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Guangdong Institute of Microbiology, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangzhou 510070, China
Correspondence
Guoping Sun
guopingsun{at}163.com
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JBT is EF125186.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Veiga et al., 1983). During the course of collecting environmental micro-organisms from various sources, sediments from along the Qijiang River, Zhongshan City, China, were sampled and used to isolate micro-organisms that decompose the cellulosic compounds of municipal solid waste. A sucrose manufacturing plant is located near the river and the sediment is rich in cellulose. One of the cellulose-decomposing micro-organisms was characterized. The taxonomic position of the isolate was determined. On the basis of morphological, biochemical, chemotaxonomic and molecular taxonomic characteristics, it was concluded that the isolate represents a novel species of the genus Brachybacterium.
The sediment samples were shaken for 2 h on a rotary shaker at 250 r.p.m. to disperse them and then suspensions were serially diluted with 0.85 % (w/v) NaCl solution. Each dilution (0.1 ml) was plated into Congo red agar medium, pH 7.0, modified from Hendrick et al. (1995) containing (g l–1): KH2PO4 (0.5), MgSO4 . 7H2O (0.25), cellulose powder (1.88), Congo red (0.2), gelatin (2.0) and agar (16). After 7 days incubation at 30 °C, isolated colonies showing distinct red circles were selected and purified further on Congo red plates. Pure cultures were stored in 15 % (w/v) glycerol at –20 °C and used for further testing. A number of the isolates had nearly identical results in Biolog GP2 MicroPlate assays after incubation for 1 day; one of these was selected as the type strain.
The reference strains used were: Brachybacterium rhamnosum LMG 19848T, Brachybacterium fresconis LMG 20336T, Brachybacterium nesterenkovii LMG 19549T (=DSM 9573T), Brachybacterium paraconglomeratum LMG 19861T and Brachybacterium sacelli LMG 20345T, all obtained from the Laboratorium voor Microbiologie, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000, Gent, Belgium; and Brachybacterium muris DSM 15460T, which was obtained from the DSMZ, Braunschweig, Germany.
Cellulose-degrading ability was measured by following the loss in dry weight of either rice straw stalk or its powder. Either 2 g rice straw stalk or 2 g powder was mixed into 100 ml zymolytic medium, pH 7.2, containing (g l–1): peptone, 0.5; yeast extract, 0.5; (NH4)2SO4, 2.0; KH2PO4, 4.0; CaCl2 . 2H2O, 0.3; and MgSO4 . 7H2O, 0.3. A 5 % inoculum was added and cultures were incubated at 30 °C with shaking at 150 r.p.m. (three replicates). After 6 days, the residual rice straw stalk or powder was harvested by centrifugation and washed repeatedly. The cellulose content was determined colorimetrically with K2Cr2O7 as described by Halliwell (1974). The cellulose-decomposing ratio (%) was calculated according to the following formula: cellulose decomposed (%)=[(A–B)/A]x100, in which A is the initial weight of straw stalk (or powder)xcellulose content and B is straw stalk (or powder) remainingxcellulose content of residual straw stalk (or powder).
For morphological, physiological and biochemical analysis, strain JBT was grown aerobically on Luria–Bertani (LB) plates (Sambrook et al., 1989) overnight at 30 °C. Microaerophilic conditions were maintained in an oxygen range varying between 0.01 and 0.5 mg l–1 without aeration in flasks at 30 °C for 36 h. Anaerobic growth (80 % N2, 10 % CO2, 10 % H2) on LB plates at 30 °C for 36 h was analysed by using a Bug Box anaerobic workstation (Ruskim). The Hucker method (Murray et al., 1994) was used for Gram staining. Gram staining was observed by normal light microscopy and cell morphology was examined by transmission electron microscopy after negative staining with 4 % phosphotungstic acid, pH 7.0. Oxidase activity was determined by using 1 % (w/v) tetramethyl-p-phenylene-diamine as the substrate. Catalase activity was determined by the detection of bubble formation in 3 % (w/v) hydrogen peroxide solution after incubation in LB medium for 18–24 h (Smibert & Krieg, 1994). Carbon source utilization was tested by using Biolog GP2 MicroPlates. Overnight cultures were used to inoculate the GP2 MicroPlates, according to the manufacturer's instructions, and they were incubated at 30 °C for 24 h; the colour result in each well was recorded by the Biolog MicroStation. Some biochemical tests were performed by using the Microbial Biochemical Identification Tube System (GuangDong Huankai Microbial Sci & Tech). Tolerance to different NaCl concentrations (0–15 %, w/v) and pH values (4–14) was measured spectrophotometrically (Bachman U 640) at OD550 in LB medium after 18–24 h incubation at 30 °C; results were scored as positive if the change in OD550 was greater than 0.300 (Heyrman et al., 2002).
