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Burkholderia symbiotica sp nov isolated from root nodules of Mimosa spp

native to North East Brazil

Article in International Journal of Systematic and Evolutionary Microbiology · November 2011

DOI: 10.1099/ijs.0.037408-0 · Source: PubMed

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International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2272–2278 DOI 10.1099/ijs.0.037408-0

Burkholderia symbiotica sp. nov., isolated from root

nodules of Mimosa spp. native to north-east Brazil
Shih-Yi Sheu,1 Jui-Hsing Chou,2 Cyril Bontemps,3 Geoffrey N. Elliott,4
Eduardo Gross,5 Euan K. James,6 Janet I. Sprent,7 J. Peter W. Young8
and Wen-Ming Chen2
Correspondence 1
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142,
Wen-Ming Chen Hai-Chuan Road Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
p62365@ms28.hinet.net 2
Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine
University, No. 142, Hai-Chuan Road Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
3
Génétique et Microbiologie, UMR UHP-INRA 1128, IFR 110 EFABA, Université de Lorraine,
Faculté des Sciences et Technologies, BP 239, 54506, Vandœuvre-lès-Nancy, France
4
The James Hutton Institute, Craigiebuckler, Aberdeen AB15 8QH, UK
5
Depto. de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, km 16,
Ilhéus 45662-900, Bahia, Brazil
6
The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
7
Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee DD2 5DA, UK
8
Department of Biology, University of York, Heslington, York YO10 5DD, UK

Four strains, designated JPY-345T, JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-
fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera,
that are native to North East Brazil, and their taxonomic positions were investigated by using a
polyphasic approach. All four strains grew at 15–43 6C (optimum 35 6C), at pH 4–7 (optimum
pH 5) and with 0–2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene
sequence analysis, strain JPY-345T showed 97.3 % sequence similarity to the closest related
species Burkholderia soli GP25-8T, 97.3 % sequence similarity to Burkholderia caryophylli
ATCC25418T and 97.1 % sequence similarity to Burkholderia kururiensis KP23T. The
predominant fatty acids of the strains were C18 : 1v7c (36.1 %), C16 : 0 (19.8 %) and summed
feature 3, comprising C16 : 1v7c and/or C16 : 1v6c (11.5 %). The major isoprenoid quinone was
Q-8 and the DNA G+C content of the strains was 64.2–65.7 mol%. The polar lipid profile
consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol
and several uncharacterized aminophospholipids and phospholipids. DNA–DNA hybridizations
between the novel strain and recognized species of the genus Burkholderia yielded relatedness
values of ,51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and
chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus
Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is
JPY-345T (5LMG 26032T 5BCRC 80258T 5KCTC 23309T).

Members of the class Betaproteobacteria, including Cupria-
vidus taiwanensis (syn. Ralstonia taiwanensis), Burkholderia
tuberum, Burkholderia phymatum, Burkholderia mimo-
sarum, Burkholderia nodosa and Burkholderia sabiae have
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequences of strains JPY-345T, JPY-347, JPY-366 and JPY-581 are
HM357233, HM357232, HM357231 and HM357234, respectively.
Three supplementary figures are available with the online version of this
paper.
been isolated from root nodules of legumes and have all
been shown to nodulate their hosts (Chen et al., 2001,
2003, 2005a, b, 2006, 2007, 2008; Barrett & Parker, 2005,
2006; Bontemps et al., 2010; Gyaneshwar et al., 2011; Liu
et al., 2011).
With the exception of B. tuberum, which nodulates the
legume Cyclopia in the subfamily Papilionoideae (Elliott
et al., 2007a), all of the so far described nodulating species
of the genus Burkholderia have been shown to nodulate

