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Journal of Bacteriology, November 1998, p. 5540-5546, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mechanism of Osmotic Activation of the Quaternary
Ammonium Compound Transporter (QacT) of Lactobacillus
plantarum
Erwin
Glaasker,
Esther
H. M. L.
Heuberger,
Wil N.
Konings, and
Bert
Poolman*
Department of Microbiology, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, NL-9751 NN Haren, The Netherlands
Received 10 June 1998/Accepted 19 August 1998
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ABSTRACT |
The accumulation of quaternary ammonium compounds in
Lactobacillus plantarum is mediated via a single transport
system with a high affinity for glycine betaine (apparent
Km of 18 µM) and carnitine and a low affinity
for proline (apparent Km of 950 µM) and other
analogues. Mutants defective in the uptake of glycine betaine were
generated by UV irradiation and selected on the basis of resistance to
dehydroproline (DHP), a toxic proline analogue. Three independent
DHP-resistant mutants showed reduced glycine betaine uptake rates and
accumulation levels but behaved similarly to the wild type in terms of
direct activation of uptake by high-osmolality conditions. Kinetic
analysis of glycine betaine uptake and efflux in the wild-type and
mutant cells is consistent with one uptake system for quaternary
ammonium compounds in L. plantarum and a separate system(s)
for their excretion. The mechanism of osmotic activation of the
quaternary ammonium compound transport system (QacT) was studied. It
was observed that the uptake rates were inhibited by the presence of
internal substrate. Upon raising of the medium osmolality, the QacT
system was rapidly activated (increase in maximal velocity) through a
diminished inhibition by trans substrate as well as an
effect that is independent of intracellular substrate. We also studied
the effects of the cationic amphipath chlorpromazine, which inserts
into the cytoplasmic membrane and thereby influences the uptake and
efflux of glycine betaine. The results provide further evidence for the
notion that the rapid efflux of glycine betaine upon osmotic downshock
is mediated by a channel protein that is responding to membrane stretch
or tension. The activation of QacT upon osmotic upshock seems to be
brought about by a turgor-related parameter other than membrane stretch or tension.
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INTRODUCTION |
Bacteria protect themselves against
high external osmolality by the uptake or synthesis of a limited
number of so-called compatible solutes. The predominant compatible
solute in many organisms is glycine betaine, which usually is
accumulated through an osmoregulated uptake system. Analogues of
glycine betaine have been found in several bacteria, and many glycine
betaine uptake systems facilitate their uptake as well. The osmotic
regulation of the transport systems may occur at the genetic or
enzymatic level or both, and these aspects have been studied in most
detail with enteric bacteria. In Escherichia coli glycine
betaine (and proline) is taken up via a low-affinity secondary
transport protein (ProP) and a high-affinity ATP-binding cassette
transport system (ProU) (1). The transport activity of both
ProP and ProU proteins is stimulated by an increase in external
osmolality, although the mechanisms of osmosensing most likely are
different (2, 9, 15, 19). Homologues of ProU have been
identified in the gram-positive bacterium Bacillus subtilis
(6, 7), whereas a homologue of ProP is present in
Erwinia chrysanthemi (5). Important structural
information regarding the nature of the osmosensing domain has recently
been obtained for the BetP protein of Corynebacterium
glutamicum, a member of the third family of osmoregulated
uptake systems for glycine betaine and proline (18). There
is clear evidence that the carboxyl-terminal region (55 amino acids)
has a central role in osmosensing.
Glycine betaine is the major compatible solute in the cytoplasm of
Lactobacillus plantarum grown in chemically defined
high-salt media containing glycine betaine. L. plantarum
is unable to synthesize or metabolize glycine betaine, and the
final accumulation levels of glycine betaine are thus determined
solely by the relative rates of uptake and efflux (3).
Previous studies have indicated that osmotic regulation of glycine
betaine uptake acted mainly on the transporter activity, whereas
changes in protein synthesis were relatively small compared to those
for systems such as ProU (13). However, the possibility that
more than one system effected the uptake could not be excluded,
whereas efflux of glycine betaine upon osmotic downshock seemed to
be mediated by more than one efflux system (4). Uptake
of glycine betaine in L. plantarum is driven by ATP and
is most likely mediated by a binding-protein-dependent system(s)
(unpublished results). In this study, mutants defective in glycine
betaine uptake were generated and characterized to elucidate the
contribution of the transport systems to the overall flux of glycine
betaine. We also describe the substrate specificity and the kinetics of
the glycine betaine uptake system under high- and low-osmolality
conditions, as well as the effect of a cationic amphipath on the uptake
and efflux activities in L. plantarum.
