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Korean Collection for Type Cultures, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong-ku, Daejon 305-333, Republic of Korea
Correspondence
Kyung Sook Bae
ksbae{at}mail.kribb.re.kr
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ABSTRACT |
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-7-cis-octadecenoic acid in their cell envelopes. Strain KCTC 12081T contained ubiquinone-8 as the major isoprenoid quinone and the G+C content of its genomic DNA was 61·3 mol%. Morphological and chemotaxonomic properties of the strains were consistent with classification in the genus Burkholderia. In a comparison of 16S rDNA sequence, KCTC 12081T shared 100 % similarity with KCTC 12082 and both strains formed a distinct phylogenetic lineage within the genus Burkholderia. The two strains were also differentiated from other species of this genus by fatty acid composition and phenotypic properties. DNA–DNA relatedness data further supported the separation of the new isolates from closely related species. It is therefore proposed that strains KCTC 12081T (=JCM 11778T) and KCTC 12082 be recognized as a novel species, for which the name Burkholderia sordidicola sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains KCTC 12081T and KCTC 12082 are AF512826 and AF512827, respectively.
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; Lim et al., 2000). White-rot fungi degrade lignin more extensively and rapidly than any other organism known (Kondo et al., 1994; Garzillo et al., 1998; Novotn
et al., 2000). P. sordida has been applied to a variety of industrial processes, such as biopulping and pulp bleaching, and also used in bioremediation, due to its enzymic capabilities (Lamar et al., 1990).
A range of bacteria that belong to different subdivisions of the Proteobacteria, e.g. Burkholderia fungorum, have been found in association with the white-rot fungus Phanerochaete chrysosporium (Seigle-Murandi et al., 1996; Coenye et al., 2001a).
Burkholderia is a group of metabolically versatile Gram-negative bacteria. The genus was first proposed by Yabuuchi et al. (1992), who transferred seven species of rRNA group II of the genus Pseudomonas to the novel genus on the basis of a polyphasic taxonomic study. Members of the genus Burkholderia are often found in contaminated soil and water, as well as in natural soil, water and the rhizosphere of plants, and are capable of degrading numerous low-molecular-mass organic compounds including aromatic compounds, halogenated derivatives and various recalcitrant organic residues (Friedrich et al., 2000; Nogales et al., 2001; Parke & Gurian-Sherman, 2001).
In an isolation study of P. sordida strains collected from the plants Quercus acutissima and Prunus serrulata, bacterial strains were found to be associated with P. sordida cultures. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains belonged to the genus Burkholderia and that they were apparently distinct from any known species in the genus. Detailed phenotypic and genotypic features of these bacterial isolates are presented in this study.
Strains KCTC 12081T (=JCM 11778T) and KCTC 12082 (=JCM 11779) were isolated from cultures of the white-rot fungi P. sordida KCTC 26213 (collected from Quercus acutissima, Kangwha, Inchon, Korea) and P. sordida KCTC 26214 (collected from Prunus serrulata, Seoul, Korea), respectively. Bacterial strains were subcultured on tryptic soy agar [tryptic soy broth (Difco) supplemented with 1·5 % agar] and grown aerobically at 30 °C. 16S rDNA was PCR-amplified by using primers 27f and 1492r (Lane, 1991). PCR products were purified by using an AccuPrep PCR Purification kit (Bioneer) and sequenced by using primers 27f, 803f, 907r and 1492r (Lane, 1991). Nearly complete 16S rRNA gene sequences (corresponding to positions 63–1453 of the Escherichia coli numbering system) were obtained and aligned with those of 23 Burkholderia species with validly published names that were retrieved from GenBank (http://www.ncbi.nlm.nih.gov). Phylogenetic trees based on the neighbour-joining, Fitch–Margoliash and maximum-likelihood methods were inferred by using the PHYLIP package (Felsenstein, 1993). Distances for the first two algorithms were calculated by using the Jukes–Cantor distances option.
DNA–DNA relatedness was determined by using a DIG-High Prime DNA Labeling and Detection kit (Roche Applied Science) and Bio-Dot SF slot-blotting apparatus (Bio-Rad). Hybridization was performed at 42 °C; labelling of probes, blotting, hybridization and detection were carried out according to the manufacturers' instructions.
The molar G+C content of genomic DNA was determined by the thermal denaturation (Tm) method described by Marmur & Doty (1962). Major isoprenoid quinones were detected by using the method of Yamada (1998). The Biolog GN system was used according to the manufacturer's instructions to test the degradation of 95 carbon substrates for the two test strains and type strains of eight related species. All tests were run in duplicate.
