INTRODUCTION

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Vibrio cholerae is the causative agent of the diarrheal disease cholera, which is usually acquired by oral ingestion of the bacterium with contaminated water or food (10). In response to specific environmental conditions, such as temperature, pH, or osmolarity (11, 23), V. cholerae expresses several virulence determinants, including the cholera toxin (CTX), a toxin-coregulated pilus (TCP), the accessory colonization factor, and a major outer membrane protein (OmpU) (24, 28, 34, 36). CTX is the best-characterized virulence factor and is composed of a single A subunit and five identical B subunits (12, 27). The enzymatically active A subunit is predominantly responsible for fluid loss through an ADP-ribosylation mechanism that results in constitutive cyclic-AMP (cAMP) production in host cells, leading to the opening of normally gated channels (1). Environmental signals optimal for CTX production also stimulate the expression of TcpA (17, 19) and OmpU, a porin that may also function as an adhesin (4, 33, 34). TCP is a pilus in the type IV family that is essential for colonization and virulence. It is made up of a single protein encoded by the tcpA gene, which is part of a pathogenicity island that includes other tcp genes whose products are involved in the biogenesis of the pilus structure, as well as the acf genes (18, 19, 26, 28).

Coordinate expression of ctxAB, tcpA to F, and some acf genes is due to the action of several regulatory proteins. In the current model, these proteins function in a branched regulatory cascade in which two activator proteins, ToxR and TcpP, are required for activation of toxT transcription (13, 32) and ToxT, a member of the AraC family of transcriptional activators (15), activates the expression of other virulence genes, including ctxAB and tcpA to F (9, 32). The ompU gene is in a ToxT-independent branch of this cascade and is activated directly by ToxR (5, 6).

Both ToxR and TcpP are inner membrane proteins with cytoplasmic DNA-binding domains homologous to members of the two-component family of transcriptional activators found in various species of bacteria (13, 24, 25). Each of these proteins is encoded by an operon by which another membrane protein is encoded. For ToxR, this protein is ToxS, and for TcpP, it is TcpH (3, 8, 21). ToxS and TcpH act as effector proteins for ToxR and TcpH, respectively, through a mechanism that likely involves periplasmic interaction between the regulator and the effector (3, 8). The precise mechanism by which ToxRS and TcpPH control toxT transcription is not understood. One observation that may eventually contribute to a better understanding of this system is that overexpression of TcpP suppresses a toxR mutant for toxT expression, while overexpression of ToxR does not suppress a tcpP mutant (13, 14, 19a).

This work examined the transcription of toxT in the context of its location at the end of the tcp gene cluster. Previous genetic analysis demonstrated that insertion mutations in the tcpA gene resulted in downregulation of the production of both TCP and CTX (2). This was interpreted as being due to the polar effects of the insertions on toxT, because RNase protection experiments showed that the tcp gene cluster, including toxT, was transcribed as a long polycistronic message (2). In addition, it was recently shown that the cAMP-cAMP receptor protein complex plays a negative role in regulation of toxin production, potentially through repression of tcpA transcription, which may result in decreased toxT transcription through transcriptional polarity (31, 32). Other work demonstrated that ToxR-dependent activation of toxT occurs at a promoter that is immediately upstream of toxT (14) and that a transcription terminator with 80% efficiency precedes the toxT gene downstream of tcpF.

In one model that would account for these observations, ToxR (in conjunction with TcpP) activates toxT from the proximal promoter and ToxT activation of the tcpA promoter contributes to subsequent expression of the gene through readthrough of the relatively inefficient transcription terminator (compared to a well-characterized  terminator) between tcpF and toxT (14). This model makes specific predictions about the contributions of different regulatory elements within the tcp gene cluster, and in this work, we tested some of those predictions.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. V. cholerae and Escherichia coli strains were grown in Luria-Bertani (LB) medium at 30 or 37°C. Strains were maintained at 70°C in LB medium with 20% glycerol. Antibiotics were used at the following concentrations (unless otherwise stated): ampicillin, 100 µg/ml; streptomycin, 100 µg/ml; kanamycin, 30 µg/ml. Plasmids were introduced into E. coli strains by either transformation or electroporation and into V. cholerae strains by conjugation.

