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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), Microbial Genetics Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Republic of Korea
3 Environment and Ecology Division, National Institute of Agricultural Science and Technology, RDA, Suwon 441-707, Republic of Korea
4 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 the major isoprenoid quinone was Q-8. The DNA G+C contents of GH34-4T and GH41-7T were 62.5 and 66.6 mol%, respectively. On the basis of the polyphasic taxonomic data presented, it is evident that each of these strains represents a novel species of the genus Lysobacter, for which the names Lysobacter niabensis sp. nov. (type strain GH34-4T=KACC 11587T=DSM 18244T) and Lysobacter niastensis sp. nov. (type strain GH41-7T=KACC 11588T=DSM 18481T) are proposed.
A table giving the fatty acid compositions of strains GH34-4T and GH41-7T is available as supplementary material in IJSEM Online.
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to accommodate non-fruiting, gliding bacteria with a high G+C content. Three novel species of the genus, Lysobacter koreensis (Lee et al., 2006), Lysobacter daejeonensis and Lysobacter yangpyeongensis (Weon et al., 2006), isolated from soils, have recently been described. At the time of writing, the genus Lysobacter comprises eight species with validly published names, Lysobacter enzymogenes, L. antibioticus, L. brunescens, L. concretionis, L. daejeonensis, L. gummosus, L. koreensis and L. yangpyeongensis. In 2005, soil samples were collected from greenhouse soil cultivated with cucumber (Cucumis sativus L.) from the Yongin and Sanju regions, Korea. The samples were serially diluted with 0.85 % NaCl (w/v) and suitable 10-fold dilutions were plated onto R2A agar (Difco). The plates were incubated at 28 °C for 4 days and strains GH34-4T and GH41-7T were subsequently isolated.
For strains GH34-4T and GH41-7T, cell morphology was determined by using phase-contrast microscopy of 2-day-old cultures. Gliding motility was observed via oil-immersion phase-contrast microscopy of the edge of colonies in exponential growth phase. The temperature range (5–50 °C), pH range (pH 4–10 at intervals of 1 pH unit) and requirement for 0, 1, 2, 3, 5 or 7 % NaCl (w/v) were determined by using R2A medium. Tests for Gram staining, catalase, oxidase and hydrolysis of casein, DNA and starch were conducted according to the methods of Smibert & Krieg (1994). Tests were also made for hydrolysis of CM-cellulose (0.1 %, w/v), chitin from crab shells (1 %, w/v) and tyrosine (0.5 %, w/v). The commercially available API 20NE and API ID 32 GN (bioMérieux) systems were used to determine biochemical properties, utilization of carbohydrates and enzyme activities according to the manufacturer's instructions. The API ZYM tests were read after 4 h incubation at 37 °C, and other API tests after 72 h at 28 °C.
Isoprenoid quinones were analysed by HPLC as described by Groth et al. (1996). DNA–DNA hybridization was carried out as described by Seldin & Dubnau (1985). Probe labelling was conducted by using the non-radioactive DIG-High prime system (Roche). Reassociation was conducted at 60 °C. Hybridized DNAs were visualized using the DIG luminescent detection kit (Roche). Levels of DNA–DNA relatedness were quantified by using a densitometer (Bio-Rad). For fatty acid methyl ester analysis, cell mass was harvested from R2A agar after cultivation for 48 h at 28 °C. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Determination of DNA G+C contents was performed according to Mesbah et al. (1989) by using a reversed-phase column (Supelcosil LC-18-S; Supelco).
The 16S rRNA gene was amplified from colonies by PCR using primers fD1 and rP2 (Weisburg et al., 1991) and the entire PCR fragment was directly sequenced (Hiraishi, 1992). The 16S rRNA gene sequences were aligned by using the MEGALIGN program of DNASTAR. A phylogenetic tree was reconstructed with the neighbour-joining method of Saitou & Nei (1987) on MEGA version 2.1 (Kumar et al., 2001). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data based on 1000 resamplings.
Strains GH34-4T and GH41-7T were aerobic, Gram-negative, rod-shaped and catalase- and oxidase-positive. They grew well on R2A, trypticase soy agar (Difco) and nutrient agar (Difco), but did not grow on MacConkey agar (Difco). The phenotypic characteristics of strains GH34-4T and GH41-7T are given in Table 1 and in the species descriptions below. Differential properties among GH34-4T, GH41-7T and recognized species of the genus Lysobacter are given in Table 1.
