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Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, South Korea
Correspondence
Deok-Chun Yang
dcyang{at}khu.ac.kr
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9c as the predominant iso-branched fatty acids, all of which corroborated the assignment of the strain to the genus Lysobacter. Results of DNA–DNA hybridization and physiological and biochemical tests clearly demonstrated that strain Dae16T represents a distinct species. Based on these data, it is proposed that Dae16T (=KCTC 12204T=NBRC 101156T) should be classified as the type strain of a novel Lysobacter species, Lysobacter koreensis sp. nov.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Dae16T is AB166878.
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for gliding bacteria with high G+C contents that do not produce fruiting bodies, with Lysobacter enzymogenes as the type species. At the time of writing, the genus Lysobacter comprises Lysobacter antibioticus, L. brunescens, L. concretionis, L. enzymogenes and L. gummosus. Except for L. concretionis (Bae et al., 2005), these species were proposed by Christensen & Cook (1978) on the basis of phenotypic characteristics; their taxonomic positions were confirmed by phylogenetic and chemotaxonomic features (Bae et al., 2005). The names of two subspecies of L. enzymogenes proposed by Christensen & Cook (1978), L. enzymogenes subsp. cookii and L. enzymogenes subsp. enzymogenes, were inadvertently omitted from the Approved Lists and these names are therefore not validly published. In a series of studies, micro-organisms have been isolated from a ginseng field in order to investigate the community structure based on a culture-dependent method. In this study, one strain was isolated from the soil of a ginseng field in Daejeon city, South Korea, and characterized by a polyphasic approach. The polyphasic approach, including phylogenetic analysis based on 16S rRNA gene sequences, genomic relatedness, and chemotaxonomic and phenotypic properties, was conducted to determine the precise taxonomic position of strain Dae16T. The results obtained in this study indicated that Dae16T is a member of the genus Lysobacter, but it is clearly distinguishable from all Lysobacter species. Here, it is proposed that Dae16T should be classified as the type strain of a novel species.
Strain Dae16T was isolated from soil of a ginseng field near Daechung lake 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. Purified colonies were tentatively identified using partial 16S rRNA gene sequences.
Cell morphology and motility were observed with a Nikon light microscope (1000x magnification), with the cells being allowed to grow for 3 days at 30 °C on R2A agar. 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 measurements of bubble production after the application of 3 % (v/v) hydrogen peroxide solution. Acid production from carbohydrates was assessed using procedures outlined by Cappuccino & Sherman (2002). Growth at various temperatures (4, 15, 25, 30, 37 and 42 °C) was assessed on R2A agar and growth at various pH values was assessed on R2A broth. Growth on nutrient agar, trypticase soy agar (TSA) and MacConkey agar was also evaluated at 30 °C. The API 20NE and API ID32 GN microtest systems were employed in these tests, according to the recommendations of the manufacturer (bioMérieux).
Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v), purified by TLC and subsequently analysed by HPLC, as described previously (Collins & Jones, 1981; Shin et al., 1996). In order to perform fatty acid methyl ester analysis, the strains were allowed to grow on TSA for 48 h at 30 °C and then two loops of the well-grown cells were harvested. Fatty acid methyl esters were prepared, separated and identified with the Sherlock Microbial Identification System (MIDI; Sasser, 1990).
The genomic DNA of strain Dae16T was extracted and purified with the QIAGEN Genomic-tip system 100/G; it was then enzymically degraded into nucleosides, as described previously (Tamaoka & Komagata, 1984; 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 micro-dilution wells. Hybridization was conducted with five replications for each sample. The highest and lowest values obtained for each sample were excluded and the means of the remaining three values are quoted as DNA relatedness values.
Genomic DNA was extracted and purified with the Genomic DNA Isolation kit (Core Bio System). The 16S rRNA gene was amplified from chromosomal DNA of strain Dae16T using the universal bacterial primer set 9F and 1512R (Weisburg et al., 1991) and the purified PCR products were sequenced by Genotec (Daejeon, Korea) (Kim et al., 2005). The full sequence of the 16S rRNA gene was compiled with SeqMan software and the 16S rRNA gene sequences of the test strain were edited using the program BioEdit (Hall, 1999). The 16S rRNA gene sequences of related taxa were obtained from GenBank/EMBL. Multiple alignments were performed with the program CLUSTAL_X (Thompson et al., 1997). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983). The phylogenetic tree was constructed via the neighbour-joining method (Saitou & Nei, 1987) in the program MEGA2 (Kumar et al., 2001). Bootstrap analysis with 1000 replicates was also conducted in order to obtain confidence levels for the branches (Felsenstein, 1985). All of the species in the genus Lysobacter were included in the phylogenetic tree.
