(Received for publication, January 17, 1996; and in revised form, February 14, 1996)
From the
Bacteria respond to changes in medium osmolarity by varying the
concentrations of specific solutes in order to maintain constant
turgor. The primary response of Lactobacillus plantarum to an
osmotic upshock involves the accumulation of compatible solutes such as
glycine betaine, proline, and glutamate. We have studied the osmotic
regulation of glycine betaine transport in L. plantarum by
measuring the overall and unidirectional rates of glycine betaine
uptake and exit at osmostasis, and under conditions of osmotic upshock
and downshock. At steady state conditions, a basal flux of glycine
betaine (but no net uptake or efflux) is observed that amounts to about
20% of the rate of ``activated'' uptake (uptake at high
osmolarity). No direct exchange of C-labeled glycine
betaine in the medium for unlabeled glycine betaine in the cytoplasm
was observed in glucose metabolizing and resting cells, indicating that
a separate glycine betaine efflux system is responsible for the exit of
glycine betaine. Upon osmotic upshock, the uptake system for glycine
betaine is rapidly activated (within seconds), whereas the basal efflux
is inhibited. These two responses account for a rapid accumulation of
glycine betaine until osmostasis is reached. Upon osmotic downshock,
glycine betaine is rapidly released by the cells in a process that has
two kinetic components, i.e. one with a half-life of less than
2 s which is unaffected by the metabolic status of the cells, the other
with a half-life of 4-5 min in glucose-metabolizing cells which
is dependent on internal pH or a related parameter. We speculate that
the former activity corresponds to a stretch-activated channel, whereas
the latter may be facilitated by a carrier protein. Glycine betaine
uptake is strongly inhibited immediately after an osmotic downshock,
but slowly recovers in time. These studies demonstrate that in L.
plantarum osmostasis is maintained through positive and negative
regulation of both glycine betaine uptake and efflux, of which
activation of uptake upon osmotic upshock and activation of a
``channel-like'' activity upon osmotic downshock are
quantitatively most important.
In bacteria the intracellular concentration of compatible solutes is regulated by the osmolarity of the environment, which involves changes in transport activities as well as synthesis and/or degradation of these compounds(1) . The regulation occurs both at the genetic (transcription) and enzyme (activation) level. For instance, in Escherichia coli and Salmonella typhimurium glycine betaine and proline are taken up via the constitutive, low affinity ProP system and the inducible, high affinity ProU system(1) . The transport of proline and glycine betaine via ProP and ProU is stimulated by an increase in external osmolarity(2, 3) . In addition, the ProP and ProU expression levels are also increased when the medium osmolarity is raised(2) . Exit of glycine betaine and proline in E. coli and S. typhimurium is thought to be mediated by specific efflux systems, but the molecular evidence is limited(4, 5, 6) .
Upon moderate osmotic
downshock (from 0.5-0.8 to 0.2-0.3 M NaCl or KCl),
bacteria specifically release compatible solutes such as
K, trehalose, glutamate, proline, and glycine betaine,
whereas other low molecular weight compounds are retained by the
cells(4, 5, 6, 7) . It has been
suggested that a severe osmotic downshock leads to a more aspecific
efflux of solutes(6, 8) . Experiments with artificial
membranes that were loaded with carboxyfluorescein indicated that low
molecular weight solutes are able to cross (aspecifically) the lipid
bilayer under conditions of osmotic downshock(9, 10) ,
but no data are available about the significance of such events in
vivo. Mechanosensitive channels that respond to changes in turgor
pressure are thought to be involved in the specific release of solutes
upon osmotic downshock(4, 6, 11) . The
activity of these channels has been shown in patch clamp experiments
using giant azolectin liposomes that were fused with bacterial
membranes or spheroplasts. In both Gram-negative and -positive
bacteria, the opening and closing of channels with different
conductivities can be triggered by suction in patch clamp experiments (12, 13, 14) . Recently, the gene encoding
one of the channels, MscL, of E. coli was cloned and
sequenced, and the channel activity of the purified protein was
demonstrated after reconstitution into artificial
liposomes(8, 15) .
A number of inhibitors of
mechanosensitive channels have been described in eukaryotic
cells(16, 17, 18) . Some of these inhibitors
have been used to modulate channel activity in bacteria, of which only
gadolinium was found to inhibit the release of specific solutes upon
osmotic downshock (6, 11) . In patch clamp experiments
with giant liposomes fused with membranes of E. coli,
gadolinium (Gd) specifically inhibited the
mechanosensitive channel activities, although about 10-fold higher
concentrations of Gd
were needed to inhibit the
bacterial channels than the eukaryotic ones(11) . Gadolinium
also inhibited the purified and reconstituted MscL channel of E. coli in liposomes (15) and the ion is therefore
considered to be a relatively specific inhibitor of mechanosensitive
channels in bacteria.
