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Journal of Bacteriology, March 2000, p. 1748-1753, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Glutathione Is Involved in Environmental Stress
Responses in Rhizobium tropici, Including Acid
Tolerance
Pablo M.
Riccillo,1,2
Cecilia I.
Muglia,1
Frans J.
de
Bruijn,2
Andrew J.
Roe,3
Ian R.
Booth,3 and
O. Mario
Aguilar1,*
Instituto de Bioquímica y Biologia
Molecular, Facultad de Ciencias Exactas, Universidad Nacional de La
Plata, La Plata, Argentina1; MSU-DOE
Plant Research Laboratory and Department of Microbiology, Michigan
State University, East Lansing, MI 488242; and
Department of Molecular and Cell Biology, Institute of
Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25
2ZD, United Kingdom3
Received 20 September 1999/Accepted 20 December 1999
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ABSTRACT |
The isolation of rhizobial strains which exhibit an intrinsic
tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance.
Rhizobium tropici strain CIAT899 displays a high intrinsic
tolerance to acidity and therefore was used in this work to study the
molecular basis of bacterial responses to acid conditions and other
environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using
Tn5-luxAB mutagenesis, and one mutant strain
(CIAT899-13T2), which fails to grow under acid conditions, was
characterized in detail. Strain CIAT899-13T2 was found to contain a
single Tn5-luxAB insertion in a gene showing a high degree
of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and
intracellular pH levels were found to be lower in the mutant than in
the parent. The glutathione-deficient mutant was shown to be sensitive
to weak organic acids, osmotic and oxidative stresses, and the presence
of methylglyoxal. Glutathione restores responses to these stresses
almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in
extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by
the wild-type strain in coinoculation experiments.
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INTRODUCTION |
Microbes are constantly challenged
by a variety of stresses in their natural environments, including
nutrient limitation and/or exposure to physical stresses, such as
elevated temperature, acidity, high osmolarity, or oxidative shock. To
adapt to these adverse conditions, microbes have evolved mechanisms to
monitor the environment and to alter gene expression patterns and the
activity of enzymes and transport proteins that adapt them to the new
environment. Microbial differentiation into stress-resistant forms,
such as endospores and cysts, constitutes one of the best-studied
stress response mechanisms (18, 42). However, the majority
of soil microbes are gram-negative bacteria that do not differentiate in the same fashion in response to stress conditions. Most of the
present knowledge about stress survival in nondifferentiating bacteria
derives from studies on enteric bacteria, such as Escherichia coli or Salmonella typhimurium, and marine
bacteria, such as Vibrio spp. (27).
The responses of soil bacteria, such as Pseudomonas spp. and
rhizobia, to environmental stress factors have been investigated (22, 34). For example, to identify genes which are expressed under nutrient or oxygen limitation conditions, transposon
Tn5-luxAB mutagenesis has been employed to generate
transcriptional fusions of lux luciferase reporter gene to
genes in Pseudomona putida (28) and
Sinorhizobium meliloti (33, 34). Several of the Tn5-luxAB-tagged S. meliloti genes have been
shown to play a role in persistence in the soil and/or competition for
nodulation of the alfalfa host plant (33). A similar
approach has been used by others (40) to identify S. meliloti genes induced by alternative N or C sources such as
stachydrine used under nutrient limitation conditions, which has also
led to the identification of genes important for root colonization.
It is known that soil acidity, temperature, and salinity affect
rhizobial persistence in the soil and rhizosphere of plants, as well as
the efficiency of nodulation, especially in tropical areas (5, 9,
25). The isolation of rhizobial strains that exhibit an intrinsic
acid tolerance has been described and has facilitated studies on the
basic mechanisms underlying acid tolerance. Rhizobium
tropici strain CIAT899 is one of the strains identified that
displays a high intrinsic tolerance to acidity and therefore constitutes a suitable system to study the molecular basis of bacterial
responses to acid conditions and other environmental stresses (23,
31). Different mechanisms have been proposed to be involved in
the ability of microbes to survive and grow at low pH levels. The
regulation of cytoplasmic pH (intracellular pH [pHi]) requires both
passive and active elements (10, 11). The importance of pH
homeostasis for growth of both acid-tolerant and acid-resistant
Rhizobium meliloti strains has been demonstrated (36). In particular, the acid-sensitive mutants examined
were unable to maintain their cytoplasmic pHs at alkaline levels under acid conditions (36).