2272 037408 G 2012 IUMS Printed in Great Britain


plants belonging to the large genus Mimosa (subfamily
Mimosoideae) (Chen et al., 2005a, b; Elliott et al., 2007b;
dos Reis et al., 2010). Recently, 143 nodule symbionts from
47 native species of Mimosa in Brazil were sampled and
their symbiotic diversity was assessed (Bontemps et al.,
2010). Analysis of 16S rRNA and recA gene sequences
showed that 141 isolates were strains of Burkholderia and
these isolates were grouped in seven species complexes
(SC1–SC7). The isolates in SC1, SC3 and SC7 almost
certainly represent novel species of the genus Burkholderia,
as their 16S rRNA/recA gene sequences are sufficiently dis-
similar to those of all type strains of recognized species in
this genus. In this study, four strains reported to be mem-
bers of SC1 by Bontemps et al. (2010) were selected and
subjected to a polyphasic taxonomical analysis. Three of
the strains, JPY-345T, JPY-347 and JPY-581, were isolated
from root nodules of Mimosa cordistipula, a species that is
endemic to the Chapada Diamantina highland region in
the state of Bahia, and one strain, JPY-366, was isolated
from nodules of Mimosa misera, which is widespread in
Brazil (dos Reis et al., 2010).
All strains were grown on yeast extract–mannitol (YEM)
agar plates (Vincent, 1970) and incubated at 25 uC, unless
otherwise indicated. Subculturing was performed on YEM at
25 uC for 2 days and all cultures were preserved at 280 uC in
YEM broth with 20 % (v/v) glycerol or by lyophiliza-
tion. Burkholderia soli DSM 18235T, Burkholderia caryo-
phylli BCRC 12862T, Burkholderia kururiensis LMG 19447T
and Burkholderia cepacia ATCC 25416T were obtained from
the Deutsche Sammlung von Mikroorganismen und Zellkul-
turen (DSMZ), the Bioresource Collection and Research
Center (BCRC), the Belgian Co-ordinated Collections of
Micro-organisms (BCCM) and American Type Culture
Collection (ATCC), respectively. All type strains were used
as reference strains for phenotypic and genotypic tests.
The bacterial cells were observed by using phase-contrast
microscopy (DM2000; Leica) using cells grown on YEM agar
at 25 uC for 2 days. Cellular motility was tested by using the
hanging drop method. The Gram Stain Set S kit (BD Difco)
and the Ryu non-staining KOH method (Powers, 1995)
were used for testing the Gram reaction. Poly-b-hydro-
xybutyrate granule accumulation was observed by using
light microscopy after staining of the cells with Sudan black.
Colony morphology was observed on YEM agar by using
stereoscopic microscopy (SMZ 800; Nikon).
Liquid cultures of strains JPY-345T, JPY-347 JPY-366 and
JPY-581 were incubated at 25 uC for 2 days with vigorous
agitation. The physiological characterizations were exam-
ined by using 100 ml inocula of the incubated cultures in
6 ml fresh culture. All tests were run in duplicate. The
optimum pH range for growth was examined in nutrient
broth (NB; BD Difco) using appropriate biological buffers;
glycine/HCl, citrate/Na2HPO4, phosphate buffer and gly-
cine/NaOH were used for adjusting the pH to 3–4,
4–6, 6–8 and 9–11 (at intervals of 1 pH unit), respectively.
The pH values were adjusted prior to sterilization and
http://ijs.sgmjournals.org
Burkholderia symbiotica sp. nov.
post-sterilization controls revealed only minor changes in
pH. The NaCl requirement was determined using NB
containing 0, 0.5 and 1.0–10.0 % (w/v) NaCl (at intervals
of 1.0 % NaCl). Growth at 4–50 uC was examined in YEM
broth. Cellular growth was determined by measuring the
turbidity (OD600) of cultures grown at various pH values,
NaCl concentrations and temperatures. Anaerobic growth
was determined after incubating the strains on YEM agar in
the Oxoid AnaeroGen system (Miller et al., 1995). Strains
were examined using a broad range of phenotypic pro-
perties. Activities of catalase, oxidase, DNase, urease and
lipase (corn oil), as well as the hydrolysis of starch, casein
and Tweens 20, 40, 60 and 80 were determined using
standard methods (Smibert & Krieg, 1994). Additional
biochemical tests were performed using API 20 NE and
API ZYM kits (bioMérieux) and carbon source utilization
was evaluated using the GN2 microplate (Biolog) system.
All commercial phenotypic tests were performed according
to the manufacturers’ recommendations.
The sensitivity of strains JPY-345T, JPY-347 JPY-366,
JPY-581 and the reference strains to antibiotics was analysed
by the diffusion method after spreading cell suspensions (0.5
McFarland) on nutrient agar (NA; Difco). The following
antibiotic discs (Oxoid) were used (mg per disc): ampicillin
(10), chloramphenicol (30), gentamicin (10), kanamycin
(30), nalidixic acid (30), novobiocin (30), rifampicin (5),
penicillin G (10), streptomycin (10), sulfamethoxazole
(23.75) plus trimethoprim (1.25), and tetracycline (30).
The effects of the antibiotics on cell growth were assessed
after 2 days of incubation at 28 uC; susceptibility was scored
based on the distance from the edge of the clear zone of
inhibition to the disc.
The 16S rRNA and recA gene sequences of strains JPY-345T,
JPY-347 JPY-366 and JPY-581 were previously reported by
Bontemps et al. (2010). The 16S rRNA gene sequences were
compared against 16S rRNA gene sequences available from
the EzTaxon server (Chun et al., 2007), the Ribosomal
Database Project (Maidak et al., 2001) and the GenBank
database (http://blast.ncbi.nlm.nih.gov/Blast.cgi.). Analysis
of the sequence data was performed by using the software
packages BioEdit (Hall, 1999) and MEGA version 5 (Tamura
et al., 2011), after multiple alignments of the data by
CLUSTAL_X (Thompson et al., 1997). Distances, corrected
according to Kimura’s two-parameter model (Kimura,
1983), were calculated and clustering was performed using
the neighbour-joining method (Saitou & Nei, 1987). The
maximum-likelihood (Felsenstein, 1981) and maximum-
parsimony (Kluge & Farris, 1969) trees were generated by
using the treeing algorithms contained in the PHYLIP software
package (Felsenstein, 1993). In each case, bootstrap values
were calculated based on 1000 replications.
The phylogenetic trees based on 16S rRNA gene sequence
comparisons (Fig. 1 and Fig. S1, available in IJSEM Online)
showed that strains JPY-345T, JPY-347 JPY-366 and JPY-
581 formed an independent phylogenetic line within the
genus Burkholderia in the family Burkholderiaceae of the
2273
S.-Y. Sheu and others

