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1 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Republic of Korea
2 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Republic of Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr
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ABSTRACT |
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9c and iso-C11 : 0 3-OH and the major components of strain GH19-3T were iso-C16 : 0, iso-C15 : 0, C16 : 17c alcohol, iso-C17 : 19c and iso-C11 : 0 3-OH. None of the species of the genus Lysobacter with validly published names showed 16S rRNA gene sequence similarity values of more than 97 % with respect to the novel isolates. The closest sequence similarity of strain GH1-9T was with Lysobacter concretionis DSM 16239T (96.4 %), whereas strain GH19-3T showed the highest sequence similarity with Lysobacter enzymogenes DSM 2043T (96.6 %). Polyphasic taxonomic studies indicated that the two strains should be classified as representing novel members of the genus Lysobacter. The names Lysobacter daejeonensis sp. nov. and Lysobacter yangpyeongensis sp. nov. are proposed, with strains GH1-9T (=KACC 11406T=DSM 17634T) and GH19-3T (=KACC 11407T=DSM 17635T), respectively, as the type strains.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains GH1-9T and GH19-3T are DQ191178 and DQ191179, respectively.
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, the taxonomic properties of its species have not been fully characterized. Recently, Bae et al. (2005) evaluated these properties more deeply and carried out physiological, biochemical, chemotaxonomic and phylogenetic analyses. At the time of writing, the genus Lysobacter includes six species, Lysobacter enzymogenes, Lysobacter antibioticus, Lysobacter brunescens, Lysobacter concretionis, Lysobacter gummosus and Lysobacter koreensis.
Two novel bacterial strains, GH1-9T and GH19-3T, were isolated from greenhouse soil cultivated with lettuce (Lactuca sativa L.). Soil samples were suspended in sterilized water and diluted solutions were spread on R2A agar (Difco) and incubated at 28 °C. Purified colonies were obtained from subcultures. Cellular morphology was determined by using phase-contrast microscopy with 2-day-old cells. Motility was examined by using 1/10 strength R2A medium, and gliding motility was observed by oil-immersion phase-contrast microscopy of the edges of colonies of cells in exponential phase. The temperature range (4–50 °C), pH range (4–10 at intervals of 1 pH unit) and the requirement for 0, 1, 2, 3, 5 and 7 % NaCl (w/v) for growth were determined using R2A medium. Gram staining, and tests for catalase and oxidase, indole production and hydrolysis of casein, chitin, DNA, gelatin and starch were conducted according to the methods of Smibert & Krieg (1994). Carboxymethylcellulose (CM-cellulose; 0.1 %, w/v; Sigma) and Whatman powder CF11 (0.1 %, w/v) were used to test for cellulase. Hydrolysis of chitin from crab shells (1 %, w/v; Sigma) and tyrosine (0.5 %, w/v) was also tested. API 20NE, API ID 32 GN and API ZYM kits (bioMérieux) were used to determine biochemical properties, utilization of carbohydrates and enzymic activities, according to the manufacturer's instructions. The API ZYM tests were read after 4 h incubation at 37 °C and the other API tests after 72 h at 28 °C.
The two strains were aerobic, with rod-shaped cells (0.4–0.6x3.0–4.0 µm). Colonies of both strains were yellow, circular and convex, with clear margins after 2 days incubation on R2A agar. Both strains grew well on R2A and nutrient agar (Difco), but did not grow on MacConkey agar (Difco). Growth on trypticase soy agar (Difco) was observed on initial inoculation, but no growth was observed after the strains had been subcultured three or four times. With a longer incubation time (>1 week), the centre of the colonies of strain GH19-3T developed a brown colour. Using the API 20NE and API ID 32 GN kits, strain GH1-9T tested positive for nitrate reduction and aesculin hydrolysis, and assimilated D-maltose, sodium acetate, glycogen, D-glucose, valeric acid and 3-hydroxybutyric acid. Strain GH19-3T tested positive for gelatin hydrolysis and assimilated glycogen and 3-hydroxybutyric acid. Using API ZYM, strain GH1-9T was positive for alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and 
-glucosidase, and weakly positive for lipase C14, valine arylamidase and 
-glucosidase. Strain GH19-3T was positive for alkaline phosphatase, esterase C4, esterase lipase C8, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and -glucosidase, and weakly positive for -chymotrypsin and N-acetyl--glucosaminidase. Phenotypic characteristics of the two strains are given in the species descriptions. Phenotypic characteristics that differentiated the two strains and other related Lysobacter species are shown in Table 1.
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. Analysis of the respiratory lipoquinones indicated that the two isolates contained ubiquinone-8 (Q-8). This quinone system is a characteristic feature of members of the genus Lysobacter (Bae et al., 2005). The DNA G+C content was determined as described by Mesbah et al. (1989) using a reversed-phase column (Supelcosil LC-18-S; Supelco). The G+C contents of strains GH1-9T and GH19-3T were 61.7 and 67.3 mol%, respectively.