In this work, we describe the generation and characterization of a
collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis. One mutant strain
(CIAT899-13T2) fails to grow under acid conditions and exhibits
extreme stress sensitivity. Strain CIAT899-13T2 was found to contain a
single Tn5-luxAB insertion in a gene showing a high degree
of similarity with the E. coli gshB gene, encoding the
enzyme glutathione synthetase (Gsh). Correspondingly, glutathione
essentially reverses the stress phenotype of the mutant. Although the
mutant efficiently nodulates beans, it fails to compete when
coinoculated with the parent strain.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth media.
R.
tropici CIAT899 (Streptomycin resistant [Smr]), a
bean-nodulating strain described previously (31), was grown
at 28°C in TY medium (6) or minimal GTS medium
(26). Strain E. coli DH5
(23)
derivatives harboring the Tn5-luxAB-containing plasmid pRL1063a (46) or the helper plasmid pRK2013 (16)
were grown at 37°C in Luria broth supplemented with 50 µg of
kanamycin · ml
1.
Random transposon mutagenesis.
Transposon
Tn5-luxAB mutagenesis of R. tropici CIAT899 was
carried out using the protocol for Tn5 mutagenesis of
Rhizobium spp. described by De Bruijn and Rossbach
(15). Cells of the donor strain E. coli DH5,
harboring the suicide plasmid pRL1063a or the helper plasmid pRK2013,
and the recipient strain, R. tropici CIAT899, were grown in
Luria broth supplemented with kanamycin and TY medium, respectively,
were washed with fresh TY medium, and were concentrated 10-fold in TY
medium. Equal amounts (100 µl) of donor, helper, and recipient cells
were mixed and spotted on TY plates. After 24 h of incubation at
28°C, the mating mixtures were resuspended in sterile distilled water
and plated on selective TY medium, supplemented with streptomycin (400 µg · ml1) and neomycin (100 µg · ml1).
Screen for acid-sensitive mutants.
R. tropici strains
carrying Tn5-luxAB insertions were screened for acid
sensitivity by using toothpicks to streak individual colonies onto GTS
and GMS plates (GMS medium is similar to GTS medium, except that Tris
was substituted for morpholonethanesulfonic acid and the medium was
acidified to a pH of 5.0 with HCl). Transconjugants that grew poorly or
not at all on GMS medium but grew well on GTS medium were selected for
further analysis.
Growth in the presence and absence of glutathione.
Starter
cultures were generated in GTS medium, supplemented with antibiotics,
and grown to saturation. Duplicate flasks containing 100 ml of GTS or
GMS medium were inoculated with an aliquot of starter culture to yield
an initial optical density at 600 nm (OD600) of
approximately 0.06 (equivalent to about 103 cells · ml1). The cultures were incubated under aeration at
28°C, and growth was monitored by periodically measuring the
OD600 and determining the viable cell counts on TY medium.
Glutathione was added to a final concentration of 80 µM. Since
methylglyoxal reacts with glutathione, cells were first grown in GTS
medium supplemented with glutathione to an OD600 of 0.2 U
(approximately 108 cells · ml1). The
cultures were centrifuged, and the pellet was resuspended in an equal
volume of fresh medium plus 2.5 mM methylglyoxal. Incubation and growth
were continued as before. For the cytoplasm acidification experiments,
sodium acetate (2.5 M, pH 5.5, stock solution) was added to GTS medium
to a final concentration of 20 mM (38). The experiments were
replicated at least twice.
Measurement of glutathione content.
The glutathione content
in rhizobia cells was measured by the method of Anderson
(4). Baker yeast-glutathione reductase was supplied by
Sigma-Aldrich. The protein concentration of the samples was determined
by the bicinchoninic acid assay (Sigma-Aldrich).
pHi measurements.