In Lactobacillus plantarum, the intracellular concentration of glycine betaine, proline, glutamate, and alanine are specifically affected under conditions of osmotic imbalance(7) . In this study, we report on the fluxes of glycine betaine in L. plantarum using dual label experiments. This allows us to follow the inward and outward fluxes simultaneously, under osmostasis as well as under conditions of changing osmolarity (osmotic upshock and downshock). These studies provide, for the first time, a rather complete picture of the intricacies of the regulation of transport of compatible solutes in bacteria.
Figure 1:
Glycine betaine
exit in glucose metabolizing cells of L. plantarum. Cells were
grown on chemically defined medium containing 0.8 M KCl,
washed, and resuspended in potassium phosphate, pH 6.5. After 6 min of
preenergization with 10 mM glucose, uptake was initiated at
zero time by the addition of [C]glycine betaine
(final concentration of 1.3 mM) and KCl (final concentration
of 0.8 M). After 46 min of uptake (indicated by the arrow), a 77-fold excess of unlabeled glycine betaine (squares), carnitine (triangles), or an equivalent
volume of water (circles) was added. The final protein
concentration was 0.15 mg/ml.
Figure 2:
Glycine betaine exit in resting cells
under iso-osmotic and hypo-osmotic conditions. Cells were grown on
chemically defined medium containing 0.8 M KCl, washed, and
resuspended in potassium phosphate, pH 6.5, to a final protein
concentration of 0.61 mg/ml. Cells were energized with 10 mM glucose and allowed to take up [C]glycine
betaine (final concentration of 1.3 mM) at high osmolarity
(0.8 M KCl) for 60 min. The loaded cells were washed
thoroughly with potassium phosphate, pH 6.5, containing 0.8 M KCl. The experiment was initiated by diluting
[
C]glycine betaine loaded cells into potassium
phosphate with (circles) or without (triangles) 0.8 M KCl. Open and closed symbols represent the
exit of [
C]glycine betaine in the presence of
100 mM unlabeled glycine betaine or 100 mM KCl,
respectively. The final protein concentration was 0.15
mg/ml.
Figure 3:
Unidirectional rates of
[H]glycine betaine uptake at osmostasis and upon
osmotic downshock. Cells were grown on chemically defined medium
containing 0.8 M KCl, washed and resuspended in potassium
phosphate, pH 6.5. After 5 min of preenergization with 10 mM glucose, uptake was initiated by the addition of
[
C]glycine betaine (final concentration of 1.3
mM) and KCl (final concentration of 0.8 M). After
50.5 min, the samples were diluted 5-fold with potassium phosphate with (squares) or without (circles) 0.8 M KCl,
but containing 10 mM glucose, 1.3 mM [
C]glycine betaine, and 1 µM [
H]glycine betaine. Transport of
C- and
H-labeled glycine betaine was monitored (open and closed symbols, respectively). The final
protein concentration was 0.60 mg/ml.
Figure 4:
Unidirectional rates of
[H]glycine betaine uptake upon hyperosmotic
shock. Cells were grown on chemically defined medium containing 0.8 M KCl, washed and resuspended in potassium phosphate, pH 6.5.
After 5 min of preenergization with 10 mM glucose, uptake was
initiated at zero time by the addition of
[
C]glycine betaine (final concentration of 1.3
mM). [
H]Glycine betaine (final
concentration of 1 µM) and KCl (final concentration of 0.8 M) were added after 0, 20.5, and 40.5 min of
[
C]glycine betaine uptake (closed
squares). Open squares and circles represent the
uptake of
C- and
H-labeled glycine betaine
upon hyperosmotic shock, respectively. The final protein concentration
was 0.39 mg/ml. The dotted lines represent the unidirectional
rate of glycine betaine uptake from which the steady state efflux rate
was subtracted.
Figure 5:
Efflux of
[C]glycine betaine upon hypo-osmotic shock.
Cells were grown on chemically defined medium containing 0.8 M KCl, washed, and resuspended in CKC buffer, pH 6.5. After 5 min of
preenergization with 10 mM glucose, uptake was initiated at
zero time by the addition of [
C]glycine betaine
(final concentration of 1.3 mM) and KCl (final concentration
of 0.8 M). After 5.5, 10.5, 15.5, 20.5, and 25.5 min the
samples were diluted 5-fold with CKC buffer, pH 6.5, containing 10
mM glucose and 1.3 mM [
C]glycine betaine, but no KCl (indicated
by the arrows and numbers). The efflux rates of the
second phase of efflux are presented in the inset (the numbers in the inset correspond to the numbers in the graph). The final
protein concentration was 0.28 mg/ml.
