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Journal of Bacteriology, October 1998, p. 5044-5051, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sucrose Is a Nonaccumulated Osmoprotectant in
Sinorhizobium meliloti
Kamila
Gouffi,1
Vianney
Pichereau,1
Jean-Paul
Rolland,2
Daniel
Thomas,2
Théophile
Bernard,1 and
Carlos
Blanco1,*
Groupe Membranes et
Osmorégulation1 and
Groupe Canaux
et Récepteurs Membranaires,2 UPRES-A
CNRS 6026, Université de Rennes 1, Campus de Beaulieu,
F35042, Rennes, France
Received 8 May 1998/Accepted 22 July 1998
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ABSTRACT |
Intracellular accumulation of sucrose in response to lowered water
activity seems to occur only in photosynthetic organisms. Here we
demonstrate, for the first time, the potent ability of this common
sugar, supplied exogenously, to reduce growth inhibition of
Sinorhizobium meliloti cells in media of inhibitory
osmolarity. Independently of the nature of the growth substrates and
the osmotic agent, sucrose appears particularly efficient in promoting
the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main
endogenous osmolytes glutamate and
N-acetylglutaminylglutamine amide (NAGGN). Examining cell
volume changes during the hyperosmotic treatment, we found a close
correlation between the enhancement of the osmotically active solute
pool and the increase in cell volume. Sucrose shares several features
with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti
appears to be strongly divergent from that in most bacteria.
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INTRODUCTION |
Water availability is crucial for
the development of all living cells. Various physical and chemical
parameters such as desiccation and hyperosmotic stress generate
cellular dehydration. The mechanisms of cellular adaptation preventing
water loss under hyperosmotic conditions (osmoregulation) have been
extensively studied in bacteria, fungi, algae, plants, and animals
(4, 6, 10, 13, 36, 37). A consensus strategy of
osmoregulation leads to the intracellular accumulation of inorganic
and/or organic low-molecular-weight compounds known as compatible
solutes. As a rule, the working capacity of the metabolic machinery is
limited by the ionic strength in the cytosolic medium. This latter must
be maintained at a relatively low level. However, one exception is
provided by the halophilic organisms, which must accumulate high
cellular levels of inorganic ions to cope with extremely high salt
concentrations in their environment (16).
For most osmotically stressed cells, turgor adjustment is achieved via
the cytoplasmic accumulation of relatively few organic solutes:
carbohydrates (sugars and polyols such as trehalose and glycerol),
amino acids or imino acids (glutamate, proline, pipecolate, and
ectoine), and betaines and their analogues (5, 9, 22). Most
of these solutes behave as noncharged compounds at physiological pH and
are highly soluble in aqueous solutions. Some of them have been
demonstrated to protect cell macromolecular structures against the
destabilizing effects of salts and urea (27, 37).
Compatible solutes are accumulated by de novo biosynthesis in many
organisms subjected to an elevated osmolarity, and their intracellular
content remains at a high level as long as the stressing conditions are
maintained. After a sudden decrease in osmolarity or cell decay,
accumulated compatible compounds may be liberated into the surrounding
environment and subsequently taken up, via an active transport process,
by other organisms under osmotic stress (5, 18, 20). Such
organic compounds, taken up and accumulated by organisms unable to
synthesize them de novo and able to improve growth under inhibitory
osmolarities, are called osmoprotectants. Hence, in natural
environments, the concept of osmoprotectant supposes an ecological
cycle in which the compatible solutes are shuttled from producers to
consumers injured by a sudden change in the osmotic strength of their
medium. Glycine betaine (GB) provides substantial evidence supporting
this concept. GB is synthesized by only a few organisms (mainly plants,
algae, and cyanobacteria) (12, 28, 36) and is actively
transported and accumulated as an osmoprotectant by a large variety of
cells (6). It is generally accepted that an osmoprotectant
must be accumulated durably within the cell to be effective. However, this concept, which was established on the basis of studies of members
of the family Enterobacteriaceae and several gram-positive and gram-negative bacteria, is not entirely applicable to
Sinorhizobium meliloti, which catabolizes most of the
osmoprotectants known to date, including GB and ectoine (2,
34). Indeed, ectoine is a compatible solute that is synthesized
de novo by many halotolerant bacteria (1, 9); it acts as a
potent osmoprotectant for both Escherichia coli and S. meliloti but displays highly contrasting behaviors in these two
bacteria: ectoine is accumulated at high intracellular concentrations
in enteric bacteria but is never accumulated by stressed cells of
S. meliloti (33). This intriguing observation led
us to hypothesize that ectoine may belong to a new class of
nonaccumulated osmoprotectants that are not detectable by the usual
methods, such as natural-abundance 13C nuclear magnetic
resonance (NMR) spectroscopy.
Previous work demonstrated that high concentrations of sucrose were
much less effective than iso-osmotic concentrations of NaCl in
inhibiting the growth of S. meliloti (25). In the
present report, we provide evidence that sucrose acts as a powerful
osmoprotectant for S. meliloti in media of inhibitory
osmolarity.
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MATERIALS AND METHODS |
Bacterial strains and media.
