Burkholderia nodosa sp. nov., isolated from root
nodules of the woody Brazilian legumes Mimosa
bimucronata and Mimosa scabrella
Wen-Ming Chen,1Sergio M. de Faria,2Euan K. James,3Geoffrey N. Elliott,3
Kuan-Yin Lin,1Jui-Hsing Chou,4Shih-Yi Sheu,5M. Cnockaert,6
Janet I. Sprent3and Peter Vandamme6
1Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine
University, 142 Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan
2EMBRAPA-Agrobiologia, km 47, Seropedica, 23851-970 Rio de Janeiro, Brazil
3School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
4Department of Soil Environmental Science, College of Agriculture and Natural Resources,
National Chung Hsing University, Taichung, Taiwan
5Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung,
6Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent,
Three strains, Br3437T, Br3461 and Br3470, were isolated from nitrogen-fixing nodules on the
roots of Mimosa scabrella (Br3437T) and Mimosa bimucronata (Br3461, Br3470), both of which
are woody legumes native to Brazil. On the basis of 16S rRNA gene sequence similarities, all the
strains were shown previously to belong to the genus Burkholderia. A polyphasic approach,
including DNA–DNA hybridizations, PFGE of whole-genome DNA profiles, whole-cell protein
analyses, fatty acid methyl ester analysis and extensive biochemical characterization, was used
to clarify the taxonomic position of these strains further; the strains are here classified within a novel
species, for which the name Burkholderia nodosa sp. nov. is proposed. The type strain, Br3437T
(=LMG 23741T=BCRC 17575T), was isolated from nodules of M. scabrella.
Recently, a considerable body of evidence has accumulated
to show that legumes, particularly those in the genus
Mimosa in the Mimosoideae, are not nodulated exclusively
by members of the Rhizobiaceae in the Alphaproteobacteria,
but may also be nodulated by members of the Betaproteo-
bacteria. These so-called ‘legume-nodulating b-proteobac-
teria’ or ‘b-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 b-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 STM815Tand 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
The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene
sequences of strains Br3437T, Br3470 and Br3461 are respectively
AY533861 and AM284970 (two determinations for each strain).
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
64873 G 2007 IUMSPrinted in Great Britain1055
International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1055–1059
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 Br3437Twas isolated
from nodules on Mimosa scabrella (Chenetal.,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 28uC unless indicated otherwise. Burkholderia reference
strains have been described previously (Vandamme et al.,
The 16S rRNA gene sequences of strains Br3437T, Br3470
and Br3461 have been reported previously by Chen et al.
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 Betaproteo-
bacteria (Fig. 1 and Supplementary Fig. S1 available in
IJSEM Online). 16S rRNA gene sequence comparison of
strain Br3437Tand 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%.
DNA samples were prepared from strains Br3437T, Br3470
and Br3461 as described by Pitcher et al. (1989). 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 50uC 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
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
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). Br3437Tcontained four replicons with a total genome
size of 9.0 Mb (Supplementary Table S1 and Fig. 2).
Differentiation of the proposed novel taxon from its closest
approaches. For the analysis of protein electrophoretic
patterns, strains were grown on nutrient agar (Oxoid CM3)
supplemented with 0.04% (w/v) KH2PO4and 0.24% (w/v)
Na2HPO4.12H2O (pH 6.8) and incubated for 48 h at 28uC.
Preparation of whole-cell proteins and SDS-PAGE were
performed as described by Pot et al. (1994). 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,
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).
Fig. 1. Phylogenetic tree of strains Br3437T, Br3470 and
Br3461 (Burkholderia nodosa sp. nov.) and related Burkhold-
eria type strains based on 16S rRNA gene sequence compari-
sons. Distanceswere calculated
neighbour-joining method was performed by using the software
package BioEdit. Numbers at nodes are percentage bootstrap
values based on 1000 resampled datasets; only values ¢50%
are given. Bar, 1% sequence dissimilarity. The sequence of
Burkholderia cepacia ATCC 25416Twas used as an outgroup.