Strain Dae16T was cultured on R2A agar (Difco) at 30 °C, yielding colonies that were yellow-coloured, circular and glossy in appearance. Strain Dae16T was an aerobic, Gram-negative, non-motile, rod-shaped bacterium. Strain Dae16T was also able to grow at 20–30 °C, but did not grow at 4 or 37 °C. Growth at 30 °C was not observed on nutrient agar or TSA. The physiological characteristics of strain Dae16T 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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. The major cellular fatty acids in strain Dae16T included iso-hexadecanoic acid (C16 : 0 iso, 33·0 %), iso-pentadecanoic acid (C15 : 0 iso, 17·0 %) and iso-heptadecenoic acid (C17 : 1 iso 9c, 19·9 %). Minor amounts of the iso-branched fatty acids C11 : 0 iso (3·5 %), C14 : 0 iso (2·7 %), C15 : 0 iso AT5 (2·7 %) and C17 : 0 iso (2·5 %) were present and minor amounts of the hydroxy fatty acids C11 : 0 iso 3-OH (5·6 %) and C16 : 1 7c alcohol (4·1 %) were also found. The presence of C15 : 0 iso, C16 : 0 iso and C17 : 1 iso 9c as the major fatty acids is a characteristic composition of genera in the Xanthomonas branch containing the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Xylella and Luteimonas (Assih et al., 2002; Roumagnac et al., 2004; Yang et al., 2005). Significant differences in fatty acid profiles were found between different species in the genus Lysobacter.
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The 16S rRNA gene sequence of strain Dae16T was a continuous stretch of 1474 nt. The 16S rRNA gene sequences of related taxa were obtained from GenBank/EMBL. Strain Dae16T belonged to the Gammaproteobacteria and had the highest degree of sequence similarity to L. gummosus ATCC 29489T (97·1 %), L. antibioticus DSM 2044T (96·6 %), L. enzymogenes DSM 2043T (96·2 %), L. concretionis KCTC 12205T (94·7 %) and L. brunescens ATCC 29482T (93·7 %). In the phylogenetic tree (Fig. 1), strain Dae16T clearly belonged to the Lysobacter lineage, as shown by the high bootstrap value.
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), which is the threshold value that delineates a genomic species. Our results support the designation of strain Dae16T as a representative of a separate, novel species within the genus Lysobacter, for which the name Lysobacter koreensis sp. nov. is proposed.
Description of Lysobacter koreensis sp. nov.
Lysobacter koreensis sp. nov. (ko.re.en'sis. N.L. masc. adj. koreensis pertaining to Korea, the location of the soil sample from which the type strain was isolated).
Gram-negative, aerobic rods (1·5–2·0x0·5–0·8 µm) after growth on R2A agar (Difco) at 25 °C for 10 days. Does not move by flagella. Colonies grown on R2A agar for 2 days are yellow-coloured, glossy circles. Optimal temperature and pH for growth are 30 °C and pH 6·8–8·0. Growth can occur in a salt concentration of 1 %, but not above 2 %. Catalase-positive and oxidase-negative. Produces protease, but does not produce arginine dihydrolase, urease, 
-glucosidase or -galactosidase. Assimilates 3-hydroxybenzoate, citrate, D-mannitol, D-sorbitol, L-arabinose, L-rhamnose, L-serine, propionate and valerate. Does not assimilate 2-ketogluconate, 3-hydroxybutyrate, 4-hydroxybenzoate, 5-ketogluconate, acetate, adipate, caprate, D-glucose, D-maltose, D-mannose, D-melibiose, D-ribose, sucrose, gluconate, glycogen, itaconate, L-alanine, L-fucose, L-histidine, L-proline, lactate, malate, malonate, myo-inositol, N-acetylglucosamine, phenylacetate, salicin or suberate. Does not produce any biopolymer-hydrolysing enzymes, e.g. amylase, cellulase, chitinase, DNase, lipase, protease or xylanase. DNA G+C content of the type strain is 68·9 mol%, as determined by HPLC. Q-8 is the predominant quinone. The major cellular fatty acids are C16 : 0 iso (33·0 %), C15 : 0 iso, (17·0 %) and C17 : 1 iso 9c (19·9 %). Minor fatty acids are C11 : 0 iso (3·5 %) and C11 : 0 iso 3-OH. Does not reduce nitrate. Can liquefy gelatin. Other phenotypic characteristics, such as substrate utilization and enzyme production, are summarized in Table 1
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The type strain is Dae16T (=KCTC 12204T=NBRC 101156T), isolated from soil of a ginseng field in Daejeon, South Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bae, H.-S., Im, W.-T. & Lee, S.-T. (2005). Lysobacter concretionis sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket reactor. Int J Syst Evol Microbiol 55, 1155–1161.
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