The following rhizobial strains
were used in this study: S. meliloti wild-type strains
102F34 (this laboratory), M5N1, and SU47 (both provided by J. N. Barbotin) and Bradyrhizobium japonicum USDA 110spc4,
Rhizobium leguminosarum bv. phaseoli H132, R. leguminosarum bv. viciae ATCC 10006, S. fredii USDA
205T, and Mesorhizobium huakuii CCBAU
2609T (which were provided by D. Le Rudulier). Cells were
grown aerobically at 30°C in MSY medium (21) to
mid-exponential phase (optical density at 570 nm [OD570]
of 1), harvested by centrifugation (5,000 × g, 10 min), washed once with minimal salt medium S (26),
concentrated at an OD570 of 10 in the same minimal medium,
and inoculated, at a final OD570 of 0.1, in S medium
supplied with carbon and nitrogen sources at a final concentration of
10 mM each. The standard minimal medium (LAS) contains
DL-lactate and L-aspartate as carbon and
nitrogen sources, respectively. The protein content of the culture was
determined by the method of Lowry et al. (17), with serum
albumin as the standard.
Cryoelectron microscopy.
Copper grids were overlaid with a
carbon film freshly glow discharged; 5 µl of bacterial culture was
allowed to adhere to the grid for 1 min and blotted with filter paper.
The grid was quickly immersed into liquid ethane and transferred to a
Gatan cryoholder in a Philips CM12 electron microscope operating at 100 kV. The sample was maintained below 
174°C throughout the observation.
Cell volume measurements.
Cells were harvested in the
exponential phase of growth. Cell volume measurements were performed on
digitized electron images, using the MACS program (29).
Briefly, in cryoelectron microscopy, cells are embedded into a thin
film of ice and then oriented parallel to the grid plan. Thus, in the
projected image, both length and width are available. For each cell,
the area and volume of the total cell and the cytoplasm were
calculated.
Cell volume was also determined as described by Stock et al.
(31) by measuring the differential retention of
3H2O (400 MBq/ml; Amersham, Les Ulis, France)
and [carboxyl-14C]inulin (308 MBq/mmol;
Amersham). Bacterial cells were collected by centrifugation and
concentrated at an OD570 of 10 in their growth medium.
[14C]inulin (2 × 105 dpm) and
3H2O (2 × 106 dpm) were added
to 500 µl of this suspension. Cells were allowed to equilibrate for
30 min at 30°C prior to centrifugation (12,000 × g,
5 min). [14C] and [3H] from the supernatant
and the pellet were simultaneously determined by liquid scintillation
counting by using the dual dpm mode of a Packard Tri-Carb 1600-TR
spectrometer.
Extraction of cellular solutes.
The pellet of freshly
harvested cells was washed with S isotonic medium and extracted at
least twice with 80% (vol/vol) ethanol-water under vigorous magnetic
stirring at room temperature for 30 min. After centrifugation
(8,000 × g, 15 min), the supernatants constituting the
ethanol-soluble fraction (ESF) were pooled and evaporated to dryness at
40°C, and the dry residue was dissolved in deionized water. The
pellet, called the ethanol-insoluble fraction (EIF), contained the
intracellular macromolecules and cell envelopes.
NMR spectroscopy.
To identify the major intracellular
compounds, the ESF extracted from about 2 × 1012
cells (350 mg of protein) was evaporated to dryness and dissolved in 1 ml of D2O. The natural-abundance 13C NMR
spectra were recorded in the pulsed Fourier transform mode at an
operational frequency of 75.4 MHz as previously described (34).
Chromatographic and quantitative analysis of endogenous
osmolytes.
The N-acetylglutaminylglutamine amide
(NAGGN) content was determined after passage of the ESF through a
cation-exchange column (Bio-Rad AG 50 × 8, H+ form).
NAGGN was converted into glutamate by chemical hydrolysis with 9 M KOH
(110°C, 14 h). K+ ions were removed by titrating the
hydrolyzed solution with 13 M perchloric acid to pH 9. Glutamate and
trehalose were then quantified as already described (11,
33).
Uptake assays.
Cells grown in LAS medium with or without 0.5 M NaCl and with or without 0.5 mM sucrose were harvested by
centrifugation, washed twice with isotonic S medium deprived of
sucrose, and then concentrated to an OD570 of 1 in isotonic
growth medium. Uptake experiments were performed as previously reported
(33). [U-14C]sucrose (23.3 GBq/mmol; Amersham)
was used at a final concentration of 500 µM in 400 µl of bacterial
suspension.
Radiolabelling assays.
Cells were grown in LAS without or
with 0.5 M NaCl or 0.5 mM sucrose or both. [14C]sucrose
was introduced in the growth medium at a specific radioactivity of 0.2 MBq/mmol. At predetermined intervals, the OD570 of the culture was measured and 1 to 2 ml of cell suspension was harvested by
centrifugation (12,000 × g, 2 min). The pellets were
immediately extracted with 80% ethanol as described above.
CO2 was trapped on a strip of filter paper (0.5 by 3 cm)
moistened with 30 µl of 5 M KOH. This filter was changed at each
sampling time. The radioactivity of subsamples of the ESF, the EIF, and
CO2 was measured by scintillation counting. The remainder
of the ESF and EIF were analyzed for composition. The ESF was analyzed
by paper chromatography and high-voltage paper electrophoresis
(2). To determine the nature and the amount of the labelled
compounds, the chromatograms and electrophoregrams were analyzed by
autoradiography. Identified spots were cut out, and their radioactivity
was quantified by scintillation counting. The EIF was treated with 9 M
KOH at 110°C for 20 h, neutralized with perchloric acid, and
centrifuged (12,000 × g, 2 min). The supernatant was
then analyzed by the same methods as used for the ESF.
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RESULTS |
Sucrose improves the growth of S. meliloti 102F34 in
media of high osmolarity.
