Báo cáo khoa học: Connection of transport and sensing by UhpC, the sensor for external glucose-6-phosphate in Escherichia coli ppt

Connection of transport and sensing by UhpC, the sensor
for external glucose-6-phosphate in
Escherichia coli
Christian Schwo¨ ppe
1
, Herbert H. Winkler
2
and H. Ekkehard Neuhaus
1
1
Pflanzenphysiologie, Universita
¨
t Kaiserslautern, Kaiserslautern, Germany;
2
Department of Microbiology and Immunology,
College of Medicine, University of South Alabama, Mobile, AL, USA
UhpC is a membrane-bound sensor protein in Escherichia
coli required for recognizing external glucose-6-phosphate
(Glc6P) and induction of the transport protein UhpT.
Recently, it was shown that UhpC is also able to transport
Glc6P. In this study we investigated whether these transport
and sensing activities are obligatorily coupled in UhpC. We
expressed a His-UhpC protein in a UhpC-deficient E. coli
strain and verified that this construct does not alter the basic
biochemical properties of the Glc6P sensor system. The
effects of arginine replacements, mutations of the central
loop, and introduction of a salt bridge in UhpC on transport
and sensing were compared. The exchanges R46C, R266C
and R149C moderately affected transport by UhpC but
strongly decreased the sensing ability. This suggested that the
affinity for Glc6P as a transported substrate is uncoupled in

UhpC from its affinity for Glc6P as an inducer. Four of the
11 arginine mutants showed a constitutive phenotype but
had near wild-type transport activity suggesting that Glc6P
can be transported by a molecule locked in the inducing
conformation. Introduction of an intrahelical salt bridge
increased the transport activity of UhpC but abolished
sensing. Three conserved residues from the central loop were
mutated and although none of these showed transport, one
exhibited increased affinity for sensing. Taken together, these
data show that transport by UhpC is not required for
sensing, that conserved arginine residues are important
for sensing and not for transport, and that residues located
in the central hydrophilic loop are critical for transport and
for sensing.
Keywords: Escherichia coli; glucose-6-phosphate transport;
sensing; signalling; site-directed mutagenesis.
For maximal efficiency a cell fully expresses the proteins
required for transport only when the substrate of that
transport system is available in the medium. The presence of
low levels of substrates in the cytosol that are not normal
components of intermediary metabolism can signal the
transcription system that a nutrient is available in the
extracellular milieu and needs to be transported. However,
if the substrate is a standard metabolite, transcription
cannot be signalled by an omnipresent cytosolic substrate
but must respond to the presence of external substrate.
The metabolic intermediate glucose-6-phosphate (Glc6P)
istakenupbyEscherichia coli via an inducible hexose
phosphate transporter (UhpT). The inducer/substrate
Glc6P must be in the medium, not just the cytoplasm, to

function as an inducer [1]. In addition to UhpT, the genomic
locus uhp encodes UhpB, UhpA and UhpC [2,3]. After
recognition of extracellular Glc6P by the constitutively
expressed sensor UhpC, this protein most likely interacts
with the membrane-bound UhpB and stimulates its kinase
activity. Finally, a phosphate group is transferred to UhpA,
a soluble transcription activator that governs the expression
of the uhpT gene [4].
The sensor membrane protein and the transport protein
are homologous molecules sharing about 32% identity [2]
and both are members of the Major Facilitator Superfamily
[5–7]. One postulates that the primordial unregulated gene
that encoded the transport protein was duplicated and then
modified to gain sensor function and lose transport
function.
Strikingly, in Chlamydia pneumoniae the system which
transports hexose phosphates [8] is structurally more similar
to UhpC than to UhpT. Besides, no genes for sensing or
regulation (uhp elements) have been identified in this
species [9]. For an obligate intracellular bacterium such as
Chlamydia there was probably no driving force for the
establishment of a sensor/regulatory system as Glc6P was
always present in the host cell cytosol ready to be
transported. Previous experiments by others led to the
conclusion that UhpC is unlikely to transport Glc6P [2] but
recent analysis demonstrated that UhpC from E. coli can
act not only as a sensor but also as a carrier that facilitates a
Glc6P/P
i
antiport mode of transport [8]. The transport

