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(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
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 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 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 [
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 [
Figure 3:
Unidirectional rates of
[
Figure 4:
Unidirectional rates of
[
Figure 5:
Efflux of
[
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 [
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 [
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 When the osmolarity of glucose metabolizing cells in potassium
phosphate containing 0.8 M KCl is lowered, whereas the
external concentration [ 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
[ When an osmotic downshock is given,
uptake of [ 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.
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10060-10065
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
, 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) .
) 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.
Bacterial Strains and Culture Conditions
L. plantarum was grown in chemically defined medium
at pH 6.7, containing 0.5% (w/v) glucose at 30 °C as described
previously(7) . High osmolarity media were obtained by adding
0.8 M KCl to the chemically defined medium.Transport Assays
Uptake and Efflux in Glucose-metabolizing
Cells
Unless specified otherwise, cells were harvested by
centrifugation, washed twice with 50 mM potassium phosphate,
pH 6.5, and resuspended to a protein concentration of approximately 10
mg/ml. Prior to transport, cells were diluted to a protein
concentration of 0.1-0.6 mg/ml in 50 mM potassium
phosphate, pH 6.5. Cells were preenergized at 30 °C for 5 min by
the addition of 10 mM glucose, after which transport was
initiated by the addition of radiolabeled substrate. To increase the
medium osmolarity aliquots of 3 M KCl or buffer were added
simultaneously with the radiolabeled substrate. Osmotic downshock was
performed by diluting the assay medium with buffer (preheated at 30
°C) containing 10 mM glucose and radiolabeled
substrate(s). Differential labeling
([N-methyl-
H] glycine betaine and
[N-methyl-C]glycine betaine) was used
to follow the unidirectional rates of uptake and efflux. The pH profile
of glycine betaine efflux was determined in 30 mM citric acid,
30 mM K
HPO
, and 30 mM CHES, (
)adjusted to the appropriate pH with KOH (CKC buffer). At
given time intervals, samples of 100-500 µl were taken and
diluted into 2 ml of ice-cold LiCl (0.1-0.8 M, identical
to the osmolarity of the buffer). The samples were rapidly filtered
through 0.45-µm pore-size cellulose-nitrate filters (Schleicher
& Schuell GmbH, Dassel, Germany) and washed with 2 ml of LiCl
(0.1-0.8 M).Exchange and Efflux in Resting Cells
Cells (1 mg
of protein/ml) were loaded with
[N-methyl-C]glycine betaine in the
presence of 10 mM glucose under conditions of high osmolarity
(50 mM potassium phosphate, pH 6.5, plus 0.8 M KCl).
After 60 min of uptake, the cells were centrifuged, washed twice with
potassium phosphate, pH 6.5, containing 0.8 M KCl, and finally
resuspended to a protein concentration of 6-10 mg/ml. To monitor
exchange and/or efflux, the cells were diluted 40-fold into potassium
phosphate, pH 6.5, and of varying osmolarity with or without 100 mM unlabeled glycine betaine. All experiments were carried out at 30
°C. Further handlings were the same as described under
``Uptake and Efflux in Glucose-metabolizing Cells.''Preparation and Loading of Membrane Vesicles and
(Proteo)liposomes
Membrane vesicles of L. plantarum were isolated as
described by Otto et al.(19) using 0.3 mg of
deoxyribonuclease and ribonuclease/ml (final concentration). Liposomes
were prepared from egg phosphatidylcholine and purified E. coli phospholipids (Sigma) in a ratio of 1:3(20) . Cytochrome c oxidase-containing proteoliposomes were prepared according
to Driessen et al.(20) . Fusion of liposomes with
membrane vesicles and loading with [C]glycine
betaine was done by freeze-thaw-extrusion. The mixture of liposomes and
membrane vesicles was rapidly frozen in liquid nitrogen, thawed slowly
at room temperature, and subsequently extruded 11 times through 400-nm
pore-size polycarbonate filters (Avestin)(21) . Transport was
assayed as described above.Miscellaneous
Protein was determined by the method of Lowry et al.(22) with bovine serum albumin as a standard. The
osmolarities of media and buffers were measured by freezing point
depression with an Osmomat 030 (Gonotec, Berlin, Germany).Synthesis of [N-methyl-
Radiolabeled glycine betaine was synthesized enzymatically
from [N-methyl-C]Glycine
Betaine and [N-methyl-H]Glycine BetaineC]- or
[N-methyl-H]choline chloride
(40-60 mCi/mmol and 60-90 Ci/mmol, respectively) as
described by Landfald and Strøhm (23) . The purity of
the glycine betaine was checked by thin layer chromatography according
to Speed and Richardson(24) . Radioactivity on the TLC plates
showed a 100% conversion of choline to a product that co-migrated with C-labeled genuine glycine betaine (made on request by
Amersham Corp., 55 mCi/mmol).Chemicals
Radiolabeled
[N-methyl-C]choline-chloride and
[N-methyl-H]choline-chloride (55
mCi/mmol and 80 Ci/mmol, respectively) were obtained from DuPont NEN.