Sucrose is usually used as a nonionic
osmotic agent to increase the osmotic strength of bacterial growth
media (6). A solution of 0.84 M sucrose develops the same
osmotic pressure as a solution with 0.65 M NaCl. Nevertheless, growth
inhibition of S. meliloti 102F34 by iso-osmotic
concentrations of these two solutes differed remarkably (Table
1). NaCl strongly reduced the growth rate
and growth yield (9- and 6-fold, respectively), whereas sucrose reduced the growth rate only 2.4-fold; 1 mM GB, which is a powerful
osmoprotectant for S. meliloti (2), partially
reversed growth inhibition by NaCl but was unable to improve the growth
of sucrose-stressed S. meliloti cells (Table 1). In
addition, the growth parameters observed in LAS medium containing 0.65 M NaCl plus 1 mM GB were similar to those obtained for cultures grown
in LAS medium containing 0.84 M sucrose, with or without 1 mM GB.
Hence, the apparently weak osmotic effect of sucrose, compared to that
of NaCl, could result from a concomitant osmoprotective effect
displayed by this sugar. This might explain the observation that
addition of a known osmoprotective solute, such as GB, to a
sucrose-stressed culture did not improve sinorhizobial growth. To
confirm this hypothesis, GB was used as an osmotic agent at a
concentration of 0.9 M, which is iso-osmotic with 0.65 M NaCl. As
expected, the growth parameters of S. meliloti 102F34 in LAS
with 0.9 M GB were similar to those observed with 0.84 M sucrose. In
contrast, when 0.9 M mannitol was used to increase the osmotic strength
of the medium, the growth rate was inhibited by a factor of 5, and the
addition of 1 mM GB significantly reduced growth inhibition caused by
mannitol (Table 1). All of these data suggest that sucrose may act as an osmoprotectant for S. meliloti.
The putative osmoprotective activity of sucrose was assayed in LAS
medium, with or without 0.5 M NaCl. Low concentrations
of sucrose (1 µM to 1 mM) were used to minimize the utilization
of this sugar as a
carbon source (
32). Sucrose added at these
concentrations
did not modify the growth parameters of unstressed
cultures. In
contrast, in media containing 0.5 M NaCl, the generation
time and
growth yield were improved for sucrose concentrations
as low as 50 µM
(Fig.
1). The greatest beneficial effect
was reached
with 0.5 mM sucrose (Fig.
1). Hence, sucrose was used at
0.5 mM
for further experiments.
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FIG. 1.
Effect of increasing concentrations of exogenous sucrose
on the doubling time (in hours/generation) and the maximal cell yield
(OD570 max) of stressed cultures of S. meliloti
102F34. Cells were inoculated in LAS minimal medium containing 0.5 M
NaCl plus the indicated concentrations of sucrose.
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KCl, K
2SO
4, and mannitol were added to LAS
medium at the appropriate concentrations to obtain an osmolarity
equivalent to
that developed by 0.5 M NaCl. These compounds, like NaCl,
impaired
the proliferation of
S. meliloti 102F34 (Table
2).
The incorporation
of 0.5 mM sucrose to these media also led to
increases in both
growth rates and growth yields (two- to threefold
increases for
KCl-, K
2SO
4-, and
mannitol-stressed cultures [Table
2]).
In contrast,
sinorhizobial growth was not inhibited by the addition of
glycerol,
used as a control because it reduces water activity but does
not
cause osmotic stress; this compound diffuses freely across
biological
membranes (
5). Sucrose had no effects when it was
added to
LAS with 0.9 M glycerol. Thus, sucrose specifically relieved
osmotic
stress rather than ionic stress.
The improvement of cell growth by sucrose under high-osmolality
conditions was also observed when
S. meliloti was grown with
0.5 M NaCl in S minimal medium containing urea, ammonium, or glutamate
as the nitrogen source and containing glucose, fructose, mannitol,
succinate, malate, fumarate, or glycerol as the carbon source.
Thus,
sucrose acted in the same way, whatever the nitrogen and
carbon sources
used in the medium. Moreover, its osmoprotective
effect did not result
from its extracellular cleavage since its
two constitutive hexoses,
glucose and fructose, supplied separately
or in combination at
concentrations ranging from 1 µM to 10 mM,
did not allow for any
growth improvement (data not shown).
Because the beneficial effect of sucrose was observed at low
concentrations of this disaccharide only when cells were subjected
to
an hyperosmotic stress, and independently of the medium composition,
sucrose could be considered an osmoprotectant for
S. meliloti 102F34.
Sucrose allows stressed cells to recover a normal cytoplasmic
volume.
The cell volume of S. meliloti 102F34 grown in
stressed or unstressed LAS minimal medium, with or without 0.5 mM
sucrose, was calculated by the radioisotopic technique of Stock et al. (31). A total mean cell volume of 0.55 ± 0.07 10
9 µl/cell was obtained whatever the osmotic strength
of the growth medium, in the presence or absence of sucrose. However,
the size of the periplasmic space of these cells could not be
determined because a suitable radioactive compound allowing the size of
this compartment of S. meliloti to be determined was not
available.
Cryoelectron microscopy offers the unique possibility of observing
bacteria in their native state without the use of any fixative
agent,
dehydration, or embedding procedures which would alter
the osmotic
equilibrium of the cells (
29). Therefore, this technique
was
used to measure the dimensions and calculate the volumes of
the
periplasmic space and the cytoplasm of
S. meliloti 102F34
cells. Figure
2a shows that the
cytoplasmic compartment of unstressed
cells was pressed against the
cell wall. The total volume of these
cells was 0.57 ± 0.02 10
9 µl/cell (
n = 90), and the volume of
the periplasmic space was
0.12 ± 0.05 10
9
µl/cell. The mean cell volumes of stressed (0.5 M NaCl) bacteria
grown with and without 0.5 mM sucrose were quite similar, 0.58
± 0.02 10
9 (
n = 75) and 0.56 ± 0.02 10
9 (
n = 57) µl/cell, respectively.