activity of UhpC from E. coli is much less than that of
UhpT, cannot be observed when the gene encoding UhpC
is present only on the chromosome, and is inadequate to
supply the amount of Glc6P required for growth [2,8].
The ability to both transport and sense is not limited to
UhpC as similar observations have been made for a
range of transporters in bacteria and eukaryotes [7,10–13].
For some glucose and sucrose sensors from yeast, human
Correspondence to E. Neuhaus, Universita
¨
t Kaiserslautern,
Pflanzenphysiologie, Postfach 3049, D-67653 Kaiserslautern,
Germany. Tel.: + 0631/205 2372,
E-mail:
Abbreviations: UhpC, glucose-6-phosphate sensor from E. coli;
Glc6P, glucose-6-phosphate; IPTG, isopropyl thio-b-
D
-galactoside.
(Received 18 December 2002, accepted 10 February 2003)
Eur. J. Biochem. 270, 1450–1457 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03507.x
cells, and plant sieve elements [14,15] the ability to import
carbohydrates has clearly been shown [14,16,17]. However,
it is not known if carbohydrate transport is required in
these systems for sensing activity. The expression of amino
acid and peptide transporters in bakers yeast is controlled
by the amino acid permease homologue Ssy1p [13]. For
this process, transport of amino acids is not required
because binding suffices to induce gene expression [13].
In this study we investigated whether the low level of
transport of Glc6P via UhpC is required for sensing of

external Glc6P by UhpC. One might postulate that the
two activities are obligatorily linked because the transport
of a few molecules of substrate changes the conformation
of UhpC and that this change is required for UhpC to
initiate the transcription of the uhpT gene. Alternatively,
the binding of Glc6P could change the conformation of
UhpC with no requirement for translocation of substrate.
We compared the effects of site-specific mutations of the
UhpC protein on both the transport and sensing
functions of this molecule. We mutated arginine residues
as these are known to be involved in binding of anions
to proteins [18] and as some of these are conserved and
essential for function in proteins homologous to UhpC
[19]. In addition, we introduced an intrahelical salt bridge
into UhpC, a bridge identified as necessary for UhpT
function [20], but that is absent in UhpC [21]. Finally, we
changed three residues that are conserved in proteins
similar to UhpC [19] and that are located in the central
hydrophilic loop between transmembrane domains 6 and
7, a loop that was shown to be essential for UhpT
activity but was thought to be less important for sensing
[22]. The very low level of transport by UhpC precluded
doing these experiments with just the chromosomal copy
of uhpC, thus UhpC had to be over-expressed from a
plasmid-borne gene. This changed the ratios of the uhp
operon products, so extrapolation to the normal E. coli
situation with all uhp genes in an operon may not be
valid and such extrapolation was not our goal. However,
we were able to clearly separate the sensing and
transport activities of UhpC membrane protein.

Materials and methods
DNA constructs for heterologous expression in
E. coli
DNA manipulations and construction of the uhpC/pET16b
plasmid were performed essentially as described previously
[8,23]. Oligonucleotide site-directed mutagenesis was per-
formed using the Quick Change
TM
mutagenesis kit (Stra-
tagene) according to supplier’s advice with oligonucleotide
primers from MWG-Biotech (Ebersberg, Germany). To
verify that modifications were correctly introduced into
uhpC all constructs were sequenced (DNA sequencing
service of SeqLab, Go
¨
ttingen, Germany).
Strains and growth conditions
E. coli strain XL1-Blue (Stratagene) was used for all cloning
steps. Strains RK7245 (uhpC::Tn1000 (Tet
r
) and RK7251
(uhpT::Tn1000 (Tet
r
) (kindly provided by R. Kadner,
University of Virginia, Charlottesville, USA) were used as
donor strains for P1 transduction of E. coli BL21(DE3) to
create UhpC- and UhpT-deficient BL21(DE3) mutants
as described previously [8]. The transformation of the
UhpC- and UhpT-deficient E. coli strains BL21(DE3)
(uhpC::Tn1000 and uhpT::Tn1000, respectively) with the

modified pET16b constructs was carried out according to
standard protocols.
Determination of transport activities of the over-
expressed UhpC mutants was carried out using the
UhpT-deficient E. coli strain BL21(DE3) (uhpT::Tn1000).
Overnight cultures were diluted 100-fold into YT medium
plus antibiotics and grown at 37 °C to a turbidity (D
578
)
of 0.5. After induction of T7-RNA polymerase activity
by the addition of isopropyl thio-b-
D
-galactoside (IPTG)
(final concentration 0.012%), cells were grown for a
further 90 min, collected by centrifugation, resuspended
in Mops buffer solution (50 m
M
,pH7.5)andstoredon
ice until use.
Sensing activities of the UhpC mutants were determined
by using the UhpC-deficient E. coli strain BL21(DE3)
(uhpC::Tn1000). When the turbidity of the growing culture
reached 0.5, Glc6P was added and the cells were grown for
additional 15 min. The maximal Glc6P concentration
added during the induction period was 400 l
M
because at
higher concentrations catabolite repression occurs. The
induction of uhpT was analysed by uptake of [
14