In analogy with the observed inhibition of glycine betaine efflux
upon osmotic upshock, we have tested whether the uptake of glycine
betaine is inhibited upon osmotic downshock. Cells were allowed to take
up [C]glycine betaine at high osmolarity. When
the steady state was reached, an osmotic downshock was given by
diluting with buffer containing [
H]glycine
betaine. The unidirectional rate of [
H]glycine
betaine uptake was almost zero in the first 2 min following the osmotic
downshock (Fig. 3, closed circles). After this period,
the uptake slowly recovered in time, probably as a result of
restoration of the turgor pressure. The internal amounts of
[
C]glycine betaine under iso-osmotic conditions
and following osmotic downshock are shown for comparison (Fig. 3, open symbols).
To test whether or not the rapid phase of efflux was caused by lysis of cells in the suspension, the integrity of the cells was monitored with the membrane impermeant fluorescent dye ethidiumbromide homodimer. Ethidiumbromide homodimer becomes fluorescent upon binding to DNA and can be used as an indicator of cell lysis(26, 27) . When the indicator was added to cells that were subjected to osmotic downshock, no increase in fluorescence was observed (data not shown). However, an increase in fluorescence was observed upon disruption of the cells by sonication. These results show that cell lysis does not occur to an extent that can explain the rapid phase of efflux.
Figure 6:
Effect of inhibitors of mechanosensitive
channels on [C]glycine betaine efflux. Cells
were grown on chemically defined medium containing 0.8 M KCl,
washed, and resuspended in HEPES, pH 6.5. After 5 min of
preenergization with 10 mM glucose, uptake was initiated at
zero time by the addition of [
C]glycine betaine
(final concentration of 1.3 mM) and KCl (final concentration
of 0.8 M). After 50.5 min of uptake the samples were diluted
5-fold with HEPES, pH 6.5, containing 10 mM glucose and 1.3
mM [
C]glycine betaine (circles) with 1
mM GdCl
(
), 2 mM quinidinechloride
(
), 1 mM amiloride (
), or 100 mM tetraethylammonium chloride (
). The final protein
concentration was 0.34 mg/ml.
When bacteria are faced with changes in external osmolarity, they respond by raising (upon hyperosmotic shock) or lowering (upon hypo-osmotic shock) the cytoplasmic pools of specific molecules, termed compatible solutes. Glycine betaine is not only used as a compatible solute by many prokaryotic and eukaryotic (micro)organisms, it is also the main osmolyte in the cytoplasm of most bacteria that are grown at high osmolarities in the presence of glycine betaine(1) . Some bacteria are able to synthesize glycine betaine from choline via a two step oxidation pathway(1, 23, 30) , but L. plantarum cannot synthesize this compound(7) . Therefore, the cytoplasmic pools of glycine betaine in L. plantarum are solely determined by the net transport rates (uptake and efflux) of glycine betaine. We have studied the regulation of the glycine betaine pools by dissecting the uptake and efflux activities at osmostasis, and hyperosmotic and hypo-osmotic shock.
When the osmolarity is raised
by adding 0.8 M KCl to glucose-metabolizing cells in 50 mM potassium phosphate, pH 6.5, the glycine betaine uptake rates
increase instantaneously from 15 to about 80 nmol/min mg of
protein, and the final accumulation levels rise from 400 to about 1500
nmol/mg of protein(7) . Since this increase in transport rates
and final accumulation levels was observed in the presence of
chloramphenicol, it must be related to changes in activity rather than
expression levels of glycine betaine transport system(s). When
[
C]glycine betaine was taken up to steady state
levels, the addition of tracer amounts of
[
H]glycine betaine allowed us to estimate the
steady state fluxes of uptake and efflux. The stationary exit flux is
inhibited directly after osmotic upshock, but this
``constitutive'' efflux activity is not the major site of
regulation; the increased glycine betaine uptake is more than can be
explained by the inhibition of efflux. This notion is confirmed by
experiments with cells grown on chemically defined medium containing
0.8 M KCl (but no glycine betaine), which were washed and
resuspended in potassium phosphate (low osmolarity), and subsequently,
at different moments in time, subjected to an osmotic upshock. When the
osmotic upshock was given at t = 0, simultaneously with
the addition of [
C]glycine betaine (so no
internal glycine betaine is present), a large increase in the initial
rate of glycine betaine uptake is already observed (Fig. 4).
When the osmolarity of glucose metabolizing cells in potassium
phosphate containing 0.8 M KCl is lowered, whereas the
external concentration [C]glycine betaine is
kept constant, a rapid loss (within 1 s) of accumulated glycine betaine
is observed. After this rapid initial loss, a second slower phase of
glycine betaine efflux is observed (Fig. 5). The rapid phase of
efflux is independent of pH and the metabolic energy status of the
cell, and is inhibited by gadolinium. This rapid efflux is therefore
reminiscent of that of mechanosensitive
channels(11, 16, 17, 18) .
