Download Biosynthesis of branched-chain amino acids is

Microbiology (2012), 158, 1758–1766
DOI 10.1099/mic.0.058370-0
Biosynthesis of branched-chain amino acids is
essential for effective symbioses between
betarhizobia and Mimosa pudica
Wen-Ming Chen,1 Jurgen Prell,2 Euan K. James,3 Der-Shyan Sheu4
and Shih-Yi Sheu4
Correspondence
Shih-Yi Sheu
[email protected]
1
Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine
University, No. 142, Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
2
Soil Ecology, Department of Botany, RWTH Aachen, 52056 Aachen, Germany
3
The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
4
Department of Marine Biotechnology, National Kaohsiung Marine University, No. 142,
Hai-Chuan Rd, Nan-Tzu, Kaohsiung City 811, Taiwan, ROC
Received 6 February 2012
Revised 4 April 2012
Accepted 25 April 2012
Burkholderia phymatum STM815 and Cupriavidus taiwanensis LMG19424 are
betaproteobacterial strains that can effectively nodulate several species of the large legume genus
Mimosa. A Tn5 mutant, derived from B. phymatum STM815 (KM60), and another derived from
C. taiwanensis LMG19424 (KM184-55) induced Fix” nodules on Mimosa pudica. The Tn5interrupted genes of the mutants showed strong homologies to ilvE, which encodes a branchedchain amino acid aminotransferase, and leuC, which encodes the large subunit of isopropylmalate
isomerase. Both enzymes are known to be involved in the biosynthetic pathways for branchedchain amino acids (BCAAs) (leucine, valine and isoleucine). The B. phymatum ilvE mutant, KM60,
was not auxotrophic for BCAAs and could grow well on minimal medium with pyruvate as a
carbon source and ammonia as a nitrogen source. However, it grew less efficiently than the wildtype (WT) strain when ammonia was substituted with valine or isoleucine as a nitrogen source.
The BCAA aminotransferase activity of KM60 was significantly reduced relative to the WT strain,
especially with isoleucine and valine as amino group donors. The C. taiwanensis leuC mutant,
KM184-55, could not grow on a minimal medium with pyruvate as a carbon source and ammonia
as a nitrogen source, but its growth was restored when leucine was added to the medium. The
isopropylmalate isomerase activity of KM184-55 was completely lost compared with the WT
strain. Both mutants recovered their respective enzyme activities after complementation with the
WT ilvE or leuC genes and were subsequently able to grow as well as their parental strains on
minimal medium. They were also able to form nitrogen-fixing nodules on M. pudica. We conclude
that the biosynthesis of BCAAs is essential for the free-living growth of betarhizobia, as well as for
their ability to form effective symbioses with their host plant.
INTRODUCTION
Nitrogen fixation by the legume–rhizobium symbiosis
produces a significant amount of the available nitrogen
in the biosphere, making it agronomically and ecologically
important. Although it was generally accepted that legumes
were nodulated exclusively by relatives of Rhizobium in the
class Alphaproteobacteria, the so-called ‘alpharhizobia’,
over the last 10 years there have been an increasing number
of reports of legumes being nodulated by members of the
Betaproteobacteria (the so-called ‘betarhizobia’) (Gyaneshwar
Abbreviations: BCAA, branched-chain amino acid; IPM, isopropylmalate;
WT, wild-type.
1758
et al., 2011). Cupriavidus taiwanensis LMG19424 and
Burkholderia phymatum STM815 were among the first identified nodulating betaproteobacterial strains (Gyaneshwar
et al., 2011) and both are highly effective nitrogen-fixing
symbionts of Mimosa species (Chen et al., 2003, 2005; Elliott
et al., 2007; dos Reis et al., 2010).
Nodulation is a multistep process (van Rhijn &
Vanderleyden, 1995; Chen et al., 2003; Gibson et al.,
2008; Oldroyd & Downie, 2008), in which rhizobia (alpha
or beta) in the rhizosphere infect legume roots via root
hairs and are ultimately released into cells of the developing
nodule primordium via endocytosis. Inside a nodule cell,
the bacteria are enclosed in symbiosomes, in which they
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BCAA metabolic mutants of betarhizobia
differentiate into bacteroids (Chen et al., 2003). The C4dicarboxylate intermediates of the tricarboxylic acid cycle,
succinate, fumarate and malate, have generally been
accepted as the principal carbon and energy sources
supplied by the plant to bacteroids (Stowers, 1985). It is
within the symbiosomes that the bacteria fix nitrogen in
the form of ammonia, which is then released to the host
plant (Brewin, 1991). In addition, it has been proposed
that nitrogen fixation requires the cycling of amino acids
between the bacteroid and plant compartments (Lodwig
et al., 2003; Prell & Poole, 2006). However, more recently,
Prell et al. (2009) have emphasized that plants control
branched-chain amino acid (BCAA) supply to bacteroids,
regulating their development and persistence, at least in
the symbioses of Rhizobium leguminosarum with Pisum
sativum and Phaseolus vulgaris (Prell et al., 2010).
Several mutants of members of the genus Rhizobium (and
related alpharhizobia genera) that are affected in their
biosynthesis of isoleucine, valine and leucine have repeatedly been demonstrated to be defective in either nodulating
their host plants or fixing nitrogen when nodules are
formed (Aguilar & Grasso, 1991; de las Nieves Peltzer et al.,
2008; Ferraioli et al., 2002; Hassani et al., 2002; Kerppola &
Kahn, 1988; López et al., 2001; Pobigaylo et al., 2008;
Sanjuán-Pinilla et al., 2002; Truchet et al., 1980). This is
often generally the case for auxotrophic mutants, because
the compounds that cannot be synthesized by the rhizobium
might also not be provided by the plant in the rhizosphere or
during the nodulation process. The symbiotic defect can
often be corrected by adding the missing compound to the
plant nutrient medium. However, in betarhizobia the link
between BCAA biosynthesis and the nodulation process has
not yet been demonstrated.
