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1 Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Road, Nan-Tzu, Kaohsiung City 811, Taiwan
2 School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
3 Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
4 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
5 Tropical Soil Biology and Fertility Institute of Centro Internacional de Agricultura Tropical (TSBF-CIAT), A.A. 6713, Cali, Colombia
6 EMBRAPA-Agrobiologia, km 47, Seropedica, 23851-970 Rio de Janeiro, Brazil
7 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
Correspondence
Wen-Ming Chen
p62365{at}ms28.hinet.net
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Tables showing the DNA–DNA binding values, G+C contents and fatty acid compositions of strain PAS44T and related taxa, together with an extended neighbour-joining phylogenetic tree, are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-proteobacteria’ or ‘-rhizobia’) (Moulin et al., 2001
). These include Cupriavidus taiwanensis (Chen et al., 2001, 2003a, b; Vandamme & Coenye, 2004) and various Burkholderia strains (Moulin et al., 2001; Vandamme et al., 2002), two of which were classified as the novel species Burkholderia tuberum and Burkholderia phymatum (Vandamme et al., 2002). Until recently, the best evidence for nodulation of legumes by -rhizobia had come from work with strains of C. taiwanensis. This species has been isolated from nodules of Mimosa pudica, Mimosa diplotricha and Mimosa pigra (synonym Mimosa pellita) in Taiwan (Chen et al., 2001, 2003a, 2005b) and from M. pudica in northern and southern India (Verma et al., 2004), and the type strain, LMG 19424T, has been shown to nodulate M. pudica and M. diplotricha effectively (Chen et al., 2003b). More recently, there has been a greater focus on -rhizobia in the genus Burkholderia, as these are being isolated from Mimosa and related species with much greater frequency than is C. taiwanensis, particularly in South America and Central America (Barrett & Parker, 2005, 2006; Chen et al., 2005a), but also in Taiwan from the invasive legume M. pigra (Chen et al., 2005b). However, with the exception of Burkholderia caribensis TJ182, B. phymatum STM815T and B. tuberum STM678T (Vandamme et al., 2002), the taxonomic positions of Burkholderia legume symbionts have not yet been described. The aim of the present study was to clarify the taxonomic affiliation of a group of strains – isolated from Mimosa nodules in Taiwan and South America – that have previously been shown via 16S rRNA gene sequence analyses to be very closely related (Chen et al., 2005a, b).
The strains used in these studies are listed in Table 1. All 14 strains were isolated from root nodules of M. pigra, except for Br3454, which was isolated from Mimosa scabrella (de Faria et al., 1988). Details of the geographical origins of the strains have been described previously (Chen et al., 2005a, b). All were grown on yeast extract-mannitol agar plates (Vincent, 1970) and incubated at 28 °C unless otherwise indicated. The Burkholderia reference strains have been described previously (Vandamme et al., 2002).
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, b) (see Fig. 1). These sequences were compared with published 16S rRNA gene sequences of other Burkholderia species, as described previously (Chen et al., 2001, 2005a). The phylogenetic analysis showed that these nine strains form a single cluster (99.5–100.0 % 16S rRNA gene sequence similarity) and belong to the genus Burkholderia within the Betaproteobacteria. Comparison of the 16S rRNA gene sequences of the nine strains with those of their closest neighbours, Burkholderia sacchari, Burkholderia unamae and Burkholderia tropica, revealed 97.1–97.9, 96.5–97.5 and 96.0–96.5 % similarity, respectively (Fig. 1). Sequence similarities with respect to other Burkholderia species were below 96 %.
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. For determination of the DNA G+C contents, DNA was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixture obtained was separated by HPLC using a Waters Symmetry Shield C8 column at 37 °C. The solvent was 0.02 M NH4H2PO4 (pH 4.0) with 1.5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference. DNA–DNA hybridizations were performed with photobiotin-labelled probes as described by Ezaki et al. (1989). The hybridization temperature was 50 °C and the reaction was carried out in 30 % formamide. Each value obtained was the mean of two hybridization experiments. The DNA G+C contents of the strains examined were in the range 63.8–64.8 mol%; the DNA–DNA binding values among these nine strains varied between 79 and 100 % (see Supplementary Table S1 in IJSEM Online). The mean binding values with respect to the closest phylogenetic neighbours, B. unamae, B. tropica and B. sacchari, were found to be 32, 46 and 53 %, respectively (Fig. 1
), and values of 56 % or less were calculated in relation to the type strains of other Burkholderia species (Supplementary Table S1).