For analysis of cellular fatty acids, strain JBT was incubated for 36 h in LB medium at 30 °C. Sample preparation was performed by the method of Song et al. (2000); extracts and analysis of cellular fatty acid methyl esters were as described by Xu et al. (2005).
The 16S rRNA gene was amplified from genomic DNA of the isolate by using forward primer 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). PCR conditions were as described by Hurek et al. (1997). The purified PCR product was sequenced directly by using the sequencing primers 35f (5'-CTKAAGAGTTTGATCMTGGCTCAGATTGAACG-3'), 342f (5'-CTCCTACGGGAGGCAG-3') and 930f (5'-GGTTAAAACTYAAAKGAATTGACGGGGA-3'). Sequencing was carried out by Shanghai Invitrogen Biotechnology and the nearly full-length 16S rRNA gene sequence was compiled using SEQMAN software (DNASTAR).The determined sequence and some related sequences selected from GenBank with the program BLAST were aligned by using the RDP program (Maidak et al., 1999). Alignment gaps and ambiguous bases were excluded from similarity calculations. Tree topology was inferred by using the neighbour-joining method (Saitou & Nei, 1987) and the phylogenetic tree was visualized and bootstrapped by using the TREECON software package (Van de Peer & De Wachter, 1994). The topology of the phylogenetic tree was evaluated by performing bootstrap analysis with 100 replicates.
Insertion sequence (IS)-PCR fingerprinting patterns were analysed to evaluate genotypic differences between strains JBT, DSM 15460T and DSM 9573T. The primer and reaction mixtures were as described by Peng et al. (2006). PCR began with denaturation at 94 °C for 7 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 68 °C for 1 min and extension at 68 °C for 2 min. The final extension cycle was at 65 °C for 15 min. PCR products were separated in 1.2 % agarose gels in 1x TAE buffer (40 mM Tris/acetate and 1 mM EDTA at pH 8.3).
Genomic DNA from strain JBT and reference strains was isolated and purified as described by Marmur (1961) and DNA base composition was determined spectrophotometrically (De Ley et al., 1970). DNA from Escherichia coli K-12 was used as the standard for estimation of G+C content. DNA–DNA relatedness was determined by using the initial renaturation rate method (De Ley et al., 1970) in 2xSSC as modified by Tan et al. (2001). Hybridization experiments were carried out under optimal and stringent temperatures calculated from the melting temperature (Tm) based on the G+C content of each test strain (three replicates for each test).
Cells of strain JBT were Gram-positive and mainly short rods, 0.48–0.65 µm wide and 0.57–0.92 µm long; any coccoid cells were 1 µm in diameter (Fig. 1). Colonies on LB plates at 30 °C for 24 h were cream-coloured, smooth, glistening and grew well in an anaerobic chamber on LB plates under microaerophilic conditions at 30 °C for 36 h.
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Phenotypic properties of strain JBT were consistent with its classification in the genus Brachybacterium. The major phenotypic properties of the isolate and the type strains of closely related species of the genus Brachybacterium (i.e. those with 16S rRNA gene sequence similarity above 97 %) are listed in Table 1; other characters are given in the species description.
