Activity regulation of the betaine transporter BetP of Corynebacterium
glutamicum in response to osmotic compensation
Johannes Botzenhardt, Susanne Morbach, Reinhard Kr7mer*
Institute of Biochemistry, University of Ko ¨ln, Zu ¨lpicher Str. 47, 50674 Ko ¨ln, Germany
Received 19 August 2004; received in revised form 18 October 2004; accepted 29 October 2004
Available online 11 November 2004
As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation
of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the
pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial
osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in
Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia
coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP,
which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake,
unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The
observed decrease in net uptake was thus mainly due to a decrease of Vmaxof BetP and not a consequence of the presence of separate efflux
system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also
different from previously described adaptation mechanisms in other organisms. Cytoplasmic K+, which was shown to be responsible for
activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results
thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Transport; Osmoregulation; Corynebacterium; Betaine; Uptake
Maintenance of cytoplasmic water activity and cell
turgor is essential for growth and viability in prokaryotic
and eukaryotic cells. Changes in environmental osmolality
lead to water flux into or out of the cell, consequently,
cells have developed a variety of mechanisms to react to
these changes by synthesis, as well as uptake and release
of so-called compatible solutes . One of the first
responses of bacterial cells to hyperosmotic stress is
uptake of compatible solutes which are accumulated to
balance the increased external osmolality. For this
purpose, the transport systems involved must be properly
regulated, osmoreactive transport system(s) should thus be
able to sense the extent of osmotic stress to respond with
an adapted regulation of activity.
In the last years, a number of triggers have been
described as stimuli for osmosensitive transport proteins
in bacteria. Mechanosensitive channels, like MscL, open
upon changes in membrane tension leading to release of
intracellular solutes . With respect to osmoregulated
uptake carriers, three different systems have mainly been
studied. ProP of Escherichia coli, a secondary carrier for
zwitterionic compatible solutes, has first been proven by
reconstitution to harbor both osmosensory and osmor-
egulatory properties [3,4]. For BetP of Corynebacterium
glutamicum, a Na+-dependent secondary betaine uptake
carrier, changes in the lumenal K+concentration were
shown to be an important stimulus related to osmotic
stress when analyzed in proteoliposomes [5,6]. Finally,
0005-2736/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +49 221 470 6461; fax: +49 221 470
E-mail address: firstname.lastname@example.org (R. Kr7mer).
Biochimica et Biophysica Acta 1667 (2004) 229–240
OpuA of Lactococcus lactis, an ABC transporter, was
found to be regulated in the dependence of the lumenal
ion concentration, and this was correlated to changes in
surface properties of the lipid membrane .
Once the hyperosmotic stress is compensated by
accumulation of compatible solutes, active net uptake of
these solutes should cease. Thus, an appropriate mecha-
nism regulating the pool size of compatible solutes by
regulation of uptake and/or efflux systems is necessary.
In contrast to detailed studies on the kind of stimuli
related to carrier activation in response to a hyperosmotic
shock, down-regulation of uptake activity in response to
osmotic compensation has not been studied equally well.
In principle, two different mechanism can be imagined.
Either the activity of the uptake carrier is reduced or a
counteracting efflux mechanism mediated by other
carriers or by efflux channels compensates the uptake
Adaptation of net uptake after compensation of
osmotic stress has been explained in terms of feedback
regulation of the respective transporter. This mechanism
was first postulated for the high-affinity, though not
osmoregulated glycine betaine uptake system of Staph-
ylococcus aureus [8,9]. In Listeria monocytogenes an
osmosensitive secondary betaine uptake carrier was
shown to be inhibited by internal glycine betaine and
carnitine . As an alternative mechanism, in Lactoba-
cillus plantarum separate systems for (primary) uptake
and efflux of betaine were discriminated and the efflux
system was postulated to be inactive under hyperosmotic
conditions . Most of these studies, however, were
done in cells harboring also other transport systems for
these solutes in addition to those which were analyzed
In this work, for studying the response of BetP to
osmotic adaptation, we used cells of C. glutamicum in
which BetP is the only carrier catalyzing uptake of
compatible solutes. We determined betaine fluxes medi-
ated by BetP in different phases of hyperosmotic stress
by dissecting net flux, unidirectional influx and unidirec-
tional efflux. In addition, steady-state betaine concen-
trations after different extents of hyperosmotic shock
were investigated. A number of possible factors and
mechanisms including feedback inhibition were studied in
terms of their influence in triggering adaptation of
transport activity. On the basis of these results, the
response of BetP to increasing osmotic compensation in
the cell was elucidated.
