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1 College of Life Sciences, Capital Normal University, Beijing 100037, People's Republic of China
2 Beijing Key Laboratory for Resources Environment and Geography Information System, College of Resources Environment and Tourism, Capital Normal University, Beijing 100037, People's Republic of China
3 Agricultural Culture Collection Center of China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100080, People's Republic of China
4 College of Life Sciences, Hebei University, Baoding 071002, People's Republic of China
Correspondence
Feiyun Zhang
feiyun39{at}126.com
Huili Gong
gonghl{at}263.net
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These authors contributed equally to this work. 
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, and the genus currently comprises three species: Dyadobacter fermentans, isolated from surface-sterilized Zea mays stems (Chelius & Triplett, 2000), Dyadobacter crusticola, from biological soil crusts (Reddy & Garcia-Pichel, 2005), and Dyadobacter hamtensis, from the ‘snout’ of the Hamta glacier (Chaturvedi et al., 2005). Recently, using culture-dependent methods, we examined rhizosphere-associated bacterial communities of the perennial grass Poa pratensis L. irrigated with reclaimed water, a practice which has been in place for more than 4 years. A total of 138 isolates were obtained from Luria–Bertani (LB) medium seeded with a rhizospheric soil sample. Most isolates belonged to the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes and Actinobacteria by 16S rRNA gene sequencing (X. Guo and Z. Dong, unpublished results). In the present study, one of the isolates, designated A54T, was assigned to the genus Dyadobacter on the basis of phenotypic, chemotaxonomic and phylogenetic analyses. Strain A54T and D. fermentans ATCC 700827T, as a reference strain, were cultivated on LB medium or R2A medium at 28 °C and maintained in R2A medium at 4 °C or as a glycerol suspension (20 %, v/v) at –20 °C.
R2A was used for determination of growth of strain A54T at various temperatures, at different pH values and in the presence of various concentrations of NaCl. Phenotypic characteristics such as colony morphology, cell morphology, various enzyme activities (Hugh & Leifson, 1953), growth in various media such as YM (Beringer, 1974), R2A and peptone water (Chelius & Triplett, 2000) and sensitivity to antibiotics at 28 °C were ascertained by using standard methods (Lanyi, 1987; Smibert & Krieg, 1994). API 50CH (bioMérieux) was employed to test carbon assimilation according to the manufacturer's instructions. The catalase test was done with cells scraped from R2A and treated with 3 % (w/v) hydrogen peroxide. The presence of flexirubin-like pigments was tested by measuring the absorbance spectrum of absolute ethanol and alkaline-ethanol extracts of lysed cells (Weeks, 1981). Fatty acid analysis was performed using standard methods and compared to the database of fatty acids in the MIDI Sherlock Microbial Identification System (Microbial ID). Genomic DNA was extracted and purified from cells using the procedure of Marmur & Doty (1962). DNA base compositions were determined using the thermal melting protocol (De Ley, 1970) with Escherichia coli K-12 as a reference strain. Levels of DNA relatedness were estimated by measurement of the initial reassociation rate (De Ley et al., 1970).
The 16S rRNA gene was amplified by PCR using universal primers 27f and 1492r (Lane, 1991). The amplified 16S rRNA gene fragment was purified from agarose gels by using a Tiangen kit and cloned into the pGEM-T Easy vector (Promega) according to the technical manual. Sequence data were obtained by single-pass double-stranded analysis using primers T7 and SP6, which flank the cloning region in the pGEM-T Easy vector. The sequence of A54T was aligned with those of closely related species of Dyadobacter and members of other related genera using CLUSTAL X version 1.8 (Thompson et al., 1997) and phylogenetic affiliations were inferred using TREECON (version 1.3b) and the PHYLIP package. The Kimura two-parameter method was used for distance calculations (Kimura, 1980) and the resultant tree topologies were evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings.
Morphological, physiological, biochemical and chemotaxonomic characteristics of strain A54T are given in the species description below and in Table 1. Strain A54T is Gram-negative, aerobic, oxidase- and catalase-positive and ferments glucose but not sucrose and cells are non-motile, rod-shaped in both exponential and stationary phases and appear in pairs. The DNA G+C content of strain A54T was 49.2 mol%, which is in the range for known Dyadobacter species (Table 1).
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) (Chelius & Triplett, 2000; Chaturvedi et al., 2005; Reddy & Garcia-Pichel, 2005). The addition of alkali (20 % KOH) changed the colour of the pigment to orange and broadened the peak, thus confirming that it is a flexirubin-type pigment (Weeks, 1981). Strain A54T contained the following major fatty acids: iso-C15 : 0 (19.22 %), C16 : 1
7c (17.46 %), iso-C15 : 0 2-OH (23.38 %), C16 : 15c (10.26 %), C16 : 0 3-OH (3.09 %) and iso-C17 : 0 3-OH (12.41 %). In D. fermentans NS114T, these fatty acids were also present; however, the level of iso-C17 : 0 3-OH was lower, while C16 : 15c and C16 : 17c were more abundant [although for D. fermentans NS114T, Chelius & Triplett (2000) reported C16 : 17c as summed feature 3, which may also contain iso-C15 : 0 2-OH] (Table 2).
