Identification of the TcpP-binding site in the toxT promoter of Vibrio cholerae and the role of ToxR in TcpP-mediated activation

Abstract

ToxR-dependent recruitment of TcpP to the toxT promoter facilitates toxT transcription in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the toxT promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of toxT promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne toxT-lacZ fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal toxT promoter. When present in ΔtoxR V. cholerae overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N(6)-TGTAA) caused defects in TcpP-dependent toxT-lacZ fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the toxT promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient toxT activation, even in the presence of ToxR. These results suggest that the toxT promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the toxT promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for toxT promoter activation.

FIG. 1.

DNA sequence of the V. cholerae classical strain O395 toxT promoter region from position −172 to +45, numbered relative to the start of transcription (5). Nucleotides corresponding to the −35 and −10 hexamers are underlined, and gray horizontal arrows below the sequence and the gray boxed regions indicate the locations of inverted repeat sequences containing ToxR binding sites (5). The region of the promoter protected by TcpP from DNase I digestion (11) and subjected to transversion mutagenesis is indicated below the DNA sequence, with the substituted nucleotides in italic. The pentameric repeats identified as the refined TcpP-binding sites are boxed in black.

FIG. 2.

Effects of TcpP-binding site mutations on toxT-lacZ and CT reporter activities in a ΔtoxR ΔtcpP background with TcpP-HSV overexpression. The positions of single- and multiple-base-pair transversions in the parental promoter are indicated relative to the toxT transcription start site. Black bars, values for ΔtoxR ΔtcpP/pMMB207 strains; white bars, values for ΔtoxR ΔtcpP/pEK41 strains overexpressing TcpP-HSV. (A) β-Galactosidase activities for 3.5-h cultures of strains carrying either pTG24 alone (empty vector) or toxT-lacZ fusions in which the parental promoter or mutant derivatives drive the expression of the lacZ reporter. Values represent the averages from at least three independent cultures. (B) CtxB levels secreted by 20-h cultures from strains carrying various chromosomal toxT promoter derivatives. toxTΔpro is a chromosomal deletion of the toxT promoter region from position −112 to +1. Values represent the averages from at least six independent cultures. ND, not determined. Strains were grown under ToxR-inducing conditions in the presence of 1 mM IPTG. Error bars represent the standard deviation for each data set. *, P < 0.05; **, P < 0.005 (relative to the parental toxT promoter).

FIG. 3.

Electrophoretic mobility shift assay for the binding of toxT promoter derivatives to TcpP-HSV in purified membrane preparations. The ³²P-end-labeled toxT promoter targets used in each lane in all panels carry the parental promoter (lanes 1, 2, and 3) or single-transversion derivatives with mutations at positions −56 to −38 (lanes 4 to 22, respectively), as indicated at the top of the figure. The horizontal arrows above each panel indicate the positions of the pentameric repeat motif (TGTAA-N₆-TGTAA) within the TcpP-footprinted region of the toxT promoter. For lanes 3 to 22, the DNA-binding solutions containing end-labeled DNA targets were mixed with membrane buffer only (A) or TcpP-containing E. coli membrane preparation at either 0.083 mg/ml (B), 0.17 mg/ml (C), 0.33 mg/ml (D), or 0.66 mg/ml (E), as indicated on the right. The positions of free and shifted end-labeled DNA target migration through the gel are indicated in the column between lanes 2 and 3. The percent shifted values given below the unshifted target band for each sample indicate the percentage of shifted signal, relative to the sum of free and shifted signals, as quantified by densitometry. Control samples containing the parental target mixed with either membrane buffer only (lane 1) or negative-control E. coli TcpP⁻ membrane preparation (lane 2), present at the same protein concentration as the experimental membrane preparation used for lanes 3 to 22 as indicated on the right side of the figure, were included in each gel run where appropriate.

FIG. 4.

Effects of TcpP-binding site mutations on toxT-lacZ and CT reporter activities in a ΔtcpP background (toxR⁺) with TcpP-HSV overexpression. The positions of single- and multiple-base-pair transversions in the parental promoter are indicated relative to the toxT transcription start site. Black bars, values for ΔtcpP/pMMB207 strains; gray bars, values for O395/pMMB207 strains; white bars, values for ΔtcpP/pEK41 strains overexpressing TcpP-HSV. (A) β-Galactosidase activities for 3.5-h cultures of strains carrying either pTG24 alone (empty vector) or derivatives in which the parental promoter or its transversion derivatives drive the expression of the lacZ reporter. Values represent the averages from at least three independent cultures (n ≥ 3). (B) CtxB levels secreted by 20-h cultures of strains carrying modified chromosomal toxT promoters. toxTΔpro is a chromosomal deletion of the toxT promoter region from position −112 to +1. Values represent the averages from at least six independent cultures (n ≥ 6). N, not determined. Error bars represent the standard deviation for each data set. Statistical variance (Student's t test) from parental promoter values: *, P ≤ 0.05; **, P ≤ 0.005. Strains were grown under ToxR-inducing conditions in the presence of 1 mM IPTG.

FIG. 5.

Models of toxT promoter activation by TcpP and ToxR. (A) Binding of ToxR to its upstream binding site (potentially centered at nucleotide −63 [12]) brings TcpP to the toxT promoter via a wing-wing interaction between ToxR and TcpP (; E. S. Krukonis et al., submitted for publication). TcpP is then released to bind its promoter-binding site centered at nucleotides −51 and −40 (the critical nucleotides of the TGTAA-N₆-TGTAA repeat). In this model, wing-wing interactions may be maintained even while ToxR and TcpP engage the DNA. (B) Based on putative assignments of the precise ToxR-binding site (12), ToxR may bind to a position further upstream, centered at nucleotide −73. In this case, ToxR could recruit TcpP to the toxT promoter via a protein-protein interaction(s), but it would then release TcpP so that TcpP could then bind its more promoter-proximal binding site (C). In both cases, TcpP binding to the center position of the TGTAA-N₆-TGTAA direct repeat (nucleotides −51 and −40) is required for toxT activation.