The pathways for biosynthesis of BCAAs, outlined in Fig. 1,
are based primarily on the KEGG pathway analysis for B.
phymatum and C. taiwanensis. The metabolic pathway for
the synthesis of valine and isoleucine consists of four successive steps, which are catalysed by acetolactate synthase
(EC 2.2.1.6, ilvHI gene product), ketol-acid reductoisomerase (EC 1.1.1.86, ilvC gene product), dihydroxy acid
dehydratase (EC 4.2.1.9, ilvD gene product) and BCAA
aminotransferase (EC 2.6.1.42, ilvE gene product). Leucine
is synthesized by the isopropylmalate (IPM) pathway.
Three enzymes convert a-ketoisovalerate, an intermediate
in valine biosynthesis, to a-ketoisocaproate, the immediate
precursor of leucine. The first enzyme in the pathway is aIPM synthase (EC 2.3.3.13), which is encoded by leuA. The
next enzymes unique to leucine synthesis are IPM
isomerase (EC 4.2.1.33), encoded by leuC (large subunit)
and leuD (small subunit), and b-IPM dehydrogenase (EC
1.1.1.85), encoded by leuB. For the formation of leucine,
BCAA aminotransferase reversibly transfers one a-amino
group from glutamate to a-ketoisocaproate, finally forming
leucine.
In this study, we present the successful use of transposon
mutagenesis in betarhizobia and show that both KM60, a
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Pyruvate
ilvHI
ilvC
ilvD
ilvC
ilvD
ilvE
Ile
–Ketobutyrate
Pyruvate
ilvHI
Pyruvate
Acetyl-CoA
ilvE
Val
leuA
leuC/leuD leuB ilvE
Leu
Fig. 1. Predicted BCAA biosynthesis in B. phymatum and C.
taiwanensis. Structural gene designations and the encoded
enzymes are: ilvHI, acetolactate synthase; ilvC, ketol-acid
reductoisomerase; ilvD, dihydroxy acid dehydratase; ilvE, BCAA
aminotransferase; leuA, a-isopropylmalate synthase; leuC, isopropylmalate isomerase large subunit; leuD, isopropylmalate
isomerase small subunit; leuB, b-isopropylmalate dehydrogenase.
Ile, isoleucine; Leu, leucine; Val, valine.
B. phymatum mutant with reduced BCAA aminotransferase activity, and KM184-55, a C. taiwanensis mutant
lacking IPM isomerase activity, are deficient in nitrogen
fixation. Our data imply that the biosynthesis of BCAAs is
important for either nodule invasion, bacteroid formation
or bacteroid maturation, and mutations affecting BCAA
biosynthesis lead to non-nitrogen-fixing nodules. We
provide genetic evidence to demonstrate that BCAA
aminotransferase activity and IPM isomerase activity are
essential for the formation and function of effective
symbioses by B. phymatum and C. taiwanensis with their
host plant, M. pudica.
METHODS
Bacterial strains, plasmids and media. Bacterial strains and
plasmids used are listed in Table 1. B. phymatum strains STM815
(wild-type, WT) and KM60, and C. taiwanensis strains LMG19424
(WT) and KM184-55 were grown in yeast extract mannitol (YEM)
medium (Vincent, 1970) at 28 uC and Escherichia coli strains were
grown in Luria–Bertani medium at 37 uC. Antibiotics were used for
these bacterial strains at the following final concentrations: 34 mg
chloramphenicol ml21, 12.5 mg tetracycline ml21, 50 mg kanamycin
ml21 and 50 mg ampicillin ml21.
Tn5 mutagenesis. Introduction of a Tn5 transposon into B.
phymatum STM815 and C. taiwanensis LMG19424 was achieved by
triparental mating (Figurski & Helinski, 1979; Matthysse et al., 1996).
Equal amounts of the donor E. coli S17-1 l pir with pUTmini-Tn5gfp
(Matthysse et al., 1996), the helper strain E. coli HB101 with pRK2013
(Boyer & Roulland-Dussoix, 1969) and the recipient (either B.
phymatum STM815 or C. taiwanensis LMG19424) were mixed and
spotted onto a nitrocellulose filter. After incubation on YEM plates,
chloramphenicol- and tetracycline-resistant colonies were selected.
Plant tests. M. pudica cultivation and nodulation tests were carried
out using the tube methods of Gibson (1963) and Somasegaran &
Hoben (1994). After germination, the seedlings were inoculated with
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W.-M. Chen and others
Table 1. Bacterial strains and plasmids used in this study
Abbreviations for antibiotics are as follows: Cm, chloramphenicol; Tc, tetracycline; Km, kanamycin; Ap, ampicillin.