C]Glc6P
(NEN). Although IPTG is unnecessary to obtain induction
mediated by the wild-type UhpC when it is expressed from
the plasmid-borne gene [8], IPTG is mandated in the
transport assays to strongly increase the levels of UhpC. We
confirmed that the increased expression (following the
addition of IPTG) of UhpC with the mutations that resulted
in the lack of sensing did not result in the induction of uhpT
(data not shown). We determined the half-maximal con-
centration of Glc6P required for maximal induction of
UhpT activity, named K
(induction)
.
Transport assays
Cells suspensions were allowed to equilibrate at 30 °C
and subsequently mixed with an equal volume of
prewarmed transport medium containing [
14
C]Glc6P.
We always checked the linearity with time of [
14
C]Glc6P
uptake catalysed by the corresponding mutant protein.
Determinations of biochemical transport constants
(apparent K
m
and V
max
values) were performed at the
1-min time points. [

14
C]Glc6P transport was stopped by
transfer of the cells to membrane filters (25 mm diameter,
0.45 lm pore size; Pall Life Science, Dreieich, Germany)
prewetted with Mops buffer solution and under vacuum.
After washing with ice-cold buffer solution the filters
were placed in vials containing scintillation cocktail (Quick-
safe A, Zinsser Analytic, Frankfurt/Main, Germany).
The radioactivity was quantified in a Canberra-Packard
Tricarb-2500 counter. The kinetic constants of transport
were estimated using the method of Hanes. All data
represent means of at least three independent experi-
ments. The standard deviation was always less than 9%
of the given mean. The background activity of IPTG-
induced E. coli cells harbouring the empty vector plasmid
pET16b has always been subtracted [8]. Protein content
of E. coli samples was quantified using Coomassie
brilliant blue [24].
Ó FEBS 2003 Bacterial hexose-phosphate transport protein (Eur. J. Biochem. 270) 1451
Cytoplasmic membrane preparation and Western blot
analysis
Site-directed mutations of a membrane protein can influ-
ence the efficiency of integration into the native membrane
and thus influence the apparent transport activity. The
efficiency of protein incorporation into the E. coli
cell membrane was quantified by Western blot analysis
[8,25]. For Western blot analysis E. coli BL21(DE3)
(uhpT::Tn1000) (harbouring the corresponding pET16b
construct) described above for transport assays was used.
Cytoplasmic membrane preparations were carried out

according to Alexeyev and Winkler [26]. Essentially, the
cells were disrupted by ultrasonication (250 W, 3 · 30 s,
4 °C) and membranes were collected by centrifugation [8].
The resulting membrane protein fractions were separated by
SDS/PAGE and Western blots were developed using a
histidine-tag specific antiserum (Qiagen) with chemilumi-
nescent detection (Roche). Expression levels were deter-
mined by densitometry of digitized images [25] after
confirming the linearity of densitometry by applying various
amounts of protein. The V
max
values of the mutated UhpC
proteins are calculated based on total protein without
regard to the level of UhpC expression. On the other hand,
specific activity was calculated as V
max
divided by the
expression level as determined normalized Western blot
values (nmol substrate transported)/(normalized Western
blot UhpC density) [8].
Results
Characterization of the expression of His-UhpC
in the
E. coli
uhpC::Tn1000 mutant
An N-terminally located histidine extension was necessary
for quantification of membrane insertion of both the wild-
type and mutated UhpC. However, this extension might
influence the interaction between UhpC and the down-
stream elements of the Uhp signalling system. Therefore, we

compared the K
(induction)
observed with chromosomally
encoded UhpC and with plasmid-encoded UhpC with a
histidine tag. In both systems increasing concentrations of
external Glc6P induced the Glc6P uptake system (UhpT)
with a K
(induction)
of 3.8 l
M
(Fig. 1A,B) which is close to the
concentration dependence of induction observed by others
[27]. Thus, the over-expression of the wild-type UhpC with a
histidine tag did not alter the concentration of Glc6P
required for half-maximal induction of UhpT.
Site-directed mutations of conserved arginine residues
Arginine residues in proteins are excellent candidates for the
binding of negatively charged substrates like Glc6P [18].
Maloney and coworkers showed that two of 14 arginine
residues in UhpT are critical for its function [19]. To identify
conserved arginine residues in UhpC we aligned several
UhpC- and UhpT-like proteins including the Glc6P trans-
porter from C. pneumoniae (HPTcp [8]); that exhibits a
higher degree of structural identity to the E. coli UhpC
protein than to UhpT [9]. UhpC and UhpT proteins have
been taken from the genomes of E. coli, Salmonella enterica,
Pasteurella multocida, Yersina pestis and Vibrio cholerae. It
should be emphasized that the function of UhpC in Yersinia
is doubtful because Y. pestis contains Uhp A, B, and C but
lacks UhpT (RefSeq: NC003143; GenBank: NC003143). In

