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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, Republic of Korea
2 Key Laboratory of Natural Resources of the Changbai Mountain and Functional Molecules (Yanbian University), Ministry of Education, Yanji 133002, China
3 Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong-gu, Daejeon 305-333, Republic of Korea
Correspondence
Wan-Taek Im
wandra{at}kaist.ac.kr
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Gsoil 161T is AB245387.
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TOPABSTRACTMAIN TEXTREFERENCES |
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. According to the original description, the genus comprised non-mycelial, non-sporulating actinomycetes that produced the macrolide antibiotic erythromycin; the type species is Aeromicrobium erythreum. In 1994, Nocardioides fastidiosa was transferred to the genus as Aeromicrobium fastidiosum (Tamura & Yokota, 1994). Two further species, Aeromicrobium marinum (Bruns et al., 2003) and Aeromicrobium alkaliterrae (Yoon et al., 2005), were described recently. In this study, taxonomic characterization is reported of an Aeromicrobium-like strain, Gsoil 161T, which was isolated from soil of a ginseng field in South Korea during a study on the culturable aerobic bacterial community.
Strain Gsoil 161T was originally isolated from soil of a ginseng field in Pocheon Province, South Korea. This soil sample was thoroughly suspended in 50 mM phosphate buffer (pH 7.0) and the suspension was spread on one-tenth-strength modified R2A (l–1: 0.25 g tryptone, 0.25 g peptone, 0.25 g yeast extract, 0.125 g malt extract, 0.125 g beef extract, 0.25 g Casamino acid, 0.25 g soytone, 0.5 g glucose, 0.3 g soluble starch, 0.2 g xylan, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g MgSO4, 0.05 g CaCl2, 15 g agar) agar plates after serial dilution with 50 mM phosphate buffer (pH 7.0). Plates were incubated at 30 °C for 1 month. Single colonies on the plates were purified by transferring them onto new plates and these were incubated again on modified R2A or half-strength modified R2A. Purified colonies were tentatively identified by partial 16S rRNA gene sequences (Im et al., 2005). Strain Gsoil 161T was one of the isolates that appeared on the modified R2A agar plates under aerobic conditions. As it could grow well on full-strength modified R2A agar and commercial R2A agar (Difco), strain Gsoil 161T was routinely cultured on R2A agar at 30 °C and maintained as a glycerol suspension (20 %, w/v) at –70 °C.
Gram-reaction testing was performed by the non-staining method as described by Buck (1982). Cell morphology was observed at x1000 magnification with a light microscope (Nikon) using cells grown for 24 h at 30 °C on modified R2A agar. Catalase activity was determined by checking bubble production using 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethyl p-phenylenediamine. For single-carbon-source assimilation studies, a defined liquid medium containing basal salts was used, containing (l–1) K2HPO4 (1.8 g), KH2PO4 (1.08 g), NaNO3 (0.5 g), NH4Cl (0.5 g), KCl (0.1 g), MgSO4 (0.1 g) and CaCl2 (0.05 g). A vitamin solution (Widdel & Bak, 1992), trace element solution SL-10 (Widdel et al., 1983) and selenite/tungstate solution (Tschech & Pfennig, 1984) were added to this medium and the pH was adjusted to 6.8. This liquid medium was added to 96-well trays and filter-sterilized carbon sources were added to each well (individually at 0.1 %, w/v). Growth was examined visually after incubation at 30 °C for up to 7 days on 96-well plates. A negative control well containing no carbon source was included. A positive control culture was grown in a well containing R2A broth.
