From the Institut für Biotechnologie 1, Forschungszentrum
Jülich GmbH, D-52425 Jülich, Germany
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INTRODUCTION |
Bacterial membranes are readily permeable to water but constitute
an effective barrier to most other solutes. An increase in the external
osmolality thus leads to instant efflux of water and to a decrease in
cell turgor. To avoid dehydration of the cytoplasm, bacteria have
developed adaptation mechanisms to hyperosmotic stress, both long term
responses, manifested during growth at high osmolality, and short term
responses, which occur instantly after changes in the external
osmolality (1-3). A general defense mechanism against high osmolality
is the uptake and accumulation of compatible solutes like glycine
betaine, proline, or ectoine. Unlike other solutes, these substances do
not interfer with vital cellular functions when present at high
concentration. The uptake of compatible solutes is mediated by
transport systems that are regulated in dependent of the change in
external osmolality.
Mechanisms and systems of osmo-adaption have been intensively studied
in the Gram-negative Escherichia coli and Salmonella typhimurium. Two uptake systems for compatible solutes have been identified in these organisms, namely the binding
protein-dependent transporter ProU and the secondary
carrier ProP. Whereas ProU is regulated both on the level of
transcription and activity (4, 5), ProP is mainly regulated on the
activity level (6-8). Another well studied organism in this respect is
the Gram-positive Bacillus subtilis. In this organism,
glycine betaine is taken up via three osmo-regulated transport systems,
belonging either to the ABC-type or to the class of secondary carriers
(9-11). Regulation of these transport systems in various organisms was studied in detail on the genetic level; however, less information is
available concerning their regulation on the level of activity (12).
Corynebacterium glutamicum carries several osmo-responsive
uptake systems for compatible solutes. The specific, high affinity glycine betaine transporter BetP was cloned and sequenced (13). Its
function and its response to osmotic changes has been analyzed in
detail (14). In addition, further osmo-regulated uptake systems for
compatible solutes have been identified (15). As for other bacteria,
however, nothing is known so far about the mechanism of osmo-sensing
and regulation on the activity level that triggers the immediate
response of the carrier protein to osmotic stress.
In the present communication we report molecular studies on the
mechanism of osmo-sensing by the glycine betaine uptake system BetP of
C. glutamicum. It has been shown that BetP is effectively regulated in dependence of external osmolality. Whereas the carrier is
completely inactive under isoosmotic conditions, its activity increases
on hyperosmotic stress in a time range of seconds or less (14). A
similar regulation pattern, although shifted in its optimum, was
observed after expression of betP in E. coli (13). We thus concluded that BetP functions not only as an
osmo-regulated carrier but also as a sensor of osmotic stress. The
secondary structure of BetP was predicted to carry two extensions at
the cytoplasmic face, a negatively charged N-terminal and a positively charged C-terminal extension (13). The function of these domains in
sensing osmotic stress was studied by construction of deletion mutants
and functional characterization of the gene products.
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MATERIALS AND METHODS |
Bacterial Strains, Plasmids, and Growth Conditions--
The
bacterial strains and plasmids used are described in Table
I. E. coli strain DH5
mcr
was grown at 37 °C in Luria Bertani (LB) medium (16). C. glutamicum was grown in brain heart infusion medium
(BHI1; Difco, Detroit, MI.)
at 30 °C.
General DNA Techniques--
Genomic DNA was isolated from
C. glutamicum according to Eikmanns et al. (17).
Plasmid DNA was isolated using the QIAGEN plasmid kit (QIAGEN, Hilden,
FRG). E. coli was transformed using standard methods (18).
To make C. glutamicum competent, strains were grown in LB in
the presence of 0.4% isonicotinic acid hydrazide, 2.5% glycine, and
0.1% Tween 80 (19). Cells were washed four times with ice-cold 10%
glycerol. Transformation was carried out by electroporation (19). Cells
were immediately transferred to BHI medium containing 0.5 M
sorbitol (19), incubated for 1 h at 30 °C in a shaking flask,
and plated on BHI medium containing 20 mg/liter kanamycin.