A tree including a wider selection of reference sequences is
available as Supplementary Fig. S1 in IJSEM Online.
andclustering with the
1056 International Journal of Systematic and Evolutionary Microbiology 57
W.-M. Chen and others
For fatty acid methylester analysis, cells were harvested after
an incubation period of 48 h at 28uC; fatty acid methyl
Microbial Identification System (Microbial ID) as described
previously (Vandamme et al., 2002). 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, 18:1v7c, 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
16:1v7c 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 pro-
files, which were therefore not useful for species discrimina-
For biochemical characterization, the API 20NE and API
ZYM microtest systems were used according to the
recommendations of the manufacturer (bioMe ´rieux). 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 b-galactosidase
and assimilation of glucose, arabinose, mannose, mannitol,
N-acetylglucosamine, gluconate, caprate, adipate, citrate,
uniformly absent: indole production, glucose fermentation,
aesculin hydrolysis, gelatin hydrolysis and assimilation of
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 aryl-
amidase, cystine arylamidase, trypsin, a-chymotrypsin,
a-galactosidase, b-galactosidase, b-glucuronidase, a-gluco-
sidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-man-
nosidase and a-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, a-D-glucose, myo-inositol, D-mannitol, D-man-
xylitol, methyl pyruvate, acetic acid, citrate, formic acid,
Fig. 2. PFGE of undigested whole-genome DNA profiles.
Lanes: 1 and 5, B. mimosarum PAS44T; 2, B. tropica LMG
22274T; 3, strain Br3437T; 4, B. phymatum STM815T; 6, B.
unamae LMG 22722T; 7, B. sacchari LMG 19450T. Molecular
markers were Saccharomyces cerevisiae Marker (Bio-Rad)
(lane 8) and B. phymatum STM815T(3.5, 2.8, 2.1 and 0.5 Mb;
Chen et al., 2003b) (lane 4).
LMG 23256TB. mimosarum
Fig. 3. Dendrogram based on numerical analysis of the whole-cell protein profiles of Mimosa isolates and type strains of
closely related Burkholderia species.
Burkholderia nodosa sp. nov.
D-galactonic acid lactone, D-galacturonic acid, D-gluconic
acid, D-glucosaminic acid, b-hydroxybutyric acid, p-hydro-
xyphenylacetic acid, a-ketoglutaric acid, DL-lactate, quinic
acid, D-saccharic acid, sebacic acid, succinic acid, bromo-
succinic 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
L-pyroglutamic acid, L-serine, c-aminobutyric acid and
urocanic acid. None of the strains oxidized a-cyclodextrin,
D-melibiose, methyl b-D-glucoside,
D-raffinose, sucrose, turanose, D-glucuronic acid, c-hydro-
xybutyric acid, itaconic acid, a-hydroxybutyric acid, a-
ketovaleric acid, malonic acid, glucuronamide, L-ornithine,
inosine, uridine, thymidine, 2-aminoethanol, 2,3-butane-
diol, DL-a-glycerol phosphate or glucose 1-phosphate.
Oxidation of the remaining substrates (monomethyl
succinate, cis-aconitic acid, a-ketobutyric acid, propionic
acid, hydroxy-L-proline, D-serine, L-threonine, DL-carni-
tine, 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
Br3437Tcan be differentiated from B. mimosarum by
the activity of b-galactosidase and oxidation of adipate,
adonitol, caprate, rhamnose, trehalose and xylitol; from
B. sacchari by the activity of urease, b-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 canproduce
N2-fixing nodules on Mimosa species (Chen et al., 2005a).
In conclusion, the present study demonstrated that three
isolates from root nodules of M. bimucronata and M.
scabrella from Brazil represent a single species that is readily
distinguished from its nearest phylogenetic neighbours by
whole-genome PFGE patterns (Fig. 2), whole-cell protein
profiles (Fig. 3), DNA–DNA reassociation experiments
(Supplementary Table S1), nodulation ability on Mimosa
species(Table 1)andbiochemicalcharacterization(Table 1).
We propose to name this organism Burkholderia nodosa sp.
nov. Moreover, isolates Br3437T, Br3461 and Br3470 pro-
duced 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.do9sa. L. fem. adj. nodosa knotty or
swollen, indicating that the type strain was isolated from
Cells are Gram-negative, non-spore-forming rods. After 24 h
growth on yeast extract-mannitol agar at 28uC, the mean cell
size is about 0.5–0.860.8–2.2 mm. Growth is observed at 28,
30 and 37uC. Catalase- and oxidase-positive. Assimilation of
gluconate, caprate, adipate, citrate, malate and phenylacetate
hydrolysis, glucose fermentation or assimilation of maltose is
observed. Additional characteristics are listed above. Known
strains were isolated from root nodules of Mimosa bimucro-
nata 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.
W.-M.C. was supported by grants from the National Science Council,
Taipei, Taiwan, Republic of China (NSC 95-2320-B-022-001-MY2 and
95-2313-B-022-001), and E.K.J., G.N.E. and J.I.S. were supported by
the Natural Environment Research Council (NERC), UK.
Barrett, C. F. & Parker, M. A. (2005). Prevalence of Burkholderia sp.
nodule symbionts on four mimosoid legumes from Barro Colorado
Island, Panama. Syst Appl Microbiol 28, 57–65.