The
slow phase of glycine betaine efflux is observed under conditions of
osmostasis as well as under conditions of osmotic downshock, i.e. the efflux under osmostatic conditions is kinetically similar to
the second phase of efflux that follows an osmotic downshock. The
following observations pertinent to the slow phase of efflux are
relevant. (i) The pH dependence of the process and the inhibition by
nigericin suggest that the slow phase of efflux is inhibited by a low
internal pH and therefore protein mediated. Since no direct exchange of
[C]glycine betaine (in the cytoplasm) for
unlabeled glycine betaine (in the medium) is observed, this efflux of
glycine betaine is driven by the concentration gradient (downhill
efflux). (ii) Upon osmotic upshock this efflux is inhibited as shown by
the experiment presented in Fig. 4. (iii) Upon osmotic
downshock, the kinetics of the second phase of efflux is dependent on
the metabolic status of the cells. In both glucose-metabolizing and
resting cells at a medium pH of 6.5, a constitutive glycine betaine
efflux of about 10 nmol/min
mg of protein is observed under
iso-osmotic conditions (Fig. 1Fig. 2Fig. 3). Upon
osmotic downshock, the slow phase of glycine betaine efflux remains 10
nmol/min
mg of protein in resting cells (Fig. 2),
whereas in glucose-metabolizing cells at pH 6.5 the overall efflux
rates can be as high as 40 nmol/min
mg of protein ( Fig. 5and Fig. 6). Although in the latter experiments
overall fluxes are measured, the increased rates can only partly be
accounted for by the inhibition of uptake (
10 nmol/min
mg
of protein). The additional increase in efflux rate seems therefore to
be a consequence of the osmotic downshock. (iv) The rate of efflux in
the second phase, following an osmotic downshock in glucose
metabolizing cells, is dependent on the intracellular glycine betaine
concentration (Fig. 5).
When an osmotic downshock is given,
uptake of [H]glycine betaine is inhibited in the
first 2 min following osmotic downshock (Fig. 3). Subsequently,
the rate of glycine betaine uptake gradually increases to normal values
in parallel with the restoration of the osmotic imbalance. Overall, the
experiments indicate that the glycine betaine uptake system(s) are not
only activated upon osmotic upshock (see above), but also inhibited
upon osmotic downshock. Similar to the stimulation of glycine betaine
uptake upon osmotic upshock, the inhibition of glycine betaine uptake
upon osmotic downshock is instantaneous. The quick response of the
glycine betaine uptake system(s) to changes in osmolarity (upshock as
well as downshock) suggests that membrane tension or turgor pressure is
sensed by the transporter. Since the regulation of glycine betaine
uptake occurs over a wide range of osmolarities, i.e. the
``activated'' rate of uptake is independent of the extent of
the osmotic upshock, we speculate that the regulation involves an
on/off mechanism, rather than one in which the activity varies
gradually with the external osmolarity.
The main findings of this study are summarized in Fig. 7, in which the putative glycine betaine transport systems and the corresponding fluxes at iso-osmotic, hyperosmotic, and hypo-osmotic conditions are shown. The sizes of the arrows reflect the magnitude of the corresponding fluxes through the uptake system(s) (black arrows) and efflux systems (gray arrows). The steady state of glycine betaine uptake represents an equilibrium between uptake and efflux of glycine betaine that is mediated by independent systems. The steady state fluxes of glycine betaine uptake and exit are similar at high and low osmolarities. Upon a hyperosmotic shock the uptake of glycine betaine is increased, whereas efflux is inhibited. When an osmotic downshock is given to the cells, an overall exit of glycine betaine is observed. Glycine betaine efflux consists of a rapid initial phase and a slower second phase; the unidirectional rate of uptake is lowered upon osmotic downshock. We speculate that the rapid efflux takes place via a mechanosensitive channel-like activity in the first second following the osmotic downshock, whereas the second phase of efflux is due to downhill efflux via a carrier-like mechanism (uniporter). The channel closes when the turgor pressure has decreased sufficiently. In later stages, the slower phase of glycine betaine efflux may serve to fine-tune the turgor pressure. It has a component that is regulated by the medium osmolarity, but also occurs to some extent under conditions of osmostasis (constitutive efflux activity, see above).
Figure 7: Schematic representation of the glycine betaine fluxes in L. plantarum. A denotes the factor that activates the glycine betaine uptake system(s) upon osmotic upshock. The relative fluxes are indicated by the size of the arrows.