DNA Manipulations and Sequence Analyses--
Restriction
enzymes, DNA polymerase I (Klenow fragment), proteinase K, RNase, and
T4 DNA ligase were obtained from Boehringer, Mannheim (Federal Republic
of Germany (FRG)) or from Promega (Heidelberg, FRG) and used as
recommended by the manufacturers. Restriction analyses were carried out
by separation of DNA fragments in 0.8% agarose gels. DNA fragments
were isolated by use of QIAex (QIAGEN, Hilden, FRG). For DNA
sequencing, the automated laser fluorescence DNA sequencer from
Pharmacia (Freiburg, FRG) was used. Sequence reactions were carried out
using the AutoRead Sequencing kit (Pharmacia).
Polymerase Chain Reaction--
Deletion mutants were constructed
by polymerase chain reaction amplification using Taq
polymerase (Boehringer, Mannheim) and a Thermo-Cycler 480 (Perkin-Elmer). The primers are listed in Table
II. A denaturating temperature of
94 °C for 30 s, an annealing time of 30 s, and an
extension temperature of 72 °C for 120 s was used for 30 cycles
(Table II). Each primer was added to a final concentration of 10 pM. As template, plasmid pHP2 was used in a final
concentration of 1 nM. Polymerase chain reaction products were ligated to the vector pUC18/SmaI using the
ochre codon directly after the SmaI site as a
stop signal. All clones were sequenced to verify the desired mutation,
and correct mutants were ligated with the IPTG-inducible plasmid pEKEX2
using the BamHI/EcoRI restriction sites.
Synthesis of [14C]Glycine Betaine--
Synthesis
of [14C]glycine betaine by oxidation of
[14C]choline (specific activity 52 µCi/µmol) using
choline oxidase was performed as described earlier (20). To dilute the
ethanolic [14C]choline solution, the volume was increased
to 500 µl and 30 units of choline oxidase were used. Radiolabeled
[14C]choline was purchased from Amersham International
(Amersham, Buckinghamshire, UK).
Transport Assays--
C. glutamicum cells were grown
overnight in BHI medium. IPTG (0.2 mM) was added when
indicated. The cells were washed with buffer containing 50 mM potassium phosphate (pH 7.5) and 10 mM NaCl,
energized with 10 mM glucose, and, where indicated,
osmotically stressed by the addition of solutes, e.g. NaCl
or sorbitol for 3 min at 30 °C. For determination of the
Na+ affinity, cells were washed with 50 mM
morpholine propanesulfonic acid (MOPS)-KOH buffer (pH 7.0),
resuspended, and measured in the same buffer containing 10 mM glucose. The osmolality in the uptake assay was adjusted
to 600 mosmol/kg by the addition of sorbitol. Uptake was started by
adding [14C]glycine betaine (0.1 µCi/ml) at a final
concentration of 100 or 200 µM. Aliquots of 200 µl were
transferred to glass fiber filters (type F, 0.45 µm pore size,
Millipore, Eschborn, FRG) at time intervals of 15 or 30 s. At
least five samples at different times were taken for each uptake
measurement. The filters were washed twice with 2.5 ml of 0.1 M LiCl solution, and the radioactivity retained on the
filters was determined by liquid scintillation counting.
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RESULTS |
Regulation of BetP by Osmotic Stress on the Level of
Activity--
We recently characterized the glycine betaine
uptake system BetP of C. glutamicum as being strongly
regulated on the level of activity by the external NaCl concentration
(14). For a more general analysis with respect to the question of
whether BetP is regulated by the external osmolality and/or by the
ionic strength, we compared betaine uptake of C. glutamicum
wild type strain ATCC 13032 independent of either NaCl or sorbitol
(Fig. 1A). The regulation pattern was identical in the two experiments, indicating that in fact
the change in osmolality is the true signal for carrier activation. To
exclude a general salt effect on transport systems, the activity of the
related Na+-dependent proline uptake system
PutP is also shown in Fig. 1A as a control (15).
Furthermore, we have previously shown that the major driving force,
i.e. the membrane potential, is not significantly changed
under these conditions (14).

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Fig. 1.