Fermentative and oxidative acid production from carbohydrates was tested by growth in O-F basal medium with bromothymol blue (Atlas, 1993) supplemented with 1 % carbohydrate [soft-agar stabs with (fermentative) and without (oxidative) sterile mineral oil overlay]. The O-F medium tubes were incubated at 30 °C for 5 days. Some physiological characteristics such as nitrate reduction, acid production from carbohydrates and activity of some enzymes were determined with API 20E galleries according to the instructions of the manufacturer (bioMérieux). Anaerobic growth was tested in serum bottles containing thioglycolate (1 g l–1) added to R2A broth and replacing the upper air layer with nitrogen gas. The anaerobic nitrate reduction test to determine the final electron acceptor was performed in serum bottles by adding 1 g thioglycolate l–1 and 10 mM KNO3 to R2A broth, under nitrogen gas. Aerobic nitrate reduction was later confirmed by inoculation into 12 ml R2A broth supplemented with 10 mM KNO3 in three 25 ml serum bottles. The reduction of nitrate was monitored by an ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and an anion exchange column (Metrosep Anion Supp 4; Metrohm). Tests for the degradation of DNA [plates of DNase agar (Scharlau) flooded with 1 M HCl], casein, chitin and starch (Atlas, 1993), lipid (Kouker & Jaeger, 1987), and xylan and cellulose (Ten et al., 2004) were performed and evaluated after 10 days. Growth at different temperatures (4, 15, 20, 25, 30, 37, 42 and 45 °C) and various pH values (pH 5.0–10.0 at intervals of 0.5 pH units) was assessed after 5 days incubation. Salt tolerance was tested on modified R2A agar supplemented with 1–10 % (w/v) NaCl after 5 days incubation. Growth on nutrient agar (5.0 g peptone, 3.0 g beef extract, 15.0 g agar in 1 l; Difco), trypticase soy agar (TSA) and MacConkey agar was also evaluated at 30 °C.
For phylogenetic analysis of strain Gsoil 161T, DNA was extracted using a genomic DNA extraction kit (Solgent). The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primer set 9F and 1512R (Weisburg et al., 1991) and purified PCR products were sequenced by Solgent (Daejeon, South Korea) (Kim et al., 2005). Full sequences of the 16S rRNA gene were compiled using SeqMan software (DNASTAR). The 16S rRNA gene sequences of related taxa were obtained from GenBank. Multiple alignments were performed using CLUSTAL X (Thompson et al., 1997). Gaps were edited in the program BIOEDIT (Hall, 1999). Evolutionary distances were calculated 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 program MEGA3 (Kumar et al., 2004) with bootstrap values based on 1000 replications (Felsenstein, 1985).
For the measurement of G+C content, genomic DNA of the novel strain was extracted and purified as described by Moore & Dowhan (1995). The DNA was enzymically degraded into nucleosides and G+C content was determined as described by Mesbah et al. (1989) using reverse-phase HPLC. Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), evaporated under vacuum and re-extracted in n-hexane/water (1 : 1, v/v). The crude n-hexane/quinone solution was purified using Sep-Pak Vac cartridges silica (Waters) and subsequently analysed by HPLC as described previously (Hiraishi et al., 1996). Cellular fatty acid profiles were determined for strains grown on modified R2A broth for 6 days. Cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by GC (Hewlett Packard 6890) were identified by the Microbial Identification software package (Sasser, 1990). The presence of diaminopimelic acid (DAP) isomers in the cell wall peptidoglycan was determined by using TLC after hydrolysis with 6 M HCl at 100 °C for 18 h (Komagata & Suzuki, 1987).
Strain Gsoil 161T was Gram-positive, strictly aerobic, non-spore-forming, non-motile and rod-shaped. Colonies grown on R2A agar plates (Difco) for 5 days were smooth, circular, light-yellowish white in colour, convex and 1–3 mm in diameter. After 5 days incubation, the novel strain did not grow well on TSA; it showed very thin layer growth on the surface of TSA plates. However, it did grow well on nutrient agar and modified R2A agar. Strain Gsoil 161T was able to grow at 15–30 °C, but not at 4 or 35 °C or above. Physiological characteristics of strain Gsoil 161T are summarized in the species description and a comparison of selective characteristics with related type strains is given in Table 1.
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, using sequences of over 1300 bp). Strain Gsoil 161T and the four Aeromicrobium species mentioned above formed a monophyletic clade with a bootstrap value of 100 %, which was supported by the two tree-making methods used in this study.
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using photobiotin-labelled DNA probes and microdilution wells. DNA–DNA relatedness values of strain Gsoil 161T with the four most closely related Aeromicrobium species were 21 % with A. marinum DSM 15272T, 3 % with A. alkaliterrae KCTC 19073T, 18 % with A. fastidiosum KCTC 9576T and 10 % with A. erythreum DSM 8599T, values that are low enough to propose strain Gsoil 161T as a representative of a novel species of genus Aeromicrobium (Wayne et al., 1987).