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; Gvozdyak et al., 1992; Takeuchi et al., 1995; Schubert et al., 1996; Heyrman et al., 2002; Buczolits et al., 2003). Additionally, considerable amounts of iso-C16 : 0 (13.13 %), iso-C15 : 0 (9.22 %), C16 : 0 (2.23 %), C20 : 1
7c (2.20 %), iso-C14 : 0 (1.45 %) and C14 : 0 (1.42 %) and minor amounts of an unknown lipid (0.57 %) were also present.
16S rRNA gene sequence analyses clearly demonstrated that strain JBT was affiliated to the genus Brachybacterium, as shown in Fig. 2. Data show that strain JBT formed a close cluster with B. muris (97.9 %), B. nesterenkovii (97.7 %), B. rhamnosum (97.7 %), B. sacelli (97.6 %), B. fresconis (97.5 %) and B. paraconglomeratum (97.1 %). Sequence similarities between strain JBT and other type strains of members of the genus Brachybacterium were in the narrow range 96.1–96.7 %; sequence similarity to another species of the family Dermabacteraceae, Dermabacter hominis DSM 7083T, was 93.6 % (bootstrap value of 100 %). However, the precise position of strain JBT within the genus is not very clear since the branching order among strain JBT, B. muris and B. rhamnosum was not supported by a high bootstrap value (<50 %).
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). The three strains had 2–4 fragments between 250 bp and 2000 bp and the pattern for strain JBT differed distinctly from those of strains DSM 9573T and DSM 15460T. This difference in genomic organization supports classification of strain JBT as a representative of a novel species of the genus Brachybacterium.
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It is necessary to perform DNA–DNA hybridization to judge whether a novel isolate belongs to a species when the isolate and species representatives share more than 97 % 16S rRNA gene sequence similarity (Stackebrandt & Goebel, 1994). Thus, six species (B. muris, B. sacelli, B. nesterenkovii, B. rhamnosum, B. fresconis and B. paraconglomeratum) were selected for DNA–DNA hybridization. Levels of DNA–DNA relatedness at optimal temperatures among these related species were 41–54 % (Table 2). All DNA–DNA relatedness values were below the threshold value that has been suggested as delineating a bacterial species (approx. 70 %) under optimal hybridization conditions (Tm of 25 °C) (Grimont, 1999; Wayne et al., 1987). This confirmed the view that strain JBT represents a novel species of the genus Brachybacterium.
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Cells vary in shape from coccoid forms (irregular) in the stationary phase to short rods in the exponential phase. Cells are non-motile, Gram-positive and do not form endospores. Colonies on LB plates are cream-coloured, smooth, glistening and grow well in an anaerobic chamber on LB plates. Temperature range for good growth is 25–40 °C; no growth occurs at 4 or 45 °C. Grows in 0–10 % (w/v) NaCl. Grows well at pH 5–8 and weakly at pH 9–11; no growth is observed at pH 12–14. Catalase and urease are produced. Oxidase is not produced. Aesculin and gelatin are hydrolysed, but starch is not. Nitrate is reduced to nitrite. Indole is not produced. Positive for arginine dihydrolase and denitrification. Acid is produced from glucose. Utilizes the following carbon sources: 
-cyclodextrin, 
-cyclodextrin, dextrin, glycogen, inulin, amygdalin, L-arabinose, arbutin, D-cellobiose, D-fructose, D-galactose, D-galacturonic acid, gentiobiose, -D-glucose, -D-lactose, lactulose, maltose, maltotriose, D-mannose, D-melezitose, D-melibiose, methyl -D-galactoside, methyl -D-galactoside, methyl -D-glucoside, palatinose, D-psicose, D-raffinose, stachyose, sucrose, D-tagatose, trehalose, turanose, xylitol, D-xylose, acetic acid, L-lactic acid, pyruvic acid methyl ester, pyruvic acid, glycerol, adenosine, inosine and thymidine. Predominant fatty acids are anteiso-C15 : 0 and anteiso-C17 : 0.
The type strain is JBT (=LMG 23926T=CGMCC 1.6508T=DSM 18832T), which was isolated from sediments along the Qijiang River, Zhongshan City, China. The DNA G+C content of strain JBT is 71.2 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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