1Abteilung Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany 2Present address: Cytos Biotechnology AG, Wagisstrasse 25, CH-8952 Zürich-Schlieren, Switzerland 3Present address: Bioinformatics, Sanofi Aventis SA, Centre de Recherche de Paris, 13 quai Jules Guesdes, 94400 Vitry-sur-Seine, France 4Present address: Medizinische Hochschule Hannover, Tissue Engineering Network, Podbielski Strasse 380, 30659 Hannover, Germany 5Present address: diaDexus Inc., 343 Oyster Point Boulevard, South San Francisco, CA 94080, USA
6 To whom correspondence should be addressed. E-mail: sbehren1{at}gwdg.de
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Abstract |
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Keywords: DNA binding/leucine zipper/proteinprotein interaction/ToxR/two-hybrid system
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Introduction |
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The transcriptional activation of the ctxAB genes in V.cholerae only indirectly involves ToxR (DiRita et al., 1991; Higgins et al., 1992
). In the heterologous host Escherichia coli, in contrast, ToxR is capable of directly activating transcription at ctx (Miller and Mekalanos, 1984
) (Figure 1). The ctx promoter is characterized by a heptameric DNA element (TTTTGAT) located 56 bp upstream of the transcription start site and directly repeated 38 times, depending on the V.cholerae strain. At least three copies of the heptad and additional sequences near the 35 promoter region are required for binding and transcriptional activation of ctx by ToxR (Miller et al., 1987
; Pfau and Taylor, 1996
). In E.coli, ToxR's function as a transcriptional activator of ctx furthermore requires the dimerization of its N-terminal DNA-binding domain. This was first deduced from the finding that the homodimeric enzyme alkaline phosphatase could functionally substitute for the periplasmic ToxR domain (Miller et al., 1987
; DiRita and Mekalanos, 1991
). Later studies supported the dimerization model by showing that the DNA-binding domain of phage
cI repressor dimerizes and represses transcription from a lambda OR1PR:lacZY reporter fusion in E.coli when N-terminally fused to ToxR (Dziejman and Mekalanos, 1994
).
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Allowing the assessment of protein interactions in the inner membrane and in both cellular compartments of E.coli, ToxR provides a broader potential application range than existing prokaryotic two-hybrid systems. So far, however, ToxR's technical utility to detect proteinprotein interactions in the cytoplasmic and periplasmic compartments has only been demonstrated using homodimerizing model proteins (Kolmar et al., 1995a). In this study, we show that the ToxR-system is also applicable to monitor asymmetric interactions between heterologous fusion partners in both cellular compartments of E.coli. Furthermore, to enhance ToxR's applicability as a prokaryotic two-hybrid system, a better understanding of its mode of action at the ctx promoter in E.coli is required. Therefore, we have investigated the minimal requirements for its function as a transcriptional activator of ctx in E.coli. We show that a short hinge region of ToxR, which lies between the wHTH domain and the transmembrane segment, is responsible for mediating ToxRToxR interactions in vitro but is not required either for specific DNA binding in vitro or for ctx activation in E.coli. Hence the conserved wHTH motif of ToxR's N-terminal DNA-binding domain defines the minimal structural element required for ToxR-mediated, dimerization-dependent transcriptional activation at ctx in E.coli.
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Materials and methods |
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LuriaBertani (LB) and dYT media were prepared as described (Sambrook et al., 1989) and supplemented with 100 µg/ml ampicillin (Ap) and 25 µg/ml chloramphenicol (Cm) if required.
Bacterial strains and plasmids
All strains and plasmids used in this study are listed in Table I. A detailed description of their construction is given in the Supplementary data available at PEDS Online under Plasmid constructions. To study ToxR-mediated transcriptional activation in vivo, vectors of the pHK series were used in which the chimeric toxR genes are under transcriptional control of the V.cholerae toxR promoter (Kolmar et al., 1994). To study asymmetric proteinprotein interactions, pHK-based vectors carrying two chimeric toxR' fusion genes including their up- and downstream transcriptional regulatory sequences were used, which allow the co-production of both ToxR' proteins at balanced molar levels within each cell.