Strain or plasmid
B. phymatum
STM815
KM60
KM60/ilvE
C. taiwanensis
LMG19424
KM184-55
KM184-55/leuC
E. coli
S17-1 l pir
HB101
DH5a
Plasmids
gfp
pRK2013
pBluescript II SK+
pBBR1MCS-2
pBBR/ilvE
pBBR/leuC
Characteristics
Wild-type, Cmr, Nod+ on M. pudica
STM815 : : Tn5, Tcr
KM60 containing pBBR/ilvE, Tcr Kmr
Elliott et al. (2007); Vandamme et al. (2002)
This study
This study
Wild-type, Cmr, Nod+ on M. pudica
LMG19424 : : Tn5, Tcr
KM184-55 containing pBBR/leuC, Tcr Kmr
Chen et al. (2001)
This study
This study
Containing pUTmini-Tn5gfp
Containing pRK2013
Transformation strain
de Lorenzo et al. (1990)
Boyer & Roulland-Dussoix (1969)
Gibco-BRL
Tn5-based delivery plasmid with gfp gene, Apr Tcr
Helper plasmid used in triparental conjugation, Kmr
Cloning and sequencing vector, Apr
Broad-host-range cloning vector, Kmr
pBBR1MCS-2 containing the ilvE ORF
pBBR1MCS-2 containing the leuC ORF
Matthysse et al. (1996)
Figurski & Helinski (1979)
Stratagene
Kovach et al. (1995)
This study
This study
approximately 105 cells of B. phymatum STM815, C. taiwanensis
LMG19424 or the Tn5-induced mutants. Roots were checked for the
presence or absence of nodules for up to 56 days post-inoculation.
Nitrogen-fixation assays (acetylene reduction assays) were carried out
on plants 28 days post-inoculation according to the method of James
& Crawford (1998). Histology of any nodules formed was studied
using the methods described by Chen et al. (2003). To test the effect
of the addition of amino acids on the nodulation ability of Tn5induced mutants, the appropriate amino acids (0.2, 0.5 and 1 mM)
were added to the plant nutrient medium.
Identification and cloning of the Tn5-interrupted genes. Total
DNA from mutant strains was prepared and digested with EcoRI.
Southern blotting was carried out by standard methodology
(Sambrook & Fritsch, 1989) to confirm that the mutant strains were
carrying a single copy of Tn5. The genomic DNA was prepared and
digested with ApaI and it was ligated to pBluescript II SK+. After
transformation of E. coli DH5a, tetracycline-resistant colonies were
selected. The Tn5-containing plasmids were purified and then
subjected to restriction enzyme analysis and DNA sequencing.
Growth studies. Washed bacterial suspensions (100 ml containing
26109 cells) were used to start 10 ml minimal medium cultures
containing 0.05 % K2HPO4.3H2O, 0.08 % MgSO4.7H2O, 0.02 %
NaCl, 0.025 % CaCl2, 0.1 % (NH4)2SO4 and 0.01 % FeCl3.6H2O
(pH 7.0) supplemented with pyruvate at 0.2 % (w/v). The cultures
were incubated at 28 uC for 24 or 60 h. The OD600 values were
recorded at intervals and represented as the mean of four independent
experiments. To test the ability of mutant strains to grow on various
nitrogen sources, growth studies were performed in a minimal
medium containing isoleucine, valine or leucine at 0.02 % (w/v) as
substitutes for (NH4)2SO4.
Enzyme assay. The BCAA aminotransferase or the IPM isomerase
activity in cells was measured. Cells were grown to exponential phase
1760
Source or reference
(26109 cells ml21) and then harvested by centrifugation, after which
they were resuspended in 2 ml lysis buffer (25 mM Tris/HCl, pH 7.5;
1 mM EDTA). Cells were lysed by sonication and the lysate was cleared
by centrifugation (30 000 g, 4 uC, 10 min). The BCAA aminotransferase activity was assayed by an aminotransferase reaction as described by
Thage et al. (2004). Reaction mixtures with a final volume of 250 ml
contained 56 mmol potassium phosphate buffer l21 (pH 7.4),
0.05 mmol pyridoxal-59-phosphate l21, 6 mmol a-ketoglutaric acid
l21, 1 mmol leucine, isoleucine or valine l21 and 50 ml (8.2 mg protein
ml21) of the bacterial lysate. The enzymic reaction was performed for
15 min at 37 uC. The reaction was stopped by heating at 80 uC for
15 min. The amount of glutamic acid formed during the reaction was
measured using a colorimetric L-glutamic acid kit from Roche (RBiopharm; formerly Boehringer Mannheim) according to the manufacturer’s instructions. The glutamic acid content was measured after
30 min at room temperature using a spectrophotometer.
IPM isomerase was assayed with a-IPM as the substrate and by
following the appearance of the intermediate dimethyl citraconate at
235 nm using a spectrophotometer. The procedure of Gross et al.
(1963) was modified such that the reaction mixture contained 5 mmol
neutralized a-IPM, 20 mmol potassium phosphate (pH 7.0) and 50 ml
cell-free extract in a total volume of 1 ml. The enzymic reaction was
performed for 5 min at room temperature.
Gene complementation. The ilvE gene was amplified by PCR using
VentR exonuclease proofreading DNA polymerase (New England
Biolabs) from B. phymatum STM815, using these oligonucleotide
primers: 59-ACGTGGGCCCTCGCCGCATGGCGCGCC-39 (ilvEApa, forward primer, ApaI site in bold) and 59-CATGTCTAGATCAGATCTTCGTCAGCCAG-39 (ilvE-Xba, reverse primer, XbaI site
in bold). After restriction enzyme digestion, the ilvE gene was cloned
into pBBR1MCS-2 (Kovach et al., 1995). The resulting plasmid
pBBR/ilvE was used to transform B. phymatum KM60 according to
the method described by Vincze & Bowra (2006). The plasmid pBBR/
leuC was constructed following the same procedure but using the
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Microbiology 158
BCAA metabolic mutants of betarhizobia
Fig. 2. Nodulation test on the roots of M. pudica with the parental and Tn5-induced mutant strains of B. phymatum and C.
taiwanensis 28 days post-inoculation. (a, c) Fix+ nodules induced by B. phymatum STM815 and C. taiwanensis LMG19424,
respectively. (b, d) Fix” nodules induced by the mutant strains KM60 and KM184-55, respectively. Bars, 5 mm.
gene amplified from C. taiwanensis LMG19424 with the primers 59ACGTGGGCCCTTGAAATAACGCCCGCTC-39 (leuC-Apa, forward
primer, ApaI site in bold) and 59-CATGTCTAGATCAGCCCAGCTTGCGGA-39 (leuC-Xba, reverse primer, XbaI site in bold).