0.01 Burkholderia unamae ATCC BAA-744 T (AY221956)

Burkholderia tropica Ppe8 T (AJ420332)
99 Burkholderia nodosa Br3437 T (AM284971)
95
Burkholderia mimosarum PAS44 T (AY752958)
Burkholderia sacchari CIP 107211 T (AF263278)
Burkholderia tuberum STM678 T (AJ302311)
98 Burkholderia sabiae Br3407 T (AY773186)
84 Burkholderia phymatum STM815 T (AJ302312)
Burkholderia caribensis CCUG 42847 T (Y17009)
Burkholderia kururiensis KP23 T (AB024310)
86 Burkholderia soli GP25-8 T (DQ465451)
74
73 Burkholderia caryophylli ATCC 25418 T (AB021423)
Burkholderia symbiotica JPY-366 (HM357231)
100
100 Burkholderia symbiotica JPY-345 (HM357233)
T

91 Burkholderia symbiotica JPY-347 (HM357232)

Burkholderia symbiotica JPY-581 (HM357234)
Cupriavidus taiwanensis LMG 19424 T (AF300324)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequence comparisons showing the positions of the four
novel strains and related bacteria. Numbers at nodes are bootstrap percentages .70 % (based on 1000 replicates) obtained
using the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles
indicate branches of the tree that were recovered using the maximum-likelihood and maximum-parsimony algorithms. Open
circles indicate corresponding nodes that were only recovered in the tree generated with the maximum-parsimony algorithm.
Cupriavidus taiwanensis LMG 19424T was used as an outgroup. Bar, 0.01 substitutions per nucleotide position.