However, the cytoplasmic volume
of stressed cells grown without added
sucrose was drastically
reduced; therefore, the periplasmic space of
these cells increased
to 0.35 ± 0.01 10
9 µl/cell
(Fig.
2b). In contrast, the periplasmic space of stressed
cells grown
in the presence of 0.5 mM sucrose was quite similar
to that observed
for unstressed cells (Fig.
2a and c).
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FIG. 2.
Restoration of cell turgor in S. meliloti
102F34 grown under osmotic stress in the presence of exogenous sucrose.
Cultures were grown for 16 h in LAS minimal medium (a), in LAS
plus 0.5 M NaCl (b), and in LAS supplemented with 0.5 M NaCl and 0.5 mM
sucrose (c). Samples were processed for cryoelectron microscopy as
described in Materials and Methods. Scale bar correspond to 1 µm.
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External sucrose is not accumulated in S. meliloti.
Most
cells attempt to increase their cytoplasmic volume by regulating the
concentrations of intracellular osmolytes. Previous studies (30,
33, 34) showed that S. meliloti, in response to an
osmotic upshift, synthesizes de novo three major osmolytes, glutamate,
NAGGN, and trehalose, which is significant only during the stationary
phase of growth.
Osmolytes accumulated by
S. meliloti grown in low- or
high-osmolarity medium, in the presence or absence of 0.5 mM sucrose,
were characterized by natural-abundance
13C NMR
spectroscopy. No major solute was detected in cells cultivated
in LAS
medium deprived of NaCl. The presence of 0.5 M NaCl in
the medium
induced the accumulation of glutamate and NAGGN during
exponential
growth, whereas trehalose content started to increase
in the late
exponential growth phase (Fig.
3A and B).
An identical
spectrum was obtained from cells cultivated in LAS medium
with
0.5 M NaCl and 0.5 mM sucrose (Fig.
3C and D). None of the peaks
attributable to sucrose was observed. Thus, sucrose appears as
a solute
that is not accumulated in osmotically significant amounts
by stressed
cells of
S. meliloti 102F34.
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FIG. 3.
Natural-abundance 13C NMR of extracts of
salt-stressed cultures of S. meliloti 102F34. Cells were
grown in LAS medium containing 0.5 M NaCl without (A and B) or with (C
and D) 0.5 mM sucrose. Samples were harvested in the early (A and C)
and late (B and D) log phases of growth and processed as described in
the text. All spectra (1,024 scans) were obtained from 2 × 1012 cells (2,000 OD570 units). Resonance
(peaks) from L-glutamate (g), the dipeptide NAGGN (d), and
trehalose (t) are indicated when these osmolytes were detected in the
cytosolic extracts.
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However,
13C NMR spectroscopy does not allow for the
detection of solutes that are present in low cellular concentrations.
To
follow the behavior of sucrose, the
14C-labelled
molecule was supplied (0.5 mM) to stressed or unstressed
cultures of
S. meliloti (Fig.
4).
Extracellular sucrose was readily
taken up at a constant velocity of 33 nmol/min/mg of protein in
the low-osmolarity medium. About 80% of the
supplied [
14C]sucrose was transported in 10 h. At
this time, 75% of the imported
radiocarbon was incorporated into
insoluble materials (the EIF),
whereas 15% was released as
[
14C]CO
2 and only 10% remained in the
soluble pool (the ESF). Interestingly,
no radiolabelled sucrose was
detected in the ESF fraction, and
the radiocarbon was distributed over
several primary metabolites.
The radioactivity of the EIF and the ESF
started to decrease as
soon as the external sucrose disappeared from
the medium (Fig.
4A).
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FIG. 4.
Fate of [14C]sucrose in S. meliloti 102F34. [U-14C]sucrose (0.5 mM, 0.2 MBq/mmol) was supplied to unstressed cells grown in LAS medium (A) and
stressed cells grown in LAS plus 0.5 M NaCl (B). The distribution of
the radioactivity in the growth medium and the cellular fractions was
monitored as described in Materials and Methods. Symbols:  , growth
expressed as OD570;  , radioactivity in the medium;  ,
radioactivity in 14CO2;  , radioactivity in
the EIF;  , radioactivity in the ESF.
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The stressed cultures grown in the presence of 0.5 M NaCl, supplied
with 0.5 mM [
14C]sucrose, incorporated the labelled
disaccharide at an initial
velocity of only 3.7 nmol/min/mg of protein
(which is ninefold
lower than that observed in cells grown at low
osmolarity) during
the first 6 h following the osmotic upshift.
Then the rate of
the radiocarbon influx increased steadily, reaching
5.4, 16, and
34 nmol/min/mg of protein 10, 15, and 20 h,
respectively, after
inoculation. Thus, the rate of sucrose uptake,
which was severely
inhibited during the lag phase, was progressively
restored to
the level observed in unstressed cells (Fig.
4B). The
imported
radiocarbon was again found primarily in the insoluble
materials.
Indeed, 50% of the radioactivity that was incorporated
during
the first 10 h was recovered in the EIF and 70% was
recovered
in the mid-exponential growth phase, whereas the level
decreased
as the external [
14C]sucrose became limiting.
The soluble pool never exceeded a maximum
of 20% of the total imported
radiocarbon. Interestingly, chromatographic
analysis of this fraction
enabled us to detect radiolabelled sucrose,
which represented about
45% of the ESF radiocarbon during the
lag phase; then, the sucrose
ratio of the ESF decreased at the
beginning of the exponential
phase. Only traces of [
14C]glutamate,
[
14C]NAGGN, and [
14C]trehalose were
detected. The incorporation of radioactivity
in CO
2 was
reduced, suggesting that sucrose was not used as a
preferential
energetic substrate.