The pHi was determined by using the
distribution of a radiolabeled weak acid following the centrifugation
method described previously (29), using bromodecane to
separate the cell pellet from the supernatant (41). The pHi
was determined using [7-14C]benzoic acid (4.5 µM; 0.1 µCi · ml1) and [3H]-inulin (1.0 µM; 1 µCi · ml1) as the extracellular marker.
The pHi value was calculated as described previously (41).
Measurements of intracellular potassium.
Cultures were grown
to mid-exponential phase (OD600 = 0.5) and 1-ml
samples were used for the analyses. Levels of potassium ions were
quantified by preparing the samples as described for pHi measurements.
The K+ content was determined by flame photometry (Corning
420) after the samples had been boiled and allowed to cool
(21).
DNA isolation and manipulations.
Plasmid DNA was prepared by
the alkaline method as described by Kragelund et al. (28).
Total genomic R. tropici DNA isolation, restriction enzyme
digestions, ligations, and Southern blotting experiments were carried
out according to procedures previously described (2, 43).
DNA sequence analysis.
Sequencing of double-stranded plasmid
DNA was performed using the dideoxy method of Sanger, using Sequenase
kits (U.S. Biochemicals). To determine the R. tropici DNA
sequence on both sides of the transposon insertion, the procedure
described by Milcamps et al. (33) was used. DNA sequence
data were analyzed using the Wisconsin Package Version 9.0 program
(Genetics Computer Group). Similarities were examined with the BLAST
program (3).
Nodulation assays.
R. tropici strains carrying
Tn5-1063 insertions were screened for their symbiotic
phenotype by inoculation on beans or on leucaena seedling roots.
Leucaena leucocephala seeds were first scarred by heat
treatment (80°C, 10 min). Seeds were sterilized by soaking for 3 min
in 95% (vol/vol) ethanol, followed by soaking for 15 min in sodium
hypochloride (8.25 g · liter1), and rinsed
thoroughly with sterile distilled water. Sterilized seeds were placed
on top of sterile agar-water and incubated in darkness at 28°C.
The inoculation with rhizobial suspensions was performed on roots of
5-day-old seedlings. Seedlings were transferred into pots containing
sterile vermiculite at a neutral pH level and incubated in a glass
house with a temperature range of 25 ± 5°C. Four to five weeks
after inoculation, the plants were examined for the presence or absence
of root nodules.
Nodule competition experiments.
Ten bean seedlings were
inoculated with a 1:1 ratio of mutant strain CIAT899-13T2 and wild-type
bacteria. Four weeks after inoculation, nodules were harvested and
individually analyzed. The nodules were surface-sterilized with 95%
ethanol and 30% (vol/vol) hydrogen peroxide, rinsed with sterile
distilled water, and crushed in 100 µl of sterile water, and the
macerate was spotted onto plates containing either streptomycin (400 µg · ml1) or neomycin (100 µg · ml1) to determine the ratio of wild-type versus mutant
bacteria. About 100 nodules from five independent randomly selected
plants were examined.
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RESULTS |
Isolation and characterization of the acid-sensitive mutant
CIAT899-13T2.
Acid sensitivity was defined experimentally as the
inability to grow on minimal medium buffered to a pH of 5.0. A
total of 6,000 Tn5-luxAB-containing strains were analyzed,
of which three mutants were found to be unable to grow on acidified GMS
medium. One of the mutant strains (CIAT899-13T2), was further
characterized. Using Southern hybridization analysis, it was shown that
strain CIAT899-13T2 carries a single Tn5-luxAB insertion
(data not shown).
In order to identify the gene carrying the Tn
5-luxAB
insertion in strain CIAT899-13T2, the tagged locus was cloned from the
rhizobial genome. This was facilitated by the presence of an origin
of
replication within the Tn
5-luxAB transposon (46).