addition, V. cholerae contains two membrane proteins
annotated as UhpC (RefSeq: NC002506; GenBank:
AE003853) so a functional distinction between both
proteins is difficult. Fig. 2 clearly illustrates that UhpC
proteins and UhpT proteins substantially similar. Arginine
204 is present in all UhpC proteins and in the Glc6P
transporter HPTcp, whereas R437 is present only in the
UhpC proteins from E. coli and S. enterica, but both
residues are absent in the transporters (Fig. 2). Arginine 149
is present in all proteins with the exception of the putative
transporter from V. cholerae and the HPTcp protein. In
contrast, R46, R152, R266 and R318 are conserved in all of
these proteins (Fig. 2).
A change in a conserved arginine residue in UhpC could
affect: (a) the ability of UhpC to interact with external
Glc6P (either as a substrate or signal molecule or both); (b)
the translocation pathway in UhpC for the transport of
Glc6P; (c) the ability of UhpC to interact with UhpB in the
transmission of the induction signal; (d) the insertion of
UhpC into the membrane; and (e) various combinations of
Fig. 1. Determination of the K
(induction)
of the UhpT-inducing system.
E. coli cells BL21(DE3) (A) and E. coli cells BL21(DE3) (uhpC::
Tn1000) harbouring plasmid uhpC/pET16b (B) were induced for
15minwithgivenGlc6P concentrations. For quantification of uptake,
cells were incubated for 1 min with 10 l
M
[
14

C]Glc6P.Insets:The
Hanes analysis (only hyperbolic parts) revealed in both cases an
apparent K
(induction)
of 3.8 l
M
and a V
max(induction)
of 160 nmolÆmg
protein
)1
Æh
)1
.
1452 C. Schwo
¨
ppe et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 2. Multiple alignment of UhpC- and UhpT-related protein amino acid sequences. The UhpC proteins share 86.9% (S. enterica) to 56.7%
(V. cholerae), the HPT protein from C. pneumoniae shares 45.3%, and the UhpT proteins share 32.6% (E. coli) to 30.0% (V. cholerae)identityto
the E. coli UhpC protein (for details see text). The multiple alignment was performed using
CLUSTALW
(default settings). The asterisks indicate the
positions of the mutated amino acids of the E. coli UhpC protein.
Ó FEBS 2003 Bacterial hexose-phosphate transport protein (Eur. J. Biochem. 270) 1453
these effects. We constructed 11 mutants of E. coli UhpC in
which we exchanged single arginine residues and attempted
to classify the effects into the above categories based on
changes in the K
M
or V

max
of transport, the concentration of
Glc6P that causes half-maximal induction (K
(induction)
), the
ability to transmit an activating signal to UhpB in the
absence of exogenous Glc6P (constitutive induction), and
the insertion of UhpC into the membrane as determined by
Western blot analysis.
As shown in Table 1, five mutations at four positions
(R152C, R152A, R149C, R204C, R318C) caused a
modest twofold increase compared to wild-type in the
affinity for Glc6P in the transport aspect of UhpC. The
effect of these five mutations on the K
(induction)
was
remarkably varied. While mutants R152A and R204C
changed little with respect to the sensor values in the
wild-type, the K
(induction)
of R152C decreased fivefold,
that of R149C increased almost 700-fold, and R318C
became constitutive. Unfortunately, the affinity for Glc6P
in the sensor aspect of UhpC cannot be evaluated in the
constitutive mutants. We confirmed the constitutive
induction also for cells that were grown in minimal
medium proving that residual Glc6P,whichmightbe
present in the complete growing medium, was not the
cause of this effect (data not shown). In contrast with
K

m
determinations that are independent of the amount
of protein, the effect of these mutations on the trans-
location pathway required that V
max
and the relative
insertion of UhpC into the membrane be measured. This
composite value is shown as Ôspecific activityÕ in Table 1.
These five mutants ranged from a fivefold decrease to a
3.6-fold increase with respect to wild-type activity.
Similarly, six mutations at five positions (R318A, R152K,
R437C, R46C, R266C, R318K) caused the same modest
decrease in the affinity for Glc6P in the transport aspect
of UhpC. Again, the effect of these five mutations on
K
(induction)
was remarkably variable. While R437C changed
only fourfold with respect to the wild-type, the K
(induction)
of
R266C increased 245-fold, three mutants (R318A, R318K,
R152K) became constitutive, and the K
(induction)
of R46C
became so high (low affinity) that it was not measurable.
The specific activities measured ranged from a 0.7-fold
decrease to a sixfold increase with respect to wild-type
activity (Table 1). Interestingly, Maloney and coworkers
showed that R46 is critical for transport function of UhpT
[19], but the major effect of the R46C mutation in UhpC