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In vivo assay for ToxR-mediated transcriptional activation and western blot analysis
ToxR-mediated transcriptional activation was assayed by monitoring ß-galactosidase activity from the chromosomal ctx::lacZ reporter fusion in E.coli FHK12 (Kolmar et al., 1995a). FHK12 was freshly transformed with equal amounts of the pHKToxR' plasmids and plated on LB/Cm plates. Single colonies were taken and transferred into 5 ml of LB/Cm supplemented with 0.3 mM isopropyl-ß-thiogalactoside (IPTG) and grown for 1012 h at 30°C. ß-Galactosidase activity was then determined from 15 µl of culture as described by Kolmar et al. (1995a)
. In addition, samples of these cultures containing 108 cells were analyzed by western blotting using MalE-specific antibodies (New England Biolabs; 1:10 000 dilution). Anti-rabbit alkaline phosphatase (Sigma-Aldrich; 1:10 000 dilution) was used as the secondary antibody. The blots were developed by incubation in reaction buffer (100 mM TrisHCl pH 8.8, 100 mM NaCl, 5 mM MgCl2, 37.5 µg/ml NBT, 150 µg/ml BCIP).
Protein production and purification
MalE, MalEGCN, ToxR'(TM)MalE and ToxR'(
TM)MalEGCN were produced from the respective pMal-c and pBSIISK() plasmids in E.coli PD28 (Table I). Cultures were grown in LB/Ap at 30°C and production of the recombinant proteins was induced with 0.5 mM IPTG at an OD600 of 0.30.5 followed by further incubation for 35 h. Cells were pelleted by centrifugation (4000 g, 40 min, 4°C), resuspended in amylose A buffer (50 mM TrisHCl, pH 7.5, 2.5 mM ß-mercaptoethanol) with 1 mM PMSF and lysed by French press treatment and sonication. The cell extract was cleared by centrifugation (15 000 g, 40 min, 4°C) and mixed with amylose resin (New England Biolabs) pre-equilibrated with amylose A buffer. After 30 min of incubation on ice the mix was centrifuged (4000 g, 10 min, 4°C) and the supernatant removed. The amylose resin was washed three times in amylose-wash buffer (amylose A buffer with 0.5 M NaCl) and once with amylose A buffer. The MalE fusion proteins were then eluted by incubation in amylose B buffer (amylose A buffer with 10 mM maltose) for 30 min on ice and subsequent centrifugation. The protein containing supernatant was dialyzed and concentrated. The typical protein yield from this protocol was
8 mg of MalE or MalEGCN protein per 50 ml of cell culture and
0.25 mg of the ToxR'(
TM) fusion proteins per liter of cell culture.
The ToxR'(TM), ToxR'(
TM)GCN, ToxR'(
TM)(
X), ToxR'(
TM)(
X)GCN, ToxR'(
139181)(
TM)(
X) and ToxR'(
139181)(
TM)(
X)GCN proteins were produced and purified using the IMPACT expression system (New England Biolabs). The proteins were produced in fusion with the Intein-CBD (Intein-chitin-binding domain) protein from pASK-plasmids in E.coli HS3018 (Table I). Cultures were grown in LB/Ap at 37°C to an OD600 of 0.5 and the production of the recombinant proteins was induced with 0.2 µg/ml anhydrotetracycline. After growth for a further 23 h, cells were harvested by centrifugation (4000 g, 40 min, 4°C), resuspended in CB buffer (40 mM TrisHCl, pH 8.5, 500 mM NaCl, 1 mM EDTA, 0.1% Triton X-100) supplemented with 75 units of Benzonase (Merck) and lysed by French press treatment and subsequent sonication. The cell extract was cleared by centrifugation (15 000 g, 40 min, 4°C) and applied to a 68 ml chitin beads column (New England Biolabs) pre-equilibrated with CB buffer. The column was washed with CB-wash buffer (CB buffer with 1 M NaCl), sealed and incubated for 30 min at room temperature to release ToxR-bound DNA, which was then removed with 10 volumes of CB-wash buffer. The column was equilibrated with 10 volumes of DNase buffer (50 mM TrisHCl, pH 8.0, 3 mM MgCl2) and incubated in 1 volume of DNase-buffer with 75 units of Benzonase for 30 min at 37°C to digest residual DNA. After washing the column with 10 volumes of CB buffer, three volumes of cleavage buffer (CB buffer with 150 mM DTT) were applied and the sealed column was incubated at room temperature for 1624 h. Finally, the proteins were eluted with 5 volumes of CB buffer, dialyzed and concentrated to 510 mg/ml. Typical protein yield from this protocol was 36 mg of ToxR'(
TM) fusion protein per liter of cell culture.