class Betaproteobacteria. The overall topology of the phylo-
genetic trees obtained with the maximum-likelihood and
maximum-parsimony methods was similar. The 16S rRNA
gene sequences of the four strains showed high levels of
similarity (. 99.7 %) to each other. Sequence similarity
calculations (over 1400 bp) indicated that strain JPY-345T
was closely related to B. soli GP25-8T (97.3 % 16S rRNA
gene sequence similarity), B. caryophylli ATCC 25418T
(97.3 %) and B. kururiensis KP23T (97.1 %). Lower sequence
similarity values (,97 %) were found with the representa-
tive members of other species listed in Fig. 1.
According to pairwise recA gene sequence comparisons,
similarity values between strain JPY-345T and the other
three strains JPY-347, JPY-366 and JPY-581 ranged from
99 to 100 %. The highest sequence similarity value between
the representative strain JPY-345T (91 %) and closely
related species was with B. caryophylli ATCC 25418T. In
addition, the levels of recA gene sequence similarity
between strain JPY-345T and species with validly published
names within the class Betaproteobacteria were ,90.5 %. A
phylogenetic tree was reconstructed as described above
based on recA gene sequences and this also showed that the
four strains (JPY-345T, JPY-347 JPY-366 and JPY-581)
formed a deep monophyletic cluster within the genus
Burkholderia (Fig. S2).
Whole genome DNA–DNA hybridization experiments
were performed with photobiotin-labelled probes as
described by Ezaki et al. (1989). DNA–DNA hybridization
experiments were performed for strains JPY-345T, JPY-347,
JPY-366 and JPY-581, as well as for JPY-345T and the three
closest relatives, B. soli DSM 18235T, B. caryophylli BCRC
12862T and B. kururiensis LMG 19447T. The degree of
DNA–DNA relatedness was calculated from triplicate
experiments. The DNA–DNA relatedness values between
strains JPY-345T, JPY-347, JPY-366 and JPY-581 were
88.5–100 %, indicating that the four strains are members of
the same genomic species (Wayne et al., 1987). In addition,
strain JPY-345T showed 29.8 %, 51.8 % and 36.3 % DNA–
DNA relatedness with B. soli DSM 18235T, B. caryophylli
BCRC 12862T and B. kururiensis LMG 19447T, respectively.
Since the recommended DNA–DNA relatedness threshold
for the delineation of bacterial species is 70 % (Wayne
et al., 1987), these results indicate that strain JPY-345T does
not belong to any known species of the genus Burkholderia.
The fatty acid profiles of strains JPY-345T, JPY-347,
JPY-366, JPY-581, B. soli DSM 18235T, B. caryophylli BCRC
12862T and B. kururiensis LMG 19447T were determined
using cells grown on YEM agar at 25 uC for 3 days. The fatty
acid methyl esters were prepared and separated according
to the instructions of the Microbial Identification System
(MIDI; Sasser, 1990) and identified by using MIDI version
6.0 and the RTSBA6.00 database. The fatty acid profiles of
strains JPY-345T, JPY-347 JPY-366 and JPY-581 were simi-
lar to those of the other species of the genus Burkholderia,
although there were differences in the proportions of some
components (Table 1). The major fatty acids (.10 %) of
strains JPY-345T, JPY-347, JPY-366 and JPY-581 were
C18 : 1v7c (36.1±2.6 %), C16 : 0 (19.8±1.5 %) and summed
feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c; 11.5±
1.3 %). In addition, substantive amounts of C12 : 0 (3.4±
0.6 %), C14 : 0 (1.2±0.2 %), C16 : 0 2-OH (1.4±0.1 %), C16 : 0
3-OH (5.3±0.3 %), C16 : 1 2-OH (2.7±0.1 %), C17 : 0 cyclo
(3.3±0.5 %), C18 : 0 (2.9±0.4 %), C18 : 1 2-OH (2.7±0.3 %),
C19 : 0 cyclo v8c (2.9±0.2 %) and summed feature 2
(comprising iso-C16 : 1 I and/or C14 : 0 3-OH; 5.3±0.4 %)
were detected.

2274 International Journal of Systematic and Evolutionary Microbiology 62

 Burkholderia symbiotica sp. nov.

