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1 Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, 142 Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan
2 EMBRAPA-Agrobiologia, km 47, Seropedica, 23851-970 Rio de Janeiro, Brazil
3 School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
4 Department of Soil Environmental Science, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung, Taiwan
5 Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung, Taiwan
6 Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
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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An extended phylogenetic tree and details of genome sizes, DNA–DNA binding values and fatty acid compositions of the novel strains and related species 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’ include Cupriavidus taiwanensis (Chen et al., 2001
, 2003a, b; Vandamme & Coenye, 2004), which has been isolated from nodules of Mimosa pudica, M. diplotricha and M. pigra (syn. M. pellita) in Taiwan (Chen et al., 2001, 2003a, b, 2005b), from M. pudica in India (Verma et al., 2004) and from M. pudica and M. pigra in Costa Rica (Barrett & Parker, 2006). More recently, however, there has been a greater focus on -rhizobia in the genus Burkholderia, as these are being isolated from Mimosa and related genera with much higher frequency than C. taiwanensis, particularly in South and Central America (Barrett & Parker, 2005, 2006; Chen et al., 2005a), but also from the invasive legume M. pigra in Taiwan (Chen et al., 2005b). However, with the exception of Burkholderia caribensis TJ182 and TJ183 (isolated from M. pudica and M. diplotricha in Taiwan; Chen et al., 2003b; Vandamme et al., 2002), Burkholderia tuberum STM678T (isolated from Aspalathus carnosa in South Africa; Moulin et al., 2001), Burkholderia phymatum STM815T and NGR195A (isolated, respectively, from Machaerium lunatum in French Guiana and Mimosa invisa in New Guinea; Moulin et al., 2001; Vandamme et al., 2002; Elliott et al., 2007) and various strains of Burkholderia mimosarum (isolated from M. pigra in Taiwan and Brazil; Chen et al., 2006), the taxonomic positions of most Burkholderia legume symbionts have not yet been described. The aim of the present study is to clarify the taxonomic positions of three strains isolated from Mimosa nodules in Brazil that have previously been shown by 16S rRNA gene sequence analyses to belong to the genus Burkholderia (Chen et al., 2005a).
Strains Br3470 and Br3461 were isolated from root nodules on Mimosa bimucronata and strain Br3437T was isolated from nodules on Mimosa scabrella (Chen et al., 2005a). Both Mimosa species are woody legumes native to Brazil, and the geographical origins of the strains have been described previously (Chen et al., 2005a). All were grown on yeast extract-mannitol agar plates (Vincent, 1970) and incubated at 28 °C unless indicated otherwise. Burkholderia reference strains have been described previously (Vandamme et al., 2002).
The 16S rRNA gene sequences of strains Br3437T, Br3470 and Br3461 have been reported previously by Chen et al. (2005a) (GenBank accession numbers AY773189, AY773198 and AY533861). However, whereas the sequences for strains Br3470 and Br3461 are >99 % similar, there is a difference of 1.5–2 % between them and that of strain Br3437T. As subsequent analyses revealed all three strains to represent a single species (see below), we repeated the 16S rRNA gene sequence analyses for all three strains. The latter sequences were deposited as GenBank accession numbers AM284971 (Br3437T), AM284972 (Br3470) and AM284970 (Br3461). All repeat analyses revealed virtually identical sequences (data not shown).
A phylogenetic analysis of the 16S rRNA gene sequences showed that strains Br3437T, Br3461 and Br3470 formed a single cluster with 99.7–98.1 % similarity and that they belonged to the genus Burkholderia within the Betaproteobacteria (Fig. 1 and Supplementary Fig. S1 available in IJSEM Online). 16S rRNA gene sequence comparison of strain Br3437T and its closest neighbours, Burkholderia unamae, B. mimosarum, B. silvatlantica, B. sacchari and B. tropica, showed it to have 97.9, 97.1, 96.8, 96.5 and 96.4 % similarity, respectively, to the type strains of these species. The similarity levels of strains Br3461, Br3470 and Br3437T to other Burkholderia species were less than 96.0 %.
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. The DNA base composition was determined as described by Mesbah et al. (1989). 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. The DNA G+C content of strains Br3437T, Br3470 and Br3461 was between 62 and 63 mol% (Supplementary Table S1). The DNA–DNA binding values among strains Br3437T, Br3470 and Br3461 were between 73 and 100 % (Supplementary Table S1), whereas mean binding values of strains Br3437T and Br3461 of 15–54 % were calculated towards their closest phylogenetic neighbours, the type strains of B. mimosarum, B. unamae, B. sacchari and B. tropica (Supplementary Table S1). The finding that these three strains represent a single species was unexpected, given the considerable divergence in 16S rRNA gene sequences. However, the high DNA–DNA binding value was further supported by the high similarity in whole-cell protein content (see below), and a repeat analysis of the sequences indeed confirmed the initial sequences. Although not unique in prokaryotic taxonomy, such a large intraspecies divergence in 16S rRNA gene sequences has, so far, not been documented in the genus Burkholderia.
For PFGE genome organization analysis as described by Chen et al. (2003b), intact genomic DNA in agarose plugs was electrophoresed on a 0.8 % agarose gel in TAE for 41 h with a pulse time of 500 s at 100 V (CHEF-III system; Bio-Rad). Br3437T contained four replicons with a total genome size of 9.0 Mb (Supplementary Table S1 and Fig. 2).