Protein preparations were confirmed to be free of contaminating DNA and of residual DNase activity. Protein concentrations were determined spectrophotometrically with absorption coefficients calculated according to Pace et al. (1995).
Gel filtration analysis
Apparent molecular protein weights were determined by gel filtration on an FPLC Superdex 200 HR 10/30 column (Pharmacia) at 4°C in gel filtration buffer (40 mM TrisHCl pH 7.5, 500 mM NaCl, 1 mM EDTA) and a flow rate of 0.5 ml/min. The proteins were applied to the column in 50100 µl samples at the concentrations indicated in Table II. Molecular weight standard proteins (Pharmacia) were used for column calibration.
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The ctx- (209 bp) and hel- (410 bp) DNA fragments were amplified from the pBSK()ctx-21 and pBSK()HELE35Q plasmids by standard PCR using the primer pairs Suctxup (5'-GGAATTCTAGAAGTGAAACGGGGT-3')/Suctxsig (5'-GGAATTCTAGAAGTGAAACGGGGT-3') and Hellup (5'- GTCGACCCCGGGAAAGTC-3')/Hello (5'-CGCGGTTCTAGATTATCACAGCCGGCAGCCTCTG-3'), respectively. The fragments were purified from agarose gels and subsequently cleaved with EcoRI and XbaI, respectively (restriction sites underlined), to generate 5'-overhangs. After inactivation of the enzymes by heat treatment, the DNA fragments were radioactively labeled in a standard fill-in reaction using Klenow DNA polymerase and
[32P]dATP (Sambrook et al., 1989
).
For gel retardation analysis, 50 fmol of radiolabeled DNA was incubated in 50 µl of shift buffer (40 mM TrisHCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 5% glycerol, 50 µg/ml BSA) with 500 ng of salmon sperm DNA (in 56- and 37-fold excess with regard to ctx- and hel-DNA, respectively) and 080 pmol of protein for 30 min at room temperature. The samples were loaded directly on a 6% TBE polyacrylamide gel (200x250x1.5 mm) and run overnight with 0.5x TBE running buffer at room temperature and 48 mA. Radiolabeled DNA was visualized by exposure to X-ray film for 212 h at 70°C.
Analysis of proteinDNA interactions by surface plasmon resonance spectroscopy
The interaction of soluble ToxR proteins with ctx- and hel-DNA was analyzed by surface plasmon resonance spectroscopy (SPR) on a Biacore system 1000 (BIAcore AB) with biotinylated DNA fragments immobilized on a streptavidin chip (SA5, BIAcore AB). Interaction was detected by monitoring the mass concentration-dependent changes of the refractive index on the sensor surface, expressed as resonance units (RU).
Biotinylated Bio-ctx- (204 bp) and Bio-hel- (417 bp) DNA fragments were amplified from the pBSK()ctx21 and pBSK()HELE35Q plasmids by standard PCR using the primer pairs Bioctxup (5'-biotin-TCTAGAAGTGAAACGGGG-3')/Suctxsig and Biohelup (5'-biotin-GTCGACCCCGGGAAAGTC-3')/Hello, respectively. The fragments were purified from agarose gels and immobilized on the SA5 sensor chip by injecting 25 µl of HBS buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween-20) containing 2.1 pmol/µl Bio-ctx or Bio-hel at a flow rate of 2 µl/min. Injection of Bio-ctx resulted in 306 RU, injection of Bio-hel in 253 RU, corresponding to final DNA densities on the chip surfaces of about 2.1 and 1.0 fmol/mm2, respectively. The Bio-ctx fragment was immobilized in 2-fold molar excess to compensate for the 2-fold length of the Bio-hel fragment and thus to display comparable numbers of potential DNA binding sites on both chip surfaces.
For interaction studies, 20 pmol of protein in 10 µl of shift buffer (without BSA) were injected at a constant flow rate of 2 µl/min. The chip surfaces were regenerated by flushing with 10 ml of 1 M NaCl. All solutions used for SPR were filtered (0.22 µM) and degassed.