Table 1. Cellular fatty acid compositions of strains JPY-345T, B. caryophylli BCRC 12862T and B. kururiensis LMG 19447T,
JPY-347, JPY-366 and JPY-581 and related species of genus all of which contained PE, PG, DPG and APL1. However,
Burkholderia APL2 was present in strain JPY-345T but absent in the other
three closest relatives. In addition, APL3 was present in
Strains: 1, JPY-345T, JPY-347, JPY-366 and JPY-581; 2, B. soli DSM
strain JPY-345T and B. caryophylli BCRC 12862T but was
18235T; 3, B. caryophylli BCRC 12862T; 4, B. kururiensis LMG 19447T.
absent in B. soli DSM 18235T and B. kururiensis LMG
All data were obtained from this study. All strains were grown on
19447T. Furthermore, two uncharacterized aminolipids, AL1
YEM agar at 25 uC for 3 days. 2, Not detected. Fatty acids amounting
and AL2, were detected in strains B. soli DSM 18235T and B.
to ,1 % are not shown.
kururiensis LMG 19447T but were not detected in strain JPY-
345T or B. caryophylli BCRC 12862T. These results show that,
Fatty acid 1* 2 3 4
despite belonging to the same genus and having very similar
C12 : 0 3.4±0.6 1.0 2 2 profiles, there are some differences in the polar lipid profiles
C14 : 0 1.2±0.2 1.1 3.9 5.0 amongst these species/strains.
C16 : 0 19.8±1.5 18.1 19.7 16.9
C16 : 0 2-OH 1.4±0.1 2.0 3.3 3.0 Detailed results of the biochemical characterization and
C16 : 0 3-OH 5.3±0.3 5.3 5.5 5.5 antibiotic sensitivity tests are given in the species description
C16 : 1 2-OH 2.7±0.1 4.0 2.4 2.3 and in Table 2. The novel species can be distinguished from
C17 : 0 cyclo 3.3±0.5 5.5 8.3 6.8 representatives of phylogenetically closely related species by
C18 : 0 2.9±0.4 2.8 1.1 1.3 a combination of its phenotypic attributes, in particular,
C18 : 1v7c 36.1±2.6 26.6 24.0 28.6 nitrate reduction, activity of urease and oxidation of various
C18 : 1 2-OH 2.7±0.3 2.9 2.8 1.7 substrates.
C19 : 0 cyclo v8c 2.9±0.2 9.4 13.1 12.1
On the basis of the data obtained from 16S rRNA and recA
Summed feature 2D 5.3±0.4 5.6 5.0 5.6
Summed feature 3D 11.5±1.3 17.2 4.5 7.3
gene sequence comparisons, the novel species occupies a
distinct position within the genus Burkholderia. The results
*Values are mean percentages±SD of total fatty acids of the four
of the phylogenetic analysis are supported by the unique
strains.
combination of chemotaxonomic and biochemical charac-
DSummed features are groups of two or three fatty acids that cannot teristics of the novel strains. It is clear from the phylogenetic
be separated by GLC using the MIDI system. Summed feature 2 and phenotypic data that strains JPY-345T, JPY-347, JPY-
comprises C14 : 0 3-OH and/or iso-C16 : 1 I, summed feature 3 366 and JPY-581 represent a novel species of the genus
comprises C16 : 1v7c and/or C16 : 1v6c. Burkholderia, for which the name Burkholderia symbiotica
sp. nov., is proposed.