RESULTS
Tn5-induced mutants form Fix” root nodules on
M. pudica
Two WT strains, B. phymatum STM815 and C. taiwanensis
LMG19424, together with about 6000 presumptive Tn5
insertion mutants were screened for their symbiotic
phenotypes by inoculation onto M. pudica. The parental
strains STM815 and LMG19424 could induce Fix+ nodules
on the roots of M. pudica 28 days post-inoculation (Fig. 2a,
c). Twenty-eight and 56 days post-inoculation, two of the
Tn5-induced mutants, designated KM60 and KM184-55,
derived from B. phymatum STM815 and C. taiwanensis
LMG19424, respectively, had only produced white nodules
(Fig. 2b, d). The mean dry weights of M. pudica inoculated
with strain KM60 or KM184-55 were significantly lower
than those of the plants inoculated with their parental
strains (data not shown). When comparing the roots of M.
pudica inoculated with strain KM60 or KM184-55 and
those inoculated with their parental strains, it could be seen
that both mutant strains induced fewer nodules than their
parental strains and neither showed any nitrogenase
(acetylene reduction) activity (Table 2). Mature nodules
from M. pudica inoculated with the parental strains were as
described previously (Chen et al., 2003; Elliott et al., 2007)
i.e. they were typically indeterminate, with a distinct meristem, invasion zone and nitrogen fixation zone, and the
infected cells were packed with nitrogen-fixing bacteroids.
However, in contrast with those induced by the parental
strains, nodules induced by the two mutants were devoid of
invasion and nitrogen-fixation zones and they contained
no bacteroids (data not shown).
Table 2. Symbiotic features of Tn5-induced mutants and of the WT
Values are means±SD for three trials. Results are numbers of nodules per plant 28 days after inoculation of
three plants. Activity is detected as reduction of acetylene to ethylene and expressed as nmol C2H4 h21 per plant.
Strain
Genotype
No. of nodules
Nitrogenase activity
STM815
KM60
KM60/ilvE
LMG19424
KM184-55
KM184-55/leuC
Uninoculated control
Wild-type
ilvE : : Tn5
pBBR/ilvE
Wild-type
leuC : : Tn5
pBBR/leuC
56±11
29±3
55±15
46±7
23±6
56±12
0
571±27.4
0
582±23.5
72±5.4
0
70±7.2
0
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W.-M. Chen and others
(a)
Tn5 insertion
Bphy_
0458
Bphy_0457
ilvE; Bphy_0456
Bphy_0454
1 kb
Tn5 insertion
(b)
RALTA_A2118
RALTA_
A2119
leuC1; RALTA_A2120
RALTA_A2121
1 kb
Fig. 3. Genetic map of the mutated regions in B. phymatum (a) and C. taiwanensis (b). (a) Interruption of the gene encoding
BCAA aminotransferase (ilvE gene; Bphy_0456). The ORFs located upstream of the ilvE gene are those encoding the BCAA
transporter (Bphy_0458) and the BCAA efflux pump (Bphy_0457). The ORF located downstream of the ilvE gene encodes a
lipopolysaccharide heptosyltransferase II (Bphy_0454). (b) Interruption of the gene encoding the IPM dehydratase large subunit
(leuC1 gene; RALTA_A2120). The ORF located upstream of the leuC1 gene encodes a high-affinity BCAA transport protein
(RALTA_A2121). The ORFs located downstream of the leuC1 gene are those encoding the IPM dehydratase small subunit
(RALTA_A2119) and b-IPM dehydrogenase (RALTA_A2118). Arrows indicate the direction of transcription.
Identification of the Tn5-interrupted genes
Cloning and sequencing revealed that mutant KM60 carried
a single Tn5 insertion in a gene identified as Bphy_0456 (B.
phymatum STM815 chromosome 1, CP001043) (Fig. 3a)
and encoding a BCAA aminotransferase (92–95 % amino
acid identity to Burkholderia spp., 77–78 % identity to
Cupriavidus/Ralstonia spp., 45 % identity to E. coli and 44 %
identity to Mesorhizobium loti). This putative ilvE gene is
924 bp long and encodes a protein of 307 amino acids.
Mutant KM60 contained a transposon insertion at base 308
of the 924 bp ilvE gene. The two ORFs located upstream of
the putative ilvE gene were Bphy_0458 and Bphy_0457 and
they encode the BCAA transporter and BCAA efflux pump,
respectively, whereas the ORF located downstream was
Bphy_0454 which encodes the lipopolysaccharide heptosyltransferase II. In addition, in B. phymatum STM815 only
one ilvE gene was identified and no paralogue could be
found.
In mutant KM184-55, the sequence flanking Tn5 identified
the disrupted gene as RALTA_A2120 (C. taiwanensis
LMG19424 chromosome 1, NC_010528) (Fig. 3b), which
encodes a putative IPM isomerase large subunit (90–96 %
amino acid identity to Cupriavidus/Ralstonia spp. and 83–
84 % identity to Burkholderia spp.). This putative leuC1
1762
gene is 1410 bp and encodes a protein of 469 amino acids.