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Results |
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Using the Bence Jones protein REIv and the leucine zipper domain of the Saccharomyces cerevisiae transcriptional activator GCN4 (Hope and Struhl, 1987) as model proteins for homodimerization, it has previously been demonstrated that the ToxR-system allows the analysis of protein stability and the detection of symmetric protein interactions in both the cytoplasm and the periplasm of E.coli (Kolmar et al., 1994
, 1995a
,b
). To examine whether the ToxR system would also be applicable to detect asymmetric protein interactions in both cellular compartments, we chose here the leucine zippers of the transcriptional activators Jun and Fos (O'Shea et al., 1989
) as model proteins for heterodimerization. Since Jun has been reported also to form homodimers in absence of Fos, we used the homodimerization-deficient JunLeu14Phe/Leu21His (JunL14F/L21H) protein (Smeal et al., 1989
) as a control. Furthermore, because it has been shown that ToxR derivatives in which the periplasmic ToxR domain is substituted only by a short leucine zipper peptide are not stably inserted into the inner membrane (Kolmar et al., 1995a
), we generated tripartite fusion proteins by placing the monomeric maltose-binding protein MalE between the transmembrane (ToxR') or cytoplasmic [ToxR'(
TM)] ToxR modules and the C-terminal leucine zipper peptides (Figure 2B). The ability of these ToxR chimeras to initiate transcription at the ctx promoter was then analyzed by measuring the ß-galactosidase activity resulting from a chromosomal ctx::lacZ fusion in the E.coli reporter strain FHK12 (Kolmar et al., 1995a
) (Figure 2C). As a positive control for dimerization, we also re-analyzed the corresponding membrane-anchored and soluble GCN4 fusion proteins (Kolmar et al., 1995a
; Figure 2B and C). Finally, to allow a quantitative comparison of the transcriptional activities, we examined the stability and the cellular levels of the ToxR fusion proteins by immunoblotting (Figure 2D).
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The cytoplasmic DNA-binding domain of ToxR promotes oligomer formation in vitro
The soluble ToxR'(TM)MalE and ToxR'(
TM)MalEGCN proteins and the corresponding variants lacking the MalE moiety, ToxR'(
TM) and ToxR'(
TM)GCN, respectively, were purified and their apparent molecular weights determined by gel filtration to confirm their monomeric and dimeric states, respectively. The maltose-binding protein MalE and a MalEGCN fusion protein, which forms homodimers with a KD below 0.1 µM (Hope and Struhl, 1987
; O'Shea et al., 1989
), served as monomeric and dimeric control proteins (Table II). The analysis confirmed a monomeric state of ToxR'(
TM)MalE and a dimeric state of ToxR'(
TM)MalEGCN. The ToxR'(
TM) and ToxR'(
TM)GCN proteins, however, revealed apparent Mr values that correspond to dimeric and pentameric states, respectively (Table II), suggesting that additional interaction sites may exist in the ToxR portion of the fusion proteins. In the ToxR'(
TM)MalE and ToxR'(
TM)MalEGCN proteins the voluminous MalE moiety may sterically hinder the association of these interaction sites.
The hinge region of ToxR is required for oligomer formation in vitro but not for activation of ctx transcription in E.coli
In the soluble ToxR fusion proteins, two regions are possible candidates for mediating the oligomerization that was observed in vitro. Firstly, the fusion proteins have been constructed in such a way that they retain a small portion of the C-terminal periplasmic domain of ToxR (Kolmar et al., 1995a; amino acids 199210 indicated by a black box in Figure 2B and Figure 3B and referred to as region X). The periplasmic domain, however, has been suggested to be important for the dimerization of ToxR in vivo (Dziejman and Mekalanos, 1994
) and therefore it may be region X that is responsible for the in vitro oligomerization of the soluble ToxR fusion proteins. Alternatively, interaction sites responsible for oligomer formation may exist in the N-terminal cytoplasmic ToxR module. While its amino acids 20123 show sequence similarity to the DNA binding/transcription activation wHTH domain of OmpR-like transcriptional activators (Martínez-Hackert and Stock, 1997
; Krukonis et al., 2000
) (Figure 3A), its amino acids 124181 link the wHTH domain to the transmembrane segment (amino acids 182198) of ToxR. This short hinge region is present in the soluble ToxR fusion proteins and might also mediate their oligomerization.