Stimulation of glycine betaine uptake by
NaCl, sorbitol, and by the local anesthetic tetracaine. Glycine
betaine uptake activity was measured independent of the external
osmolality (A) and of added tetracaine (B).
C. glutamicum cells were grown overnight in BHI complex
medium. Uptake was started by the addition of 100 µM
labeled glycine betaine. A, the osmolality of the uptake
assay was increased either by the addition of NaCl (solid
circles) or by the addition of sorbitol (open circles)
in the presence of 50 mM NaCl. The value for uptake at
1,350 mosmol/kg external osmolality was set to 100%, and the absolute
values at 1,350 mosmol/kg were 105 nmol·min 1·(mg of
dry weight) 1 for NaCl and 38 nmol·min 1·(mg of dry weight) 1 for
sorbitol stimulation. As a control, proline uptake by the PutP system
of C. glutamicum independent of external NaCl is shown (stars). The absolute value at 1,380 mosmol/kg was 9.5 nmol·min 1·(mg of dry weight) 1.
B, glycine betaine uptake of C. glutamicum wild
type (solid symbols) and C. glutamicum DHP1
( betP) (open symbols) was measured. The
osmolality of the uptake assay was adjusted with NaCl to a final value
of 400 mosmol/kg (circles), 600 mosmol/kg
(squares), or 1,000 mosmol/kg (triangles).
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For a more fundamental approach, we investigated whether the change in
the physical state of the membrane (mechanostress) is the direct
signal for carrier activation. We applied a method known from
biophysical studies on membranes, i.e. the addition of local
anesthetics, to influence the membrane tension in a defined manner
(21-25). For this purpose, the influence of tetracaine on betaine
uptake in the wild type strain C. glutamicum ATCC 13032 as
well as in the betP deletion strain DHP1 was determined
(Fig. 1B). The osmolality was adjusted with NaCl to a final
value of 400, 650, or 1,000 mosmol/kg, respectively, and tetracaine was varied from 0 to 1.25 mM. Whereas at 400 mosmol/kg the
uptake activity of the wild type reached its optimum between 0.5 and 0.75 mM added tetracaine, no increase in activity was
observed in strain DHP1. At higher osmolalities (650 and 1,000 mosmol/kg) the tetracaine-induced increase in uptake activity of the
wild type was less pronounced and reached optimal values between 0.75 and 1 mM tetracaine. The results of Fig. 1B
demonstrate that BetP can be stimulated by the addition of tetracaine
alone and this stimulation is related to the activation of the carrier
protein by hyperosmotic stress.
Construction of N- and C-terminal Deletion Mutants of
BetP--
From the nucleotide sequence of the betP gene, a
protein of 595 amino acids was derived that shares a high degree of
identity to two other osmotically regulated bacterial carriers, OpuD of B. subtilis and BetT of E. coli (9, 26). The
secondary structure prediction (PHDhtmtop program; EMBL, Heidelberg,
FRG) of these three proteins revealed a common structure of 12 transmembrane segments as well as a common hydrophilic C-terminal
extension. In contrast to the other two carriers, BetP possesses an
additional hydrophilic N-terminal domain, which is longer than the
C-terminal one. Interestingly, the two hydrophilic domains, which are
predicted to face the cytoplasm, are both highly charged; however, with different polarity. Whereas 15 negatively charged and only two positively charged residues are located within the 62 amino acids of
the N-terminal part, 21 positively charged and 8 negatively charged
amino acid residues are located within the 55 amino acid residues of
the C-terminal domain (Fig. 2). We
investigated the role of these two extensions in osmo-sensing and
regulation by constructing mutant genes of betP coding for
proteins with deletions in one or both extensions. The two domains were
truncated stepwise, resulting in four mutants of each domain. In the
case of a full deletion of the extensions (C0 and N0) only two and
three amino acid residues, respectively, were left in front of the
first putative transmembrane segment or at the end of the last segment.
In addition, three double mutants lacking the N-terminal and part or
all of the C-terminal domain were constructed (C0N0, C1N0, C2N0) (Fig. 2).

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Fig. 2.