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. Densitometric analysis, normalization and interpolation of the protein profiles and numerical analysis using Pearson's product-moment correlation coefficient were performed using the GelCompar 4.2 software package (Applied Maths). Whole-cell protein extracts were prepared from strains Br3437T, Br3470 and Br3461 and compared with others present in our database. Strains Br3437T, Br3470 and Br3461 formed a single cluster with similarities of >92 %, in comparison with similarities of less than 85 % to other Burkholderia species (Fig. 3).
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). Fatty acid profiles of strains Br3437T, Br3461 and Br3470 were determined and compared with those of other Burkholderia species. Fatty acid profiles of strains Br3437T, Br3461, Br3470 and other reference strains were similar, and were predominated by 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 of 10.928 or 12 : 0 ALDE, or any combination of these fatty acids) and summed feature 3 (comprising 16 : 17c and/or 15 : 0 iso 2-OH). Details of the cellular fatty acid compositions and those of closely related Burkholderia species are shown in Supplementary Table S2. In general, all these organisms had very similar whole-cell fatty acid profiles, which were therefore not useful for species discrimination. For biochemical characterization, the API 20NE and API ZYM microtest systems were used according to the recommendations of the manufacturer (bioMérieux). For carbon substrate assimilation tests, Biolog GN2 microtitre test plates were used.
When using the API 20NE microtest gallery, the following characteristics were present in all strains: nitrate reduction, activity of oxidase, catalase, urease and -galactosidase and assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate, caprate, adipate, citrate, malate and phenylacetate. The following characteristics were uniformly absent: indole production, glucose fermentation, aesculin hydrolysis, gelatin hydrolysis and assimilation of maltose.
When using the API ZYM microtest gallery, activities of alkaline phosphatase, C4 esterase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase were present in all strains. Activities of C14 lipase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase were uniformly absent.
When using the Biolog GN2 microtitre test system, the following substrates were oxidized: glycogen, Tween 40, Tween 80, N-acetyl-D-glucosamine, adonitol, arabinose, arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, -D-glucose, myo-inositol, D-mannitol, D-mannose, D-psicose, L-rhamnose, D-sorbitol, D-trehalose, xylitol, methyl pyruvate, acetic acid, citrate, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, DL-lactate, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, L-leucine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, 
-aminobutyric acid and urocanic acid. None of the strains oxidized -cyclodextrin, dextrin, N-acetyl-D-galactosamine, gentiobiose, -D-lactose, lactulose, maltose, D-melibiose, methyl -D-glucoside, D-raffinose, sucrose, turanose, D-glucuronic acid, -hydroxybutyric acid, itaconic acid, -hydroxybutyric acid, -ketovaleric acid, malonic acid, glucuronamide, L-ornithine, inosine, uridine, thymidine, 2-aminoethanol, 2,3-butanediol, DL--glycerol phosphate or glucose 1-phosphate. Oxidation of the remaining substrates (monomethyl succinate, cis-aconitic acid, -ketobutyric acid, propionic acid, hydroxy-L-proline, D-serine, L-threonine, DL-carnitine, phenylethylamine, putrescine, glycerol and glucose 6-phosphate) was strain dependent.
A comparison of the phenotypic characteristics of the type strain of the novel taxon with those of the type strains of related Burkholderia species is shown in Table 1. Strain Br3437T can be differentiated from B. mimosarum by the activity of -galactosidase and oxidation of adipate, adonitol, caprate, rhamnose, trehalose and xylitol; from B. sacchari by the activity of urease, -galactosidase and oxidation of caprate, rhamnose, sucrose, trehalose and xylitol; and from B. unamae and B. tropica by the activity of urease and oxidation of trehalose and xylitol. Only strains Br3437T, Br3461 and Br3470 and B. mimosarum can produce N2-fixing nodules on Mimosa species (Chen et al., 2005a).
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), whole-cell protein profiles (Fig. 3), DNA–DNA reassociation experiments (Supplementary Table S1), nodulation ability on Mimosa species (Table 1) and biochemical characterization (Table 1). We propose to name this organism Burkholderia nodosa sp. nov. Moreover, isolates Br3437T, Br3461 and Br3470 produced N2-fixing nodules on Mimosa species. These results strongly confirm that these Burkholderia strains can form effective symbioses with legumes of Mimosa species (Chen et al., 2005a, b).
Description of Burkholderia nodosa sp. nov.
Burkholderia nodosa (no.do'sa. L. fem. adj. nodosa knotty or swollen, indicating that the type strain was isolated from root nodules).
Cells are Gram-negative, non-spore-forming rods. After 24 h growth on yeast extract-mannitol agar at 28 °C, the mean cell size is about 0.5–0.8x0.8–2.2 µm. Growth is observed at 28, 30 and 37 °C. Catalase- and oxidase-positive. Assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate, caprate, adipate, citrate, malate and phenylacetate is observed. No indole production, gelatin hydrolysis, aesculin hydrolysis, glucose fermentation or assimilation of maltose is observed. Additional characteristics are listed above. Known strains were isolated from root nodules of Mimosa bimucronata and Mimosa scabrella.
The type strain is strain Br3437T (=BCRC 17575T=LMG 23741T). Phenotypic characteristics of the type strain are the same as described for the species. Its DNA G+C content is 62.8 mol% and the genome size is approximately 9.0 Mb. Strains Br3461 (=R-22632) and Br3470 (=R-25486) are also assigned to this species.
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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.
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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50 % are given. Bar, 1 % sequence dissimilarity. The sequence of Burkholderia cepacia ATCC 25416T was used as an outgroup. A tree including a wider selection of reference sequences is available as Supplementary Fig. S1 in IJSEM Online.