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Analysis of DNA binding by soluble ToxR derivatives
To study the requirements for specific DNA binding by ToxR we analyzed the interaction of ToxR'(TM) and ToxR'(
TM)GCN with DNA in electrophoretic mobility shift assays (Figure 5) and by surface plasmon resonance (SPR) (Figure 6). For both experimental approaches, an about 200 bp ctx promoter fragment and an about 410 bp hel fragment were used as specific and non-specific DNA probes, respectively. The core sequence (138 bp) in the ctx fragment is identical with the chromosomal ctx promoter region in the E.coli reporter strain FHK12 (Kolmar et al., 1995a
) but, in contrast to the ctx promoter region of V.cholerae (Miller et al., 1987
; GenEMBL accession number 00171), carries an only 7-fold instead of an 8-fold direct repeat of the heptanucleotide sequence TTTTGAT. The flanking regions of ctx (
30 bp) originate from pMc vector DNA (see Supplementary data, Plasmid constructions). The hel-DNA fragment corresponds to nucleotides 79471 of GenBank accession number V00428, encoding mature hen egg lysozyme with a G to C mutation in codon 35 (helE35Q). The fragment is flanked on either side by eight base pairs originating from the primers used for its PCR amplification (see Supplementary data, Plasmid constructions).
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In contrast to ToxR'(TM)GCN, eight times more protein (40 pmol) was required to observe complex formation between ToxR'(
TM) and ctx-DNA. At 40 pmol, however, both ToxR'(
TM) and ToxR'(
TM)GCN already interacted with the helE35Q control DNA (Figure 5B), ToxR'(
TM)GCN again forming high molecular weight aggregates. This suggests that these complexes rather reflect non-specific binding of the proteins to ctx and hel DNA, which might occur possibly because at higher protein concentrations all potential non-specific binding sites provided by the cold salmon sperm competitor DNA have been bound. Still, a further increase in protein concentration resulted in a further shift of ToxR'(
TM)ctx complexes (Figure 5A) but not of ToxR'(
TM)helE35Q complexes (Figure 6B), suggesting that ToxR'(
TM) exhibits higher binding specificity for ctx- than for helE35Q-DNA. Finally, the mobility shift patterns obtained for binding of the oligomerization-deficient ToxR'(
139181)(
TM)(
X)GCN protein to ctx- and hel-DNA were similar to those obtained for ToxR'(
TM)GCN binding to these DNA fragments (data not shown and Figure 5). Hence the ability to form higher order oligomers is not required for specific DNA binding by ToxR dimers and does not affect the binding mode.
The interaction of ToxR'(TM)GCN and ToxR'(
TM) with ctx- and helE35Q-DNA was also analyzed by SPR (Figure 6). The biotinylated DNA-fragments were immobilized on a streptavidin sensor chip as described in Materials and methods and the proteins (20 pmol in 10 µl of sample buffer) passed over the chip surface at a constant flow rate of 2 µl/min. Injection of ToxR'(
TM)GCN to the ctx chip resulted in a fast initial association phase that plateaued at about 2000 response units (RU), indicating that the protein rapidly bound to the ctx fragment until steady state (Figure 6A). ToxR'(
TM)GCN then dissociated in two phases. Unbound or loosely attached protein was washed off the surface in a first rapid dissociation phase, which was followed by a second, slow dissociation phase that started at
750 RU and very slowly approached the baseline, indicative of a fairly strong interaction of ToxR'(
TM)GCN with the ctx fragment. In contrast, the sensorgram of ToxR'(
TM) binding to the ctx chip hardly differs from the buffer control in that a plateau was reached immediately upon injection, indicating that no or only little protein bound to the chip surface. Accordingly, dissociation occurred rapidly back to the baseline level. Hence ToxR'(
TM)GCN but not ToxR'(
TM) binds with high affinity to ctx, again suggesting dimerization as the critical parameter for specific ToxRDNA interaction. To rule out the possibility that the ToxR'(
TM)GCNDNA interaction was mediated by the GCN component, we also examined ctx binding by a BlaGCN fusion protein. As for ToxR'(
TM), the resulting sensorgram did not reveal any specific interaction of BlaGCN with the ctx-DNA chip surface. Hence the GCN component does not mediate DNA binding.
Finally, all three proteins showed very similar sensorgrams when passed over the helE35Q-DNA chip (Figure 6B). Their traces did not reveal any significant proteinDNA interactions and differed only in their signal heights. Only a very slight retardation in the dissociation of ToxR'(TM)GCN from the hel chip indicates that some non-specific ToxR'(
TM)GCNhelE35Q interactions might have occurred and appear to be even more stable than ToxR'(
TM)ctx interactions.
Overall, the mobility shift and SPR data show that the cytoplasmic ToxR module ToxR'(TM) binds DNA with only low specificity. In contrast, ToxR'(
TM)GCN exhibits strong specificity for ctx-DNA, corroborating the notion that dimerization is a prerequisite for efficient and specific DNA binding by ToxR.