In summary, the osmotic upshift occurring at inoculation of
S. meliloti in high-osmolarity LAS led to a lag phase of growth
during which sucrose was accumulated temporarily at low levels.
The
major part of sucrose-derived radiocarbon was detected in
insoluble
materials, whereas only a low amount of radioactivity
was found in the
dominant osmolytes (i.e., glutamate, NAGGN, and
trehalose) that were
revealed by NMR analysis. Moreover, the stimulation
of sucrose uptake
and catabolism corresponded with the resumption
of growth of the
stressed cells.
Kinetics of endogenous osmolyte pool.
The amounts of
intracellular osmolytes were determined during the growth cycle of
S. meliloti cultivated in LAS medium deprived of NaCl. As
previously described (33, 34), the levels of intracellular glutamate, NAGGN, and trehalose remained very low (below 0.05 µmol/mg
of protein).
Two hours after the application of the osmotic upshift (0.5 M NaCl),
the intracellular glutamate level had reached 0.55 µmol/mg
of
protein, a value about 10 times higher than the value measured
in
unstressed cells. This level remained constant during 24 h
and
then decreased progressively (Fig.
5A).
In contrast, the level
of NAGGN increased progressively throughout the
growth period,
reaching a maximum of 0.5 µmol/mg of protein after
30 h of growth.
Trehalose remained at low levels (about 0.1 µmol/mg of protein)
during the exponential growth and began to
increase as glutamate
and NAGGN decreased.
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FIG. 5.
Effect of exogenous sucrose on the internal osmolyte
composition of salt-stressed S. meliloti 102F34. Cells were
grown in LAS medium containing 0.5 M NaCl without (A) or with (B) 0.5 mM sucrose supplied as an osmoprotectant. Endogenous osmolytes were
quantified as described in the text. Symbols: , growth expressed as
OD570; , glutamate; , NAGGN; , trehalose;  ,
sucrose.
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The osmolyte levels in a stressed culture grown in the presence of 0.5 mM sucrose were also determined (Fig.
5B). The profile
of osmolytes
accumulation of these cells was remarkably different
from that observed
in a stressed culture grown without sucrose
(Fig.
5A). Indeed,
glutamate levels (which were similar in the
two cultures for 8 h
after inoculation) increased sharply during
the exponential growth in
the sucrose-supplemented culture (Fig.
5B). Consequently, the maximal
level of this amino acid was about
twofold higher in this culture
(about 1 µmol/mg of protein in
the late exponential phase) than in
the stressed culture grown
without sucrose. Furthermore, during the
late exponential growth
phase, cytoplasmic glutamate decreased faster
in the sucrose-supplemented
culture than in the culture grown without
sucrose (Fig.
5). Interestingly,
the level of cytosolic NAGGN also
increased sharply (about sixfold)
during the exponential phase of the
culture grown with 0.5 mM
sucrose, but NAGGN, in contrast to glutamate,
did not decrease
thereafter (Fig.
5B). The trehalose content was very
similar in
the stressed cultures grown with or without sucrose. It
reached
a steady-state level of about 0.2 µmol/mg of protein during
the
stationary phase (Fig.
5). The intracellular sucrose level was
very
similar to that of trehalose for about 24 h in stressed cells
grown in the presence of 0.5 mM sucrose. Later, cytosolic sucrose
(which never exceeded 75 nmol/mg of protein) decreased and became
negligible (Fig.
5B).
To determine whether the cytosolic solutes described above played a
part in osmotic adjustment, it was necessary to measure
their specific
cytoplasmic concentrations. Taking into account
the different
cytoplasmic volumes of the stressed cells grown
with or without 0.5 mM
sucrose (Fig.
2), the concentrations of
the two major endogenous
osmolytes were 187 mM for glutamate and
120 mM for NAGGN in stressed
cells grown without sucrose and 247
mM for glutamate and 107 mM for
NAGGN when 0.5 mM sucrose was
added. The cytoplasmic concentration of
sucrose was always much
lower than that of glutamate and NAGGN and
reached a maximal concentration
of 21 mM, which explains why sucrose
signals were never observed
in the NMR spectra.
In conclusion, sucrose is not accumulated to osmotically significant
levels in salt-stressed cells of
S. meliloti. Consequently,
this highly beneficial osmoprotectant cannot be considered a true
compatible solute in this species. Moreover, this disaccharide
contributes significantly to enhance glutamate and NAGGN levels
in
stressed cells and subsequently to promote cytoplasmic expansion
and
turgor recovery.
Effect of sucrose on other rhizobial strains.
To determine
whether osmoprotection by sucrose was specific to S. meliloti 102F34, the osmoprotective activity of this compound was
evaluated in several other strains of rhizobia. Growth rates and growth
yields were also improved in NaCl-containing media in the presence of
0.5 mM sucrose for S. meliloti wild-type strains SU47 and
M5N1 and for R. leguminosarum bv. phaseoli H132 (Table 3). In contrast, no beneficial effect of
sucrose was observed for R. leguminosarum bv. viciae,
S. fredii USDA 205T, M. huakuii CCBAU
2609T, and B. japonicum USDA 110spc4 (data not
shown). The uptake and the fate of [14C]sucrose in
S. meliloti strains SU47 and M5N1 grown in NaCl-containing medium were the same as those observed above for strain 102F34; i.e.,
sucrose was never accumulated in significant amounts and sucrose-derived radiocarbon was recovered essentially in the EIF fraction (Table 4). Sucrose uptake in
R. leguminosarum bv. phaseoli was stimulated by NaCl, and
most of the radiocarbon from [14C]sucrose was
incorporated into CO2 but not into the EIF as observed in
S. meliloti (Table 4). In all of these strains, the ESF
represented the minor labelled fraction, which demonstrates that
sucrose was not accumulated to significant levels.