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). The fatty acid compositions of the novel strains and closely related Lysobacter species are given in Supplementary Table S1 available in IJSEM Online. The major fatty acids detected (percentages of the total cellular fatty acids) from strains GH34-4T and GH41-7T were iso-C16 : 0 (23.7 and 23.3 %, respectively), iso-C15 : 0 (12.7 and 21.9 %) and iso-C17 : 19c (10.0 and 10.9 %). The DNA G+C contents of strains GH34-4T and GH41-7T were 62.5 and 66.6 mol%, respectively.
The phylogenetic tree using the almost-complete 16S rRNA gene sequence (approximately 1450 bp) of strains GH34-4T and GH41-7T showed clearly that the two strains were located within the genus Lysobacter (Fig. 1). Strain GH34-4T was most closely related to L. yangpyeongensis GH19-3T (97.5 % 16S rRNA gene sequence similarity). Strain GH41-7T was grouped with several Lysobacter species, showing highest sequence similarity to L. antibioticus DSM 2044T (97.5 %), L. enzymogenes DSM 2043T (97.5 %) and L. gummosus ATCC 29489T (97.4 %). The level of DNA–DNA relatedness between strain GH34-4T and L. yangpyeongensis GH19-3T was 25 %, and levels between strain GH41-7T and the type strains of L. enzymogenes, L. antibioticus and L. gummosus were 42, 39 and 32 %, respectively.
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Description of Lysobacter niabensis sp. nov.
Lysobacter niabensis (ni.ab.en'sis. N.L. masc. adj. niabensis pertaining to NIAB, National Institute of Agricultural Biotechnology, where taxonomic studies of this taxon were conducted).
Cells are aerobic, Gram-negative rods (0.5x2.0–5.0 µm). Growth occurs at 5–37 °C (optimum 28 °C), pH 5–8 (optimum pH 6–7) and 0–1 % NaCl. Colonies are yellow and irregular after 48 h of cultivation at 28 °C on R2A medium. Casein, starch and tyrosine are hydrolysed, but chitin, CM-cellulose, DNA and urea are not. Major cellular fatty acids are iso-C16 : 0, iso-C15 : 0 and iso-C17 : 19c. The detailed cellular fatty acid composition is given in Supplementary Table S1 available in IJSEM Online. The major isoprenoid quinone is Q-8. The DNA G+C content of the type strain is 62.5 mol%.
The type strain, GH34-4T (=KACC 11587T=DSM 18244T), was isolated from greenhouse soil in the Republic of Korea.
Description of Lysobacter niastensis sp. nov.
Lysobacter niastensis (ni.as.ten'sis. N.L. masc. adj. niastensis pertaining to NIAST, National Institute of Agricultural Science and Technology, where taxonomic studies of this taxon were conducted).
Cells are aerobic, Gram-negative rods (0.5–0.6x2.0–4.0 µm). Growth occurs at 10–40 °C (optimum 28 °C), pH 4–9 (optimum pH 6–8) and 0–1 % NaCl. Motile by gliding. Colonies are light beige, convex, round with clear margins after 48 h of cultivation at 28 °C on R2A medium. Casein, starch and tyrosine are hydrolysed, but chitin, CM-cellulose, DNA and urea are not. Major cellular fatty acids are iso-C16 : 0, iso-C15 : 0 and iso-C17 : 19c. The detailed cellular fatty acid composition is given in Supplementary Table S1 available in IJSEM Online. The major isoprenoid quinone is Q-8. The DNA G+C content of the type strain is 66.6 mol%.
The type strain, GH41-7T (=KACC 11588T=DSM 18481T), was isolated from greenhouse soil in the Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Christensen, P. & Cook, F. D. (1978). Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol 28, 367–393.
Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, 234–239.
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Weon, H.-Y., Kim, B.-Y., Baek, Y.-K., Yoo, S.-H., Kwon, S.-W., Stackebrandt, E. & Go, S.-J. (2006). Two novel species, Lysobacter daejeonensis sp. nov. and Lysobacter yangpyeongensis sp. nov., isolated from Korean greenhouse soils. Int J Syst Evol Microbiol 56, 947–951.
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