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MATERIALS AND METHODS |
Bacterial strains, culture conditions, and media.
L.
plantarum ATCC 14917 was grown at 30°C and pH 6.7 in a
chemically defined medium (CDM) or modified CDM (without proline) containing 0.5% (wt/vol) glucose, as described previously
(3). High-osmolality media were obtained by adding 0.8 M KCl
to the standard CDM.
Isolation of mutants defective in glycine betaine uptake.
A
3-ml aliquot of exponentially growing cells
(A660 of 0.2 to 0.6) in low-osmolality CDM was
dispersed over a petri dish (diameter, 9 cm) and irradiated for 1 min
with UV light (254 nm) at a distance of 24 cm from the petri dishes.
The survival rate was around 1% as estimated from the plating of
irradiated and nonirradiated samples on MRS agar plates. The irradiated
culture was washed and concentrated in CDM without proline and
subsequently plated confluently on CDM agar plates without proline. The
toxic proline analogue dehydroproline (DHP) (20 µl of a 100 mM
solution) was spotted in the middle of the plates (17).
After 48 h of incubation, putative DHP-resistant mutants were
picked from the colony-free zone around the DHP spots. DHP is a toxic
proline analogue that was found to competitively inhibit the uptake of
glycine betaine and proline. Transport of leucine and glutamic acid was
not affected by DHP (data not shown). Several independently isolated
DHP-resistant mutants were able to grow on CDM agar without proline in
the presence of 1 mM DHP. The protein patterns of three mutants
(DHPR-38.1, -38.2, and -38.3) were analyzed on a Coomassie
blue-stained sodium dodecyl sulfate-polyacrylamide gel and were found
to be similar to each other. The patterns of the mutants differed from that of the wild type in four protein bands with apparent molecular masses of 120, 90, 75, and 31 kDa (data not shown). These protein bands
are missing, or at least significantly reduced in the three mutants.
Transport assays.
Transport assays, enzymatic synthesis of
[14C]glycine betaine from
[N-methyl-14C]choline (40 to 60 mCi/mmol), and
assays of uptake of [14C]proline (260 mCi/mmol) were
performed as described previously (3, 4). Briefly, the cells
were washed and resuspended in 50 mM potassium phosphate (pH 6.5) plus
0.8 M KCl or in 50 mM potassium phosphate (pH 6.5). The latter buffer
system was used to release most of the compatible solutes that were
accumulated during the growth under high-osmolality conditions. Prior
to the transport assays, cells were diluted to a protein concentration of 0.1 to 0.4 mg/ml in 50 mM potassium phosphate (pH 6.5) plus 0.8 M
KCl or in 50 mM potassium phosphate (pH 6.5) containing 10 mM glucose,
and the mixtures were incubated at 30°C. Following a period of
preenergization (see figure legends), radiolabeled substrate was added
at time zero, and the reactions were terminated at given time intervals
by rapid filtering of 100- to 200-µl samples on 0.45-µm-pore-size
cellulose nitrate filters. The filters were washed with 2 ml of LiCl
(0.1 to 0.9 M, depending on the osmolality of the assay medium). To
monitor the exit of [14C]glycine betaine or
[14C]proline upon osmotic downshock, the cells were
allowed to accumulate these compounds for a given period of time, after
which the media were diluted with buffer (30°C) containing glucose
plus [14C]glycine betaine or [14C]proline;
care was taken to keep all of the parameters except the osmolality
(i.e., KCl concentration) constant. The data from the kinetic
experiments were fitted with the Michaelis-Menten equation, from which
the apparent affinity constant (Km) and maximal rate of uptake (Vmax) were calculated.
Miscellaneous.
Protein was determined by the method of Lowry
et al. (12) with bovine serum albumin as a standard. Total
protein extracts of wild-type and DHP-resistant mutants of
L. plantarum were subjected to sodium dodecyl
sulfate-polyacrylamide (10% wt/vol) gel electrophoresis after lysis of
the cells by sonication. The osmolalities of media and buffers were
measured by freezing-point depression with an Osmomat 030 apparatus
(Gonotec, Berlin, Germany). Growth experiments were performed in
sterile low-protein-binding microplates. Plate wells containing 200 µl of culture were sealed by adding 75 µl of sterile silicone oil
(1.03 g/ml), and growth rates were determined from
A620 increases by using a Multiscan MCC/340 MKII
instrument (Flow Laboratories, Lugano, Switzerland). For the
calculation of intracellular concentrations, a value of 3 µl/mg of
protein for the specific internal volume was used.
