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: Neuhaus@rhrk.uni-kl.de 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 . 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. 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.