Total genomic
DNA of strain CIAT899-13T2 was digested with
EcoRI, self-ligated,
and introduced into
E. coli
via transformation. This plasmid was
used to confirm that the
Tn
5-luxAB insertion correlates with the
phenotype found in
mutant CIAT899-13T2. The mutated region was
recombined in wild-type
CIAT899 by marker exchange. Randomly chosen
exchange mutants were
assayed for growth on GTS and GMS media,
and found to be unable to grow
at a pH of 5.0. Finally, the DNA
sequence of the Tn
5-luxAB
target junctions was determined and
an open reading frame of 945 nucleotides was identified. The deduced
amino acid sequence of this
open reading frame was compared to
sequences in the databases. It was
found that the Tn
5-luxAB-tagged
locus shares a high degree
of similarity with glutathione synthetase
(Gsh), which is encoded by
the
gshB gene, of
E. coli (39% identity,
72%
similarity) (
8) and other bacteria, such as a
Sinechococcus sp. (40% identity, 73% similarity)
(
37), a
Synechocystis sp.
(37% identity, 72%
similarity) (
35), an
Anabaena sp. (36% identity,
73% similarity) (
17), and
Anaplasma centrale
(35% identity,
68% similarity) (
39). Glutathione
synthetase catalyzes the second
step of glutathione biosynthesis and
adds glycine to the C-terminal
site of
-glutamylcysteine to form
glutathione.
In order to confirm that strain CIAT899-13T2 is a glutathione-deficient
mutant, we measured the content of glutathione in
the cell extracts of
the wild-type strain and the mutant strain.
In the cell extracts
prepared from the wild-type strain, the glutathione
content was 14 nmol · mg of protein
1, whereas in the cell
extracts prepared from the mutant strain
the glutathione content was
very low, about 3% of the wild-type
level.
Glutathione is important for acid tolerance.
Strain
CIAT899-13T2 was found to grow at the same rate as the parent strain in
liquid media buffered to a pH of 7.5, with a mean generation time of
approximately 3 h. However, after dilution in fresh GTS medium
from late-exponential-phase cultures, strain CIAT899-13T2 exhibited a
slower adaptation to the new medium before achieving a growth rate
similar to that of the parent. Contrarily, the growth yield of the
mutant was substantially reduced in acidic medium GMS, with a cessation
of growth after four generations (Fig.
1). Growth was also affected, but to a
lesser extent, in the pH range of 5.0 to 6.0.
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FIG. 1.
Recovery of acid tolerance by R. tropici
CIAT899-13T2. At time zero, rhizobia cells were diluted with GTS or GMS
medium to an OD600 of 0.08. Growth of wild-type strain
CIAT899 (open symbols) and mutant strain CIAT 899-13T2 (closed symbols)
in GTS at a pH of 7.5 (circles), in GMS at a pH of 5.0 (squares), and
in GMS (pH 5.0) supplemented with 80 µM glutathione at time zero
(triangles) was monitored by measuring the OD600. The data
are the means from three replicate experiments, with standard
deviations of less than 10%.
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To determine whether endogenous glutathione was involved in providing
protection against low external pH stress, cells of
R. tropici CIAT 899-13T2 were diluted in fresh GMS medium (pH
5.0)
supplemented with 80 µM glutathione, and bacterial growth
was
monitored (Fig.
1). It was found that mutant CIAT899-13T2
recovered the
ability to grow at a pH of 5.0. In addition, strain
CIAT899-13T2
exhibited an extended lag before growth was initiated
at a pH of 7.5 and this could be shortened by incubation with
glutathione (data not
shown), which is similar to observations
made for
E. coli
glutathione-deficient mutants (
19).
In order to examine if glutathione played a role in the growth response
of
R. tropici CIAT899 to changes in pHi, the effect
of
adding sodium acetate (NaAc) to cultures of strains CIAT899-13T2
and
CIAT899 was examined. Acetic acid in equilibrium with NaAc
is a weak
acid capable of crossing the bacterial membrane in its
undissociated
form, but it dissociates and liberates protons once
inside the cells,
causing cytoplasmatic acidification (
38,
41).
The wild-type
strain CIAT899 was found to display a reduced growth
rate at a pH of
7.5 in the presence of NaAc, but it eventually
achieved the same final
growth yield (Fig.
2). Supplementation
of
the growth medium with glutathione enhanced the growth of the
wild-type
strain (Fig.
2). Incubation of mutant strain CIAT 899-13T2
with NaAc
provoked a 40-h lag prior to the initiation of exponential
growth (Fig.
