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Int J Syst Evol Microbiol 55 (2005), 1625-1628; DOI  10.1099/ijs.0.63695-0
© 2005 International Union of Microbiological Societies

Dyella koreensis sp. nov., a {beta}-glucosidase-producing bacterium

Dong-Shan An1, Wan-Taek Im1, Hee-Chan Yang1, Deok-Chun Yang2 and Sung-Taik Lee1

1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Guseong-dong 373-1, Yuseong-gu, Daejeon 305-701, South Korea
2 Department of Oriental Medicinal Material & Processing, College of Life Science, Kyung Hee University, 1 Seocheon, Kihung Yongin, Kyunggi 449-701, South Korea

Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr


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A bacterial strain (designated BB4T), which has {beta}-glucosidase activity, was isolated from soil around the roots of bamboo plants. Cells were Gram-negative, aerobic, non-motile and straight-rod-shaped. Phylogenetic analysis of 16S rRNA gene sequences revealed a clear affiliation with members of the family ‘Xanthomonadaceae’. The 16S rRNA gene sequence of strain BB4T showed the following sequence similarities: 97·7 % to Dyella japonica XD53T, 97·1 % to Frateuria aurantia LMG 1558T, 96·2 % to Fulvimonas soli LMG 19981T, 94·3 % to Rhodanobacter lindaniclasticus RP5575T and <90 % to other members of the ‘Gammaproteobacteria’. The G+C content of the genomic DNA was 63·8 mol%. The major fatty acids were branched forms, especially large proportions of iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1{omega}9c, similar to the profile of the genus Dyella. The results of DNA–DNA hybridization with D. japonica XD53T and Frateuria aurantia LMG 1558T, in combination with phenotypic characteristics and 16S rRNA gene sequence analysis, demonstrated that strain BB4T should be classified as a novel Dyella species. The name Dyella koreensis sp. nov. is proposed, with strain BB4T (=KCTC 12359T=NBRC 100831T) as the type strain.

Published online ahead of print on 14 March 2005 as DOI 10.1099/ijs.0.63695-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BB4T is AY884571.


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During a study of bacterial populations that metabolize ginsenoside Rb1 to Rg3, Rh2 or compound-K, 100 preliminary bacteria producing {beta}-glucosidase were isolated from a number of sources such as human faeces, environmental soils, wastewater sludges and different types of ginseng. One of these isolates, designated strain BB4T, represented a member of the genus Dyella in the family ‘Xanthomonadaceae’ on the basis of 16S RNA gene sequence data; this strain was subjected to a taxonomic investigation.

The aim of this study was to determine the taxonomic position of strain BB4T using a polyphasic approach that included physiological and DNA–DNA hybridization analyses. The results suggested that BB4T is a representative of a novel species within the genus Dyella.

Strain BB4T was isolated on aesculin agar (Atlas, 1993Go) at 30 °C from a soil sample around bamboo roots at the Korea Advanced Institute of Science and Technology (Daejeon, South Korea). Cell morphology and motility were observed under a Nikon light microscope (1000x magnification), with cells grown on R2A agar for 3 days at 30 °C. Growth at different temperatures and different pH was assessed after 5 days of incubation. Salt tolerance was tested on R2A medium supplemented with 1–10 % (w/v) NaCl after 5 days of incubation. Growth was determined by monitoring the OD600. The yellow cellular pigments of our isolates were extracted with absolute ethanol from freeze-dried biomass and analysed spectrophotometrically (Gauthier & Breittmayer, 1992Go). The absorption spectra of crude extracts were determined in the range 200–700 nm using a spectrophotometer (DU 650; Beckman). Anaerobic growth was observed in serum bottles by adding thioglycolate (1 g l–1) to R2A broth and substituting the upper air layer with nitrogen gas. Carbon-source utilization and some enzyme activities were tested by using the API 20 NE, API ID32 and API 50CHL test kits (bioMérieux). Catalase activity was determined by using 3 % (v/v) H2O2 and oxidase activity was determined by using 1 % (w/v) tetramethyl p-phenylenediamine. Degradation of DNA (using DNA agar from Difco, supplemented with 0·01 % toluidine blue from Merck), degradation of casein, cellulose and starch (Atlas, 1993Go), degradation of lipid (Kouker & Jaeger, 1987Go) and degradation of xylan (Ten et al., 2004Go) were also investigated; reactions were read after 5 days. Duplicate antibiotic-sensitivity tests were performed using filter-paper discs containing the following: streptomycin (5, 10 and 15 µg ml–1), tetracycline (5, 10 and 15 µg–1), kanamycin (1·0, 1·5 and 2·0 mg ml–1) and ampicillin (20, 25 and 30 µg ml–1) (Sigma). Discs were placed on R2A plates spread with BB4T culture and were then incubated at 30 °C for 5 days. Physiological and biochemical characteristics of strain BB4T and related type strains are summarized in Table 1Go.