1. Experimental procedures
1.1. Bacterial strains and plasmids
For transport measurements of BetP, strain C. gluta-
micum DHPF (betP?putP?proP?ectP?lcoP?) was
used, which is totally deficient in compatible solute
uptake. This strain was derived from C. glutamicum
DHPE  by deletion of the lcoP gene, which codes
for a fifth uptake system for compatible solutes with
very low activity . DHPF cells were transformed
with plasmids pGTG (encoding BetP) or pC2 (encoding
BetP lacking the last C-terminal 25 amino acids) as
described previously . pGTG and pC2 are derivatives
of pEKEX2, in which the genes are under the control of
the isopropyl-1-thio-h-d-galactopyranoside (IPTG)-induci-
ble ptac promoter. In the experiment reported in Fig. 2B,
C. glutamicum strain Cgl-ProP was used, in which all
carriers except ProP had been deleted (betP?putP?
ectP?lcoP?) [12–14]. For uptake measurements in E.
coli, compatible solute uptake deficient E. coli MKH13
cells  were transformed with pASK-IBA5 strep-betP
, in which the betP gene is fused in frame at its 5V
prime end to the sequence encoding the eight-amino acid
strep tag II. The gene is under control of the tet
1.2. Growth, osmotic shock and transport assays
overnight in MM1 minimal medium , supplemented
with 1 Ag/ml desferoxamine, or BHI brain heart
infusion medium (Difco, Detroit, MI, USA), each
supplemented with0.2 mM
kanamycin. Cells were washed with buffer containing
25 mM KPi pH 7.5 and 25 mM NaPi pH 7.5 at 4 8C,
resuspended in the same buffer at a cell density of
about 0.1 g cdm/L (cell dry mass/L) including 10 mM
glucose. In the experiments shown in Fig. 5, cells were
washed and resuspended in MM1 medium. Cells could
be stored for up to 4 h before application of hyper-
osmotic shocks by addition of NaCl at the indicated
concentrations without loss of BetP transport activity. To
avoid cell growth and changes in protein synthesis
during the experiment, 25 Ag/ml chloramphenicol was
added, except in Fig. 5. E. coli MKH13 was treated
like C. glutamicum with the following alterations: cells
were grown overnight in Luria Bertani medium 
and then transferred to fresh medium. At an OD600 of
1, anhydrotetracycline (IBA, Gfttingen, Germany) was
added for induction of betP for 3 h. K+/Na+phosphate
buffer was replaced by 100 mM Tris/morpholino ethane
sulfonic acid (MES), pH 7.5. The co-solute Na+was
provided by addition of 200 mM NaCl. For transport
assays, 500 AM glycine betaine, [14C]- or [3H]-labeled
betaine, [14C]-labeled proline, or [14C]-labeld glutamine,
respectively, was added 30 s after the hyperosmotic
shock. At time intervals of 15 s after betaine addition,
samples of 200 Al were transferred to glass fiber filters
(Schleicher und Schuell, Dassel, Germany) and washed
twice with 2.5 ml 0.1 M LiCl (C. glutamicum) or 0.5
M sucrose plus 0.05 M MgCl2 (E. coli). The radio-
DHPF pGTG cells were grown
IPTG and50 mg/L
J. Botzenhardt et al. / Biochimica et Biophysica Acta 1667 (2004) 229–240
conditions, e.g., Na+or betaine concentration, were also
ruled out as possible stimuli since they were kept constant
throughout the experiment.
Finally, down-regulation of BetP activity may be
discussed in view of the above mentioned hypothesis
suggesting that internal betaine directly inhibits transport.
The fact that betaine accumulation linearly depends on the
extent of osmotic stress rules out both simple feedback
regulation by betaine and inhibition by the increasing
outward-directed chemical potential of betaine. Both param-
eters would not depend on the extent of osmotic stress. For
carrier systems in S. aureus [8,9], L. plantarum  and L.
monocytogenes , a modified model for feedback
regulation has been suggested. To explain the systems’
adapted response to osmotic stress, it was supposed that
feedback inhibition is released under hyperosmotic stress.
Two results argue against this modified model of
feedback inhibition as the explanation for down-regulation
of BetP activity. Most importantly, BetP activity is
decreased upon osmotic compensation also in the absence
of internal betaine. Furthermore, although both BetP and
BetPD25 catalyze betaine uptake, identical osmotic con-
ditions lead to different betaine accumulation levels. If
feedback inhibition was the trigger for BetP down-regu-
lation, it would have to act on the two BetP forms at
different concentrations of internal solutes. As a very remote
explanation, inhibition by internal proline or trehalose in
principle still remains a possibility to explain the results of
Fig. 4. Both trehalose and proline, however, are not
substrates of BetP, nor are they closely related to betaine
in terms of structure.