After growth of the cells on R2A agar for 48 h at 28 °C, fatty acid methyl esters were extracted and prepared by using the standard protocol of the Microbial Identification System (MIDI; Microbial ID). The major fatty acids of strain GH1-9T were iso-C16 : 0, iso-C15 : 0, iso-C14 : 0, iso-C17 : 19c and iso-C11 : 0 3-OH and the major fatty acids of strain GH19-3T were iso-C16 : 0, iso-C15 : 0, C16 : 17c alcohol, iso-C17 : 19c and iso-C11 : 0 3-OH. The characteristic fatty acid of the two isolates that differentiated them from the other Lysobacter species was iso-C12 : 0 3-OH. A unique fatty acid component of GH1-9T was iso-C15 : 1 F. Unique fatty acid components of strain GH19-3T were anteiso-C17 : 0 and C10 : 0 3-OH. A comparison of fatty acid profiles among Lysobacter species is shown in Table 2.
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). The products were sequenced directly using an ABI PRISM BigDye Primer cycle sequencing kit (Applied Biosystems) with an ABI 3700 DNA sequencer (Applied Biosystems). The 16S rRNA gene sequences of the two isolates were aligned with reference sequences of Lysobacter species and members of related genera. Escherichia coli (GenBank accession no. J01695) was used as an outgroup. Sequences were aligned using the multiple sequence alignment program CLUSTAL W (Thompson et al., 1994). Phylogenetic distances were determined according to Jukes & Cantor (1969) and a tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987) as implemented in MEGA version 2.1.
According to the phylogenetic tree (Fig. 1), the two isolates were clearly grouped in a cluster composed of Lysobacter species except for L. brunescens ATCC 29482T. The sequence similarity between strains GH1-9T and GH19-3T was 95.3 %. The neighbour-joining tree indicated that strain GH1-9T was most highly related to L. concretionis DSM 16239T (96.4 % sequence similarity) and strain GH19-3T showed the highest sequence similarity with L. enzymogenes DSM 2043T (96.6 %). The low level of 16S rRNA gene sequence similarity (<97 %) among Lysobacter species with validly published names and the novel isolates indicated that the two isolates each represented novel genomic species of the genus Lysobacter. Consequently, DNA–DNA reassociation studies were not necessary (Stackebrandt & Goebel, 1994). Therefore, based on the polyphasic taxonomic approach described here, we propose the name Lysobacter daejeonensis sp. nov. for isolate GH1-9T and Lysobacter yangpyeongensis sp. nov. for isolate GH19-3T.
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Cells are aerobic, Gram-negative, non-motile and rod-shaped (0.4–0.6x3.0–4.0 µm in size). Colonies are yellow, circular and convex, with clear margins after 2 days incubation on R2A agar. NaCl, temperature and pH ranges for growth are 0–3 % (w/v), 10–37 °C and 6–8, respectively. Does not hydrolyse chitin, CM-cellulose, Whatman powder CF11 or starch, but does hydrolyse casein, DNA, gelatin and tyrosine. Major fatty acids are iso-C16 : 0 (33.7 %), iso-C15 : 0 (13.1 %), iso-C14 : 0 (11.2 %), iso-C17 : 19c (6.7 %) and iso-C11 : 0 3-OH (6.0 %). Contains Q-8. The G+C content of the genomic DNA is 61.7 mol% (HPLC). Additional characteristics are listed in Table 1
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The type strain, GH1-9T (=KACC 11406T=DSM 17634T), was isolated from greenhouse soil in Korea.
Description of Lysobacter yangpyeongensis sp. nov.
Lysobacter yangpyeongensis (yang.pye.ong.en'sis. N.L. masc. adj. yangpyeongensis pertaining to Yangpyeong, a province in Korea, from where the type strain was isolated).
Cells are aerobic, Gram-negative, motile and rod-shaped (0.4–0.6x3.0–4.0 µm in size). Colonies are yellow, circular and convex, with clear margins after 2 days incubation on R2A agar. Temperature and pH ranges for growth are 15–40 °C and 5–8, respectively. Does not grow in 1 % (w/v) NaCl. Hydrolyses casein, DNA, gelatin, starch and tyrosine, but not chitin, CM-cellulose or Whatman powder CF11. Major fatty acids are iso-C16 : 0 (27.5 %), iso-C15 : 0 (14.5 %), C16 : 17c alcohol (8.8 %), iso-C17 : 19c (6.7 %) and iso-C11 : 0 3-OH (5.5 %). Contains Q-8. The G+C content of the genomic DNA is 67.3 mol% (HPLC). Additional characteristics are listed in Table 1.
The type strain, GH19-3T (=KACC 11407T=DSM 17635T), was isolated from greenhouse soil in Korea.
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REFERENCES |
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Christensen, P. (1989). Genus I. Lysobacter Christensen and Cook 1978, 372AL. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 2083–2089. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
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