Secondary structure model of BetP. For
the prediction, the program PHDhtmtop was used. Positively charged
amino acid residues are black, negatively charged amino acid
residues are white. The constructed truncations of the C-
and N-terminal extensions are indicated by arrows, and the
number of deleted amino acids is noted. EX, external;
IN, internal.
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Kinetic Discrimination of BetP-mediated Glycine Betaine Uptake of
C. glutamicum DHP1--
The C. glutamicum betP deletion
mutant DHP1 showed a reduced betaine uptake rate of about 10% that of
the wild type, which is due to at least one additional osmo-regulated
uptake system (15). In contrast to specific uptake via BetP, betaine
uptake by the additional transport system(s) was competitively
inhibited by ectoine or proline. To discriminate the activity of the
mutant BetP proteins in C. glutamicum DHP1, we tested
betaine uptake of strain DHP1/pGTG carrying the wild type
betP gene and of strain DHP1/pEKEX2 (vector without insert)
in the absence and presence of ectoine (Fig.
3). Betaine uptake of the betP
deletion strain DHP1/pEKEX2 was completely abolished by the addition of
ectoine in 100-fold excess. In contrast, strain DHP1/pGTG showed high betaine uptake in the presence of ectoine, the activity of which depended on the external osmolality exactly as in C. glutamicum wild type. Thus, the betaine uptake observed in the
presence of a 100-fold excess of ectoine represents the activity of
BetP only. These conditions were used in all further measurements.

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Fig. 3.
Competition of glycine betaine uptake by
ectoine. Glycine betaine uptake in strains DHP1/pGTG (solid
symbols) and DHP1/pEKEX (open symbols) was measured
without (triangles) and with (circles) the
addition of unlabeled ectoine. Cells were grown overnight in BHI
complex medium containing 0.2 mM IPTG and 20 mg/liter
kanamycin. Uptake was started by the addition of 100 µM
labeled glycine betaine. Ectoine was added in 100-fold excess, which
corresponds to a final concentration of 10 mM in the uptake
assay.
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Glycine Betaine Uptake Activity of betP Mutants in C. glutamicum
Strain DHP1--
Fig. 4 shows betaine
uptake of the various plasmid-encoded BetP mutant carrier proteins in
the presence of unlabeled ectoine in comparison to strain DHP1/pGTG.
All BetP proteins with deletions in the N-terminal extension were
active; however, the regulation pattern was altered. Whereas the wild
type reached its maximum activity at 600 mM NaCl
(corresponding to 1.35 osmol/kg), the optimal activation of the
proteins with N-terminal deletions was shifted to higher values. All
mutant proteins behaved similarly and reached their maximum of activity
at osmolalities 2-fold higher than that of the wild type. Fig.
4B summarizes the uptake rates of proteins with truncated
C-terminal part. Strain DHP1/pC0 completely lacking the extension was
not active at all. The activity of all other C-terminal deletion
mutants increased with increasing NaCl, similar to the wild type.
However, the optimum activity was shifted to lower NaCl concentrations
of about 200 mM. Furthermore, uptake activity of strains
DHP1/pC1 and DHP1/pC2 did not decrease as strongly as the wild type
activity at higher NaCl concentrations but remained nearly constant
between 200 and 1,500 mM NaCl. For further analysis, double
mutants were constructed with deletions in both, the C- and the
N-terminal domains. As expected, strain DHP1/pC0N0, i.e. the
mutant protein with deletion of both extensions, was not active, as was
strain DHP1/pC0 (Fig. 4C). The double mutants DHP1/pC1N0 and
DHP1/pC2N0 were active in transport and behaved similar to the
corresponding C-terminal deletion mutants DHP1/pC1 and DHP1/pC2.

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Fig. 4.
Glycine betaine uptake in various mutant
strains independent of the external NaCl concentration. Glycine
betaine uptake was measured in strain DHP1/pGTG (wild type)
(solid symbols) and in strains of DHP1 carrying different
mutated betP alleles on a plasmid (open symbols).