Isoprenoid quinones were extracted and purified according
to the method of Collins (1985) and were analysed by
HPLC. Strains JPY-345T, JPY-347, JPY-366 and JPY-581
had Q-8 as their main respiratory quinone. The DNA
G+C contents of strains JPY-345T, JPY-347, JPY-366 and
JPY-581, determined by HPLC according to Mesbah et al.
(1989), were 64.2–65.7 mol%.
Polar lipids were extracted and analysed by two-dimensional
TLC according to Embley & Wait (1994). Molybdophos-
phoric acid was used for detection of all lipids, ninhydrin
reagent was used for detection of lipids containing free
amino groups, zinzadze reagent was used for detection of
phosphorus-containing lipids and a-naphthol reagent was
used for detection of glycolipids. Strains JPY-345T, JPY-347,
JPY-366 and JPY-581 exhibited a complex polar lipid profile
consisting of phosphatidylethanolamine (PE), phosphati-
dylglycerol (PG), diphosphatidylglycerol (DPG) and several
uncharacterized aminophospholipids (APL1–3) and phos-
pholipids (PL1–5) (Fig. S3; data of JPY-347 JPY-366 and
JPY-581 not shown). The polar lipid content of strain JPY-
345T was generally consistent with that of B. cepacia ATCC
25416T, the type species of the genus Burkholderia, although
strain JPY-345T differed in that it contained PL3 and PL4
and lacked PL6. Strain JPY-345T also exhibited a very similar
polar lipid profile to its closest relatives, B. soli DSM 18235T,
Description of Burkholderia symbiotica sp. nov.
Burkholderia symbiotica (sym.bio9ti.ca. N.L. fem. adj. sym-
biotica from Gr. n. sumbios, a companion, partner, living
together, symbiotic).
Cells are Gram-reaction-negative, non-motile, aerobic, non-
spore-forming rods. Positive for poly-b-hydroxybutyrate
accumulation. After 24 h of growth on YEM agar at 25 uC,
the mean cell size is ~0.8–1.061.0–1.5 mm. Colonies on
YEM agar are yellow pigmented, circular, smooth and
convex with entire edges. Colony size is ~1.0–1.3 mm in
diameter on YEM agar after 48 h of incubation at 25 uC.
Growth occurs at 15–43 uC (optimum 35 uC), at pH 4–7
(optimum pH 5) and with 0–2 % (w/v) NaCl (optimum 0 %
NaCl). Catalase- and oxidase-positive. Positive for hydro-
lysis of Tweens 20, 40, 60 and 80; negative for hydrolysis of
DNA, starch, chitin, casein, gelatin, aesculin, alginate and
corn oil. In API 20 NE tests, positive reactions for nitrate
reduction, urease and b-galactosidase activities and assi-
milation of glucose, arabinose, mannose, mannitol, N-
acetylglucosamine, gluconate, malate and citrate; negative
reactions for indole production, glucose fermentation,
arginine dihydrolase activity, aesculin and gelatin hydrolysis
and assimilation of maltose, caprate, adipate and phenyla-
cetate. In the API ZYM kit, positive for alkaline phosphatase,
C4 esterase, C8 esterase lipase, leucine arylamidase, valine

http://ijs.sgmjournals.org 2275
S.-Y. Sheu and others

Table 2. Differential phenotypic characteristics of strains JPY-345T, JPY-347, JPY-366 and JPY-581 and related species of the
genus Burkholderia
Strains: 1, JPY-345T, JPY-347, JPY-366 and JPY-581; 2, B. soli DSM 18235T; 3, B. caryophylli BCRC 12862T; 4, B. kururiensis LMG 19447T. Data for
reference strains were obtained in this study with the exception of sources of isolation and DNA G+C contents, which were taken from Yoo et al.
(2007), Ballard et al. (1970) and Zhang et al. (2000). +, Positive; 2, negative; S, sensitive; R, resistant; V, variable (all strains were sensitive except
strain JPY-366). All strains were Gram-reaction-negative, non-motile, non-spore-forming, rod-shaped and positive for oxidase, catalase and b-
galactosidase activities and assimilation of glucose, mannose, mannitol, N-acetylglucosamine, gluconate and malate. All strains were negative for
arginine dihydrolase activity, hydrolysis of DNA, aesculin, starch, gelatin, casein, alginate, chitin and corn oil, indole production, glucose
fermentation, assimilation of maltose and caprate. All strains were sensitive to chloramphenicol, kanamycin, streptomycin, tetracycline, nalidixic
acid and gentamicin.

Characteristic 1* 2 3 4

Isolation source Nodules Soil Plant Trichloroethylene-polluted water

Nitrate reduction + + + 2
Urease + 2 + +
Assimilation of (API 20 NE):
Arabinose + + + 2
Adipate 2 + 2 2
Citrate + 2 + 2
Phenylacetate 2 + 2 +
Oxidation of (Biolog):
Glycogen + 2 + +
N-Acetyl-D-galactosamine 2 2 2 +
Trehalose 2 + + 2
L-Rhamnose + 2 + +
Acetic acid 2 2 + +
D-Glucuronic acid 2 2 + +
p-Hydroxy phenylacetic acid 2 2 + +
L-Histidine 2 + + +
c-Hydroxybutyric acid 2 + 2 2
DL-a-Glycerol phosphate + 2 + 2
a-D-Glucose-1-phosphate + 2 2 2
Susceptibility to:
Penicillin G R R R S
Ampicillin R R R S
Novobiocin V S R S
Rifampicin V S R R
Sulfamethoxazole plus trimethoprim S S R S
DNA G+C content (mol%) 64.2–65.7 64.9 65.3 64.8