View this table:
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|
TABLE 3.
Beneficial effect of exogenous sucrose on the growth of
salt-stressed cultures of various
rhizobial strainsa
|
|
In sharp contrast, sucrose uptake and its fate appeared to be clearly
different in strains that were not osmoprotected by
sucrose.
Salt-stressed cultures of
M. huakuii CCBAU 2609
T
were unable to transport sucrose. Moreover,
B. japonicum
USDA
110spc4 did not take up [
14C]sucrose in either low-
or high-osmolarity medium (Table
4).
This result is consistent with the
nonutilization of sucrose as
a carbon source by this strain
(
32). Last, the uptake and catabolism
of sucrose were
abolished or strongly reduced by an increase in
medium osmolarity for
S. fredii USDA 205
T and
R. leguminosarum bv. viciae ATCC 10006, respectively (Table
4).
| |
DISCUSSION |
In this study, we provide evidence that exogenously supplied
sucrose acts as an osmoprotectant in stressed cultures of S. meliloti, the root symbiont of the plant crop alfalfa. First, we
showed that a submolar concentration of sucrose (0.84 M) only weakly
inhibits the growth rate of sinorhizobial cultures in comparison to
iso-osmotic concentrations of inorganic salts and mannitol, which were
highly inhibitory. Second, micromolar levels of exogenous sucrose (50 to 500 µM) strongly stimulate the growth of salt-stressed and
mannitol-stressed cultures of S. meliloti 102F34. Moreover, sucrose appears to be as effective as GB in promoting the growth of
S. meliloti under an hyperosmotic constraint. To the best of our knowledge, this is the first report demonstrating the pivotal role
of sucrose in the alleviation of osmotic inhibition of growth in a
nonphotosynthetic bacterium.
A few examples of sucrose accumulation in response to water and salt
stresses have been reported for desiccation-tolerant (3) and
salt-stressed (24) plants and for photosynthetic eubacteria
(12, 19, 28, 35) in which sucrose is accumulated by de novo
biosynthesis. In contrast, exogenously supplied sucrose is not
accumulated as a compatible solute in osmotically stressed cells of
S. meliloti but is actively catabolized. Moreover, its intracellular concentration did not reach levels compatible with its
direct participation in the recovery of cell turgor and in the
protection of intracellular macromolecular structures against the
deleterious effects of high osmolarities. In contrast to the situation
observed with other sinorhizobial and bacterial osmoprotectants (i.e.,
stimulated uptake in stressed cells [2, 6, 33]), the
uptake of sucrose was strongly reduced by an osmotic upshift and
started to increase only when cultures entered the exponential phase of
growth. All of these features clearly indicate that sucrose, while
allowing for growth improvement in adverse conditions, cannot be
considered a classical osmoprotectant. Indeed, sucrose is neither, like
choline (5), a precursor of an accumulated osmoprotectant nor preferentially used to synthesize any endogenous osmolyte, as
observed with proline in Brevibacterium linens
(14). Moreover, the behavior of sucrose is very closely
related to that previously reported for ectoine in S. meliloti (33), because (i) both sucrose and ectoine
induce osmoprotection without being accumulated at significant levels,
(ii) no metabolites derived from these osmoprotectants were
accumulated, and (iii) sucrose and ectoine (unlike GB and dimethylsulfoniopropionate [DMSP] [23, 34]) do not
suppress the accumulation of the major endogenous compatible osmolytes (glutamate, NAGGN, and trehalose). Another interesting observation is
the spectacular increase in the level of cytosolic glutamate during the
initial steps preceding the resumption of growth. This increase seems
to be the initial event that triggers the recovery of a maximal
cytoplasmic volume. We do not know how sucrose and ectoine stimulate
the accumulation of glutamate in stressed cells of S. meliloti. However, these osmoprotectants did not act as close
precursors to glutamate. A significant correlation was observed among
rhizobia between osmoprotection by sucrose and its metabolization under
stressing conditions, suggesting that these two properties are linked.
The same observation has been previously described for ectoine
(33).
Until now, this kind of cell strategy, permitting the achievement of
osmotic equilibrium without preferential accumulation of the supplied
osmoprotectant, has not been reported for any other organism. Among the
rhizobial strains analyzed, the beneficial effect of sucrose was
observed only for S. meliloti strains and R. leguminosarum bv. phaseoli. This finding raises the question of
the acquisition of this specific pathway of osmoprotection during
evolution. One of the obvious differences between S. meliloti and the enteric bacteria is its ability to catabolize all
of the compounds that it uses as exogenous osmoprotectants except DMSP (23). This catabolic ability is shared with various other
bacteria such as the pseudomonads (15, 20), but no one has
yet demonstrated a protective effect without preferential accumulation
of an osmoprotectant in these organisms.