DNA manipulation. Plasmid DNA was purified with Qiagen columns (Qiagen, Inc.). Cloning was performed by using standard protocols (29). PCR was performed with the Expand High Fidelity PCR System (Boehringer Mannheim) by using the manufacturer's protocols. A recombinant PCR method, gene splicing by overlap extension (16), using primers within and surrounding the toxT promoter region, the tcpA promoter region, tcpP, and tcpH, was performed to create deletions of the corresponding regions (for a description of the construction of toxT_hth, see reference 5). The DNA templates (15, 36) and primers used for construction of each mutant are listed in Table 1. This procedure requires two PCR rounds, as follows (using the toxT promoter deletion as an example). Outside primer 1 is complementary to a sequence in tcpF (upstream of the toxT promoter), and primer 4 is complementary to a sequence in toxT (downstream of the toxT promoter), and they both have restriction endonuclease sites inserted to mediate cloning. Inside overlapping primers 2 and 3 were designed to delete the toxT promoter region, and they are partly complementary to the sequence upstream and partly complementary to the sequence downstream of the desired deletion. First, two PCRs were carried out, one with primers 1 and 2 and the other with primers 3 and 4. The product of each reaction was purified by agarose gel electrophoresis, followed by gel extraction with the QIAEX II gel extraction system (Qiagen, Inc.), mixed together, and used as the template for a second round of PCR with outermost primers 1 and 4. The amplified PCR fragment was cloned into the corresponding sites of positive-selection suicide plasmid pKAS32, which cannot replicate in V. cholerae in the absence of the Pir protein (30).

RNA analysis. For primer extension analysis, an overnight culture of V. cholerae grown in LB medium at 30°C to late log phase (optical density at 600 nm [OD₆₀₀], >3.0) was subcultured 1:100 into fresh LB medium. After back dilution (t = 0) and at 1-h intervals, aliquots of the cultures were removed and poured over crushed ice prior to centrifugation for cell recovery. Cell pellets were stored at 20°C until ready for RNA isolation. RNA was obtained from cultures of V. cholerae by using TRIzol Reagent (Gibco BRL) by following the manufacturer's protocols. The RNA samples were then treated with DNase I and quantified by determination of the OD₂₆₀. The same amount of RNA was used for primer extension as previously described (14), by using a toxT-specific primer (5'-CATTAGTTTGAAAAGATTTTTTCCCAATCAT-3') or a tcpA-specific primer (5'-TTCTTTTACAAATTTCTTCTTAAAAAGCTGTTTTAA-3').

Protein analysis. Total cell lysates were prepared from V. cholerae cells grown to stationary phase overnight (with 1 mM isopropyl--D-thiogalactopyranoside [IPTG] if necessary). Samples (1 ml) were removed from the cultures and centrifuged. The harvested pellet was resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer, normalized for OD₆₀₀, and boiled for 5 min. Aliquots of this lysate were subjected to electrophoresis on a 12% polyacrylamide gel with a 5% stacking gel. For Western blotting, the gels were blotted onto nitrocellulose and probed with anti-TcpA peptide 6 rabbit polyclonal antibodies kindly supplied by Ron Taylor (Dartmouth Medical School).

	RESULTS

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Transcription of toxT in the tcp gene cluster. To analyze transcription of toxT, we used derivatives of classical strain O395 carrying deletions of key elements in the tcp gene cluster (Fig. 1). We had observed that wild-type strain O395 produces little detectable toxT mRNA after overnight growth and exploited this observation to establish a time course experiment as shown in Fig. 2A. When primer extension of toxT RNA was done on samples prepared from wild-type strain O395 after 1:100 back dilution of an overnight culture, we observed that both of the primer extension products previously described (14) became detectable within an hour after back dilution. The smaller primer extension product (99 bp) has been previously shown to initiate from a ToxR-dependent promoter immediately preceding the toxT open reading frame (ORF) (14). The source of the larger product (204 bp) is less clear, but it has been speculated to arise from either an RNA-processing event or a block to reverse transcription due to potential secondary structure in the RNA at that position (14, 15). In this experiment, the smaller product was more abundant within the first hour, but by 2 h, the two products were roughly equivalent. By 5 to 9 h after back dilution, each began to diminish in prevalence (Fig. 2A) and after 9 h, they became undetectable (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of the V. cholerae tcp gene cluster. Shown in the center is the organization of the tcp gene cluster of wild-type V. cholerae. Each box represents a gene. The right-angle arrows represent promoters. Certain regions are enlarged to show the details of each deletion. Brackets indicate the deleted sequence, and the number below each bracket is the nucleotide position, relative to the +1 start site for transcription, which represents the start or the end of a deletion. Arrows at the toxT promoter region denote three pairs of inverted repeats. tcpP and tcpH are in an operon, and their ORFs overlap. The ORFs are shown above the operon. The leftward-pointing arrows beneath toxT and tcpA represent primers used in the RNA primer extension analyses. SD, Shine-Dalgarno region; HTH, helix-turn-helix motif.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   RNA primer extension analyses of V. cholerae wild-type O395 and a toxThth mutant strain (A) and toxTpro and tcpApro mutant strains (B). Samples were collected at 1-h intervals after back dilution of an overnight culture grown in LB medium at 30°C 1:100 into fresh LB medium. A radiolabeled toxT primer was used. RNA collected from wild-type strain O395 after 2 to 3 h of growth was used as a positive control for the mutant strains in panel B.