*Data were obtained from the four novel strains.

arylamidase, acid phosphatase and naphthol-AS-BI-phos-
phohydrolase activities; negative for C14 lipase, cystine
arylamidase, trypsin, a-chymotrypsin, a- and b-galactosi-
dase, b-glucuronidase, a- and b-glucosidase, N-acetyl-b-
glucosaminidase, a-mannosidase and a-fucosidase activities.
The following compounds are utilized as sole carbon sources
in the GN2 microplate: dextrin, Tween 40, Tween 80, N-
acetyl-D-glucosamine, L-arabinose, D-arabitol, D-fructose,
L-fucose, D-galactose, a-D-glucose, myo-inositol, lactulose,
D-mannitol, D-mannose, D-psicose, L-rhamnose, D-sorbitol,
pyruvic acid methyl ester, succinic acid mono-methyl-ester,
cis-aconitic acid, citric acid, formic acid, D-galactonic acid
lactone, D-gluconic acid, D-glucosaminic acid, b-hydroxy-
butyric acid, a-ketobutyric acid, DL-lactic acid, quinic acid, D-
saccharic acid, succinic acid, bromosuccinic acid, succinamic
acid, L-alanine, L-alanylglycine, L-asparagine, L-aspartic
acid, L-glutamic acid, L-phenylalanine, L-proline, glycerol,
DL-a-glycerol phosphate, a-D-glucose-1-phosphate and D-
glucose-6-phosphate. Negative for oxidization of a-cyclo-
dextrin, glycogen, N-acetyl-D-galactosamine, cellobiose,
i-erythritol, gentiobiose, a-lactose, maltose, melibiose, b-
methyl-D-glucoside, raffinose, sucrose, turanose, acetic acid,
D-galacturonic acid, D-glucuronic acid, c-hydroxybutyric
acid, p-hydroxyphenylacetic acid, itaconic acid, a-ketoglu-
taric acid, a-ketovaleric acid, sebacic acid, glucuronamide,
glycyl-L-aspartic acid, glycyl-L-glutamic acid, hydroxy-
L-proline, L-leucine, L-ornithine, D-serine, DL-carnitine,
c-aminobutyric acid, inosine, uridine, thymidine, phenyl-
ethylamine, putrescine, 2-aminoethanol and 2,3-butanediol.
Oxidation of the remaining substrates, adonitol, trehalose,

2276 International Journal of Systematic and Evolutionary Microbiology 62


xylitol, a-hydroxybutyric acid, malonic acid, propionic acid,
L-alaninamide, D-alanine, L-histidine, L-pyroglutamic acid,
L-serine, L-threoninea and urocanic acid, was strain-
dependent. Resistant to penicillin G and ampicillin; sensitive
to chloramphenicol, gentamicin, kanamycin, tetracycline,
streptomycin, sulfamethoxazole plus trimethoprim and
nalidixic acid. Sensitive to novobiocin and rifampicin except
strain JPY-366. The major fatty acids are C18 : 1v7c, C16 : 0
and summed feature 3 (comprising C16 : 1v7c and/or
C16 : 1v6c). DNA G+C content is 64.2–65.7 mol%. The
major respiratory quinone is Q-8. The polar lipid profile
consists of a mixture of phosphatidylethanolamine, phos-
phatidylglycerol, diphosphatidylglycerol and several unchar-
acterized aminophospholipids and phospholipids.
The type strain, JPY-345T (5LMG 26032T 5BCRC 80258T
5KCTC 23309T), was isolated from root nodules of Mimosa
cordistipula, a plant species endemic to the Chapada Dia-
mantina highlands in the state of Bahia, Brazil.
Acknowledgements
This work was supported by a grant from the National Science
Council, Taipei, Taiwan, Republic of China (NSC 96-2313-B-022-
001-MY3).
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