Mutant KM184-55 contained a transposon insertion at
base 1219 of the 1410 bp leuC1 gene. The ORF located
upstream of the putative leuC1 gene was RALTA_A2121
and it encodes a high-affinity BCAA transport protein, whereas the two ORFs located downstream were
RALTA_A2119 and RALTA_A2118 which encode the IPM
dehydratase small subunit and b-IPM dehydrogenase,
respectively. However, there are two paralogues in C.
taiwanensis LMG19424, one strong paralogue (70 % amino
acid identity) named leuC2 (RALTA_A0672, chromosome
1, NC_010528) and another paralogue (30 % identity)
named leuC3 (RALTA_B0053, chromosome 2, NC_010530).
Tn5-induced mutants are defective in the
metabolism of BCAAs
Both BCAA aminotransferase and the IPM isomerase large
subunit are involved in the synthetic pathway of BCAAs
(Fig. 1). The former catalyses the reversible transamination
of a-amino groups and is essential for anabolism and
catabolism of the three amino acids, leucine, isoleucine and
valine. IPM isomerase catalyses the conversion of a-IPM to
b-IPM, the second step in leucine biosynthesis. In order to
elucidate their metabolic ability, the mutants KM60 and
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BCAA metabolic mutants of betarhizobia
Growth (OD600)
2.0
mutant. Both mutants KM60 and KM184-55 were defective
in their metabolism of BCAAs.
1.5
KM60 has reduced BCAA aminotransferase
activity and KM184-55 lost IPM isomerase activity
1.0
0.5
0
Ammonia
Ile
Val
Leu
Fig. 4. Growth (maximum OD600 values within 24 h) of B.
phymatum STM815 (WT, grey bars) and KM60 (ilvE : : Tn5, white
bars) with minimal medium supplemented with pyruvate and either
ammonia or various BCAAs. Error bars show mean±SD. Ile,
isoleucine; Leu, leucine; Val, valine.
KM184-55 and their parental strains STM815 and
LMG19424 were cultured on minimal medium containing
pyruvate and ammonia as carbon and nitrogen sources,
respectively. Surprisingly, this experiment showed that
KM60 was not an auxotroph for BCAAs, as it could grow
as well as the parental strain, STM815 (Fig. 4). In contrast,
KM184-55 is a true auxotroph, as it could not grow at all
under these conditions (Fig. 5).
When isoleucine, valine or leucine were substituted as
nitrogen sources, KM60 grew less well than the parental
strain, STM815, in the presence of isoleucine and valine,
but grew as well as STM815 in the presence of leucine (Fig.
4). Furthermore, the growth of KM184-55 could be
restored when using leucine, but not isoleucine or valine,
as a nitrogen source (Fig. 5). These results indicated that
KM60 was deficient in its utilization of isoleucine and
valine, and that KM184-55 was a leucine-auxotrophic
Growth (OD600)
In the case of the C. taiwanensis mutant KM184-55, the
Tn5 insertion interrupted the gene coding for the large
subunit of IPM isomerase which is involved in the pathway
of leucine synthesis. The leucine growth requirement of
KM184-55 was consistent with this, and so we performed
an IPM isomerase assay with KM184-55 and the parental strain LMG19424 to confirm this. Compared with
LMG19424, the level of IPM isomerase activity dropped in
KM184-55 to almost undetectable levels (1.7 % of that of
LMG19424). This explains why KM184-55 is a true leucine
auxotroph.
In summary, enzyme assays confirmed that KM60 had only
partially lost BCAA aminotransferase activity and thus was
not a true auxotroph, but it was defective in its utilization
of isoleucine and valine, whereas KM184-55 had no IPM
isomerase activity and thus was a true leucine auxotroph.
BCAA aminotransferase activity and IPM
isomerase activity are essential for effective
symbioses between betarhizobia and M. pudica
2.5
2.0
1.5
1.0
0.5
0
A database search suggested that the Tn5-interrupted gene
in the B. phymatum mutant strain KM60 may code for
BCAA aminotransferase. This possibility is consistent with
the aforementioned BCAA utilization studies. In order to
confirm this, we performed an enzyme assay on KM60 in
comparison with the parental strain STM815. BCAA
aminotransferase-specific activities were detected in the
cell lysate of strain STM815 when using isoleucine, valine
or leucine as an amino group donor, but the mutant KM60
had relatively lower activities (21.4–78.6 %) (Table 3).
These residual activities explain why KM60 is not an
auxotroph and why leucine, when used as the sole nitrogen
source, allows better growth compared with valine and
isoleucine.
Ammonia
Ile
Val
Leu
Fig. 5. Growth (maximum OD600 values within 24 h) of C.
taiwanensis LMG19424 (WT, grey bars) and KM184-55
(leuC : : Tn5, white bars) with minimal medium supplemented with
pyruvate and either ammonia or various BCAAs. Error bars show
mean±SD. Ile, isoleucine; Leu, leucine; Val, valine.
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The phenotypes imparted by a mutated gene can usually be
complemented by introduction of the corresponding WT
gene into the cell. To test this, we generated two
recombinant plasmids by cloning the ilvE gene of the B.
phymatum parental strain STM815 and by cloning the leuC
gene of the C. taiwanensis parental strain LMG19424 into
the broad-host-range vector pBBR1MCS-2. The two
recombinant plasmids (pBBR/ilvE and pBBR/leuC) were
then introduced into the respective mutants KM60 and
KM184-55 and the resulting strains were named KM60/ilvE
and KM184-55/leuC. Both were then used for further
examination.
The cell lysates extracted from strains KM60/ilvE and
KM184-55/leuC were assayed for their respective enzyme
activities. The results from the aminotransferase reaction
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W.-M. Chen and others
Table 3. BCAA aminotransferase enzyme assay
Enzyme activities were assayed by the aminotransferase reaction. The specific activity using isoleucine as an
amino group donor of the parental strain STM815 was adjusted to 100 % and the activities of the mutants
were calculated relative to this. Each value is the mean of six independent experiments. ND, not determined.