Cultures of P. sordida KCTC 26213 and KCTC 26214 were always found to be associated with bacterial strains and it was not possible to obtain pure fungal cultures despite repeated trials. In contrast, the bacterial strains could be isolated easily from the mixed cultures and grew well on fungal culture media as well as on complex bacterial media, such as tryptic soy agar. A similar case on P. chrysosporium has been reported previously (Seigle-Murandi et al., 1996; Coenye et al., 2001a).
On agar plates, colonies of strains KCTC 12081T and KCTC 12082 were cream to light ochraceous in colour. No diffusible pigment was produced. Under scanning electron microscopic observation, cells appeared as ovoid to short rod-shaped, approximately 1·3–1·7 µm in length and 1·1 µm in width (Fig. 1). Cells were non-motile, non-spore-forming and Gram-negative.
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-7-cis-octadecenoic acid (C18 : 17c) as the major membrane fatty acid components, and also small amounts of 3-hydroxyhexadecanoic acid (C16 : 0 3-OH), the presence of which is characteristic for members of the genus Burkholderia (Table 1; Gillis et al., 1995). Morphological and chemotaxonomic properties of the strains were consistent with their classification within the genus Burkholderia (Yabuuchi et al., 1992; Gillis et al., 1995).
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, in which the two strains formed an independent phylogenetic lineage that was related most closely to the clade that included B. glathei and Burkholderia graminis. The two test strains and B. fungorum Coenye et al. (2001a), another species known to be associated with a white-rot fungus (Siegle-Murandi et al., 1996), were distantly related as 16S rDNA sequence similarity between the type strains was 96·1 % (57/1459 nucleotide differences). The relationship between the test strains and related species in the neighbour-joining tree was not strongly supported by bootstrap analysis and the tree topology around the test strains was not recovered in all other trees generated by different algorithms (Fig. 1
). However, the similarity level of 97·3 % between the test strains and B. glathei ATCC 29195T is comparable to the values among the 13 species of the Burkholderia cepacia clade, which range between 96·1 and 99·9 % with a mean of 98·3 %.
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). In another study, all species pairs that shared >98·1 % 16S rDNA sequence similarity resulted in DNA relatedness levels of <58 % (Coenye et al., 2001b). Thus it is apparent by comparison of 16S rDNA sequence and DNA–DNA relatedness that strains KCTC 12081T and KCTC 12082 are sufficiently distant from B. glathei ATCC 29195T, B. phenazinium LMG 2247T and other closely related species to be recognized as an independent taxospecies.
The test strains and B. glathei were also distinguished by comparison of fatty acid profiles and other phenotypic characteristics (Tables 1 and 2). One major fatty acid component, C18 : 17c, exceeded 30 % of the total amount in B. glathei, whereas the same component comprised about 23–24 % in strains KCTC 12081T and KCTC 12082 (Table 1; Vandamme et al., 1997). The two test strains and B. glathei can also be differentiated by motility, as the test strains are not motile but members of B. glathei are motile by a polar flagellum (Palleroni, 1984). The Biolog test proved helpful for species discrimination (Table 2). The test strains and closely related species were separated by at least 15 differences, with the exception of KCTC 12081T and B. phenazinium KCTC 2971T; these two strains differed in nine tests, but 32 tests out of 83 could not be compared because of the variable results for either strain (Table 2). There were 11 differences between strains KCTC 12081T and KCTC 12082 (Table 2). Tests that showed identical results for all strains, namely 2,3-butanediol, 
-D-glucose, L-glutamic acid, DL-lactic acid and methyl pyruvate (all positive) and cellobiose, i-erythritol, glycyl L-aspartic acid, -ketovaleric acid, -D-lactose, D-melibiose and thymidine (all negative) were excluded from Table 2.
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Description of Burkholderia sordidicola sp. nov.
Burkholderia sordidicola (sor.di.di'co.la. N.L. n. sordida from Phanerochaete sordida, a species of white-rot fungus; L. suff. n. -cola inhabitant; N.L masc. n. sordidicola inhabitant of Phanerochaete sordida).
Gram-negative, non-motile, non-spore-forming, ovoid to short rod-shaped cells (approx. 1·3–1·7 µm in length and 1·1 µm in width). Contains major amounts of UQ-8 and small amounts of UQ-7 and UQ-9 in the cell envelope. Major fatty acid components are C16 : 0, C17 : 0 cyclo and C18 : 17c and also smaller amounts of a summed feature (composed of iso-C15 : 0 2-OH and/or C16 : 17c); C19 : 08c cyclo and C16 : 0 3-OH are also found. Utilizes a number of carbon compounds, as shown in Table 2. Molar G+C content of genomic DNA of the type strain is 61·3 mol% (Tm method).
Two strains have been reported to date, both of which were isolated in association with cultures of white-rot fungi that belonged to the species Phanerochaete sordida. Type strain is KCTC 12081T (=JCM 11778T). Reference strain is KCTC 12082.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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