Cells were grown overnight in BHI complex medium containing 0.2 mM IPTG and 20 mg/liter kanamycin. Uptake was started by
the addition of 100 µM labeled glycine betaine and 10 mM unlabeled ectoine. Maximum uptake at optimum conditions of external osmolality was set to 100% as explained in the legend to
Fig. 1 in all cases. For absolute values, see data in Table. III.
A, uptake activity of strains DHP1/pN0 (squares),
DHP1/pN1 (rhomboids), DHP1/pN2 (circles), and DHP1/pN3 (triangles). B, uptake activity of strains DHP1/pC0 (dotted line, hexagons),
DHP1/pC1, DHP1/pC2, and DHP1/pC3 (broken line, stars,
triangles, and squares, respectively). C,
uptake activity of strains DHP1/pC0N0 (dotted line,
hexagons), DHP1/pC1N0, and DHP1/pC2N0 (broken lines,
squares, and circles, respectively).
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Na+ Dependence of Glycine Betaine Uptake of the betP
Mutants--
Glycine betaine uptake via the BetP system is coupled to
symport of two Na+ ions (14). As a consequence,
Na+ acts on betaine transport both as cosubstrate and as
osmo-stimulants. To discriminate between the two effects, we measured
betaine uptake at a constant NaCl concentration of 50 mM
and varied the external osmolality with sorbitol. 50 mM
NaCl does not stimulate uptake in the wild type and is not sufficient
to fully stimulate uptake in the respective BetP mutants. Fig.
5 shows the uptake activities of strains
DHP1/pC1, DHP1/pC2, and DHP1/pC1N0 in comparison to strain DHP1/pGTG.
Whereas the wild type carrier was stimulated by sorbitol similar as
observed for NaCl, the mutant strains did not respond to the change in
osmolality. High sorbitol concentrations led to a decrease of glycine
betaine uptake in strains DHP1/pC1 and DHP1/pC1N0, whereas uptake of
strain DHP1/pC2 remained constant. Consequently, the data of Fig.
4B must be re-interpreted. Stimulation of uptake activity of
the proteins C1 and C2 at low NaCl concentrations between 10 and 200 mM must in fact be caused by the increasing concentration
of Na+ ions but not by the increase in osmolality.

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Fig. 5.
Glycine betaine uptake independent of the
external sorbitol concentration in the presence of 50 mM
NaCl. Cells were grown overnight in BHI complex medium containing
0.2 mM IPTG and 20 mg/l kanamycin. Uptake was started by
the addition of 100 µM labeled glycine betaine and 10 mM unlabeled ectoine. Strains DHP1/pGTG (wild type)
(solid circles), DHP1/pC1 (squares), DHP1/pC2
(triangles), and DHP1/pC1N0 (stars) were
analyzed. The values of the relative uptake rates are directly
comparable to those of Fig. 4, B and C,
i.e. the relative rates of the different mutants in Fig. 5 at zero sorbitol are identical to those of Fig. 4 at 50 mM
external sodium. Consequently, also the optimum values (100%) in Figs. 4 and 5 are identical.
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It could be argued that the initial stimulation by 50 mM
NaCl is not due to the availability of Na+ ions
specifically but to the change in ionic strength. We thus measured
uptake activity of the C-terminal mutants at increasing NaCl
concentration (10-150 mM) and adjusted the ionic strength by KCl to a constant salt concentration of 1,200 mM.
Whereas the wild type and the C3 mutant reached their full activity
below 50 mM NaCl, the activity of the C1 mutant increased
up to 100 mM NaCl (data not shown). These results indicate
that neither the increase in osmolality nor in ionic strength is
responsible for uptake stimulation in these mutants. Consequently, the
Na+ affinity of the C1 mutant must be altered as compared
with the wild type. To determine the Na+ affinity of the
mutants we adjusted the osmolality of the assays by sorbitol to a
constant value of 600 mosmol/kg and varied NaCl in the range of 1-150
mM (Fig. 6). The
Na+ dependence of strain DHP1/pC3 did not differ
significantly from the wild type, whereas the affinity toward
Na+ was decreased in strains DHP1/pC1 and DHP1/pC2,
corroborating the results of Fig. 5. The dependence of uptake activity
on Na+ was found to be sigmoidal in shape. However, when
the results of all four strains are analyzed according to the Hill
equation, irrespective of the significantly changed affinity for
Na+, the best fit for the value of (Hill)n actually lies
between 2.2 and 2.35 in all four cases (plot not shown).