Radiolabeled L-[U-C]lysine (282
mCi/mmol), L-[U-C]leucine (312
mCi/mmol), and L-[U-C]glutamic acid
(266 mCi/mmol) were obtained from Amersham Corp. (Buckinghamshire, UK).
All other chemicals were of reagent grade and obtained from commercial
sources.
Characterization of Glycine Betaine Exit
Since
ATP synthesis, but not the proton motive force, was required for
glycine betaine uptake, ()we speculate that this osmolyte is
taken up by an ATP-dependent transporter rather than a secondary
transport system. ATP-dependent solute uptake systems generally operate
unidirectionally, whereas secondary transporters are able to catalyze
efflux of solutes down the concentration gradient as well as exchange
of solutes(25) . Nevertheless, when unlabeled glycine betaine
or the substrate analogue carnitine was added to cells that had
accumulated [C]glycine betaine, a slow exit of
label was observed (Fig. 1). To characterize the glycine betaine
exit activity, glucose-metabolizing cells were preloaded with
[C]glycine betaine at high osmolarity, followed
by washing with equiosmolar buffer to remove external glucose and
label. This washing procedure took about 15 min and resulted in a
depletion of cells from (endogenous) metabolic energy as the ATP levels
fell to ``zero'' and uptake of glycine betaine or proline was
no longer observed (data not shown). When
[C]glycine betaine-loaded cells were diluted
iso-osmotically, a slow but significant efflux of glycine betaine was
observed (Fig. 2, filled circles). Since efflux is
similar with or without external glycine betaine, it is unlikely that
[C]glycine betaine exit is caused by direct
exchange for external glycine betaine. These results suggest that
glycine betaine is transported out of the cells by a separate efflux
system. The observed exit rates at osmostasis are similar for glucose
metabolizing cells (Fig. 1) and resting cells (Fig. 2),
suggesting that efflux of glycine betaine takes place either passively
via the lipid bilayer or protein-mediated via a system that catalyzes
downhill glycine betaine export without the use of metabolic energy.
Upon osmotic downshock, however, a much more rapid release of label was
observed both in resting (Fig. 2) and in glucose metabolizing
cells (see section ``fluxes of glycine betaine upon osmotic
downshock''). The presence of glycine betaine in the medium also
did not affect this rapid efflux process.
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.
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.
Fluxes of Glycine Betaine under Osmostatic
Conditions
To establish how the pools of glycine betaine are
precisely regulated, the net fluxes under osmostatic (steady state)
conditions were determined first. Cells in high osmolarity media were
allowed to take up [C]glycine betaine until the
steady state was reached. At this point, a low amount of
[H]glycine betaine was added that did not
significantly affect the overall concentration of glycine betaine.
Since uptake of glycine betaine had reached steady state no net accumulation of glycine betaine is observed under these
conditions. The accumulation of H-label (Fig. 3, filled squares) now reflects the unidirectional rate of
glycine betaine uptake (and efflux!). Since a direct exchange of the
differently labeled pools of glycine betaine is unlikely (see above, Fig. 2), these results suggest that the steady state reflects an
equilibrium between uptake and efflux which are facilitated by separate
systems. Importantly, the unidirectional rates of
[H]glycine betaine uptake were similar in cells
that had reached osmostasis at different external osmolarities (data
not shown).
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.
Fluxes of Glycine Betaine upon Osmotic Upshock
In
a previous study, we have shown that an osmotic upshock increases
glycine betaine and proline uptake rates as well as the final
accumulation levels(7) . Since the steady state glycine betaine
pools are the resultant of uptake and efflux (see above), we studied
both activities separately and under conditions that osmostasis is
disturbed. It is possible that the observed activation of glycine
betaine uptake is due to an inhibition of efflux and/or to a
stimulation of uptake. To discriminate between these two possibilities,
uptake of glycine betaine was followed by using two different labels (C and H) to monitor the uptake of glycine
betaine. Uptake of [C]glycine betaine was
followed at low osmolarity (Fig. 4; closed squares). At
different time intervals, the osmolarity was raised by adding KCl
together with tracer amounts of [H]glycine
betaine and the initial uptake rates of both H- and C-labeled glycine betaine were measured. Since
[C]glycine betaine is also present
intracellularly, it is a substrate for both the efflux and the uptake
systems. When the efflux of glycine betaine is inhibited under
conditions of osmotic upshock, the rates deduced from the uptake of H- and C-labeled glycine betaine should be
identical. The data indicate that the initial uptake rates of C- and H-labeled glycine betaine are indeed
similar (Fig. 4, open symbols), suggesting that the
efflux of glycine betaine is inhibited during an osmotic upshock. The
rate constants of glycine betaine exit under high osmolarity conditions
(buffer plus 0.8 M KCl) were determined in separate
experiments (e.g.Fig. 3, filled squares) and
the corresponding rates were subtracted from the unidirectional rate of
[H]glycine betaine uptake. These hypothetical
lines (Fig. 4, dotted lines) represent uptake of
glycine betaine when efflux would not be inhibited. The difference
between this theoretical line and the actual uptake curves indicates
that even though efflux is inhibited upon osmotic upshock, the effect
of this (negative) regulation on the overall glycine betaine flux is
limited.