2). The growth rate was much slower but could be
restored to normal by
the addition of glutathione to the growth
medium. These results suggest
that glutathione synthesis in
R. tropici CIAT899 is
essential for growth at acid pH levels and
in the presence of weak
organic acids that provoke perturbations
of cytoplasmic pH levels.
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FIG. 2.
Acidification of cytoplasm and protection by glutathione
in R. tropici CIAT899. At time zero, cells of CIAT899 (open
symbols) and mutant CIAT 899-13T2 (closed symbols) were diluted in
fresh GTS medium. Growth in GTS medium (circles), in GTS medium
supplemented with 20 mM sodium acetate (squares), and in GTS
supplemented with 20 mM sodium acetate plus 80 µM glutathione
(triangles) was monitored by measuring the OD600. The data
are the means from at least two replicate experiments, with standard
deviations of less than 12%.
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pHi and potassium levels in the CIAT 899-13T2 mutant strain.
We investigated the ability of mutant strain CIAT899-13T2 to control
pHi levels and to maintain potassium pools when exposed to acid medium.
At a pH of 7.5 the pHi values for the mutant and the wild type were
identical, but 60 min after the cells were transferred from a pH of 7.5 to a pH of 5.0, the acid-sensitive CIAT 899-13T2 mutant strain was
found to have a lower pHi level than the wild-type strain had (Table
1).
Since potassium efflux activity, mediated by KefB- and KefC-generated
channels, involves glutathione (
19), we measured the
intracellular K pool in the CIAT899-13T2 mutant strain under neutral
and acid conditions (Table
1). The level of potassium was found
to be
higher in wild-type cells than in the mutant CIAT899-13T2
cells
incubated in either neutral or acid media, and in each case
the
potassium level was found to be higher under acidic conditions,
indicating that exposure to acidity stimulated potassium accumulation
(Table
1). This result indicates that the observed acid sensitivity
of
mutant strain CIAT899-13T2 may be related to the inability
to regulate
the transport of ion
potassium.
Glutathione is important for osmotic stress tolerance.
In
order to examine whether glutathione was important for the
response of R. tropici CIAT899 to other environmental
stresses, we tested the effect of osmotic shock (addition of 0.3 M NaCl) on the growth characteristics of strains CIAT899 and
CIAT899-13T2. In the presence of 0.3 M NaCl, growth of the wild-type
CIAT899 strain was found to be significantly reduced compared to growth in normal GTS medium (Fig. 3). Addition
of glutathione to the medium only slightly enhanced growth of CIAT899.
On the other hand, growth of the mutant strain CIAT899-13T2 was
completely inhibited in the presence of NaCl and the OD600
declined. This inhibition was relieved by glutathione and the growth
rate of the mutant approached that of the parent strain (Fig. 3).
Similarly, the mutant strain CIAT899-13T2 was found to be more
sensitive to incubation in the presence of the oxidative stress agent
H2O2; after 20 min of exposure in the presence
of 1.5 mM H2O2, the number of viable cells was
reduced one order of magnitude from 105 to 104
cells · ml1, whereas the parent strain was found
to be unaffected. These results suggest that glutathione is important
for both acid tolerance and responses to other environmental stresses
in R. tropici CIAT899.
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FIG. 3.
Growth inhibition by osmotic shock is relieved by
glutathione. At time zero, cells of CIAT899 (open symbols) and mutant
CIAT 899-13T2 (closed symbols) were diluted in fresh GTS medium. Growth
in GTS medium (circles), GTS medium supplemented with 0.3 M sodium
chloride (squares), and GTS medium supplemented with 0.3 M sodium
chloride plus 80 µM glutathione (triangles) was monitored by
measuring the OD600. The data are the means from two
replicate experiments, with standard deviations of less than 10%.
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Glutathione is important for protection against
methylglyoxal toxicity.
Methylglyoxal is a naturally
occurring toxic electrophilic compound, and it has been shown
previously that in E. coli glutathione is involved in its
detoxification (19, 20, 21). In order to examine if
glutathione was also involved in preventing methylglyoxal toxicity in
R. tropici CIAT899, the effect of adding methylglyoxal to
the growth medium of both CIAT899 and CIAT899-13T2 cultures was assessed.