Osmoprotection without accumulation of the osmoprotectant is not
exclusive of the classical phenomenon; in S. meliloti, both may coexist. Accumulated osmoprotectants, like GB and DMSP, inhibit the
de novo synthesis of endogenous solutes (7, 23, 34), ensuring a metabolic economy for the cell. In contrast, nonaccumulated osmoprotectants (sucrose and ectoine) stimulate the adaptation capacities of the cell. In the most highly developed bacterial model,
E. coli, the concentration of potassium glutamate is
considered the intracellular signal of osmotic stress; it acts on the
transcriptional regulation of genes involved in the osmoadaptative
response (6, 20). Osmoadaptation is achieved in S. meliloti by accumulated and nonaccumulated osmoprotectants, which
act in opposite manners on glutamate cellular content. Therefore, the
conclusions derived from the E. coli model are not
applicable to S. meliloti. Potassium glutamate cannot be
considered the only intracellular osmotic signal for this bacterium; a
novel mechanism of osmoregulation involving a different intracellular
signal could occur in S. meliloti.
Sucrose is widespread in nature; it is produced mainly in plants and
photosynthetic microorganisms. In the symbiotic association Medicago sativa-S. meliloti, sucrose is the most prominent
carbohydrate in roots and nodules; its levels have been shown to
increase in the latter under salt stress (8). Although the
peribacteroid membrane is a very selective barrier through which no
sugar is actively transported (32), sucrose could enter the
bacteroid by slow and passive diffusion. These data suggest two
consequences. First, sucrose could be released into the environment via
plant exudates and wilting plants (20) and could be absorbed
by free-living rhizosphere bacteria. Second, sucrose is available in
low amounts but is not limiting for bacteroids. Thus, sucrose could
participate in their osmoprotection rather than in their energy supply,
which is mainly ensured through active dicarboxylic acid import systems and catabolism (32). These points must be analyzed since
they are crucial for improvement of the resistance of this association to drought and, in general, to any osmotic stress.
| |
ACKNOWLEDGMENTS |
We thank M. Uguet for technical assistance, J. Hamelin for NMR
experiments, J. N. Barbotin and D. Le Rudulier for providing rhizobial strains, J.-A. Pocard for helpful discussion, and V. James
for language improvement.
This work was supported by the Direction de la Recherche et des Etudes
Doctorales and by the Centre National de la Recherche Scientifique.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Groupe Membranes
et Osmorégulation, UPRES-A CNRS 6026, Université de Rennes
1, Campus de Beaulieu, Av. du Général Leclerc, F35042
Rennes, France. Phone and fax: 33 (0)2 99 28 61 40. E-mail:
Carlos.Blanco{at}univ-rennes1.fr.
| |
REFERENCES |
| 1.
|
Bernard, T.,
M. Jebbar,
Y. Rassouli,
S. Himdi-Kabbab,
J. Hamelin, and C. Blanco.
1993.
Ectoine accumulation and osmotic regulation in Brevibacterium linens.
J. Gen. Microbiol.
139:129-138.
|
| 2.
|
Bernard, T.,
J.-A. Pocard,
B. Perroud, and D. Le Rudulier.
1986.
Variations in the response of salt-stressed Rhizobium strains to betaines.
Arch. Microbiol.
143:359-364.
|
| 3.
|
Bianchi, G.,
A. Gamba,
R. Limiroli,
N. Pozzi,
R. Elster,
F. Salamini, and D. Bartels.
1993.
The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia.
Physiol. Plant.
87:223-226.
|
| 4.
|
Brown, A. D.
1976.
Microbial water stress.
Bacteriol. Rev.
40:803-846[Free Full Text].
|
| 5.
|
Csonka, L. N.
1989.
Physiological and genetic responses of bacteria to osmotic stress.
Microbiol. Rev.
53:121-147[Abstract/Free Full Text].
|
| 6.
|
Csonka, L. N., and A. D. Hanson.
1991.
Prokaryotic osmoregulation: genetics and physiology.
Annu. Rev. Microbiol.
45:569-606[Medline].
|
| 7.
|
Dinnbier, U.,
E. Limpinsel,
R. Schmid, and E. P. Bakker.
1988.
Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations.
Arch. Microbiol.
150:348-357[Medline].
|
| 8.
|
Fougère, F.,
D. Le Rudulier, and J. G. Streeter.
1991.
Effects of salt stress on amino acid, organic acid, and carbohydrate composition of roots, bacteroids, and cytosol of alfalfa (Medicago sativa L.).
Plant Physiol.
96:1228-1236[Abstract/Free Full Text].
|
| 9.
|
Galinski, E. A., and H. G. Trüper.
1994.
Microbial behaviour in salt-stressed ecosystems.
FEMS Microbiol. Rev.
15:95-108.
|
| 10.
|
Garcia-Perez, A., and M. B. Burg.
1991.
Renal medullary organic osmolytes.
Physiol. Rev.
71:1081-1115[Abstract/Free Full Text].
|
| 11.
|
Guillouet, S., and J. M. Engasser.
1995.
Sodium and proline accumulation in Corynebacterium glutamicum as a response to an osmotic saline upshock.
Appl. Microbiol. Biotechnol.
43:315-320.
|
| 12.
|
Hellebust, J. A.
1976.
Osmoregulation.
Annu. Rev. Plant Physiol.
27:485-505.
|
| 13.
|
Imhoff, J. F.
1986.
Osmoregulation and compatible solutes in eubacteria.
FEMS Microbiol. Rev.
39:57-66.
|
| 14.
|
Jebbar, M.,
G. Gouesbet,
S. Himdi-Kabbab,
C. Blanco, and T. Bernard.
1995.
Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool.
Arch. Microbiol.
163:380-386.
|
| 15.
|
Kortstee, G. J. J.
1970.
The aerobic decomposition of choline by microorganisms. I. The ability of aerobic organisms, particularly coryneform bacteria, to utilize the choline as the sole carbon and nitrogen source.
Arch. Mikrobiol.