Amino acid substrate
as amino group donor
Isoleucine
Valine
Leucine
Relative activity (%)
STM815
KM60
KM60/ilvE
100
89.5
98.5
21.4
38.2
78.6
107.8
91.7
assay showed that KM60/ilvE had recovered its activity to a
level slightly exceeding that of the parental strain STM815
when using isoleucine or valine as amino group donors
(Table 3). In the IPM isomerase assay, KM184-55/leuC had
almost the same activity as the parental strain, LMG19424
(112 versus 100 %). In the ensuing growth studies, strain
KM60/ilvE showed that it had recovered its ability to utilize
isoleucine or valine as nitrogen sources and KM184-55/
leuC regained its prototrophy when cultured on a minimal
medium containing pyruvate and ammonia (data not
shown). To further analyse their symbiotic capacity, KM60/
ilvE and KM184-55/leuC were inoculated onto M. pudica.
The results from the nodulation tests and nitrogen fixation
assays demonstrated that KM60/ilvE and KM184-55/leuC
had the same symbiotic phenotype as their respective
parental strains, STM815 and LMG19424 (Table 2).
These studies confirmed that the phenotypes of the mutant
strains KM60 and KM184-55 resulted from the insertion of
Tn5 into the genes ilvE and leuC, respectively. They also
demonstrated that biosynthesis of BCAAs is essential for
free-living growth of betarhizobia, as well as for their
ability to form an effective symbiosis with their host
legume.
DISCUSSION
In this study, both betarhizobial mutants generated by
transposon mutagenesis produced Fix2 nodules on M.
pudica. Both mutant strains were mutated in genes
involved in BCAA biosynthesis. These data are consistent
with the results obtained from previous studies on
alpharhizobial mutants with altered BCAA biosynthesis,
which were also defective in nodulating their host legumes
(Aguilar & Grasso, 1991; de las Nieves Peltzer et al., 2008;
Ferraioli et al., 2002; Hassani et al., 2002; Kerppola & Kahn,
1988; López et al., 2001; Pobigaylo et al., 2008; SanjuánPinilla et al., 2002; Truchet et al., 1980).
The B. phymatum STM815 mutant, KM60, was mutated in
its ilvE gene, the gene encoding BCAA aminotransferase,
which is the last enzyme of the BCAA biosynthetic pathway.
Interestingly, however, we did not obtain an auxotrophic
mutant, which suggests that the parental strain STM815 may
1764
ND
have at least one functionally redundant ilvE copy. Two
BCAA aminotransferases (IlvE1 and IlvE2) catalysing the
last step of the BCAA biosynthesis pathway have been
demonstrated as being functional in Sinorhizobium meliloti
(de las Nieves Peltzer et al., 2008) and in R. leguminosarum
bv. viciae (J. Prell, unpublished data). In B. phymatum
STM815 only one ilvE gene was identified and no paralogue
could be found. So while KM60 was not an auxotroph for
BCAAs and it grew less well than the WT strain when using
minimal media with pyruvate and valine or isoleucine (Fig.
4), it still showed significant residual BCAA aminotranferase
activities (Table 3). Based on this, there must be an
unknown enzyme that partially compensates for the lost
BCAA aminotransferase activity. Therefore, it is surprising
that KM60 lost its capacity to effectively nodulate the roots
of M. pudica (Fig. 2b). The fact that these phenotypes were
caused by a mutation in the ilvE gene which could be
complemented by the introduction of the corresponding
WT gene indicates that BCAA aminotransferase activity is
essential for an effective symbiosis between B. phymatum
STM815 and its host plant. However, it is possible that the
unidentified enzyme compensating for IlvE in cultures is not
expressed during nodulation.
The C. taiwanensis LMG19424 mutant, KM184-55, is a true
leucine auxotroph due to a mutation in the leuC1 gene
encoding the enzyme catalysing the second step in leucine
biosynthesis. The mutated leuC1 gene has two paralogues,
leuC2 and leuC3, as described above. In this study, a single
mutation in the leuC1 gene led to leucine auxotrophy,
indicating that the leuC2 and leuC3 genes are not required
for the pathway under the conditions tested.
Truchet et al. (1980) reported a leucine-auxotrophic
mutant in S. meliloti L5-30 that formed ineffective nodules,
inside which mutants were sequestered within infection
threads. It was also found that the capacity of this mutant
to invade host cells and fix nitrogen was restored when
leucine was added to the plant medium. Another S. meliloti
leucine auxotroph (strain 104A14) that formed very small,
white nodules was characterized by Kerppola & Kahn
(1988); addition of leucine to the medium around plants
infected with this mutant did not restore the WT
phenotype. Two other leucine-auxotrophic mutants of S.
meliloti, strains GR4 and 1021, both defective for host
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Microbiology 158
BCAA metabolic mutants of betarhizobia
legume nodulation, were identified by Sanjuán-Pinilla et al.
(2002). Both strains were mutated in leuA1, the gene
encoding the first enzyme of the leucine biosynthesis
pathway, and the addition of leucine to the plant growth
medium allowed the mutants to nodulate alfalfa roots,
although with slower kinetics than the parental strain. Two
leucine auxotrophs of S. meliloti Rmd201 were isolated and
characterized by Hassani et al. (2002). Based on crossfeeding and intermediate accumulation studies, they were
designated leuC/leuD and leuB mutants. These two leucine
auxotrophs could induce nodules, but did not fix nitrogen,
and the addition of leucine to the plant medium did not
restore the symbiotic effectiveness of the auxotrophs.