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Fig. 6.
Na+ dependence of glycine betaine
uptake. The osmolality of all uptake assays was adjusted with
sorbitol to a final value of 600 mosm/kg. Cells were grown overnight in
BHI medium containing 0.2 mM IPTG and 20 mg/l kanamycin.
Uptake was started by the addition of 100 µM labeled
glycine betaine and 10 mM unlabeled ectoine. Strains
DHP1/pGTG (solid circles), DHP1/pC1 (stars), DHP1/pC2 (triangles), and DHP1/pC3 (squares) were
analyzed. All rates were normalized by setting the uptake rate in the
different strains at full stimulation with NaCl to 100% as described
in the legend to Fig. 1.
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For a further characterization of the mutant proteins, the
Km values for glycine betaine were determined also
(Table III). Except for strains DHP1/pC1
and DHP1/pC1N0, Km was not altered as compared with
the wild type. Strains DHP1/pC1 and DHP1/pC1N0 showed a 6- to 10-fold
lowered substrate affinity; however, the uptake rate of these strains
was quite low, thereby rendering the discrimination against additional
betaine uptake systems by use of the competitor ectoine problematic.
The Vmax values of uptake of the other strains
varied from 35 to 61 nmol·min
1·(mg of dry
weight)
1. Despite various attempts, we did not succeed in
obtaining a specific antiserum against BetP. Thus, we could not decide
whether the differences in the maximum rates resulted from a different degree of expression or whether they were caused by the mutations directly. Consequently, in contrast to the Km
values, the observed maximum rates are not a meaningful
characterization of the different mutant strains.
Regulation of BetP Mutant Proteins under Conditions of
Kinetic Equilibrium--
So far we have used initial uptake
rates to characterize the various mutant carrier proteins. To test
whether the regulation under steady state conditions is changed in the
mutant proteins, betaine uptake was analyzed under equilibrium
conditions by measuring the internal [14C]glycine
betaine for 2.5 h after a hyperosmotic shift in strain DHP1/pC2 as
compared with the wild type (Fig. 7,
A and B). Both strains accumulated betaine until
a steady state was reached at about 1 h, whereafter internal
betaine remained virtually constant (130 mM for DHP1/pGTG
and 210 mM for DHP1/pC2). The observed steady state can be
explained by at least three different models. (i) The activity of BetP
is switched off when the internal osmolality matches that of the
medium, (ii) BetP remains active, and the observed constant
concentration of betaine is the sum of uptake and efflux via BetP,
(iii) BetP remains active, and the observed constant internal label is
the sum of the uptake activity of BetP and the efflux via another
system.

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Fig. 7.
Time course of internal
[14C]glycine betaine accumulation. Strains DHP1/pGTG
(A) and DHP1/pC2 (B) were used. Cells were grown
overnight in BHI complex medium containing 0.2 mM IPTG and 20 mg/l kanamycin. The osmolality of the uptake assay was adjusted with
NaCl to a final value of 720 mosM. Uptake was started by the addition of 1.5 mM labeled glycine betaine
corresponding to a value of 10,000 dpm/mg dry weight (solid
symbols). Unlabeled glycine betaine in 33-fold excess, which
corresponds to a final concentration of 50 mM, was added to
an aliquot of cells after 5 min or after 1.5 h (open
symbols).