wastoabolishsensingactivity.
Although most of the mutations were replacements of
arginine with cysteine, at two positions (152 and 318)
additional mutations were made. Arginine at these positions
was also replaced by alanine (to prevent the putative
formation of an intramolecular disulfide bridge that might
have occurred with cysteine) and lysine. At position 318 all
three mutants became constitutive (Table 1). In contrast, at
position 152 the two neutral mutations (R152C and R152A)
retained near wild-type affinity for the inducer, but
the conservative replacement R152K became constitutive
(Table 1).
Introduction of an intrahelical salt-bridge
The amino acids D388 and K391 in transmembrane domain
11 of the UhpT protein from E. coli are proposed to rep-
resent a salt bridge that is critical for transport function [25].
Table 1. Effects of site-directed mutations on the transport and sensing activities of the Glc6P sensor UhpC from E. coli. Transport activities were
determined using the UhpT-deficient BL21(DE3) strain (uhpT::Tn1000) while sensing activities were determined using the UhpC-deficient
BL21(DE3) strain (uhpC::Tn1000) (details are given above). For calculation of the specific activity see Materials and methods. n.m., not measurable.
Mutant
Transport Sensing
K
M
(l
M
)
V
max
(nmolÆmg
)1

Æh
)1
)
Membrane
incorporation (% wild-type)
Specific activity
(nmolÆmg
)1
Æh
)1
) K
(induction)
(l
M
)
His-UhpC 63 110 100 110 3.8
Arginine mutants
R46C 135 105 22 480 n.m.
R149C 30 154 39 395 2646
R152C 23 30 13 231 0.86
R152A 46 9 37 24 3.2
R152K 82 61 9 678 Constitutive
a
R204C 33 31 32 97 7.3
R266C 145 75 93 81 932
R318C 45 145 81 179 Constitutive
a
R318A 73 152 63 241 Constitutive
a
R318K 150 106 67 158 Constitutive

a
R437C 115 100 128 78 15.2
Salt bridge
T382D/V385K 160 150 10 1500 n.m.
Loop mutants
G213V n.m. n.m. 50 – Constitutive
a
H222Q n.m. n.m. 32 – 0.53
D223K n.m. n.m. 4 – n.m.
a
See Fig. 3.
1454 C. Schwo
¨
ppe et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Only transporters (with the exception of HPTcp and the
putative transporter from V. cholerae) contain these amino
acids whereas none of the UhpC proteins contain similarly
charged residues at this position (Fig. 2). The introduction
of a putative salt bridge (T382D/V385K) in the proposed
transmembrane domain 11 of UhpC increased the specific
transport activity from 110 units (wild-type UhpC) to 1500
units (Table 1). This was accompanied by a total loss of
ability to sense Glc6P in the medium (the K
(induction)
was so
high that it could not be measured (Table 1).
Mutations of the central hydrophilic loop
The central hydrophilic loop of UhpC is represented by
amino acids 202–253 and connects transmembrane domains
6 and 7 [19]). Previous analysis of mutated UhpC proteins

with insertional mutations in the central hydrophilic loop
between TM6 and TM7 led to the assumption that this
domain, in contrast with the corresponding domain in
UhpT, is not of major importance for the UhpC phenotype
[22]. However, the conservation of the amino acid sequence
of the central hydrophilic loop in UhpC proteins is
remarkable (Fig. 2). Therefore, to investigate whether single
conserved amino acid residues in the central hydrophilic
loop are critical for sensing and/or transport by UhpC we
mutated three conserved residues in this region (Fig. 2):
G213 (that is conserved in all proteins aligned); H222 (that
only appears in UhpC proteins with a complete uhp locus-
E. coli, S. enterica and P. multocida); and D223 (that
appears in all UhpC-like proteins including HPTcp, but
not in the UhpT proteins).
The G213V exchange altered both the transport and the
sensor aspect of the mutated UhpC protein. No transport
activity was measurable with this UhpC protein and its
presence resulted in near wild-type UhpT activity that was
constitutively expressed in the absence of Glc6P during
induction (Table 1, and Fig. 3). Interestingly, the charge-
reversal mutation D223K lost both activities and was
unable to either transport Glc6P or sense Glc6P in the
medium. The H222Q mutant, like the other two loop
mutants, was unable to transport Glc6P. However, most
significantly, this mutant showed intact sensing activity and
it responded to an even lower concentration of Glc6P in the
medium than the wild-type as illustrated by the sevenfold
lower K
(induction)