Ferraioli et al. (2002) also reported a leucine-auxotrophic
mutant of Rhizobium etli CE3 which was mutated in the
leuC gene. The leucine auxotroph induced up to fivefold
fewer nodules than the parental strain and had very low
nitrogenase activity.
More recently, de las Nieves Peltzer et al. (2008) tested a set
of mutants that were auxotrophic for leucine and were
affected in almost all enzymic steps of the biosynthesis
pathway. These leucine auxotrophs, including leuA1, leuC,
leuD and leuB mutants, exhibited severe nodulation
defects. For example, nodules induced by these mutants
appeared on roots from approximately 7 to 20 days after
those induced by the parental strain, the number of nodules was low and they did not fix nitrogen. Interestingly,
however, in the presence of exogenous leucine, nodules
induced by these mutants were invaded in the normal way
and fixed nitrogen (de las Nieves Peltzer et al., 2008). A
leuD mutant of R. leguminosarum bv. viciae Rlv3841 did
not nodulate pea plants, but if 1 mM leucine was added to
the nodulation medium (Prell et al., 2009), peas infected
with the leuD mutant were indistinguishable from plants
infected with the WT strain. Interestingly, however, in
Bradyrhizobium japonicum a leucine-auxotrophic mutation
did not lead to a nodulation defect on soybean (Kummer &
Kuykendall, 1989), thus indicating that negative effects of
auxotrophy on symbioses are not universal even among the
alpharhizobia.
The leucine auxotroph of C. taiwanensis LMG19424,
KM184-55, which is the first described leucine auxotroph
among the betarhizobia, had a Nod+ Fix2 phenotype on
its host legume, M. pudica (Fig. 2d). These observations
suggest that the host cells do not provide leucine to the
invading bacteria at any point during the infection or
nodule invasion process. The fact that different leucineauxotrophic mutants have similar phenotypes indicates
that the reason for the symbiotic defect is the disruption of
the metabolic pathway in which the products of all four
genes are involved. As with studies on rhizobia and
sinorhizobia (de las Nieves Peltzer et al., 2008; SanjuánPinilla et al., 2002; Truchet et al., 1980; Prell et al., 2009),
we also observed that the nodulation deficiency of some
leucine mutants was essentially restored by the addition of
leucine to the plant growth medium. However, in our
study, the supplementation of leucine (at 0.2–1 mM) to the
http://mic.sgmjournals.org
plant medium did not restore symbiotic effectiveness to the
leucine auxotrophs (data not shown). This demonstrates
that either leucine from the nodulation medium did not
reach the invading bacteria or leucine was limiting in the
rhizosphere of their host plants. Extending this study to
other auxotrophic mutants will give a more complete
picture of the BCAAs available to betarhizobia during the
development of symbioses with compatible hosts.
ACKNOWLEDGEMENTS
This work was supported by a grant from the National Science
Council, Taipei, Taiwan, in the Republic of China (NSC 96-2313-B022-001-MY3).
REFERENCES
Aguilar, O. M. & Grasso, D. H. (1991). The product of the Rhizobium
meliloti ilvC gene is required for isoleucine and valine synthesis and
nodulation of alfalfa. J Bacteriol 173, 7756–7764.
Boyer, H. W. & Roulland-Dussoix, D. (1969). A complementation
analysis of the restriction and modification of DNA in Escherichia coli.
J Mol Biol 41, 459–472.
Brewin, N. J. (1991). Development of the legume root nodule. Annu
Rev Cell Biol 7, 191–226.
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.
(2003). Nodulation of Mimosa spp. by the b-proteobacterium
Ralstonia taiwanensis. Mol Plant Microbe Interact 16, 1051–1061.
Chen, W. M., de Faria, S. M., Straliotto, R., Pitard, R. M., SimõesAraùjo, J. L., Chou, J. H., Chou, Y. J., Barrios, E., Prescott, A. R. &
other authors (2005). 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.
de las Nieves Peltzer, M., Roques, N., Poinsot, V., Aguilar, O. M.,
Batut, J. & Capela, D. (2008). Auxotrophy accounts for nodulation
defect of most Sinorhizobium meliloti mutants in the branched-chain
amino acid biosynthesis pathway. Mol Plant Microbe Interact 21,
1232–1241.
de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990).
Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter
probing, and chromosomal insertion of cloned DNA in gramnegative eubacteria. J Bacteriol 172, 6568–6572.
dos Reis, F. B., Jr, Simon, M. F., Gross, E., Boddey, R. M., Elliott,
G. N., Neto, N. E., Loureiro, M. F., de Queiroz, L. P., Scotti, M. R. &
other authors (2010). Nodulation and nitrogen fixation by Mimosa
spp. in the Cerrado and Caatinga biomes of Brazil. New Phytol 186,
934–946.
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.
Ferraioli, S., Tatè, R., Cermola, M., Favre, R., Iaccarino, M. &
Patriarca, E. J. (2002). Auxotrophic mutant strains of Rhizobium etli
reveal new nodule development phenotypes. Mol Plant Microbe
Interact 15, 501–510.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 08 May 2017 14:17:28
1765
W.-M. Chen and others
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-
Pobigaylo, N., Szymczak, S., Nattkemper, T. W. & Becker, A. (2008).
containing derivative of plasmid RK2 dependent on a plasmid
function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.
Identification of genes relevant to symbiosis and competitiveness in
Sinorhizobium meliloti using signature-tagged mutants. Mol Plant
Microbe Interact 21, 219–231.
Gibson, A. H. (1963). Physical environment and symbiotic nitrogen
fixation. I. The effect of root temperature on recently nodulated
Trifolium subterraneum L. plants. Aust J Biol Sci 16, 28–42.