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Recently we have shown that C. glutamicum is able to release
compatible solutes via stretch-activated (mechanosensitive) channels which are tightly regulated under hypoosmotic conditions (outside low)
(27). We did not find any indication for a betaine efflux carrier, as
reported for Lactobacillus plantarum (28). To discriminate between the three different possibilities, we added unlabeled betaine
in 33-fold excess to cells 1.5 h after the addition of labeled
betaine, i.e. after the steady state of betaine accumulation had been reached (Fig. 7). The addition of 50 mM unlabeled
substrate did not lead to a major decrease of internal label in strain
DHP1/pGTG. The low efflux of glycine betaine thus indicates that both
the reverse reaction of BetP and the activity of a putative efflux channel must be very low, i.e. model (i) held true in this
case. In contrast, the addition of unlabeled betaine to strain DHP1/pC2 resulted in a rapid decrease of internal label. Consequently, the
observed steady state must be the sum of both uptake and efflux, i.e. models (ii) or (iii) are valid. Thus, either the mutant
BetP is functionally changed, being now active in a (futile) exchange under iso-osmotic conditions, or the mutant protein catalyzes uptake of
unlabeled substrate, and the label is released from the cytosol via the
efflux channel. For a discrimination, unlabeled substrate was added
after 5 min, i.e. when betaine was still taken up at the
maximum rate. As expected, the internal label remained constant in
strain DHP1/pGTG, indicating again that no efflux occurred (Fig.
7A). The uptake kinetics seems to stops at the point of
addition because of the low specific label of external betaine after
the addition of unlabeled substrate. In strain DHP1/pC2 no efflux of
label occurred within the first 30 min after addition of unlabeled
substrate (Fig. 7B). Interestingly, the onset of the efflux
occurred at the same time, when the cells without betaine addition were
approaching the steady state. On the basis of these results models (ii)
and (iii) can be discriminated. Since no initial efflux was observed
after the addition of unlabeled betaine to cells that carry the mutant
protein and were actively taking up betaine, the mutant carrier also
does not catalyze counter-exchange under these conditions, identical to
the wild type protein. Consequently, the rapid decay of internal label
on the addition of unlabeled substrate after 1.5 h is not due to
exchange activity of the modified BetP but represents the sum of uptake
activity of BetP and efflux activity via the channel.
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DISCUSSION |
Osmotic stress is a major challenge in the habitat of a soil
bacterium like C. glutamicum. Consequently, this organism is not only equipped with a number of transport systems responding to
osmotic stress, but these systems are also efficiently regulated, both
on the level of expression and, most important for an instant stress
response, on the activity level. We have previously described that the
glycine betaine carrier BetP, the major osmo-reactive uptake system in
C. glutamicum, effectively responds to osmotic changes
within a time range of seconds or less (14). We were interested in the
mechanism of how the BetP protein senses the osmotic stress. The data
presented here indicate that both the negatively charged N- and the
positively charged C-terminal extension of BetP are involved in sensing
and/or transducing osmotic changes to the domain of the protein
responsible for the translocation of the substrate glycine betaine.
The usual way to exert osmotic stress to a bacterial cell is the
addition of salts, e.g. NaCl. In the case of BetP being a Na+-coupled secondary system (14), the addition of NaCl
changes not only the osmolality but also the ionic strength as well as the concentration of the coupling ion. To discriminate between these
mechanisms, we extended our previous studies of
NaCl-dependent regulation of BetP to the uncharged solute
sorbitol and to the membrane-active amphipathic local anesthetic
tetracaine. Tetracaine is known to change the physical state of the
membrane (21-24), which is the structural basis of osmo-stress
(mechanostress) on the level of membrane structure. In a similar study
on osmo-sensing by the KdpD protein, a sensor kinase of the E. coli Kdp system, local anesthetics were found to modulate the
activity of the membrane-inserted protein (25). Both the addition of
sorbitol and of local anesthetics led to an activation of the BetP
protein, similar to that evoked by the addition of NaCl. This indicates
that in fact membrane/protein interaction is the basis of the
transduction of osmo-stress to the carrier protein. Interestingly,
additional transport systems involved in osmo-regulation in C. glutamicum do not seem to respond to addition of tetracaine (Fig.
1B), which indicates again that the effect of the local
anesthetic is not at all simply an unspecific membrane action.
The charged N- and C-terminal extensions of BetP are an obvious target
for studies on osmotic regulation, especially in view of the hypothesis
on ProP function in E. coli, which shows a comparable C-terminal extension (12). All four different deletion mutants in the
N-terminal region of BetP were active in transport. Furthermore, even
the carrier protein with a completely missing N-terminal extension did
not show a change in the binding affinity toward glycine betaine. On
the other hand, the pattern of osmo-regulation of these recombinant
proteins on the level of activity was altered. We observed an optimum
of stimulation at a 2-fold higher external osmolality, i.e.
at a NaCl concentration where the wild type protein is already largely
inhibited.