(Table 1, Fig. 4).
Discussion
A major aim of this work was to determine whether the
transport of Glc6P catalysed by UhpC or just the binding of
Glc6P to UhpC is required to signal the presence of external
Glc6P to downstream components of the uhp system. We
uncoupled transport and sensing by creating mutants of
UhpC and estimating their biochemical constants. In
addition, we determined the essentiality of conserved amino
acid residues for these two functional aspects of UhpC
activity.
In order to analyse the altered transport properties of
UhpC mutants it was necessary to quantify the level of
mutated protein in the E. coli cytoplasmic membrane by
using a histidine-specific antibody. This was necessary
because a single amino acid exchange in UhpC influences
the efficiency of membrane insertion drastically (Table 1).
Similar observations have been made for site-directed
mutated UhpT proteins [19]. The data given in Fig. 1 show
that expression of a His-UhpC protein does not negatively
affect the interaction of the sensor with the next elements of
the Uhp signal pathway. In addition, the determined
K
(induction)
for the wild-type and the His-UhpC protein of
3.8 l
M
concurs with previous determinations by others [27].
Are conserved arginine residues important for function
of UhpC as transporter and sensor?

Although most of the residues mutated in UhpC are highly
conserved in UhpC and UhpT proteins (Fig. 2), and in the
case of R46 and R266 had been shown to be critical for
transport in UhpT [19], 10 of 11 mutations had modest
Fig. 4. Determination of the K
(induction)
of the UhpT-inducing system in
UhpC-deficient E. coli cells BL21(DE3) (uhpC::Tn1000) harbouring the
pET16b construct which encodes the mutated UhpC-H222Q protein.
The cells were induced for 15 min with given Glc6P concentrations.
For quantification of uptake cells were incubated for 1 min with 10 l
M
[
14
C]Glc6P. Inset: The Hanes analysis revealed an apparent K
(induction)
of 0.53 l
M
and a V
max(induction)
of 150 nmolÆmg protein
)1
Æh
)1
.
Fig. 3. Complementation of the UhpC-deficient E. coli strain BL21
(DE3)(uhpC::Tn1000) with the pET16b constructs encoding UhpC
mutants R152C/A/K, R318C/A/K or G213V. The corresponding cul-
tures were either grown with (+) or without (–) 100 l
M

Glc6P as
inducer. For quantification of uptake cells were incubated for 1 min
with 10 l
M
[
14
C]Glc6P.
Ó FEBS 2003 Bacterial hexose-phosphate transport protein (Eur. J. Biochem. 270) 1455
effects on transport mediated by UhpC (Table 1). The K
M
for transport in the 10 mutants ranged from 23 to 150 l
M
,
less than threefold on each side of the wild-type (63 l
M
).
The V
max
ranged from 30 to 154 nmolÆmg
)1
Æh
)1
with the
wild-type value being 110 nmolÆmg
)1
Æh
)1
. The one excep-
tion was R152A which had a V
max

less than 10% of wild-
type but could be fully induced. However, because insertion
of six of the 11 mutated UhpC proteins into the cell
membrane was less than 50% of the insertion in the wild-
type, the calculated transport activity per membrane-
inserted molecule was up to sixfold more than wild-type
and was very low only in the case of R152A (Table 1).
Although the analysis of site-directed mutants of UhpT led
to the hypothesis that arginine residues R46 and R275
(corresponding to 46 and R266 in UhpC, Fig. 2) were
involved in the binding of the transport substrate Glc6P
[19], our observations demonstrate that it is not valid to
transfer data about single amino acid residues critical for
transport by UhpT to UhpC.
In contrast to the modest effects on transport, the
effects of the arginine mutations on induction were large
and varied. The concentration of Glc6P that gave 50%
induction of UhpT increased from 3.8 l
M
to 932 l
M
in
R266C, to 2646 l
M
in R149C, and was so high in R46C
that it could not be measured. This suggests that the
affinity for Glc6P as a transported substrate is uncoupled
in a UhpC molecule from its affinity for Glc6P as an
inducer; this is seen most dramatically in R149C in which
the affinity for Glc6P as the transport substrate increased

twofold and that for Glc6P as the inducer decreased 700-
fold (Table 1). Obviously, after gene duplication which led
to the generation of UhpC, the evolutionary pressure was
to optimize sensing and not transport. A surprisingly high
number, four of the 11, arginine mutants had a consti-
tutive phenotype, that is, they were fully induced for
UhpT expression in the absence of any inducer. A
constitutive mutant can be understood as a UhpC
molecule that is locked into the active, inducing confor-
mation which is maintained at all Glc6P concentrations.
The four constitutive mutants had near wild-type trans-
port activity suggesting that Glc6P can be transported by
a molecule that is locked in the inducing conformation
and which argues against the transport of Glc6P causing
an inducing conformation. For UhpC and other mem-
brane proteins acting as sensors it has been shown that
insertional mutations led to constitutive induction [11,22].
In case of the mutated bacterial iron transporter FecA it
has been postulated that the constitutive induction
demonstrates that transport of the substrate (iron citrate)
is not required for sensing [11]. However, one would
prefer a system in which a mutated protein can respond to
external Glc6P but does not transport.
Function of a newly introduced intrahelical salt bridge
on sensing
The E. coli UhpT protein exhibits an intramolecular salt
bridge located in transmembrane domain 11 [25] that
appears to be highly conserved in all the UhpT-like, but not
in the UhpC-like, proteins (Fig. 1). Introduction of a
corresponding salt bridge into UhpC (T382D/V385K