It should be pointed out here that the modified feedback
inhibition model has actually never been proven or
disproven also in organism such as L. plantarum or L.
monocytogenes, since a second model, which we prefer
because of its simplicity, has not been ruled out. Down-
regulation of BetP activity in the adaptation phase could be
due to a trigger acting on the protein directly and indicating
increasing osmotic compensation. This trigger may be
related to the physical state of the membrane (membrane
strain) or, alternatively, to the hydration state of BetP as
influenced by the amount of free water in the cytoplasm.
The latter explanation, however, does not seem to be very
likely since the measured change of free water under the
conditions studied here was not impressive (Fig. 3). On the
other hand, osmoregulated carrier proteins have in fact been
shown to be influenced by the surrounding membrane
[6,35]. It has also to be emphasized that a modified model of
feedback inhibition is necessarily more complicated. It
essentially requires, besides the presence of a second,
regulatory binding site, also a second stimulus (signal
input), which is responsible for the release of feedback
inhibition under hyperosmotic stress. The release of feed-
back inhibition, then, has to depend linearly on the extent of
stress. It furthermore requires the affinity of the putative
regulatory site for solutes which are not substrates of the
carrier. Notably, the feedback inhibition model was recently
found not to be valid for osmosensitive betaine uptake in
intact cells of L. lactis  as well as for reconstituted
OpuA from L. lactis .
We thank Dr. J. Kunte, M. Stein, and A. Wirtz for help in
the HPLC analysis of betaine. This work was financially
supported by the DFG (SP 1070).
 J.M. Wood, Osmosensing by bacteria: signals and membrane-based
sensors, Microbiol. Mol. Biol. Rev. 63 (1999) 230–262.
 S. Sukharev, M. Betanzos, C.S. Chiang, H.R. Guy, The gating
mechanism of the large mechanosensitive channel MscL, Nature 409
 K.I. Racher, R.T. Voegele, E.V. Marshall, D.E. Culham, J.M. Wood,
H. Jung, M. Bacon, M.T. Cairns, S.M. Ferguson, W.J. Liang, P.J.
Henderson, G. White, F.R. Hallett, Purification and reconstitution of
an osmosensor: transporter ProP of Escherichia coli senses and
responds to osmotic shifts, Biochemistry 38 (1999) 1676–1684.
 D.E. Culham, J. Henderson, R.A. Crane, J.M. Wood, Osmosensor
ProP of Escherichia coli responds to the concentration, chemistry, and
molecular size of osmolytes in the proteoliposome lumen, Biochem-
istry 42 (2003) 410–420.
 R. Rqbenhagen, H. Roensch, H. Jung, R. Kr7mer, S. Morbach,
Osmosensor and osmoregulator properties of the betaine carrier BetP
from Corynebacterium glutamicum in proteoliposomes, J. Biol.
Chem. 275 (2000) 735–741.
 R. Rqbenhagen, S. Morbach, R. Kr7mer, The osmoreactive betaine
carrier BetP from Corynebacterium glutamicum is a sensor for
cytoplasmic K+, EMBO J. 20 (2001) 5412–5420.
 T. van der Heide, M.C. Stuart, B. Poolman, On the osmotic signal and
osmosensing mechanism of an ABC transport system for glycine
betaine, EMBO J. 20 (2001) 7022–7032.
 B. Pourkomailian, I.R. Booth, Glycine betaine transport by Sta-
phylococcus aureus: evidence for feedback regulation of the activity
of the two transport systems, Microbiology 140 (1994) 3131–3138.
 K.W. Stimeling, J.E. Graham, A. Kaenjak, B.J. Wilkinson,
Evidence for feedback (trans) regulation of, and two systems for,
glycine betaine transport by Staphylococcus aureus, Microbiology
140 (1994) 3139–3144.
 A. Verheul, E. Glaasker, B. Poolman, T. Abee, Betaine and l-carnitine
transport by Listeria monocytogenes Scott A in response to osmotic
signals, J. Bacteriol. 179 (1997) 6979–6985.
 E. Glaasker, W.N. Konings, B. Poolman, Glycine betaine fluxes in
Lactobacillus plantarum during osmostasis and hyper- and hypo-
osmotic shock, J. Biol. Chem. 271 (1996) 10060–10065.
 H. Peter, B. Weil, A. Burkovski, R. Kr7mer, S. Morbach, Coryne-
bacterium glutamicum is equipped with four secondary carriers for
compatible solutes: identification, sequencing and characterization of
the proline/ectoine uptake system ProP, and the ectoine/proline/
glycine betaine carrier EctP, J. Bacteriol. 180 (1998) 6005–6012.
 R. Steger, M. Weinand, R. Kr7mer, S. Morbach, LcoP, an osmoregu-
lated betaine/ectoine uptake system from Corynebacterium glutami-
cum, FEBS Lett. 573, 155–160.
