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon-dong, Giheung-gu Yongin, Kyunggi-do 449-701, South Korea
2 Kanwon National University, School of Bioscience and Biotechnology, Chunchon 200-701, South Korea
3 Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA
Correspondence
Deok-Chun Yang
deokchunyang{at}yahoo.co.kr
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ABSTRACT |
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The cellular fatty acid profile of strain DCY12T is available as supplementary material with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Strain DCY12T was isolated from surface soil from an agricultural field in which ginseng was growing. A soil sample (1 g) was immersed in 50 ml saline solution, vortexed and serially diluted and then an aliquot (100 µl) was inoculated onto 10-fold-diluted R2A agar (Difco). Single colonies on these agar plates were purified by transferring them onto new plates and subjecting them to additional incubation for 5 days at 30 °C. The purified colonies were tentatively identified using a partial sequence of the 16S rRNA gene.
Cell morphology and motility were observed with a Nikon light microscope (x1000 magnification), using cells grown on R2A agar at 30 °C for 5 days. Gram reactions were determined according to the non-staining method, as described by Buck (1982)
. Oxidase activity was evaluated via 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. Growth at a various temperatures (4, 15, 25, 30, 37 and 42 °C) was assessed on R2A agar, while growth at different pHs was assessed in R2A broth. Growth on nutrient agar, Luria–Bertani (LB) agar and trypticase soy agar (TSA) was also evaluated at 30 °C. The API 20NE, API ID 32GN and API ZYM microtest systems were employed in these tests, according to the recommendations of the manufacturer (bioMérieux). Because strain DCY12T was unable to assimilate any of the single growth substrates in the API 20NE and API ID 32GN tests, LB broth was added to the AUX medium (0.1 %, v/v) and then the microbial broth was inoculated; with LB broth as a growth factor, strain DCY12T was then able to assimilate some substrates in these microtest systems.
Isoprenoid quinones were extracted using chloroform/methanol (2 : 1, v/v), purified via TLC and subsequently analysed by HPLC, as described previously (Collins & Jones, 1981; Shin et al., 1996). In order to perform a fatty acid methyl ester analysis, the strain was allowed to grow on TSA for 48 h at 30 °C and then two loops of the well-grown cells were harvested. The fatty acid methyl esters were prepared, separated and identified with the Sherlock Microbial Identification System (MIS; MIDI) (Sasser, 1990).
To determine the G+C content, genomic DNA was extracted and purified with the Genomic-tip 100/G system (Qiagen) and was then enzymically degraded into nucleosides. The nucleosides were analysed using HPLC, as described previously (Tamaoka & Komagata, 1984; Mesbah et al., 1989).
The 16S rRNA gene was amplified from the chromosomal DNA using the universal bacterial primer set 9F and 1512R (Weisburg et al., 1991) and the purified PCR product was sequenced by Genotec (Daejeon, Korea) (Kim et al., 2005). The complete sequence of the 16S rRNA gene was compiled with SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank and edited using the BioEdit program (Hall, 1999). 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) and 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 conducted in order to obtain confidence levels for the branches (Felsenstein, 1985). The closest relatives of strain DCY12T and all of the type species representing all of the orders in the Betaproteobacteria and Gammaproteobacteria were included in the phylogenetic tree.
Strain DCY12T was cultured on R2A agar at 30 °C, yielding pale-yellow, circular colonies. The strain was found to be an aerobic, Gram-negative, non-motile bacterium consisting of short rod-shaped cells. Strain DCY12T was also shown to be able to grow at 25–37 °C, but it did not grow at 4 or 42 °C. Physiological data for strain DCY12T are summarized in the species description; Table 1 lists the differential characteristics with respect to related type strains.
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5c (4.99 %), C18 : 1 (25.90 %), C19 : 0 cyclo 8c (8.59 %), summed feature 3 (C16 : 1 iso I/C14 : 0 3-OH; 13.25 %) and summed feature 4 (C15 : 0 iso 2-OH/C16 : 17c; 3.65 %). Minor amounts of saturated fatty acids C15 : 0 (1.36 %), C17 : 0 (0.85 %), C18 : 0 (0.62 %), C16 : 0 2-OH (1.31 %), C16 : 0 3-OH (0.92 %) and summed feature 2 (C15 : 1 iso H/I/C13 : 0 3-OH; 0.25 %) were also present. The strain contained a ubiquinone with eight isoprene units (Q-8) as the predominant isoprenoid quinone. Q-8 is commonly found in species belonging to the Gammaproteobacteria.