exchange) increased the specific transport activity of UhpC
about 14 times in accordance with previous findings
indicating the importance of this salt bridge for transport
by UhpT [25] (Table 1). However, UhpC with this salt
bridge was unable to sense exogenous Glc6P and induce
UhpT. Curiously, this is essentially the same phenotype seen
in the arginine mutant R46C where we removed, rather than
introduced, a residue that was essential to transport by
UhpT. Again, this suggests that in a UhpC molecule the
affinity for Glc6P as a transported substrate is not related to
its affinity for Glc6P as an inducer. It is worth mentioning
that removal of this salt bridge from UhpT does not confer
signalling activity to this transporter when expressed in a
UhpC-deficient strain (data not shown). Thus, removal of
this salt bridge from UhpC after gene duplication appears
necessary to allow sensing activity, but was not sufficient to
create a sensor.
Function of amino acid residues located in the central
loop of UhpC
The alignment reveals that UhpC-like proteins exhibit
a number of highly conserved residues located in the
predicted central hydrophilic loop that are different in
UhpT-like proteins (Fig. 2). Previous observations had
suggested that the large central hydrophilic loop of UhpC
might not be important for exhibiting the Uhp phenotype
[22]. However, the reciprocal exchange D223K abolished
both transport and sensing and the mutant G213V is
constitutive and lacks the ability to transport Glc6P
(Table 1). The mutant H222Q also lacks transport activity
but remarkably possesses an increased affinity for sensing

exogenous Glc6P and inducing UhpT (K
(induction)
decreased
about sevenfold, Table 1, Fig. 4). Our major aim was to
demonstrate whether transport and sensing by UhpC are
obligatorily connected. Our data show that the Glc6P
transport activity of UhpC is not necessary for the sensing
activity of UhpC and vice versa. Mutants of UhpC were
found that had transport and little or no sensing activity,
others that had transport and were constitutive, and still
others that had sensing activity and no transport.
Acknowledgements
Work in the laboratory of H.H.W. was supported by Public Health
Service grant AI-15035 from the National Institute of Allergy and
Infectious Diseases. Work in the laboratory of H.E.N. was supported
by the Schwerpunkt Biotechnologie des Landes Rheinland-Pfalz.
References
1. Winkler, H.H. (1966) A hexose-phosphate transport system in
E. coli. Biochim. Biophys. Acta 117, 231–240.
2. Kadner, R.J., Island, M.D., Dahl, J.L. & Webber, C.A. (1994) A
transmembrane signalling complex controls transcription of the
Uhp sugar phosphate transport system. Res. Microbiol. 145, 381–
387.
3. Island, M.D., Wei, B.Y. & Kadner, J.J. (1992) Structure and
function of the uhp genes for the sugar phosphate transport system
in E.coli and Salmonella typhimurium. J. Bacteriol. 174, 2754–
2762.
4. Wright, J.S. III & Kadner, R.J. (2001) The phosphoryl transfer
domain of UhpB interacts with the response regulator UhpA.
J. Bacteriol. 183, 3149–3159.

1456 C. Schwo
¨
ppe et al. (Eur. J. Biochem. 270) Ó FEBS 2003
5. Marger, M.D. & Saier, M.H. (1993) A major superfamily of
transmembrane facilitators that catalyse uniport, symport and
antiport. Trends Biol. Sci. 18, 13–20.
6. O
¨
zcan,S.,Dover,J.,Rosenwald,A.G.,Wo
¨
lfl, S. & Johnston, M.
(1996) Two glucose transporters in Saccharomyces cerevisiae
are glucose sensors that generate a signal for induction of gene
expression. Proc. Natl. Acad. Sci. USA 93, 1–5.
7. Lalonde,S.,Boles,E.,Hellmann,H.,Barker,L.,Patrick,J.W.,
Frommer, W.B. & Ward, J.M. (1999) The dual fucntion of sugar
carriers: transport and sugar sensing. Plant Cell 11, 707–726.
8. Schwo
¨
ppe, C., Winkler, H.H. & Neuhaus, H.E. (2002) Properties
of the glucose 6-phosphate transporter from Chlamydia pneumo-
niae (HPTcp) and the glucose 6-phosphate sensor from Escherichia
coli (UhpC). J. Bacteriol. 184, 2108–2115.
9. Stephens,R.S.,Kalman,S.,Lammel,C.,Fan,J.,Marathe,R.,
Aravind,L.,Mitchell,W.,Olinger,L.,Tatusol,R.L.,Zhao,Q.,
Koonin, E.V. & Davis, R.W. (1998) Genome sequence of an
obligate intracellular pathogen of humans: Chlamydia tracho-
matis. Science 282, 754–759.
10. Postma, P.W., Lengeler, J.W. & Jacobson, G.R. (1993) Phos-
phoenolpyruvate: carbohydrate phosphotransferase systems of