Differentiation of the novel strains from their closest phylogenetic neighbours was examined by using several approaches. For the analysis of protein electrophoretic patterns, strains were grown on nutrient agar (CM3; Oxoid) supplemented with 0.04 % (w/v) KH2PO4 and 0.24 % (w/v) Na2HPO4.12H2O (pH 6.8) and incubated for 48 h at 28 °C. The preparation of whole-cell proteins and the performance of SDS-PAGE were carried out as described by Pot et al. (1994). Densitometric analysis, normalization and interpolation of the protein profiles and numerical analysis using the Pearson product-moment correlation coefficient were performed using the GelCompar 4.2 software package (Applied Maths). Whole-cell protein extracts from the Mimosa isolates were prepared and compared with others present in our database. All of the novel strains formed a single cluster with similarities of more than 72 %; this compares with similarities of less than 68 % with other Burkholderia species (Fig. 2). For fatty acid methyl ester analyses, 10 µl cell culture was harvested after incubation at 28 °C for 24 h. Fatty acid methyl esters were then prepared, separated and identified using the Microbial Identification System (Microbial ID) as described previously (Vandamme et al., 2002). The fatty acid profiles of strains PAS44T, PTK1 and MAP3-5 were determined and then compared with those of other Burkholderia species (see Supplementary Table S2 available in IJSEM Online). The fatty acid profiles of these three Mimosa strains and other reference strains were similar and showed a predominance of the following fatty acids: 16 : 0, 18 : 1
7c, summed feature 2 (comprising 14 : 0 3-OH, 16 : 1 iso I, an unidentified fatty acid with an equivalent chain-length value of 10.928 or 12 : 0 ALDE, or any combination of these fatty acids) and summed feature 3 (comprising 16 : 17c or 15 iso 2-OH or both). The fatty acid profile of strain PAS44T consisted of the following: 14 : 0 (3.6±0.1 %), 16 : 0 (19.1±0.4 %), 16 : 0 2-OH (1.6±0.1 %), 16 : 0 3-OH (4.2±0.1 %), 16 : 1 2-OH (0.9±0.1 %), 17 : 0 cyclo (3.3±0.3 %), 18 : 17c (45.6±1.0 %), 18 : 1 2-OH (1.0±0.1 %), 19 : 0 cyclo 8c (1.8±0.2 %) and summed features 2 (5.2±0.1 %) and 3 (12.6±0.6 %). Finally, amplified rDNA restriction analysis of all 14 Mimosa strains has been described previously by Chen et al. (2005b). The Burkholderia strains isolated from Taiwan contained two amplified rDNA restriction analysis types and formed a single cluster together with the Venezuelan strains (MAP3-1, MAP3-2, MAP3-3, MAP3-4, MAP3-5) and the Brazilian strains (Br3454 and Br3467) with at least 91 % banding similarity. This compared with a figure of less than 85 % for banding similarity with other nodulating Burkholderia strains (Chen et al., 2005b).
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-galactosidase activity and the assimilation of maltose, caprate, adipate and citrate. The following characteristics were strain-dependent: nitrate reduction, urease activity and the assimilation of mannose, phenylacetate and gluconate.
When the API ZYM microtest gallery was used, the following characteristics were present in all strains: alkaline phosphatase, C8 lipase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities. The following characteristics were uniformly absent: C14 lipase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities. C4 esterase activity was strain-dependent.
When the Biolog GN2 microtitre test system was used, the following substrates were oxidized: dextrin, glycogen, Tweens 40 and 80, N-acetyl-D-glucosamine, arabinose, arabitol, i-erythritol, D-fructose, L-fucose, D-galactose, -D-glucose, myo-inositol, D-mannitol, D-mannose, D-sorbitol, methyl pyruvate, monomethyl succinate, acetic acid, cis-aconitic acid, citrate, formic acid, D-galacturonic acid, D-glucosaminic acid, - and -hydroxybutyric acids, p-hydroxyphenylacetic acid, -ketoglutaric acid, DL-lactate, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, D- and L-alanine, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, L-threonine and 
-aminobutyric acid. None of the strains oxidized -cyclodextrin, N-acetyl-D-galactosamine, adonitol, cellobiose, gentiobiose, -D-lactose, maltose, D-melibiose, methyl -D-glucoside, D-raffinose, L-rhamnose, sucrose, D-trehalose, turanose, xylitol, D-glucuronic acid, -hydroxybutyric acid, itaconic acid, -ketovaleric acid, glucuronamide, inosine, uridine, thymidine, 2,3-butanediol, DL--glycerol phosphate, glucose 1-phosphate or glucose 6-phosphate. Oxidation of the following substrates was strain-dependent: lactulose, D-psicose, D-galactonic acid lactone, D-gluconic acid, -ketobutyric acid, malonic acid, succinamic acid, alaninamide, L-alanyl glycine, L-asparagine, glycyl L-aspartic acid, glycyl L-glutamic acid, L-ornithine, D-serine, DL-carnitine, urocanic acid, phenylethylamine, putrescine, 2-aminoethanol and glycerol.