The 16S rRNA gene sequence of DCY12T was found to be a continuous stretch of 1491 nt, and the strain was found to belong to the class Gammaproteobacteria. The highest levels of sequence similarities for DCY12T occurred with Hydrocarboniphaga effusa AP103T (89.2 %), Nevskia ramosa Soe1 (88.8 %) and Pseudomonas aeruginosa ATCC 10145T (83.2 %). In the phylogenetic tree (Fig. 1), strain DCY12T clearly belonged to the lineage containing the genera Hydrocarboniphaga and Nevskia, as evidenced by the high level of bootstrap support. On the basis of the 16S rRNA gene sequencing results, the phylogenetic position of DCY12T among members of the Gammaproteobacteria was unique and distinct.
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; Palleroni et al., 2004). In our study, 16S rRNA gene sequences of the type species representing all of the orders in the Betaproteobacteria and Gammaproteobacteria were collected from GenBank and a phylogenetic tree was constructed. In Fig. 1
, the Nevskia–Solimonas–Hydrocarboniphaga cluster is grouped with the Gammaproteobacteria, although its position is not supported by high levels of bootstrap support. The G+C content of the genomic DNA of strain DCY12T was 40.5 mol%, which is much lower than those of related taxa (N. ramosa, 67.8–69.0 mol%; H. effusa, 60–61 mol%).
On the basis of the phenotypic, chemotaxonomic and phylogenetic data, we conclude that strain DCY12T represents a novel species and genus, within the Gammaproteobacteria, for which the name Solimonas soli gen. nov., sp. nov. is proposed.
Description of Solimonas gen. nov.
Solimonas (So'li.mo'nas. L. n. solum soil; L. fem. n. monas a unit, monad; N.L. fem. n. Solimonas a monad in soil).
Gram-negative, non-motile rods. Oxidase-negative and catalase-positive. The major ubiquinone is Q-8. The major cellular fatty acids are C16 : 0, C18 : 1 and summed feature 3 (C16 : 1 iso I/C14 : 0 3-OH). The DNA G+C content of the type strain of the type species is 40.5 mol%. The type, and only, species is Solimonas soli.
Description of Solimonas soli sp. nov.
Solimonas soli (so'li. L. neut. gen. n. soli of soil, the source of the type strain).
General characteristics are given above under the genus description. Cells grown on R2A agar at 30 °C for 5 days are 0.3–0.5x0.2–0.4 µm, non-motile rods. Colonies grown on R2A agar plates for 5 days are pale yellow. Grows optimally at 30 °C and at pH 7.0; growth occurs at 25–37 °C and pH 7.0–9.0 but not at temperatures above 42 °C or below 4 °C and not above pH 11 or below pH 5. Negative for indole production and acid production from glucose. Acid phosphatase, alkaline phosphatase, esterase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase and urease are produced, but N-acetyl-
-glucosaminidase, arginine dihydrolase, 
-chymotrypsin, cystine arylamidase, -fucosidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, -glucuronidase, lipase, -mannosidase, protease, trypsin and valine arylamidase are not produced. In the presence of LB broth as a growth factor, 3-hydroxybutyrate, acetate, adipate, gluconate, n-valerate, D-glucose and L-histidine are assimilated. Growth on propionate, L-arabinose, L-fucose, D-sorbitol, L-proline and L-serine, with R2A as a growth factor, is slow and very poor. No substrates are assimilated in the presence of yeast extract as a growth factor or in the absence of a growth factor. The following are not assimilated: 2-ketogluconate, 3-hydroxybenzoate, 4-hydroxybenzoate, 5-ketogluconate, caprate, citrate, itaconate, lactate, L-malate, malonate, phenylacetate, suberate, maltose, D-mannose, D-melibiose, L-rhamnose, D-ribose, sucrose, myo-inositol, D-mannitol, L-alanine, N-acetyl-D-glucosamine, salicin and glycogen. Major cellular fatty acids include C16 : 0 (21.24 %), summed feature 3 (C16 : 1 iso I/C14 : 0 3-OH; 13.25 %) and C18 : 1 (25.90 %). Nitrate is not reduced to nitrite or nitrogen gas.
The type strain, DCY12T (=KCTC 12834T =LMG 24014T), was isolated from soil from a ginseng field in South Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
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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]
Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Palleroni, N. J., Port, A. M., Chang, H.-K. & Zylstra, G. J. (2004). Hydrocarboniphaga effusa gen. nov., sp. nov., a novel member of the 
-Proteobacteria active in alkane and aromatic hydrocarbon degradation. Int J Syst Evol Microbiol 54, 1203–1207.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: MIDI Inc.
Shin, Y. K., Lee, J.-S., Chun, C. O., Kim, H.-J. & Park, Y.-H. (1996). Isoprenoid quinone profiles of the Leclercia adecarboxylata KCTC 1036T. J Microbiol Biotechnol 6, 68–69.[CrossRef]
Stürmeyer, H., Overmann, J., Babenzien, H.-D. & Cypionka, H. (1998). Ecophysiological and phylogenetic studies of Nevskia ramosa in pure culture. Appl Environ Microbiol 64, 1890–1894.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
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