View larger version (73K): [in this window] [in a new window]   FIG. 3.   RNA primer extension analyses of V. cholerae strain O395 wild-type (A) and mutant strains toxTpro and tcpApro (B). Samples were collected at 1-h intervals after back dilution of an overnight culture grown in LB medium at 30°C 1:100 into fresh LB medium. A radiolabeled tcpA primer was used. RNA collected from wild-type strain O395 after 2 to 3 h of growth was used as a positive control for the mutant strains in panel B. The position of the tcpA transcript is indicated.

toxT_hth

toxT_hth

toxT_pro

toxT_pro

toxT_pro

toxT_pro

tcpA_pro

toxT_pro

tcpA_pro

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Western blot analysis using anti-TcpA antibody. V. cholerae wild-type O395 and mutant strains toxR, toxTpro, and tcpApro (A); wild-type O395 and mutant strain toxTpro/pMMB66HE or toxTpro/ptoxT (B); and wild-type O395 and mutant strain tcpApro/pMMB66HE or tcpApro/ptoxT (C) were grown overnight in LB medium at 30°C. IPTG (1 mM) was added to induce toxT transcription in strains carrying plasmids. ptoxT harbors the toxT ORF on low-copy-number plasmid pMMB66HE under the control of the IPTG-inducible tac promoter. Protein molecular size standards (lane M) are indicated on the left. w.t., wild type.

TcpP and TcpH are required for toxT transcription and coordinate regulation. Two other gene products encoded in the tcp gene cluster, TcpP and TcpH, have been implicated in coordinate regulation in V. cholerae through their effects on toxT transcription (3, 13, 20). To characterize the role of TcpP and TcpH in the context of the regulatory loop model analyzed above, mutant strains carrying deletions in these genes (tcpP and tcpH) were constructed and their RNAs were used in primer extension reactions with a radiolabeled toxT primer. Deletion of the tcpP gene resulted in complete abolishment of both toxT-specific primer extension products (Fig. 5A) and reduction of TcpA to the background level (data not shown). Both toxT transcription and TcpA production were restored upon introduction of a tcpPH-encoding plasmid into the tcpP mutant.

View larger version (34K): [in this window] [in a new window]   FIG. 5.   RNA primer extension analyses of V. cholerae mutant strains tcpP/pMMB66EH and tcpP/ptcpPH (A) and mutant strain tcpH (B). Samples were collected at 1-h intervals after back dilution of a culture grown overnight in LB medium at 30°C 1:100 into fresh LB medium. IPTG (1 mM) was used to induce transcription in strains carrying plasmids. A radiolabeled toxT primer was used. RNA collected from wild-type strain O395 after 2 to 3 h of growth was used as a positive control for all of the mutant strains. ptcpPH harbors the tcpP and tcpH ORFs on low-copy-number plasmid pMMB66EH under the control of the IPTG-inducible tac promoter.

	DISCUSSION

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Previous studies showed that virulence gene expression in V. cholerae is controlled through a regulatory cascade by the ToxR-ToxT system (7, 9, 15). According to this model, activation of toxT expression requires ToxR, and gene fusion studies and electrophoretic mobility shift assays showed that there is a ToxR binding site between 73 and 114 relative to the toxT transcript initiation site that is required for activation of toxT transcription (14). Virulence gene expression is activated by ToxT, whose carboxyl terminus is similar to the DNA-binding domain of the AraC transcription activator of E. coli and Salmonella typhimurium (15); thus, it likely acts in a manner similar to that of AraC.

Previous work suggested that the ToxR-dependent promoter is not the only source of toxT mRNA (2, 14). This conclusion rests on the observation that insertions in the tcpA gene have a negative effect on toxin production, which suggests that a long tcp transcript initiating at the tcpA promoter also encodes toxT. RNase protection analysis supported this possibility (2). The presence of a transcription terminator between tcpF and toxT, albeit a relatively weak one compared to a well-characterized  terminator, indicates that not all transcripts initiating at the tcpA promoter would read through to toxT (14). While the results presented in this report generally support the autoregulatory loop of transcription in the tcp operon proposed by Brown and Taylor (2), there is a slight discrepancy between our results and theirs in the levels of CTX produced by strains with mutations in tcpA. The tcpA_pro strain we constructed for this study synthesizes nearly wild-type levels of CTX, with, at most, a twofold decrease compared with the wild type. In contrast, tcpA transposon insertion mutants in the study of Brown and Taylor (such as CS2-1 and RT110.21) synthesize approximately 10-fold less CTX than the wild type (2). The basis of this discrepancy is not clear.