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.
Fluxes of Glycine Betaine upon Osmotic Downshock
A
rapid exit of glycine betaine was observed when an osmotic downshock
was given to glucose metabolizing cells that had accumulated glycine
betaine to steady state levels at high osmolarity (Fig. 3). The
extent of the release of glycine betaine was dependent on the drop in
external osmolarity, i.e. 300, 450, and 700 nmol/mg of protein
was released when the external KCl concentration was lowered from 0.8
to 0.48, 0.32, and 0.16 M KCl, respectively. The efflux was
less pronounced in cells that were cultured at low osmolarity; only 100
nmol of glycine betaine/mg of protein were released when the external
KCl concentration was lowered from 0.8 to 0.16 M KCl. However,
these cells also accumulated glycine betaine to much lower
concentrations than cells cultured at high osmolarity. Therefore, the
dependence of efflux on the internal concentration of glycine betaine
was investigated next. Cells that had taken up glycine betaine for 5,
10, 15, 20, and 25 min, resulting in internal concentrations of glycine
betaine of 300, 550, 750, 900, and 1050 nmol/mg of protein (Fig. 5), respectively, were subjected to the same osmotic
downshock. It was observed that efflux of glycine betaine was biphasic,
as was observed in resting cells (Fig. 2), and that the extent
of efflux was more pronounced when more glycine betaine had accumulated (Fig. 5). Since the fast efflux was completed within 1 s (data
not shown), which is the time resolution of the experiment, it was not
possible to estimate the rate(s) (constants) of the rapid phase. The
rates of efflux in the second phase appear to be dependent on the
internal glycine betaine concentration (Fig. 5, inset).
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.
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).Characterization of Glycine Betaine Efflux
glycine
betaine efflux has at least two kinetic components (Fig. 2, Fig. 3, and Fig. 5). To separate the two events, attempts
were made to specifically inhibit one of the two phases of efflux. When
an osmotic downshock was performed at pH 4.5, 5.5, 7.5, and 8.5, the
rates of the second (slow) phase of efflux were 25, 50, 88, and 112
nmol/mg 
min, respectively, whereas the first (rapid) phase of
efflux was not affected (data not shown). Since L. plantarum is unable to maintain the internal pH constant over this pH range
(the internal pH decreased from 7.4 to 5.0 when the external pH was
varied from 7.0 to 4.5), it is possible that the inhibition
of the slow phase of efflux was caused by the decrease of the internal
rather than the external pH. Evidence for this suggestion was obtained
from experiments in which the external pH was kept constant at pH 6.5,
and the internal pH was lowered by the K/H ionophore nigericin. Since nigericin alone was able to inhibit
the slow phase of efflux, whereas dissipation of the membrane potential
by valinomycin did not affect glycine betaine efflux (data not shown),
the data are most consistent with an inhibition of efflux caused by
changes in cytoplasmic pH rather than by the dissipation of the proton
motive force or lowering of the external pH. Biphasic exit of glycine
betaine upon osmotic downshock was also observed in resting cells that
were loaded with [C]glycine betaine (Fig. 2), confirming that the efflux is indeed independent of
the proton motive force. The data of the slow phase of efflux are
consistent with carrier (uniporter) mediated transport of glycine
betaine.Inhibitor Studies
To investigate a possible role
of mechanosensitive channels in the rapid and/or slow phase of glycine
betaine efflux from L. plantarum, several known inhibitors of
these channels were studied for their effect on glycine betaine efflux
upon osmotic downshock. The following inhibitors were tested: 1 mM Gd, 100 mM tetraethylamine, 2 mM quinidine, and 1 mM amiloride. It was found that
Gd specifically inhibited the rapid phase of efflux,
whereas quinidine inhibited the slow phase (Fig. 6). The
inhibition of the rapid phase of efflux by gadolinium was specific as
both 5 mM Ca
and 5 mM Mg, which also interact with lipid head
groups(28) , did not elicit any inhibition of efflux (data not
shown). Control experiments showed that the uptake rates of glycine
betaine, glutamate, lysine, and leucine were only somewhat inhibited in
the presence of 1 mM Gd. Quinidine, on the
other hand, abolished the uptake of lysine and leucine (proton motive
force-driven), whereas the uptake of glutamate and glycine betaine
(ATP-driven) was not affected (data not shown). Therefore the
inhibition by quinidine is not necessary specific, but might be caused
by the lowering of the internal pH. Furthermore, it cannot be excluded
that partitioning of quinidine into the lipid bilayer affects the
efflux activity(12, 29) .
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.
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). 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) .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).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.
))
We thank Dr. I. R. Booth for the gift of
[C]glycine betaine (manufactured at his request
by Amersham Corp.), and Drs. P. F. ter Steeg and J. P. P. M. Smelt for
stimulating discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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