The G+C content of genomic DNA of strain Gsoil 161T was 65.5 mol%. The respiratory quinone system supports affiliation of strain Gsoil 161T to the genus Aeromicrobium, where the majority of species have MK-9(H4) as the predominant quinone. The cell wall peptidoglycan of strain Gsoil 161T contained LL-DAP. The fatty acid profile of strain Gsoil 161T was composed mainly of 10-methyl C18 : 0 (tuberculostearic acid; 14.5 %), C16 : 0 (13.0 %), C16 : 0 2-OH (12.8 %), 10-methyl C17 : 0 (11.9 %), 10-methyl C16 : 0 (11.7 %), C16 : 1
7c/iso-C15 : 0 2-OH (6.6 %), C18 : 19c (5.3 %), C15 : 0 (4.9 %), C17 : 0 (4.8 %), C17 : 18c (4.7 %), C17 : 0 2-OH (2.7 %), C15 : 0 2-OH (1.8 %), iso-C16 : 0 (1.5 %) and C17 : 16c (1.4 %). The fatty acid profile of strain Gsoil 161T differed significantly from those of the most closely related Aeromicrobium species. However, some of the major components of the fatty acid profiles of these taxa were the same, i.e. C16 : 0 and C16 : 0 2-OH.
In summary, the characteristics of strain Gsoil 161T are consistent with descriptions of the genus Aeromicrobium with regard to morphological, biochemical and chemotaxonomic properties. However, on the basis of phylogenetic distance from known Aeromicrobium species indicated by 16S rRNA gene sequence similarities and the combination of unique phenotypic characteristics (Table 1
), Gsoil 161T should be placed in the genus Aeromicrobium as a representative of a novel species, for which the name Aeromicrobium panaciterrae sp. nov. is proposed.
Description of Aeromicrobium panaciterrae sp. nov.
Aeromicrobium panaciterrae (pa.na.ci.ter'rae. N.L. n. Panax, -acis scientific name of ginseng; L. n. terra soil; N.L. gen. n. panaciterrae of soil of a ginseng field).
Cells are Gram-positive, strictly aerobic, non-spore-forming, non-motile and rod-shaped, 0.2–0.4 µm in diameter and 1.0–1.5 µm in length after culture for 3 days on modified R2A broth. Colonies grown on R2A agar (Difco) for 5 days are smooth, circular, and light yellowish in colour, convex and 1–3 mm in diameter. Grows well at 15–30 °C and pH 5.0–8.5, but does not grow at 4 or 35 °C. Growth occurs in the absence of NaCl and in the presence of 3.0 % (w/v) NaCl, but not in 4.0 % (w/v) NaCl. Grows on nutrient agar. Does not grow on MacConkey agar. Catalase-positive and oxidase-negative. Does not reduce nitrate to nitrite. H2S is not produced. Negative for production of urease, lysine decarboxylase, ornithine decarboxylase, 
-galactosidase and tryptophan deaminase. Positive for gelatinase and arginine dihydrolase. Does not produce any acid or gas from glucose, but glucose is utilized as a sole carbon source. Does not degrade xylan, chitin, cellulose, casein, DNA or starch. The following compounds are utilized as sole carbon sources: acetate, alanine, D-cellobiose, D-galactose, D-glucose, D-mannose, trehalose, glycerol, maltose, salicin, succinate, sucrose, melibiose, L-lactate, L-malate, glycine and D-glycogen. The following compounds are not utilized as sole carbon sources: citrate, D-fructose, D-xylose, lactose, L-arabinose, mannitol, rhamnose, L-serine, L-sorbose, sodium formate, D-ribose, L-lysine, D-fucose, xylitol and D-lyxose. MK-9(H4) is the predominant menaquinone, and C16 : 0, 10-methyl C18 : 0, C16 : 0 2-OH, 10-methyl C17 : 0 and 10-methyl C16 : 0 are the major components of the cellular fatty acids. The cell wall peptidoglycan contains LL-DAP.
The type strain is Gsoil 161T (=KCTC 19131T=DSM 17939T=CCUG 52476T), isolated from soil of a ginseng field in Pocheon Province, South Korea. The DNA G+C content of Gsoil 161T is 65.5 mol% (as determined by HPLC).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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