bacteria. Microbiol. Rev. 57, 543–594.
11. Ha
¨
rle,C.,Kim,I.,Angerer,A.&Braun,V.(1995)Signaltransfer
through three compartments: transcription initiation of the
Escherichia coli ferric citrate transport system from the cell surface.
EMBO J. 14, 1430–1438.
12. O
¨
zcan, S., Dover, J. & Johnston, M. (1996) Glucose sensing and
signalling by two glucose receptors in the yeast Saccharomyces
cerevisiae. EMBO J. 17, 2566–2573.
13.Didion,T.,Regenberg,B.,Jorgensen,M.U.,Kielland-Brandt,
M.C. & Andersen, H.A. (1998) The permease homologue Ssy1p
controls the expression of amino acid and peptide transporter
genes in Saccharomyces cerevisiae. Mol. Microbiol. 27, 643–650.
14. Antoine, B., Lefrancois-Martinez, A M., Le Guillou, G., Letur-
gue, A., Vandervalle, A. & Kahn, A. (1997) Role of the GLUT 2
glucose transporter in the response of the 1-type pyruvate kinase
gene to glucose in liver-derived cells. J. Biol. Chem. 272, 17937–
17943.
15. Barker, L., Kuhn, C., Weise, A., Schulz, A., Gebhardt, C., Hirner,
B.,Hellmann,H.,Schulze,W.,Ward,J.M.&Frommer,W.B.
(2000) SUT2, a putative sucrose sensor in sieve elements. Plant
Cell 12, 1153–1164.
16. Bisson,L.F.,Neigeborn,L.,Carlson,M.&Fraenkel,D.G.(1987)
The SNF3 gene is required for high-affinity glucose transport in
Saccharomyces cerevisiae. J. Bacteriol. 169, 1656–1662.
17. Meyer, S., Melzer, M., Truernit, E., Hummer, C., Besenbeck, R.,
Stadler, R. & Sauer, N. (2000) AtSUC3, a gene encoding a new

Arabidopsis sucrose transporter, is expressed in cells adjacent to
the vascular tissue and in a carpel cell layer. Plant J. 24, 869–882.
18. Riordan, J.F. (1979) Arginyl residues and anion binding in pro-
teins. Mol. Cell. Biochem. 26, 71–92.
19. Fann,M.,Davies,A.H.,Varadhachary,A.,Kuroda,T.,Sevier,
C., Tsuchiya, T. & Maloney, P.C. (1998) Identification of two
essential arginine residues in UhpT, the sugar phosphate anti-
porter of Escherichia coli. J. Memb. Biol. 164, 187–195.
20. Hall, J.A., Fann, M.C. & Maloney, P.C. (1999) Altered substrate
selectivity in a mutant of an intrahelical salt bridge in UhpT, the
sugar phosphate carrier of Eschericheria coli. J. Biol. Chem. 274,
6148–6153.
21. Friedrich, M.J. & Kadner, R.J. (1987) Nucleotide sequence of the
uhp region of Escherichia coli. J. Bacteriol. 169, 3556–3563.
22. Island, M.D. & Kadner, R.J. (1993) Interplay between the mem-
brane-associated UhpB and UhpC regulatory proteins. J. Bac-
teriol. 175, 5028–5034.
23. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, Vol. 3, 2nd edn. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
24. Bradford, M.M. (1976) A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
25. Hall, J.A. & Maloney, P.C. (2001) Transmembrane segment 11 of
UhpT, the sugar phosphate carrier of Escherichia coli,isanalpha-
helix that carries determinants of substrate selectivity. J. Biol.
Chem. 276, 25107–25113.
26. Alexeyev, M.F. & Winkler, H.H. (1999) Membrane topology of
the Rickettsia prowazekii ATP/ADP translocase revealed by novel
dual pho-lac reporters. J. Mol. Biol. 285, 1503–1513.

27. Verhamme, D.T., Postma, P.W., Crieland, W. & Hellingwerf, K.J.
(2002) Cooperativity in signal transfer through the Uhp system of
Escherichia coli. J. Bacteriol. 184, 4205–4210.
Ó FEBS 2003 Bacterial hexose-phosphate transport protein (Eur. J. Biochem. 270) 1457