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RESULTS AND DISCUSSION |
Substrate specificity of the glycine betaine uptake system.
In
several gram-positive and gram-negative bacteria, proline and glycine
betaine are taken up via the same system despite their difference in
molecular structure (15, 16, 20). In L. plantarum, a 100-fold excess of unlabeled proline did not affect glycine betaine uptake, whereas proline transport was completely inhibited by a 100-fold excess of unlabeled glycine betaine (Table 1). Experiments with other substrate
analogues showed that glycine betaine uptake was strongly inhibited by
tetramethylammonium and even by dimethylsulfonium propionic acid.
The substrate analogue carnitine abolished the uptake to the
same extent as glycine betaine itself. Other substrate analogues, like
dimethylglycine, trimethylamine, and 4-aminobutyric acid, had no
significant inhibitory effect on the glycine betaine uptake rates. In
fact, some of these solutes stimulated the glycine betaine uptake
consistently. Since the analogues were added at a concentration of 125 mM, the increased uptake most likely reflects osmotic activation of the
glycine betaine uptake system (see below), which may even have masked small inhibitory effects. The osmotic activation of the transport system by 125 nM solute makes the extent of inhibition in all cases an
underestimate; e.g., in the case of proline, the osmotic activation and
competitive inhibition cancel each other out under these conditions
(see below).
All of the tested compounds with two or three methyl groups attached to
the nitrogen or sulfur atom inhibited the uptake of
proline completely.
These results are consistent with a single
uptake system, with a
high affinity for glycine betaine (and carnitine)
and a low
affinity for proline (and other analogues). To substantiate
this
conclusion, we determined the kinetic parameters for glycine
betaine and proline uptake under various osmotic conditions (see
the
next section). Of the solutes tested, glycine betaine and
carnitine
offered the most osmoprotection in growth experiments,
proline and
dimethylglyine had lesser effects, and 4-aminobutyric
acid offered no
protection (see also references
3 and
8).
The transport system was termed QacT (for
quaternary ammonium
compound transporter), as the quaternary
ammonium compounds are
physiologically most relevant for
osmoprotection.
Kinetic analysis of glycine betaine and proline uptake.
With
cells grown in CDM and uptake assayed at low osmolality, the uptake of
glycine betaine was monophasic with an apparent Km of 18 µM and a Vmax
of 27 nmol/min per mg of protein (Table 2, line
1). Proline uptake was also monophasic
under these conditions, with an apparent Km of
950 µM and a Vmax of 21 nmol/min per mg of
protein (Table 2, line 7). The apparent Km for
glycine betaine increased to 33 µM, and the
Vmax increased to 105 nmol/min per mg of
protein, when high-osmolality assay media were used (Table 2, line 1).
Similar changes were found for the uptake of proline; the apparent
Km and Vmax for proline
increased to 1,500 µM and 150 nmol/min per mg of protein,
respectively, at high assay osmolality (Table 2, line 7). The uptake of
glycine betaine and proline in cells cultured at high osmolality was
also studied, but the kinetic parameters were not significantly
different from those of low-osmolality-grown cells (Table 2, compare
lines 1 and 5). These results indicate that the increased rate of
uptake upon an osmotic upshift mainly involves an increased
Vmax as a result of activation of QacT. Since
the effects of culture and assay conditions on
Km and Vmax are similar
for glycine betaine and proline uptake and the uptake is monophasic
under all conditions tested, the data are best explained by uptake via
a single system.
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TABLE 2.
Kinetic parameters of glycine betaine and proline uptake
in wild-type L. plantarum ATCC 14917 and a
DHP-resistant mutanta
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Effect of internal substrate.
Low- or high-osmolality growth
media do not significantly affect the expression of QacT. However, it
was observed that the final accumulation levels as well as the rates of
glycine betaine uptake were lower when the cells were cultured in the
presence of glycine betaine or proline, i.e., under conditions in which the cells contained large amounts of glycine betaine or proline (data
not shown). We also observed that the rate of activated uptake above a
certain threshold upshock (200 mM KCl) was similar irrespective of the
size of the osmolality change but that the extent of uptake (final
accumulation level) was in proportion to the shift (3).
