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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
2 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, South Korea
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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7c/iso-C15 : 0 2-OH, C18 : 17c/9t/12t and C17 : 0 cyclo. On the basis of the evidence presented, it is proposed that strain KM-45T should be placed in a novel genus and species, for which the name Silvimonas terrae gen. nov., sp. nov. is proposed. The type strain is KM-45T (=KCTC 12358T=NBRC 100961T).
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Strain KM-45T was isolated from forest soil collected near the Korea Advanced Institute of Science and Technology (KAIST) in Daejeon, South Korea. The soil was homogenized by using an Ace Homogenizer (Nihonseiki Kaisha Ltd). The suspension was spread on R2A agar plates (Difco) after being diluted serially with 50 mM phosphate buffer (pH 7·0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by transferring them onto new plates and incubating again under the same conditions. The isolate was cultured routinely on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Extraction of genomic DNA was performed by using a commercial genomic DNA extraction kit (Core Biosystem), and PCR-mediated amplification of the 16S rRNA gene and sequencing of the purified PCR product were carried out according to the methods given by Kim et al. (2005)
. The full-length 16S rRNA gene sequences were compiled by using SeqMan software (DNASTAR). 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed by using the CLUSTAL_X program (Thompson et al., 1997). Gaps were edited in the BIOEDIT program (Hall, 1999). Evolutionary distances were calculated by using the Kimura two-parameter model (Kimura, 1983). Phylogenetic trees were constructed by using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods, using the MEGA3 program (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
A nearly complete 16S rRNA gene sequence of strain KM-45T was obtained (1491 bp). Preliminary comparison against the 16S rRNA gene sequences in GenBank indicated that strain KM-45T belonged to the order ‘Neisseriales’ of the ‘Betaproteobacteria’. On the basis of 16S rRNA gene sequence similarity, the closest recognized relatives were Chitinibacter tainanensis BCRC 17254T (92·4 %), Formivibrio citricus DSM 6150T (91·2 %) and Iodobacter fluviatilis ATCC 33051T (88·9 %). This relationship between strain KM-45T and other members of the order ‘Neisseriales’ was also evident in the phylogenetic tree (Fig. 1). Strain KM-45T and the three strains discussed above formed a monophyletic clade with a high bootstrap value (99 %), which was supported by the neighbour-joining and maximum-parsimony methods employed.
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. Cell morphology was examined by light microscopy (Nikon) and transmission electron microscopy (Carl Zeiss) after negative staining with 1 % (w/v) phosphotungstic acid. Catalase and oxidase tests were performed as outlined by Cappuccino & Sherman (2002). Substrate utilization as sole carbon source and other physiological characteristics were determined with the API 32GN and API 20NE galleries according to the manufacturer's instructions (bioMérieux). Tests for anaerobic growth were performed in a serum bottle containing R2A broth supplemented with thioglycolate (1 g l–1) and the upper air layer was substituted with nitrogen gas. Nitrate- and nitrite-reduction tests were performed in serum bottles containing R2A broth supplemented with KNO3 (10 mM) and NaNO2 (10 mM), respectively, and reduction of nitrate and nitrite was monitored by using an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion-exchange column (Metrosep Anion Supp 4; Metrohm). Degradation of DNA [using DNA agar (Difco) supplemented with 0·01 % toluidine blue (Merck)], chitin, CM-cellulose, starch (Atlas, 1993), lipids (Kouker & Jaeger, 1987) and xylan (Ten et al., 2004) was also investigated; reactions were read after 5 days. Growth at different temperatures and pH was assessed after 5 days incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Duplicate antibiotic-sensitivity tests were performed by using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg ml–1), tetracycline (5, 10 and 15 µg ml–1), kanamycin (1·0, 1·5 and 2·0 mg ml–1) and ampicillin (20, 30 and 50 µg ml–1) (Sigma). Discs were placed on R2A plates spread with KM-45T culture and were then incubated at 30 °C for 5 days.
Cells of strain KM-45T are Gram-negative, rod-shaped, facultatively anaerobic and motile by means of a single polar flagellum (Fig. 2b). The colony grown on R2A agar plates (Difco) for 3 days was circular (1·3–2·0 mm in diameter), convex and milky white. On R2A agar, strain KM-45T was able to grow at 10–40 °C, but not at 4 or 45 °C. Physiological characteristics of strain KM-45T are summarized in the genus and species descriptions and a comparison of selective characteristics with those of its nearest phylogenetic neighbours is given in Table 1.
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using reversed-phase HPLC; cellular fatty acids were analysed in cells grown on trypticase soy agar (TSA, Difco) for 2 days. The 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. Mean values for each component were obtained from duplicate experiments.
Chromosomal DNA for determination of G+C content was extracted from cells grown on a nutrient agar plate (Difco) as described by Moore (1995). RNA in the DNA solution was removed by incubation with a mixture of RNases A and T1 (each at 20 U ml–1) at 30 °C for 1 h. The G+C content of the chromosomal DNA was analysed as described by Mesbah et al. (1989) using reversed-phase HPLC.