The consequences of changes in the C-terminal region were completely
different. Truncation of only 12 amino acid residues (C3 mutant) led to
activity of the carrier at low external osmolality, i.e. in
a range where the wild type protein is inactive. Deletion of 23 and 32 residues in the C2 and C1 mutants, respectively, resulted in proteins
that were not only active at low osmolality but remained active over a
broad range of osmolality. The full extent of the change in regulation
properties of BetP proteins with truncated C-terminal extensions became
obvious after a detailed functional analysis. Proteins with a
significantly shortened C-terminal extension (C2 and C1 mutants) had
completely lost regulation by osmotic stress both at high and low
osmolality. The initial rise in activity, which seemed to be similar to
the wild type, was found to be solely the consequence of a changed
affinity for the co-substrate Na+. In the presence of a low
basic concentration of Na+, we demonstrated by the use of
sorbitol instead of NaCl that these mutant BetP proteins are in fact
not sensitive to osmo-stress anymore. The dominance of the C-terminal
extension for this regulation was corroborated by the construction of
double mutants with deletions both in the N- and in the C-terminal
region. The double mutants turned out to be functionally identical to
the respective proteins with altered C-terminal region only.
In summary, the following pattern of consequences of truncating
terminal regions of BetP emerges. Deletions in the N-terminal extension
cause a shift to higher values of the optimum of osmotic stimulation. A
truncation in the C-terminal region by 12 amino acids (C3) lowers the
optimum of activation. Larger deletions of 23 (C2) or 32 amino acids
(C1) lead to a complete loss of the response to osmotic stress as well
as to a significant decrease in the Na+ affinity. In
addition, deletion of 32 amino acids (C1) results in a decrease of the
affinity toward glycine betaine. After truncation of the whole
C-terminal extension, no transport activity was detected anymore.
These findings to some extent resemble a hypothesis that was put
forward for the E. coli ProP protein (12). The E. coli proline/betaine transporter ProP also functions both as
osmo-sensor and osmo-regulator and carries a C-terminal extension of
about 50 amino acids, which was supposed to be involved in
osmo-sensing. Furthermore, a related hypothesis on the location of
sensory and regulatory domains in a membrane protein involved in the
response to osmotic stress was suggested for the KdpD sensor kinase in E. coli. In this case, however, transmembrane segments were
made responsible for the putative sensory domain (29).
The results on the BetP protein provide the first step for a molecular
analysis of membrane-protein interactions involved in osmo-sensing by a
bacterial carrier system. Moreover, our results demonstrate that the
altered regulation of the mutant proteins has severe consequences for
the response of the cell to osmostress. C. glutamicum
carrying the C2 mutant with the truncated C-terminal region reached a
significantly higher level of steady state accumulation of betaine.
This indicates that the basis for the kinetic steady state in wild type
cells and in cells with the mutant BetP carrier are different. A
kinetic analysis of the situation revealed that this is in fact the
case. By measuring exchange of labeled (internal) and unlabeled
(external) betaine in the wild type and in mutant strains, we showed
that the steady state in the wild type is based on a down-regulation of
BetP activity due to the fact that the stimulus, i.e. the
osmotic stress, is abolished after a high internal concentration of
compatible solute is reached (14). In contrast, in the mutant strain
carrying the BetP protein that is insensitive to hyperosmotic stress,
the observed steady state was found to be the result of an unchanged
uptake activity counter-balanced by glycine betaine efflux via a
different pathway, independent from BetP. Consequently, in this case, a
futile cycle is created. The efflux pathway, being a security valve
under these conditions, most likely is the mechanosensitive efflux
channel that we recently described in C. glutamicum
(27).
We thank E. Galinski for a gift of ectoine, J. Wood for stimulating exchange of information, and H. Sahm for
continuous support and helpful discussions.