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Discussion |
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Requirements for specific DNA binding and transcriptional activation by ToxR
We have shown that the soluble ToxR'(TM) and ToxR'(
TM)GCN proteins form dimers and higher order oligomers in vitro, respectively, suggesting that the N-terminal cytoplasmic ToxR module harbors previously unidentified interaction sites. We have confirmed that oligomerization is not mediated by a short fragment of the periplasmic ToxR domain (region X) present in all ToxR fusion proteins. The existence of additional interaction sites in the N-terminal DNA-binding module of ToxR has consequences for the applicability of ToxR as a genetic indicator for proteinprotein interactions, since it might cause misleadingly high transcriptional activities particularly for ToxR fusion proteins with only weakly interacting C-terminal fusion partners. Therefore, we have identified the region in the N-terminal ToxR module responsible for in vitro oligomerization and asked whether this region affects ToxR function. Our data show that in vitro ToxRToxR interactions are mediated by a short hinge region, which in the full-length ToxR protein lies between the wHTH motif and the transmembrane segment (amino acids 139181). The hinge region, however, is neither required for specific ctx-DNA binding in vitro nor for ctx promoter activation in E.coli, suggesting that the wHTH motif of the N-terminal ToxR domain in fusion with a dimerizing protein module is sufficient to activate transcription at ctx in E.coli. We have not yet tested whether the hinge region can also be deleted from membrane-anchored ToxR derivatives without loss of function. Possibly it confers flexibility on the N-terminal domain of membrane-anchored ToxR proteins that is required to allow its association into a functional dimeric state (see discussion below). Finally, the identification of the hinge region in ToxR's N-terminal domain as the site responsible for mediating ToxRToxR interactions may also be of biological relevance. Using a system that reports via transcriptional activation of a PRMlacZ fusion, it has previously been shown that ToxR dimers are capable of interacting cooperatively and that these interactions are mediated by its cytoplasmic module (Dziejman et al., 1999
). Hence the hinge region would be a primary candidate for an interaction site that is responsible for these cooperative interactions. Although they may not be important for binding and activation of ctx (our data do not support a role of cooperative ToxR interactions in ctx binding and activation in E.coli and in V.cholerae the direct involvement of ToxR in ctx activation remains uncertain), they may still be relevant for the ToxR-mediated regulation of other ToxR-regulon genes in V.cholerae. Cooperative ToxR interactions have, for example, been suggested to play a role in the binding and activation of the ompU promoter (Crawford et al., 1998
).
One question that arises from our studies is why ToxR'(TM) dimerizes in vitro but does not activate transcription at ctx in vivo. Our electrophoretic mobility shift assays show that the isolated cytoplasmic ToxR'(
TM) module in principle is capable of binding to DNA but that it acquires specificity for the ctx promoter sequence only in fusion with a dimerizing protein module (e.g. GCN4). Binding of ToxR'(
TM) to ctx required a protein concentration that also resulted in unspecific interactions with the hel control DNA (protein:DNA ratio of 800:1). In contrast, first ToxR'(
TM)GCNctx complexes were observed at a protein:DNA ratio of 100:1. Moreover, the ToxR'(
TM)GCN protein interacted more strongly with the ctx fragment than ToxR'(
TM), as indicated by its slow dissociation from ctx in SPR experiments. Hence the GCN-mediated dimerization of ToxR'(
TM) increases its DNA-binding specificity. Furthermore, ToxR'(
TM)GCN bound ctx in a concentration-dependent manner, suggesting the existence of multiple binding sites which are occupied successively by multiple ToxR molecules. The seven tandemly repeated copies of the TTTTGAT heptad in the ctx promoter fragment are primary candidates for such multiple ToxR binding sites. This would also be consistent with earlier observations that ToxR requires multiple TTTTGAT direct repeats upstream of the ctxAB structural genes for binding and that promoter activity as well as the apparent relative strength of the ToxRctx interaction increases with the number of repeats (Miller and Mekalanos, 1984
; Miller et al., 1987
; Pfau and Taylor, 1996
). Overall, we conclude that dimerization of ToxR'(
TM) and ToxR'(
TM)GCN involves different surfaces of the cytoplasmic ToxR module, such that the ToxR'(
TM)GCN but not the ToxR'(
TM) dimer is sterically qualified to interact specifically with the ctx promoter sequence. Finally, deletion of the hinge region from ToxR'(
TM)GCN did not affect its interaction with the ctx and hel fragments, indicating that the oligomerization of ToxR dimers does not alter their DNA-binding specificity and that oligomerization is not required for a rapid and complete occupation of ctx in vitro.