Thus, the larger the upshock, the longer the system remained in the
activated state. These experiments, however, were performed with washed
cell suspensions that had already accumulated glycine betaine to about
600 nmol/mg of protein. Consequently, the glycine betaine uptake
system might have already been inhibited by internal substrate, and
differences in the uptake rates as a function of the size of the
osmolality shift may have been overlooked. In a new series of
experiments, the initial rate of glycine betaine uptake in cells that
did not contain any glycine betaine was measured. Moreover, the washing
of these cells in hypotonic medium (50 mM potassium phosphate, pH 6.5)
released most compatible solutes, including proline. In these cells the rate of uptake increased with medium osmolality, in particular in the
range of 35 to 200 mM (Fig. 1). Thus,
although we concluded previously that the activation of the uptake
system occurred through an on-off mechanism, this conclusion appears to
have been somewhat premature. It was based on the rates of glycine
betaine uptake in cells already containing internal glycine betaine and
under conditions where the medium osmolality was increased by adding 0.2 to 1.2 M KCl. Since some differences in the uptake rate were also
observed in the range above 0.2 M KCl (Fig. 1), which was not seen
before, we conclude that the presence or absence of internal glycine betaine might have contributed to these variations.

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FIG. 1.
Dependence of the initial rate of glycine betaine uptake
on increases in medium osmolality imposed by KCl. Cells of
L. plantarum ATCC 14917 were grown on CDM containing
0.8 M KCl, washed, and resuspended in 50 mM potassium phosphate (pH
6.5). The concentrated cell suspensions were diluted in 50 mM potassium
phosphate (pH 6.5) to a final protein concentration of 0.13 mg/ml.
After 8 min of preenergization with 10 mM glucose, uptake was initiated
at time zero by the addition [14C]glycine betaine (final
concentration of 1.3 mM) plus KCl as indicated. The initial
uptake rates were determined and plotted against the concentration of
KCl that was added to the 50 mM potassium phosphate.
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To discriminate between possible effects on the expression of QacT and
feedback inhibition by cytoplasmic proline, cells were
grown on CDM
without proline. Half of the cells were allowed to
take up unlabeled
proline (final concentration, 1.3 mM) in the
presence of 50 µg of
chloramphenicol per ml; the other half were
treated similarly, but
proline was omitted. After 45 min of incubation,
[
14C]glycine betaine (final concentration, 1.3 mM) was
added (time
zero in Fig.
2), and the
uptake was monitored. It was observed
that cells that had accumulated
proline in the cytoplasm exhibited
a lower rate of uptake of glycine
betaine than control cells,
under both high- and low osmolality assay
conditions (Fig.
2).
In other experiments, the cells were energized for
45 min prior
to the addition of 1.3 mM unlabeled proline together with
[
14C]glycine betaine. Under these conditions, the
unlabeled proline
exerted no significant effect on the uptake of
[
14C]glycine betaine, as anticipated from the large
differences in
Km values for these substrates.
Thus, the preloading of the cells
with proline lowers the uptake rate
and final accumulation levels
of glycine betaine, which is consistent
with a mechanism that
involves a specific interaction of internal
proline with the QacT
system. To test this hypothesis further, the
effects of internal
proline on the kinetic parameters of glycine
betaine uptake under
high- and low-osmolality assay conditions in cells
that were grown
on CDM with or without proline were studied. For cells
grown in
low-osmolality media, the
Vmax of
uptake under low-osmolality
assay conditions was about fourfold lower
when proline was a component
of the growth medium, whereas these
differences were much smaller
under high-osmolality assay conditions,
i.e., upon osmotic upshock
(Table
2, lines 1 and 2). This indicates
that internal proline,
and most likely glycine betaine as well,
inhibits QacT, an inhibition
that is largely relieved upon osmotic
upshock. Similar observations
were made for cells grown in
high-osmolality media, although the
increase in
Vmax may not entirely be explained by the relief
of
trans inhibition by internal proline upon osmotic upshock
(compare
lines 5 and 6 in Table
2). In the mutant
DHP
R-38.1, exhibiting reduced glycine betaine uptake (see
below),
the internal proline affected the uptake of glycine betaine in
a manner similar to that in the wild type (Table
2, lines 3 and
4).