Strain KM-45T had Q-8 as its major quinone. The fatty acid profile of strain KM-45T included C16 : 0 (33·5±0·8 %), summed feature 4 (C16 : 17c/iso-C15 : 0 2-OH, 21·8±1·3 %), summed feature 7 (C18 : 17c/9t/12t, 14·7±0·2 %), C17 : 0 cyclo (15·3±1·6 %), C14 : 0 (6·4±0·3 %), C12 : 0 (3·6±0·3 %) and summed feature 3 (C14 : 0 3-OH/iso-C16 : 1 I, 3·4±0·2 %). The G+C content of the genomic DNA of strain KM-45T was 58 mol%.
Phylogenetically, strain KM-45T formed a novel lineage of descent within the ‘Betaproteobacteria’, and clustered with C. tainanensis BCRC 17254T (92·4 % 16S rRNA gene sequence similarity), F. citricus DSM 6150T (91·2 %) and I. fluviatilis ATCC 33051T (88·9 %). These low 16S rRNA gene sequence similarities support the affiliation of strain KM-45T to a novel genus, which is also supported by its phenotypic and chemotaxonomic characteristics (Table 1
). Strain KM-45T can be differentiated from C. tainanensis BCRC 17254T by its anaerobic growth, nitrate reduction and fatty acid profile. Distinguishing phenotypic characteristics of F. citricus DSM 6150T are inability to reduce nitrate into nitrite, inability to hydrolyse chitin and no 
-glucosidase activity. I. fluviatilis ATCC 33051T can be differentiated from strain KM-45T on the basis of its relatively low G+C content and other phenotypic characteristics (Table 1). On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rRNA gene sequence comparisons, strain KM-45T is considered to represent a novel genus and species, for which the name Silvimonas terrae gen. nov., sp. nov. is proposed.
Description of Silvimonas gen. nov.
Silvimonas (Sil.vi.mo'nas. L. fem. n. silva forest; L. fem. n. monas a unit, monad; N.L. fem. n. Silvimonas forest monad).
Cells are Gram-negative, rod-shaped (1·3–2·0 µm by 0·5–0·8 µm) and facultatively anaerobic. They occur singly or in pairs (Fig. 2a) and are motile by means of a single polar flagellum (Fig. 2b). Colonies on R2A are circular, convex and milky white. Catalase and oxidase are positive. The predominant quinone is Q-8. Major cellular fatty acids are C16 : 0, summed feature 4 (C16 : 17c/iso-C15 : 0 2-OH), summed feature 7 (C18 : 17c/9t/12t) and C17 : 0 cyclo. Based on 16S rRNA gene sequence analysis, Silvimonas belongs to the ‘Betaproteobacteria’. The type species is Silvimonas terrae.
Description of Silvimonas terrae sp. nov.
Silvimonas terrae (ter'rae. L. gen. n. terrae of the earth).
In addition to the characteristics that define the genus, it has the following characteristics. Growth is observed at temperatures of 15–40 °C (optimum 25–30 °C) and at pH values of 6·0–8·0 (optimum pH 7·0). Growth occurs in the absence of NaCl and in the presence of 2·0 % (w/v) NaCl; no growth is observed above 4·0 % (w/v) NaCl. Positive for production of acid from glucose, reduction of nitrate to nitrite and production of -glucosidase and -galactosidase. It can degrade chitin, starch, CM-cellulose and DNA, but cannot degrade lipid or xylan. Negative for reduction of nitrite to nitrogen, production of indole, arginine dihydrolase, urease and protease activities (gelatin hydrolysis). The following compounds are assimilated as sole carbon sources: L-fucose, L-arabinose, maltose, D-mannose, L-rhamnose, D-sucrose, mannitol, N-acetylglucosamine and gluconate. The following compounds are not assimilated as sole carbon sources: D-ribose, 3-hydroxybutyrate, acetate, adipate, citrate, 3-hydroxybenzoate, malate, valerate, 4-hydroxybenzoate, 5-ketogluconate, caprate, itaconate, D-sorbitol, lactate, malonate, phenylacetate, suberate, L-alanine, L-proline, L-serine, inositol, D-melibiose, glycogen and salicin. Resistant to 30 µg ampicillin ml–1, 15 µg tetracycline ml–1 and 15 µg streptomycin ml–1, but sensitive to 1 mg kanamycin ml–1. Major cellular fatty acids are C16 : 0, summed feature 4 (C16 : 17c/iso-C15 : 0 2-OH), summed feature 7 (C18 : 17c/9t/12t) and C17 : 0 cyclo. The G+C content of the genomic DNA of the type strain is 58 mol%.
The type strain, KM-45T (=KCTC 12358T=NBRC 100961T), was isolated from forest soil collected near KAIST, Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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