Gibson, K. E., Kobayashi, H. & Walker, G. C. (2008). Molecular
determinants of a symbiotic chronic infection. Annu Rev Genet 42,
413–441.
Gross, S. R., Burns, R. O. & Umbarger, H. E. (1963). The biosynthesis of
leucine. II. The enzymic isomerization of b-carboxy-b-hydroxyisocaproate and a-hydroxy-b-carboxyisocaproate. Biochemistry 2, 1046–1052.
Gyaneshwar, P., Hirsch, A. M., Moulin, L., Chen, W.-M., Elliott, G. N.,
Bontemps, C., Estrada-de Los Santos, P., Gross, E., Dos Reis, F. B.,
Jr & other authors (2011). Legume-nodulating betaproteobacteria:
diversity, host range, and future prospects. Mol Plant Microbe Interact
24, 1276–1288.
Hassani, R., Prasad, C. K., Vineetha, K. E., Vij, N., Singh, P., Sud, R.,
Yadav, S. & Randhawa, G. S. (2002). Symbiotic characterization of
isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti.
Indian J Exp Biol 40, 1110–1120.
James, E. K. & Crawford, R. M. M. (1998). Effect of oxygen availability
on nitrogen fixation by two Lotus species under flooded conditions.
J Exp Bot 49, 599–609.
Kerppola, T. K. & Kahn, M. L. (1988). Symbiotic phenotypes of
auxotrophic mutants of Rhizobium meliloti 104A14. J Gen Microbiol
134, 913–919.
Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A.,
Roop, R. M., II & Peterson, K. M. (1995). Four new derivatives of the
broad-host-range cloning vector pBBR1MCS, carrying different
antibiotic-resistance cassettes. Gene 166, 175–176.
Kummer, R. M. & Kuykendall, L. D. (1989). Symbiotic properties of
amino acid auxotrophs of Bradyrhizobium japonicum. Soil Biol
Biochem 21, 779–782.
Prell, J. & Poole, P. (2006). Metabolic changes of rhizobia in legume
nodules. Trends Microbiol 14, 161–168.
Prell, J., White, J. P., Bourdes, A., Bunnewell, S., Bongaerts, R. J. &
Poole, P. S. (2009). Legumes regulate Rhizobium bacteroid devel-
opment and persistence by the supply of branched-chain amino acids.
Proc Natl Acad Sci U S A 106, 12477–12482.
Prell, J., Bourdès, A., Kumar, S., Lodwig, E., Hosie, A., Kinghorn, S.,
White, J. & Poole, P. (2010). Role of symbiotic auxotrophy in the
Rhizobium-legume symbioses. PLoS ONE 5, e13933.
Sambrook, J. & Fritsch, E. F. (1989). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Sanjuán-Pinilla, J. M., Muñoz, S., Nogales, J., Olivares, J. & Sanjuán,
J. (2002). Involvement of the Sinorhizobium meliloti leuA gene in
activation of nodulation genes by NodD1 and luteolin. Arch Microbiol
178, 36–44.
Somasegaran, P. & Hoben, H. J. (1994). Handbook for Rhizobia:
Methods in Legume-Rhizobium Technology. New York: SpringerVerlag.
Stowers, M. D. (1985). Carbon metabolism in Rhizobium species.
Annu Rev Microbiol 39, 89–108.
Thage, B. V., Rattray, F. P., Laustsen, M. W., Ardö, Y., Barkholt, V. &
Houlberg, U. (2004). Purification and characterization of a branched-
chain amino acid aminotransferase from Lactobacillus paracasei
subsp. paracasei CHCC 2115. J Appl Microbiol 96, 593–602.
Truchet, G., Michel, M. & Dénarié, J. (1980). Sequential analysis of
the organogenesis of lucerne (Medicago sativa) root nodules using
symbiotically-defective mutants of Rhizobium meliloti. Differentiation
16, 163–172.
Lodwig, E. M., Hosie, A. H. F., Bourdès, A., Findlay, K., Allaway, D.,
Karunakaran, R., Downie, J. A. & Poole, P. S. (2003). Amino-acid
van Rhijn, P. & Vanderleyden, J. (1995). The Rhizobium-plant
cycling drives nitrogen fixation in the legume-Rhizobium symbiosis.
Nature 422, 722–726.
Vandamme, P., Goris, J., Chen, W. M., de Vos, P. & Willems, A.
(2002). Burkholderia tuberum sp. nov. and Burkholderia phymatum sp.
López, J. C., Grasso, D. H., Frugier, F., Crespi, M. D. & Aguilar, O. M.
(2001). Early symbiotic responses induced by Sinorhizobium meliloti
nov., nodulate the roots of tropical legumes. Syst Appl Microbiol 25,
507–512.
symbiosis. Microbiol Rev 59, 124–142.
iIvC mutants in alfalfa. Mol Plant Microbe Interact 14, 55–62.
Vincent, J. M. (1970). A Manual for the Practical Study of Root-Nodule
Matthysse, A. G., Stretton, S., Dandie, C., McClure, N. C. &
Goodman, A. E. (1996). Construction of GFP vectors for use in
Bacteria. IBP Handbook 15. Oxford: Blackwell Scientific Publications
Oxford.
gram-negative bacteria other than Escherichia coli. FEMS Microbiol
Lett 145, 87–94.
Vincze, E. & Bowra, S. (2006). Transformation of Rhizobia with
Oldroyd, G. E. D. & Downie, J. A. (2008). Coordinating nodule
broad-host-range plasmids by using a freeze-thaw method. Appl
Environ Microbiol 72, 2290–2293.
morphogenesis with rhizobial infection in legumes. Annu Rev Plant
Biol 59, 519–546.
Edited by: H.-M. Fischer
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