 H. Peter, A. Burkovski, R. Kr7mer, Isolation, characterization, and
expression of the Corynebacterium glutamicum betP gene, encoding
the transport system for the compatible solute glycine betaine,
J. Bacteriol. 178 (1996) 5229–5234.
J. Botzenhardt et al. / Biochimica et Biophysica Acta 1667 (2004) 229–240
 H. Peter, A. Burkovski, R. Kr7mer, Osmosensingby N- and C-terminal
extensions of the glycine betaine uptake system BetP of Corynebacte-
rium glutamicum, J. Biol. Chem. 273 (1998) 2567–2574.
 M. Haardt, B. Kempf, E. Faatz, E. Bremer, The osmoprotectant
proline betaine is a major substrate for the binding-protein-dependent
transport system ProU of Escherichia coli K-12, Mol. Gen. Genet.
246 (1995) 783–786.
 D. Nottebrock, U. Meyer, R. Kr7mer, S. Morbach, Molecular and
biochemical characterization of mechanosensitive channels in
Corynebacterium glutamicum, FEMS Microbiol. Lett. 218 (2003)
 T. Maniatis, E.F. Fritsch, J. Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, USA, 1982.
 B. Landfald, A.R. Strom, Choline-glycine betaine pathway confers a
high level of osmotic tolerance in Escherichia coli, J. Bacteriol. 165
 H. Rottenberg, The measurement of membrane potential and delta-
pH in cells, organelles, and vesicles, Methods Enzymol. 55 (1979)
 E.A. Galinski, R.M. Herzog, The role of trehalose as a substitute for
nitrogen-containing compatible solutes (Ectothiorhodospira halochlo-
ris), Arch. Microbiol. 153 (1990) 607–613.
 M. Farwick, R.M. Siewe, R. Kr7mer, Glycine betaine uptake after
hyperosmotic shift in Corynebacterium glutamicum, J. Bacteriol. 177
 R.M. Siewe, B. Weil, R. Kr7mer, Glutamine uptake by a sodium-
dependent secondary transport system in Corynebacterium glutami-
cum, Arch. Microbiol. 164 (1995) 98–103.
 S. Ruffert, C. Lambert, H. Peter, V.F. Wendisch, R. Kr7mer,
Efflux of compatible solutes in Corynebacterium glutamicum
mediated by osmoregulated channel activity, Eur. J. Biochem.
247 (1997) 572–580.
 D. McLaggan, J. Naprstek, E.T. Buurman, W. Epstein, Interdepend-
ence of K+and glutamate accumulation during osmotic adaptation of
Escherichia coli, J. Biol. Chem. 269 (1994) 1911–1917.
 E. Glaasker, E.H. Heuberger, W.N. Konings, B. Poolman, Mechanism
of osmotic activation of the quaternary ammonium compound
transporter (QacT) of Lactobacillus plantarum, J. Bacteriol. 180
 A. Wolf, R. Kr7mer, S. Morbach, Three pathways for trehalose
metabolism in Corynebacterium glutamicum ATCC13032 and their
significance in response to osmotic stress, Mol. Microbiol. 49 (2003)
 D. Schiller, R. Kr7mer, S. Morbach, Cation specificity of osmosensing
by the betaine carrier BetP of Corynebacterium glutamicum, FEBS
Lett. 563 (2004) 108–112.
 B. Poolman, E. Glaasker, Regulation of compatible solute accumu-
lation in bacteria, Mol. Microbiol. 29 (1998) 397–407.
 T. van der Heide, B. Poolman, Glycine betaine transport in
Lactococcus lactis is osmotically regulated at the level of expression
and translocation activity, J. Bacteriol. 82 (2000) 203–206.
 D.E. Culham, B. Tripet, K.I. Racher, R.T. Voegele, R.S. Hodges, J.M.
Wood, The role of the carboxyl terminal alpha-helical coiled-coil
domain in osmosensing by transport ProP of Escherichia coli, J. Mol.
Recognit. 13 (2000) 309–322.
 B. Poolman, P. Blount, J.H. Folgering, R.H. Friesen, P.C. Moe, T. van
der Heide, How do membrane proteins sense water stress? Mol.
Microbiol. 44 (2002) 889–902.
 S. Morbach, R. Kr7mer, Body shaping under water stress: osmosen-
sing and osmoregulation of solute transport in bacteria, ChemBio-
Chem 3 (2002) 384–397.
 J.S. Patzlaff, T. van der Heide, B. Poolman, The ATP/substrate
stoichiometry of the ATPbinding cassette (ABC) transporter OpuA,
J. Biol. Chem. 278 (2003) 29546–29551.
 T. van der Heide, B. Poolman, Osmoregulated ABC-transport system
of Lactococcus lactis senses water stress via changes in the physical
state of the membrane, Proc. Natl. Acad. Sci. U. S. A. 97 (2000)
J. Botzenhardt et al. / Biochimica et Biophysica Acta 1667 (2004) 229–240