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FIG. 2.
Dependence of glycine betaine uptake on the internal
proline concentration. Cells of L. plantarum ATCC 14917 were grown on CDM without proline containing 0.8 M KCl. The cells were
washed and resuspended in 50 mM potassium phosphate (pH 6.5) plus 50 µg of chloramphenicol per ml to a final protein concentration of 0.18 mg/ml. After 40 min of preenergization with 10 mM glucose, uptake was
initiated at time zero by the addition of 1.3 mM
[14C]glycine betaine (no add) or 1.3 mM
[14C]glycine betaine plus 1.3 mM unlabeled proline
[Pro(0)]. Alternatively, the cells were preenergized for 40 min with
10 mM glucose plus 1.3 mM unlabeled proline, and uptake was initiated
at time zero by the addition of 1.3 mM [14C]glycine
betaine [Pro( 40)]. The uptake was assayed without (open symbols) or
with (closed symbols) 0.8 M KCl (final concentration) added together
with the glucose.
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From all of these experiments we conclude the following. (i) Glycine
betaine and proline are most likely taken up via a single
transport
system (QacT). (ii) The QacT system is expressed semiconstitutively;
neither proline, glycine betaine, nor the osmolality of the medium
has
a large effect on the expression per se. (iii) Upon osmotic
upshift the
system is activated through diminished inhibition
by
trans substrate as well as through a turgor-related increase
in the activity. Both effects result in an increase of the
Vmax when the medium osmolality is raised.
Feedback inhibition of glycine
betaine and proline uptake has also been
described for
Staphylococcus aureus (
20,
25).
However, from these studies it is not clear
whether an osmotic upshift
relieves the
trans inhibition. Activation
of uptake through
a relief of
trans inhibition has been described
for the
glycine betaine and carnitine uptake system of
Listeria monocytogenes (
28).
Isolation and physiological properties of DHP-resistant
mutants.
Mutants that are defective in glycine betaine
uptake were isolated to test whether we could dissect the glycine
betaine uptake activity even more rigorously into one or more systems.
In low-osmolality media, the growth of wild-type L. plantarum was severely impaired by the addition of 1 mM DHP to CDM
without proline (the specific growth rate [µ] was decreased from
0.17 to 0.04 h
1), whereas the growth of the mutants was
not affected (µ of 0.17 h
1) (data not shown). The
growth inhibition of wild-type L. plantarum by DHP in
low-osmolality media was counteracted by the addition of proline or
glycine betaine to the medium, which prevents DHP from entering the
cell due to competitive inhibition of DHP uptake. In contrast to the
wild-type cells, the mutants did not grow in CDM plus 0.8 M KCl,
whereas some growth (µ ~ 0.02 h
1) was observed when
glycine betaine (2.5 mM) was present. In high-osmolality media (CDM
plus 0.8 M KCl), wild-type cells grew with a µ of about 0.14 h
1, which increased to 0.17 h
1 when glycine
betaine was present. Together, the data clearly show that the
DHP-resistant mutants are compromised in their ability to accumulate
glycine betaine, which results in an osmosensitive phenotype.
Kinetic characterization of the DHP-resistant mutants.
Uptake
experiments showed that the mutants were highly defective in their
ability to take up glycine betaine under low-osmolality assay
conditions, but the system could still be activated upon osmotic
upshock (Table 2, lines 3 and 4). In fact, the hyperosmotic activation of glycine betaine uptake by the mutants was comparable to
that by the wild type. To establish this point more firmly, we measured
the kinetics of uptake in DHPR-38.1 cells grown in CDM with
and without proline and under assay conditions of high and low
osmolality. As observed for the wild-type cells grown under high- and
low-osmolality conditions, an osmotic upshift increased the
Vmax three- to fourfold when the cells were grown in the presence of proline, whereas the effect was much smaller
for cells lacking proline (Fig. 3; Table
2, lines 3 and 4) (similar results were obtained for the other
mutants). Despite the more than 10-fold reduction of
Vmax under high-osmolality conditions (Table 2,
compare lines 1 and 2 with lines 3 and 4), the mutant cells were still
able to accumulate glycine betaine to concentrations of more than 400 nmol/mg of protein, which is only threefold lower than in the wild-type
(data not shown). Overall, the results show that glycine betaine and
proline uptake is severely impaired in each of the three mutants, most
likely as a result of a lowered expression of QacT. Importantly, the
residual uptake activity can still be activated upon hyperosmotic
shock.