The addition of methylglyoxal did not substantially alter the growth
pattern of the wild-type strain CIAT899 (Fig.
4). However,
growth of the mutant CIAT
899-13T2 strain was found to be completely
inhibited in the presence of
2.5 mM methylglyoxal (Fig.
4). The
effect of the addition of
glutathione was also examined. Since
methylglyoxal reacts with
glutathione to form hemithiolacetal
(
19), we assessed the
effect of adding glutathione by growing
the rhizobia in the presence of
glutathione alone, prior to resuspension
in a medium containing
methylglyoxal. The results are shown in
Fig.
4 and demonstrate that
glutathione prevents the inhibitory
effect of methylglyoxal, allowing
restoration of growth of CIAT899-13T2
cells. However, the lag period
before active growth was observed
was found to be greater than that for
the parent strain (Fig.
4). These data suggest that
R. tropici CIAT899 may require glutathione
for the detoxification of
methylglyoxal, by a mechanism probably
similar to that found in
E. coli (
19).
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FIG. 4.
Effect of methylglyoxal and protection by glutathione.
Cells of strain CIAT899 (closed symbols) and mutant CIAT899-13T2 (open
symbols) were grown in fresh GTS medium (circles) and in fresh GTS
medium supplemented with 80 µM glutathione (triangles). After
outgrowth for about 5 h, cells were resuspended in GTS medium with
2.5 mM methylglyoxal (squares). The data are the means from at least
three replicate experiments, with standard deviations of less than
10%.
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Symbiotic properties of mutant strain CIAT899-13T2.
Strain
CIAT899-13T2 was examined for its symbiotic properties and found to
form effective nodules on beans and leucaena. The appearance of plants
inoculated with the mutant strain was found to be similar to that of
plants inoculated with the wild-type strain. Control plants without
rhizobial inoculation showed clear symptoms of nitrogen deficiency
(data not shown). This result suggests that a mutation in the
glutathione synthetase gene (gshB) does not affect the
symbiotic ability of CIAT899 to form effective nodules. However, in a
coinoculation experiment, in which bean plants were coinoculated with a
combination of wild-type and mutant CIAT899-13T2 cells in a 1:1 ratio,
the total number of nodules harvested from five independent bean plants
were found to be occupied exclusively by the wild-type strain.
Therefore, the gshB mutation appears to affect significantly
the ability to compete during the process of bean nodulation.
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DISCUSSION |
A Tn5-luxAB insertion mutant of R. tropici
CIAT899 that is unable to grow under acidic conditions was isolated and
characterized. The mutation in this strain was shown to be due to a
single Tn5-luxAB insertion in the gshB gene,
based on the following evidence. Firstly, Southern analysis of
EcoRI-restricted DNA from strain CIAT899-13T2 with a
Tn5-luxAB probe revealed a single hybridizing
EcoRI fragment. Secondly, DNA sequence analysis of the
Tn5-luxAB flanking region revealed a high degree of
similarity with the gshB gene of several bacteria. Finally,
exogenous glutathione allowed mutant CIAT899-13T2 to recover the
wild-type tolerance to low pH levels, high osmolarity, and the effects
of weak organic acids, hydrogen peroxide, and methylglyoxal.
Different genera of rhizobia show significant variability in their
ability to grow under low pH conditions, although the molecular basis
for the observed differences is not clear. A correlation between the
bacterial cell surface, including the amount of exopolysaccharides, and
lipopolysaccharide composition and acid tolerance has been reported
(1, 23, 44, 45). Metabolic changes upon shifting cultures to
acid media have been reported, including the accumulation of cellular
polyamines and elevated levels of glutamate (23). Overall,
these responses seem to be a consequence of pH stress, rather than
factors required for survival. Here, we demonstrate that metabolic
production of glutathione is essential to protect R. tropici
against environmental stresses that are frequently found in nature,
such as acidity and osmotic or oxidative shock. Soil acidity and other
acid-related toxicity factors, including aluminum and manganese
toxicity, have previously been implicated to be major environmental
constraints affecting the symbiotic performance of legumes
(9).