71:235-244[Medline].
|
| 16.
|
Kushner, D. J.
1978.
Life in high salt and solute concentrations: halophilic bacteria, p. 317-368.
In
D. J. Kushner (ed.), Microbial life in extreme environments. Academic Press, New York, N.Y.
|
| 17.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 18.
|
Lucht, J. M., and E. Bremer.
1994.
Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system ProU.
FEMS Microbiol. Rev.
14:3-20[Medline].
|
| 19.
|
Mackay, M. A.,
R. S. Norton, and L. J. Borowitzka.
1984.
Organic osmoregulatory solutes in cyanobacteria.
J. Gen. Microbiol.
130:2177-2191.
|
| 20.
|
Miller, K. J., and J. M. Wood.
1996.
Osmoadaptation by rhizosphere bacteria.
Annu. Rev. Microbiol.
50:101-136[Medline].
|
| 21.
|
O'Gara, F., and K. T. Shanmugam.
1976.
Regulation of nitrogen fixation by Rhizobia: export of fixed N2 as NH2+.
Biochim. Biophys. Acta
437:313-321[Medline].
|
| 22.
|
Pichereau, V.,
A. Cosquer,
A. C. Gaumont, and T. Bernard.
1997.
Synthesis of trimethylated phosphonium and arsonium analogues of the osmoprotectant glycine betaine; contrasted biological activities in two bacterial species.
Bioorg. Med. Chem. Lett.
7:2893-2896.
|
| 23.
|
Pichereau, V.,
J.-A. Pocard,
J. Hamelin,
C. Blanco, and T. Bernard.
1998.
Differential effects of dimethylsulfoniopropionate, dimethylsulfonioacetate, and other S-methylated compounds on the growth of Sinorhizobium meliloti at low and high osmolarities.
Appl. Environ. Microbiol.
64:1420-1429[Abstract/Free Full Text].
|
| 24.
|
Pilon-Smits, E. A. H.,
M. J. M. Ebskamp,
M. J. Paul,
M. J. W. Jeuken,
P. J. Weisbeek, and S. C. M. Smeekens.
1995.
Improved performance of transgenic fructan-accumulating tobacco under drought stress.
Plant Physiol.
107:125-130[Abstract].
|
| 25.
|
Pocard, J.-A.
1987.
Ph.D. thesis.
Université de Rennes 1, Rennes, France.
|
| 26.
|
Rains, D. W.,
L. N. Csonka,
D. Le Rudulier,
T. P. Croughan,
S. S. Yang,
S. J. Stavarek, and R. C. Valentine.
1982.
Osmoregulation by organisms exposed to saline stress: physiological mechanisms and genetic manipulation, p. 283-302.
In
A. San Pietro (ed.), Biosaline research: a look to the future. Plenum Publishing Corp., New York, N.Y.
|
| 27.
|
Randall, K.,
M. Lever,
B. A. Peddie, and S. T. Chambers.
1996.
Natural and synthetic betaines counter the effects of high NaCl and urea concentrations.
Biochim. Biophys. Acta
1291:189-194[Medline].
|
| 28.
|
Reed, R. H., and W. D. P. Stewart.
1985.
Osmotic adjustment and organic solute accumulation in unicellular cyanobacteria from freshwater and marine habitats.
Mar. Biol.
88:1-9.
|
| 29.
|
Rolland, J. P.,
P. Bron, and D. Thomas.
1997.
MACS, automatic counting of objects based on shape recognition.
Comput. Appl. Biosci.
5:563-564.
|
| 30.
|
Smith, L. T., and G. M. Smith.
1989.
An osmoregulated dipeptide in stressed Rhizobium meliloti.
J. Bacteriol.
171:4714-4717[Abstract/Free Full Text].
|
| 31.
|
Stock, J. B.,
B. Rauch, and S. Roseman.
1977.
Periplasmic space in Salmonella typhimurium and Escherichia coli.
J. Biol. Chem.
252:7850-7861[Abstract/Free Full Text].
|
| 32.
|
Stowers, M. D.
1985.
Carbon metabolism in Rhizobium species.
Annu. Rev. Microbiol.
39:89-108[Medline].
|
| 33.
|
Talibart, R.,
M. Jebbar,
G. Gouesbet,
S. Himdi-Kabbab,
H. Wroblewski,
C. Blanco, and T. Bernard.
1994.
Osmoadaptation in rhizobia: ectoine-induced salt tolerance.
J. Bacteriol.
176:5210-5217[Abstract/Free Full Text].
|
| 34.
|
Talibart, R.,
M. Jebbar,
K. Gouffi,
V. Pichereau,
G. Gouesbet,
C. Blanco,
T. Bernard, and J.-A. Pocard.
1997.
Transient accumulation of glycine betaine and dynamics of endogenous osmolytes in salt-stressed cultures of Sinorhizobium meliloti.
Appl. Environ. Microbiol.
63:4657-4663[Abstract].
|
| 35.
|
Welsh, D. T., and R. A. Herbert.
1993.
Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy.
FEMS Microbiol. Ecol.
13:145-149.
|
| 36.
|
Wyn Jones, R. G., and J. Gorham.
1984.
Phytochemical aspects of osmotic adaptation.
Recent Adv. Phytochem.
18:55-78.
|
| 37.
|
Yancey, P. H.,
M. E. Clark,
S. C. Hand,
R. D. Bowlus, and G. N. Somero.
1982.
Living with water stress: evolution of osmolyte systems.
Science
217:1214-1222[Abstract/Free Full Text].
|
Journal of Bacteriology, October 1998, p. 5044-5051, Vol. 180, No. 19
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