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  		Table 1. Comparison of selected characteristics of Dyella koreensis sp. nov. BB4T with those of its nearest phylogenetic neighbours within the family ‘Xanthomonadaceae’

Strains: 1, Dyella koreensis BB4T; 2, Dyella japonica XD53T; 3, Frateuria aurantia LMG 1558T (data for taxa 1–3 are from this study); 4, Fulvimonas soli LMG 19981T (data from Mergaert et al., 2002Go). +, Positive; –, negative; W, weak. All strains were positive for assimilation of glucose, growth on maltose and hydrolysis of DNA and negative for assimilation of gluconate and mannitol, growth on caprate and citrate and production of urease.

 
Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987)Go. Cellular fatty acids were analysed in strain BB4T grown on trypticase soy agar (TSA; Difco) for 48 h. The cellular fatty acids were saponified, methylated, extracted and identified by using the Microbial Identification software package (Sasser, 1990Go). The G+C content of the chromosomal DNA was determined as described by Mesbah et al. (1989)Go using a reversed-phase HPLC. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989)Go, using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained for each sample were excluded, and the means of these remaining three values were used as relatedness values.

Genomic DNA was extracted and purified using the Genomic DNA isolation kit (Core Bio System Co., Ltd) and the 16S rRNA gene was PCR-amplified (Weisburg et al., 1991Go) and directly sequenced. The 16S rRNA gene sequence was compiled using SEQMAN software and 16s rrna gene sequences of the test strain and related taxa obtained from the genbank were edited using the BIOEDIT program (Hall, 1999Go). Multiple alignments were performed using the CLUSTAL_X program (Thompson et al., 1997Go). Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1983Go). The phylogenetic tree was constructed by using a neighbour-joining method (Saitou & Nei, 1987Go) in the program MEGA2 (Kumar et al., 2001Go). Bootstrap analysis with 1000 replicates was used to obtain the confidence level of the branches (Felsenstein, 1985Go).

The nearly complete 16S rRNA gene sequence (1486 bp) of strain BB4T was determined and subjected to comparative analysis. The 16S rRNA gene sequence of strain BB4T showed high similarity (>97·0 %) to that of Dyella japonica XD53T (Xie & Yokota, 2005Go) and the phylogenetic tree (Fig. 1Go) shows that these strains are clustered within the family ‘Xanthomonadaceae’ of the ‘Gammaproteobacteria’. 16S rRNA gene sequence similarity values between strain BB4T and Frateuria aurantia LMG 1558T (Swings et al., 1980Go), Fulvimonas soli LMG 19981T (Mergaert et al., 2002Go) and Rhodanobacter lindaniclasticus RP5557T (Nalin et al., 1999Go) were 97·1, 95·6 and 94·3 %, respectively. Other members of the ‘Gammaproteobacteria’, including members of the genera Xanthomonas, Stenotrophomonas and Xylella, showed less than 90 % sequence similarity.



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  		Fig. 1. Rooted phylogenetic tree based on the 16S rRNA gene sequences of strain BB4T and related bacteria in the ‘Gammaproteobacteria’. This tree was made using the neighbour-joining method (Saitou & Nei, 1987Go) with a two-parameter distance matrix (Kimura, 1983Go) and pairwise deletion. Bootstrap values (expressed as percentages of 1000 replications) greater than 70 % are shown at branch points. Bar, 20 nucleotide substitutions per 1000 nucleotides.

 
The major respiratory lipoquinone of strain BB4T was ubiquinone-8 (Q-8), which was also found in D. japonica. The fatty acid profile and G+C content of the DNA of strain BB4T were similar to those of D. japonica but different from the genera Fulvimonas and Frateuria. Table 2Go details the fatty acid profiles of strain BB4T and its closest neighbours. The major fatty acids of strain BB4T were branched forms, especially large proportions of iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1{omega}9c. The G+C content of the DNA of strain BB4T was 63·8 mol%. These phenotypic and chemotaxonomic results supported the phylogenetic analysis, which suggests that strain BB4T belongs to the genus Dyella. However, DNA–DNA hybridization experiments showed levels of relatedness of 35 and 10 % between strain BB4T and D. japonica XD53T and Frateuria aurantia LMG 1558T, respectively, suggesting that strain BB4T should be classified as representing a novel species (Wayne et al., 1987Go).