A ToxR-based prokaryotic two-hybrid system
Currently, two systems exist that use ToxR as a genetic indicator for folding stability (Kolmar et al., 1995b) and for interactions between heterologous protein modules or membrane-spanning protein domains (Kolmar et al., 1994
, 1995a
,b
; Langosch et al., 1996
; Russ and Engelman, 1999
). Both systems are based on the original notion that the activity of ToxR as a transcriptional activator of ctx in E.coli depends on the dimerization of its cytoplasmic DNA-binding domain (Miller et al., 1987
; DiRita and Mekalanos, 1991
) and both take advantage of the finding that the periplasmic domain of ToxR and its transmembrane segment can be replaced individually by heterologous protein modules without loss of function. The ToxR system developed by Kolmar et al. (1994)
and used in this study assays dimerization-induced ctx activation by monitoring ß-galactosidase activity originating from a single chromosomal copy of the ctx::lacZ reporter fusion (E.coli strain FHK12). The various ToxR constructs are constitutively produced from a phasmid vector. Besides its applicability to study symmetric interactions of heterologous fusion partners in the periplasm of E.coli, this system has also been shown to be a useful tool for the detection of protein homodimerization in the E.coli cytoplasm (Kolmar et al., 1994
, 1995a
). Using the leucine zippers Jun and Fos as model proteins for heterodimerization, we have shown here that the ToxR system also allows the detection of asymmetric protein interactions in both cellular compartments of E.coli. Consistent with similar association constants of 107 M1 for GCN and for Jun/Fos, respectively (Blondel and Bedouelle, 1991
; Pernelle et al., 1993
), the transcriptional activity of the co-produced membrane-anchored and soluble ToxR-Fos/Jun proteins was comparable to that of the respective homodimerizing ToxR-GCN fusion proteins. We were not able, however, to monitor Jun/Jun homodimerization even though at least the membrane-anchored ToxR'MalEJun protein was present in the cells at a level comparable to ToxR'MalEGCN. One possible explanation for this lack of detectable transcriptional activity may be a lower stability of Jun/Jun homodimers as compared with Fos/Jun heterodimers. Studies performed by Smeal et al. (1989)
are in support of this assumption, although to our knowledge an association constant has not yet been determined for the Jun/Jun homodimer. However, the failure to measure ToxR'MalEJun-mediated transcriptional activation of ctx suggests that it may be lower than the detectable threshold value of the ToxR system. The determination of this threshold value would therefore be a valuable contribution to the enhancement of the ToxR system.
Another aspect of ToxR's technical utility is reflected in the TOXCAT assay, which was developed to study the association of transmembrane helices in a natural membrane environment (Russ and Engelmann, 1999). TOXCAT uses a plasmid-encoded ctx:chloramphenicol acetyl transferase (CAT) reporter fusion to measure ctx activation by quantifying chloramphenicol acetylation by CAT in vitro or by acquired resistance to chloramphenicol in vivo. The chimeric toxR genes are located on the same plasmid under control of a lac promoter to allow a modulation of their cellular concentration.
Overall, the ToxR system provides the advantages that E.coli-based two-hybrid systems have over the yeast two-hybrid system (for a review, see Hu et al., 2000), but, by allowing the analysis of periplasmic and transmembrane proteinprotein interactions, offers an even broader potential application range than the prokaryotic two-hybrid systems described to date (e.g. Hu et al., 1990
; Dove et al., 1997
; Dmitrova et al., 1998
; Karimova et al., 1998
; Di Lallo et al., 2001
). Moreover, the option to study interactions in the periplasm of E.coli provides a promising basis for the expansion of the ToxR system towards screening of low molecular weight compounds to study ligand-binding or ligand-induced conformational changes, since it obviates the necessity for such compounds supplied externally with the E.coli growth medium to pass the cytoplasmic membrane. In this context, the ToxR system could furthermore be adjusted for high-throughput screening by converting it into a mating-based, conjugative system, as has recently been described for the LexA-based prokaryotic two-hybrid system (Clarke et al., 2005
).
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Acknowledgements |
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Received May 6, 2005; revised July 19, 2005; accepted July 22, 2005.
Edited by Marius Clore
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