Table 1. Comparison of phenotypic characters of strain
and the type strains of related Burkholderia
Strains: 1, strain Br3437T; 2, B. sacchari LMG 19450T; 3, B. tropica
LMG 22274T; 4, B. unamae LMG 22722T; 5, B. mimosarum
PAS44T. Data for reference strains were obtained in this study
with the exception of the G+C contents, which were taken from
Reis et al. (2004) and Chen et al. (2006).
G+C content (mol%)
64.863.7 63.5 63.5
1058 International Journal of Systematic and Evolutionary Microbiology 57
W.-M. Chen and others
Barrett, C. F. & Parker, M. A. (2006). Coexistence of Burkholderia, Download full-text
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.
Chen, W. M., James, E. K., Prescott, A. R., Kierans, M. & Sprent, J. I.
(2003a). Nodulation of Mimosa spp. by the b-proteobacterium
Ralstonia taiwanensis. Mol Plant Microbe Interact 16, 1051–1061.
Chen, W. M., Moulin, L., Bontemps, C., Vandamme, P., Be ´na, G. &
Boivin-Masson, C. (2003b). Legume symbiotic nitrogen fixation by
b-Proteobacteria is widespread in nature. J Bacteriol 185, 7266–7272.
Chen, W. M., de Faria, S. M., Straliotto, R., Pitard, R. M., Simoes-
Araujo, J. L., Chou, J. H., Chou, Y. J., Barrios, E., Prescott, A. R. &
other authors (2005a). Proof that Burkholderia strains form effective
symbioses with legumes: a study of novel Mimosa-nodulating strains
from South America. Appl Environ Microbiol 71, 7461–7471.
Chen, W. M., James, E. K., Chou, J. H., Sheu, S. Y., Yang, S. Z. &
Sprent, J. I. (2005b). b-Rhizobia from Mimosa pigra, a newly
discovered invasive plant in Taiwan. New Phytol 168, 661–675.
Chen, W. M., James, E. K., Coenye, T., Chou, J. H., Barrios, E.,
de Faria, S. M., Elliott, G. N., Sheu, S. Y., Sprent, J. I. & Vandamme,
P. (2006). Burkholderia mimosarum sp. nov., isolated from root
nodules of Mimosa spp. from Taiwan and South America. Int J Syst
Evol Microbiol 56, 1847–1851.
Elliott, G. N., Chen, W.-M., Chou, J.-H., Wang, H.-C., Sheu, S.-Y., Perin,
L., Reis, V. M., Moulin, L., Simon, M. F. & other authors (2007).
Burkholderia phymatum is a highly effective nitrogen-fixing symbiont
of Mimosa spp. and fixes nitrogen ex planta. New Phytol 173, 168–180.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric DNA-
DNA 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.
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.
Moulin, L., Munive, A., Dreyfus, B. & Boivin-Masson, C. (2001).
Nodulation of legumes by members of the b-subclass of proteo-
bacteria. Nature 411, 948–950.
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid
extraction of bacterial genomic DNA with guanidium thiocyanate.
Lett Appl Microbiol 8, 109–114.
Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electro-
phoretic whole-organism protein fingerprints. In Modern Microbial
Methods (Chemical Methods Prokaryotic Systematics Series), pp. 493–
521. Edited by M. Goodfellow & A. G. O’Donnell. Chichester: Wiley.
Reis, V. M., Estrada-de los Santos, P., Tenorio-Salgado, S., Vogel,
J., Stoffels, M., Guyon, S., Mavingui, P., Baldani, V. L., Schmid, M. &
other authors (2004). Burkholderia tropica sp. nov., a novel nitrogen-
fixing, plant-associated bacterium. Int J Syst Evol Microbiol 54,
Vandamme, P. & Coenye, T. (2004). Taxonomy of the genus
Cupriavidus: a tale of lost and found. Int J Syst Evol Microbiol 54,
Vandamme, P., Goris, J., Chen, W. M., de Vos, P. & Willems, A.
(2002). Burkholderia tuberum sp. nov. and Burkholderia phymatum
sp. nov. nodulate the roots of tropical legumes. Syst Appl Microbiol
Verma, S. C., Chowdhury, S. P. & Tripathi, A. K. (2004). Phylogeny
based on 16S rRNA gene and nifH sequences of Ralstonia taiwanensis
strains isolated from nitrogen-fixing nodules of Mimosa pudica, in
India. Can J Microbiol 50, 313–322.
Vincent, J. M. (1970). A Manual for the Practical Study of the Root-
Nodule Bacteria. Oxford: Blackwell Scientific.
Burkholderia nodosa sp. nov.