The observation that a toxT-lacZ fusion is activated to high levels in wild-type V. cholerae but that ToxR does not activate toxT-lacZ expression in E. coli led to the suggestion that ToxR is necessary but not sufficient for activation of toxT (14). The TcpP gene product had been implicated in coordinate regulation in the ToxR system by others (20, 37), including Häse and Mekalanos, who most recently demonstrated that a tcpP mutant V. cholerae strain does not activate expression of a toxT-lacZ gene fusion (13). These investigators also showed that overexpressed TcpP activates toxT transcription in the absence of ToxR, although wild-type expression of TcpP does not (13). We also note that overexpression of TcpPH in the tcpP strain led to toxT expression that was sustained and stronger than that observed in the wild type (cf. Fig. 2A and 5A) and a concomitant increase in CTX compared to the wild type (Table 2). This implies that TcpP and TcpH are limiting in this system and, if this is so, may provide evidence for another level of control in the ToxR regulatory system. One hypothesis that might account for the elevated CTX levels when TcpP and TcpH are overexpressed is that TcpP activates ctxAB transcription directly. However, overexpression of TcpP and TcpH in the toxT_pro strain did not result in detectable CTX production (data not shown), confirming that TcpP and TcpH mediate their effect on CTX expression solely through the toxT promoter. Combined with the fact that ToxR is normally required for toxT transcription, we conclude that ToxR and TcpP work together in some way to activate the toxT promoter.

These data support the following model for transcription control of toxT and coordinate regulation of virulence in V. cholerae (Fig. 6). ToxR, ToxS, TcpP, and TcpH in the inner membrane activate toxT transcription from the toxT promoter, generating the transcript represented by the smaller extension product seen in our experiments. ToxT made from this transcript activates the tcpA promoter, producing a long transcript that reads through a weak terminator between tcpF and toxT, thereby generating another source of toxT mRNA. The readthrough transcript may be processed at the site to which the longer primer extension product maps. ToxT from both transcripts is necessary for maximal expression of CTX.

View larger version (22K): [in this window] [in a new window]   FIG. 6.   Model for control of toxT transcription and coordinate regulation of virulence in V. cholerae. ToxR, ToxS, TcpP, and TcpH in the inner membrane activate toxT transcription from the toxT promoter. ToxT protein then activates transcription of the tcpA promoter, producing more ToxT from the readthrough transcript. ToxT also activates transcription of other virulence genes, including ctx, as shown here. The tcp gene cluster and the ctx operon are shown with each box representing a gene. Symbols: , promoter; , transcript of the relevant genes; stem-loop structure which is a putative RNA-processing site for the readthrough transcript initiating from the tcpA promoter.

There are alternative hypotheses that would account for our data regarding the two RNA species observed in the primer extension experiments. One is that the larger one originates from a promoter activated by a product of one of the genes coexpressed with tcpA. We do not favor this possibility because none of the ORFs downstream of tcpA encodes a protein with homology to transcription activators. Another hypothesis is that readthrough transcription could result in the utilization of another upstream toxT promoter. Although this has not been ruled out, there are no putative 35 and 10 sequences preceding the site to which the larger toxT primer extension product maps.

The fact that two different activators, ToxR and TcpP, are required for priming of the autoregulatory loop leading to toxT expression raises questions about how this system evolved. One way to view this issue is to assume that prior to acquisition of the V. cholerae pathogenicity island (18) by virulent V. cholerae, ToxR controlled only ompU expression (as well as that of the ToxR-repressed ompT gene). While the island encodes an activator of toxTTcpPthe level of toxT activation by TcpP alone in V. cholerae may not have been sufficient for a competitive advantage. However, under conditions appropriate for activation of ompU by ToxR, perhaps there was a competitive advantage for strains that also expressed CTX and TCP. This would have been a driving force allowing ToxR to take control of the TcpP-dependent activation of toxT with subsequent CTX and TCP expression. That TcpP remains a requirement in this system (i.e., that ToxR did not gain complete control over toxT transcription) may indicate that its role in toxT expression is not limited to transcription activation. One possibility, for example, is that TcpP is an important component of the signaling pathway leading to toxT expression.