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) based on the 16S rRNA gene sequence. It forms a robust clade with the type strains of species of the genus Dyadobacter. The nearly complete 16S rRNA gene sequence of strain A54T (1411 bp) showed 97.7, 94.4 and 94.7 % similarity, respectively, to those of D. fermentans NS114T, D. hamtensis HHS 11T and D. crusticola DSM 16708T. The results of DNA–DNA hybridization did not show significant relatedness (<25 % hybridization) between strain A54T and D. fermentans ATCC 700827T, indicating that strain A54T is different from D. fermentans ATCC 700827T at the species level when the recommendation of a threshold value of 70 % DNA–DNA relatedness for definition of bacterial species is considered (Wayne et al., 1987).
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Description of Dyadobacter beijingensis sp. nov.
Dyadobacter beijingensis (bei.jing.en'sis. N.L. masc. adj. beijingensis pertaining to Beijing, the geographical origin of the type strain).
Colonies on R2A agar are yellow, mucoid, flaky, convex and smooth. Cells are aerobic, non-motile, Gram-negative rods. Growth occurs at 4–35 °C (optimum 28 °C) but not at 37 °C, at pH 6–8 (optimum pH 7) and in the presence of up to 1.5 % NaCl. Growth occurs in peptone water, on LB medium and R2A medium. Positive for catalase and oxidase and negative for urease, gelatinase, hydrolysis of casein, cellulose, starch and gelatin, H2S production, the methyl red, indole and Voges–Proskauer tests and reduction of nitrate to nitrite. Acid is produced from sucrose, D-glucose and D-fructose, but not from D-arabinose, D-galactose or D-rhamnose. Utilizes erythritol, L-arabinose, D- and L-xylose, D-adonitol, methyl 
-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, dulcitol, inositol, D-mannitol, methyl 
-D-mannopyranoside, methyl -D-glucopyranoside, arbutin, aesculin, citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, gentiobiose and D-fucose as sole carbon sources. Does not utilize glycerol, D-arabinose, D-mannitol, D-ribose, lactic acid, myo-inositol, sodium acetate, tartrate, L-rhamnose, L-sorbose, D-sorbitol, N-acetylglucosamine, amygdalin, xylitol, D-turanose, D-lyxose, L-fucose, D-arabitol, L-arabitol, glycogen, D-tagatose, starch, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate or methanol as sole carbon sources. Cells are sensitive to (µg per disc) acetylspiramycin (100), amoxicillin (30), ampicillin (25), carbenicillin (100), cephalothin (30), chloramphenicol (25), erythromycin (10), gentamicin (10), kanamycin (50), lincomycin (20), norfloxacin (10), penicillin (10), roxithromycin (30), spectinomycin (10) and streptomycin (10), but resistant to levofloxacin (100), rifampicin (25), tetracycline (10) and vancomycin (30). The pigment present is of the flexirubin type, with absorption maxima at 428, 452 and 478 nm. The G+C content of the DNA is 49.2 mol%. The major cellular fatty acids are 15 : 0 iso, 16 : 17c, 15 : 0 iso 2-OH, 16 : 15c, 16 : 0 3-OH and 17 : 0 iso 3-OH.
The type strain, A54T (=CGMCC 1.6375T=JCM 14200T), was isolated from the rhizosphere of turf grasses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chaturvedi, P., Reddy, G. S. N. & Shivaji, S. (2005). Dyadobacter hamtensis sp. nov., from Hamta glacier, located in the Himalayas, India. Int J Syst Evol Microbiol 55, 2113–2117.
Chelius, M. K. & Triplett, W. E. (2000). Dyadobacter fermentans gen. nov., sp. nov., a novel Gram-negative bacterium isolated from surface-sterilized Zea mays stems. Int J Syst Evol Microbiol 50, 751–758.[Abstract]
De Ley, J. (1970). Reexamination of the association between melting point, buoyant density, and chemical base composition of deoxyribonucleic acid. J Bacteriol 101, 738–754.
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Weeks, O. B. (1981). Preliminary studies of the pigments of Flavobacterium breve NCTC 11099 and Flavobacterium odoratum NCTC 11036. In The Flavobacterium–Cytophaga Group, pp. 108–114. Edited by H. Reichenbach & O. B. Weeks. Weinheim: Gesellschaft für Biotechnologische Forschung.
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