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  		Table 2. Cellular fatty acid profiles of strain BB4T and type strains of related species

Taxa: 1, Dyella koreensis BB4T (data from this study); 2, Dyella japonica XD53T (Xie & Yokota, 2005Go); 3, Frateuria aurantia LMG 1558T (this study); 4, Fulvimonas soli LMG 19981T (Mergaert et al., 2002Go). ‘Unknown 11·798’ was an unknown peak that appeared at 11·798 min. Fatty acids that account for <0·5 % of the total are not shown; therefore, percentages do not total 100 %. –, Not detected.

 
Cells of strain BB4T were aerobic, Gram-negative, non-motile, straight rods, 0·2–0·5 µm in diameter and 1·5–2·1 µm in length. Colonies formed on R2A medium (Difco) were yellow, convex, transparent with clear edges. The absorption spectra of pigments from the strain showed a maximum peak at 425 nm. The strain was oxidase-positive and weakly catalase-positive. It did not grow anaerobically but could reduce nitrate to nitrite. Strain BB4T grew at 10–37 °C, with an optimum at 30 °C. The optimum pH for growth was in the range 7·0–8·0. As shown in Table 1Go, BB4T showed positive results for {beta}-glucosidase and {beta}-galactosidase. It showed growth in R2A medium containing 4 % (w/v) NaCl. Details of various differentiating characteristics of strain BB4T and phylogenetically related species are shown in Table 1Go; other characteristics determined are given in the species description.

On the basis of phylogenetic and physiological data, we propose that isolate BB4T should be classified as the type strain of a novel species, Dyella koreensis sp. nov.

Description of Dyella koreensis sp. nov.
Dyella koreensis (ko.re.en'sis. N.L. fem. adj. koreensis pertaining to Korea, the location of the soil sample from which the type strain was isolated).

Gram-negative, aerobic, non-motile, rod-shaped cells. Colonies on R2A agar plates are smooth, circular, transparent and yellow in colour within 5 days at 30 °C. Optimal growth occurs at 30 °C and pH 6·8–7·5. It does not grow anaerobically. Growth occurs with several carbon sources, such as acetate, D-fucose, D-glucose, 3-hydroxybutyrate, malate, maltose, mannose, D-melibiose, N-acetylglucosamine, salicin, D-sucrose and valerate. However, it shows negative results for adipate, L-alanine, L-arabinose, caprate, citrate, glycogen, gluconate, L-histidine, 3-hydroxybenzoate, 4-hydroxybenzoate, itaconate, inositol, lactate, malonate, mannitol, phenylacetate, L-proline, propionate, rhamnose, D-ribose, L-serine, D-sorbitol and suberate. It can hydrolyse aesculin, cellulose, casein and DNA, produces {beta}-galactosidase and {beta}-glucosidase and can reduce nitrate. The G+C content of the genomic DNA is 63·8 mol%. Ubiquinone-8 is the main respiratory lipoquinone. Major fatty acids are iso-C15 : 0, iso-C17 : 0 and iso-C17 : 1{omega}9c when grown on TSA for 48 h.

The type strain is BB4T (=KCTC 12359T=NBRC 100831T).


   ACKNOWLEDGEMENTS
 
This work was supported by the 2004 Agricultural R&D Promotion Center Program, Ministry of Agriculture and Forestry, South Korea, and the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (grant MG05-0101-4-0), South Korea.


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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

Felsenstein, J. (1985). Confidence limit on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]

Gauthier, M. J. & Breittmayer, V. A. (1992). The genera Alteromonas and Marinomonas. In The Prokaryotes, 2nd edn, pp. 3046–3070. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York: Springer.

Hall, T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.

Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge: Cambridge University Press.

Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.

Kouker, G. & Jaeger, K.-E. (1987). Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53, 211–213.[Abstract/Free Full Text]

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Mergaert, J., Cnockaert, M. C. & Swings, J. (2002). Fulvimonas soli gen. nov., sp. nov., a {gamma}-proteobacterium isolated from soil after enrichment on acetylated starch plastic. Int J Syst Evol Microbiol 52, 1285–1289.[Abstract]

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.

Nalin, R., Simonet, P., Vogel, T. M. & Normand, P. (1999). Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium. Int J Syst Bacteriol 49, 19–23.[Abstract/Free Full Text]

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Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: MIDI Inc.

Swings, J., Gillis, M., Kersters, K., De Vos, P., Gosselé, F. & De Ley, J. (1980). Frateuria, a new genus for "Acetobacter aurantius". Int J Syst Bacteriol 30, 547–556.[Abstract/Free Full Text]

Ten, L. N., Im, W.-T., Kim, M.-K., Kang, M.-S. & Lee, S.-T. (2004). Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates. J Microbiol Methods 56, 375–382.[CrossRef][Medline]

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