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FIG. 3.
Kinetics of glycine betaine uptake in wild-type and
DHPR-38.1 L. plantarum ATCC 14917. The
cells were grown on CDM or CDM without proline, washed, and resuspended
in 50 mM potassium phosphate (pH 6.5). After 7 min of preenergization
with 10 mM glucose, uptake was initiated by the addition of
[14C]glycine betaine with (squares) or without (circles)
0.8 M KCl. Further details are described in the footnote to Table 2.
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The DHP
R mutants behaved similarly to the wild type with
regard to the rapid and slow components of efflux when an osmotic
downshock was applied (data not shown; see also reference
4 and the next section). The uptake system for
glycine betaine (QacT),
which is significantly hampered in the mutants
(lower
Vmax and
unaltered
Km), is thus not involved in the efflux of
glycine betaine
upon osmotic downshock. This clearly
establishes the independence
of QacT and the efflux systems.
Effect of membrane strain on the uptake and efflux of glycine
betaine.
Previously, we have shown that L. plantarum releases glycine betaine and other compatible solutes in
response to an osmotic downshock (3, 4). Under these
conditions, the uptake of glycine betaine is virtually completely
inhibited. The observed rates of efflux upon osmotic downshock are far
higher than the highest rates of uptake, implying that the net efflux
upon osmotic downshock cannot be explained by the diminished uptake.
The net efflux is largely due to an increased exit of glycine betaine via a system with properties resembling those of mechanosensitive ion
channels (19, 27). Efflux of glycine betaine and other compounds in response to a hypo-osmotic shock has also been observed in
E. coli, S. aureus, C. glutamicum, and several other bacteria (9, 10, 21, 22).
For many of these systems, membrane strain is thought to trigger their
activation or to increase the open probability or the
duration of the
open state in the case of channel proteins. The
partitioning of
amphipaths into the cytoplasmic membrane also
affects the membrane
strain. Cationic amphipaths insert into the
more negatively charged
inner leaflet of the membrane, causing
the cells to form cups (convex
shapes), whereas anionic amphipaths
insert into the less negatively
charged outer leaflet, forming
concave shapes (
23,
24).
Chlorpromazine is an cationic amphipath
that increases the open
probability of mechanosensitive ion channels
in
E. coli
(
14), triggers glutamate excretion in
C. glutamicum (
11), and stimulates the activity of the
KdpD protein (
26).
The addition of 0.1 mM chlorpromazine to
cells of
L. plantarum which had accumulated glycine
betaine under high-osmolality conditions
resulted in a rapid efflux of
glycine betaine that mimics the
efflux elicited by an osmotic downshock
(Fig.
4). An important
difference between
the effluxes triggered by an osmotic downshock
and by chlorpromazine is
that the former is instantaneous (within
1 s), whereas a lag time
of about 15 s was observed for the efflux
triggered by
chlorpromazine (Fig.
4, inset). This lag time may
reflect the time that
chlorpromazine needs to partition into the
lipid bilayer. Nevertheless,
the observation that rapid glycine
betaine exit can be elicited by
osmotic downshock as well as by
chlorpromazine (an amphipatic compound)
is consistent with the
idea that the system is directly regulated via
membrane stretch
or tension. In contrast, under low-osmolality
conditions, chlorpromazine
elicited only a small, transient exit of
glycine betaine (Fig.
4). Thus, membrane strain evoked by
chlorpromazine alone is not
sufficient to activate the efflux system(s)
for long periods of
time; rather, a high intracellular osmolality is
required in order
to observe the effect of chlorpromazine.

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FIG. 4.
Hypo-osmotic shock and chlorpromazine trigger efflux of
[14C]glycine betaine. Cells of L. plantarum ATCC 14917 were grown on CDM containing 0.8 M KCl,
washed, and resuspended in 50 mM potassium phosphate (pH 6.5). The
concentrated cell suspensions were diluted in 50 mM potassium phosphate
(pH 6.5) to a final protein concentration of 0.39 mg/ml. After 6 min of
preenergization with 10 mM glucose, uptake was initiated at time zero
by the addition of [14C]glycine betaine (final
concentration of 1.3 mM) without (open symbols) or with (closed
symbols) 0.8 M KCl (final concentration). After 36.5 min, 0.1 mM
chlorpromazine (final concentration) was added (circles), or the
samples were diluted fivefold with 50 mM potassium phosphate (pH 6.5)
containing 10 mM glucose plus 1.3 mM [14C]glycine betaine
(squares). The inset shows the lag time of the efflux triggered by the
addition of 0.1 mM chlorpromazine (circles); the efflux after
hypo-osmotic shock (squares) is instantaneous, since 2 s is the
time resolution of the experiment.