An important role for glutathione has been demonstrated in E. coli. Glutathione is involved in the binding, transformation, and
detoxification of a wide variety of compounds. One recognized component
of this process of detoxification is the formation of a glutathione
adduct, which in turns activates potassium efflux systems (KefB and
KefC) (19). The KefB and KefC channels offer the cell a
means to lower the cytoplasmic pH level to effect protection against
cytotoxic chemicals such as methylglyoxal (19). Glutathione also provides protection against chlorine compounds in E. coli and against oxidants in E. coli and
Rhizobium leguminosarum bv. phaseoli (13,
14). The behavior of the glutathione-deficient CIAT899-13T2
strain of R. tropici reported here appears to be similar to
that reported for equivalent mutant strains of E. coli and
other bacteria. Therefore, the role of glutathione in bacterial stress
protection seems to be shared by a diversity of bacteria, although the
molecular basis of its action mechanism remains unknown. Furthermore,
our finding of glutathione involvement in the acid tolerance adds a
novel aspect to the observed protection of cells against environmental stresses.
pHi homeostasis is not fully understood, but it had been proposed to
involve the circulation of Na+ and K+ ions
(10, 11, 30). Higher potassium levels in CIAT899 following exposure to acid pH levels were indeed detected (1). Other studies have shown an increased expression of the E. coli
Kdp system, a high-affinity potassium transporter found in many
bacteria, under low pH conditions (6, 30). In E. coli the potassium channels KefB and KefC are inhibited by
glutathione, and in the absence of glutathione, K+ leaks
out of the cells (11). In addition, it was found that the
pHi level in a glutathione-deficient E. coli strain was
lower than that in the parent strain in medium containing a low level of K+ (19). Therefore, we hypothesized that the
CIAT899-13T2 mutant strain might be affected in its ability to maintain
K+ levels. Furthermore, if the pHi homeostasis of R. tropici also involves the control of cytoplasmic potassium levels,
then the essential requirement of glutathione may result from its
involvement in a process associated with acid tolerance rather than the
movement of H+ ions, per se. Consistent with this
hypothesis we observed that the intracellular K+ pool of
the wild-type strain indeed increased when cells were exposed to acidic
conditions and that the K+ level in the acid-sensitive
mutant CIAT899-13T2 was lower than that in the wild-type strain.
Therefore, it is reasonable to conclude that in order to tolerate
external acidity, bacterial cells respond to adjust their intracellular
potassium levels and that the capacity to generate a pH gradient,
associated with potassium uptake, may be disturbed in the CIAT899-13T2
mutant (10). Under acidic conditions, the available
mechanisms for potassium uptake, such as Kdp and Trk found in E. coli (32), may not be sufficient in Gsh
mutant strains to reverse the effect of potassium leakage via the
glutathione-dependent Kef system, resulting in an inadequate intracellular potassium level.
With regard to the observed impairment in nodule occupancy efficiency
in our R. tropici Gsh-deficient strain CIAT899-13T2, we do
not know which of the different stages of infection (e.g., root
colonization, infection thread, bacterial release, etc.) is affected in
the glutathione-deficient mutant. Competition for nodulation of legumes
is a very complex process. Both environmental influences, such as
temperature and soil characteristics, and specific rhizobial genes
impact competition (12). Clearly, glutathione is important
for the symbiotic process in R. tropici, since a glutathione-deficient strain is outcompeted by the wild-type strain.
In conclusion, the data presented here demonstrate the essential nature
of glutathione biosynthesis for R. tropici under acidic conditions and other environmental stresses. Our results provide new
evidence for the proposed role for glutathione in bacterial physiology
in general and in optimal symbiotic performance of rhizobia in particular.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from Agencia de Promoción
Científica y Tecnológica, Argentina (PICT-97 No. 0628),
from the NSF Center of Microbial Ecology, and from the Department of Energy. O.M.A. is member of the research career of the National Research Council-CONICET, Argentina. P.M.R. was supported by CIC, Buenos Aires, Argentina.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Bioquímica y Biologia Molecular, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Calles 47 y 115 (1900), La
Plata, Argentina. Phone: 54 221 4250497, ext. 61. Fax: 54 221 4226947. E-mail: aguilar{at}nahuel.biol.unlp.edu.ar.
| |
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Abstract
Introduction