A comparison of the biochemical characteristics of strain PAS44T with those of the type strains of 20 closely related Burkholderia species is shown in Table 2. Strain PAS44T can be differentiated from B. sacchari by the oxidation of adonitol, raffinose and sucrose, and from B. unamae and B. tropica by the oxidation of adonitol, arabitol, cellobiose, rhamnose and trehalose. Strain PAS44T is the only strain within the Burkholderia cluster tested in this work that is negative for the oxidation of adonitol, rhamnose, sucrose and trehalose.
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) and amplified rDNA restriction analysis profiles (Chen et al., 2005b), the results of DNA–DNA reassociation experiments (Supplementary Table S1) and the biochemical characterization (Table 2). Therefore the 14 novel strains can be classified as representing a novel species, for which we propose the name Burkholderia mimosarum sp. nov., with strain PAS44T as the type strain. Isolates PAS44T, PTK1, PTU38, PTU17, PTU10, PTK31, MAP3-5 and Br3454 effectively nodulated Mimosa species, and the presence of nif and nod genes in the genomes of strains PAS44T, MAP3-5 and Br3454 has been demonstrated (Chen et al., 2005a, b). Moreover, green fluorescent protein-expressing transconjugant derivatives of PAS44T and MAP3-5 produced N2-fixing nodules on their original host, M. pigra (Chen et al., 2005a, b). These results strongly confirm that these Burkholderia strains can form effective symbioses with legumes of Mimosa species.
Description of Burkholderia mimosarum sp. nov.
Burkholderia mimosarum [mi.mo.sa'rum. N.L. gen. pl. n. mimosarum of mimosas (of Mimosa spp.), from which all the strains, including the type strain, were isolated].
Cells are Gram-negative, non-spore-forming and rod-shaped. After 24 h growth on yeast extract-mannitol agar at 28 °C, the mean cell size is about 0.5–0.7 µm (width) x 0.8–2.0 µm (length). Growth is observed at 28, 30 and 37 °C. Catalase- and oxidase-positive. Assimilates glucose, arabinose, mannitol, N-acetylglucosamine and malate. Indole is not produced, gelatin and aesculin are not hydrolysed and glucose is not fermented. Does not assimilate maltose, caprate, adipate or citrate. Additional characteristics are listed above. The DNA G+C content is about 63.8–64.8 mol%. Strains have been isolated from root nodules of Mimosa pigra and Mimosa scabrella.
The type strain, PAS44T (=LMG 23256T=BCRC 17516T), was isolated from M. pigra nodules at Anso in south-east Taiwan. The phenotypic characteristics of the type strain are the same as those described for the species. Its DNA G+C content is 64.8 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Barrett, C. F. & Parker, M. A. (2006). Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica. Appl Environ Microbiol 72, 1198–1206.
Brämer, C. O., Vandamme, P., da Silva, L. F., Gomez, J. G. C. & Steinbüchel, A. (2001). Burkholderia sacchari sp. nov., a polyhydroxyalkanoate-accumulating bacterium isolated from soil of a sugar-cane plantation in Brazil. Int J Syst Evol Microbiol 51, 1709–1713.[Abstract]
Caballero-Mellado, J., Martinez-Aguilar, L., Paredes-Valdez, G. & Estrada-de los Santos, P. (2004). Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species. Int J Syst Evol Microbiol 54, 1165–1172.
Chen, W. M., Laevens, S., Lee, T. M., Coenye, T., de Vos, P., Mergeay, M. & Vandamme, P. (2001). Ralstonia taiwanensis sp. nov., isolated from root nodules of Mimosa species and sputum of a cystic fibrosis patient. Int J Syst Evol Microbiol 51, 1729–1735.[Abstract]
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