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As reasoned for the efflux upon osmotic downshock (see the beginning of
this section), the very rapid net efflux of glycine
betaine that is
triggered by chlorpromazine cannot be due solely
to an inhibition of
uptake. Nevertheless, it was important to
establish whether
chlorpromazine affected the uptake under osmostatic
and hyperosmotic
conditions. Chlorpromazine had no effect on the
uptake under osmostatic
conditions, but in the presence of the
amphipath the rates of uptake no
longer increased upon osmotic
upshock (Fig.
5). The final level of accumulation of
glycine betaine
under hyperosmotic conditions was not significantly
affected by
chlorpromazine, suggesting that the system is still
responding
to the lowered turgor pressure. Thus, hyperosmotic
conditions
are sensed in the presence of chlorpromazine, and as a
result,
the lowered rate of uptake does not level off as quickly as it
does under osmostatic conditions (Fig.
5).

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FIG. 5.
Effect of chlorpromazine on the uptake of
[14C]glycine betaine. Cells of L. plantarum ATCC 14917 were grown on CDM containing 0.8 M KCl,
washed, and resuspended in 50 mM potassium phosphate (pH 6.5). The
concentrated cell suspensions were diluted in 50 mM potassium phosphate
(pH 6.5) to a final protein concentration of 0.17 mg/ml. After 6 min of
preenergization with 10 mM glucose, uptake was initiated at time zero
by the addition of [14C]glycine betaine (final
concentration of 1.3 mM) without (closed symbols) or with (open
symbols) 0.1 mM chlorpromazine (final concentration). The uptake was
assayed without (circles) or with (squares) 0.8 M KCl (final
concentration) added together with the [14C]glycine
betaine.
|
|
Concluding remarks.
L. plantarum possesses a
transport system (QacT) that is regulated by turgor and has a broad
specificity for a wide range of compatible solutes. The phenotypes of
three DHP-resistant mutants were identical in terms of kinetics of
uptake of glycine betaine and proline under osmostatic and hyperosmotic
conditions. It is concluded that the expression of the QacT system is
decreased in the mutants, thereby preventing the cells from
accumulating DHP to toxic levels. Neither the kinetic analysis of
uptake in the wild type nor the properties of the mutants provide any
indication for more than one major uptake system for quaternary
ammonium compounds and proline. If more than one transporter is
operative, then all of the systems should have very similar kinetic and
regulatory properties. For instance, if the basal uptake (osmostatic
conditions) and the activated uptake (after osmotic upshift) were
mediated by separate systems, the relative contributions of these
activities to the overall flow would be different in the wild type and
DHPR mutants.
The mutant analysis clearly shows that the QacT system is also distinct
from the osmoregulated systems that mediate the efflux
of compatible
solutes. The QacT system and the putative channel
protein not only are
affected in opposing manners by osmolality
changes in the medium but
also are affected differently by the
cationic amphipath chlorpromazine.
Chlorpromazine mimics an osmotic
downshock in terms of evoking glycine
betaine efflux, which is
most likely due to an increase in the open
probability or the
duration of the open state of the putative channel
protein in
response to membrane strain. The data for the regulation of
QacT
are more ambiguous but indicate that membrane strain is not
directly
involved in the regulation of QacT. Finally, kinetic analysis
of QacT activity reveals a dual mode of regulation, since a
hyperosmotic
shock seems to affect the
Vmax
through a diminishing of the
trans inhibition as well as
through an effect that is independent of
internal substrate.
 |
ACKNOWLEDGMENTS |
This research was funded by Unilever Research Laboratories,
Vlaardingen, The Netherlands.
We thank P. F. ter Steeg and J. P. P. M. Smelt for
stimulating discussions.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands. Phone: 31-50-3632150. Fax:
31-50-3632154. E-mail: B.Poolman{at}biol.rug.nl.
 |
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Journal of Bacteriology, November 1998, p. 5540-5546, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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