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1.
PLoS One ; 14(9): e0221936, 2019.
Article in English | MEDLINE | ID: mdl-31498842

ABSTRACT

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT.


Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Helix-Turn-Helix Motifs , Transcription Factors/chemistry , Transcription Factors/metabolism , Adhesins, Bacterial/genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial/genetics , Models, Molecular , Mutation , Protein Binding , Protein Domains , Transcription Factors/genetics , Transcriptional Activation
2.
J Bacteriol ; 198(3): 498-509, 2016 02 01.
Article in English | MEDLINE | ID: mdl-26574510

ABSTRACT

UNLABELLED: TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCE: The Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.


Subject(s)
Bacterial Proteins/metabolism , Fimbriae Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Transcription Factors/metabolism , Vibrio cholerae/metabolism , Bacterial Proteins/genetics , Fimbriae Proteins/genetics , Mutation , Protein Conformation , Proteolysis , Transcription Factors/genetics
3.
Infect Immun ; 81(3): 884-95, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23297386

ABSTRACT

ToxR facilitates TcpP-mediated activation of the toxT promoter in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin secretion and toxin coregulated pilus expression. ToxR binds a region from -104 to -68 of the toxT promoter, from which ToxR recruits TcpP to the TcpP-binding site from -53 to -38. To precisely define the ToxR-binding site within the toxT promoter, promoter derivatives with single-base-pair transversions spanning the ToxR-footprinted region were tested for transcription activation and DNA binding. Nine transversions between -96 to -83 reduced toxT promoter activity 3-fold or greater, and all nine reduced the relative affinity of the toxT promoter for ToxR at least 2-fold, indicating that activation defects were due largely to reduced binding of ToxR to the toxT promoter. Nucleotides important for ToxR-dependent toxT activation revealed a consensus sequence of TNAAA-N(5)-TNAAA extending from -96 to -83, also present in other ToxR-regulated promoters. When these consensus nucleotides were mutated in the ompU, ompT, or ctxA promoters, ToxR-mediated regulation was disrupted. Thus, we have defined the core ToxR-binding site present in numerous ToxR-dependent promoters and we have precisely mapped the binding site for ToxR to a position three helical turns upstream of TcpP in the toxT promoter.


Subject(s)
Bacterial Proteins/metabolism , Vibrio cholerae/metabolism , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Bacterial Proteins/genetics , Base Sequence , Binding Sites , DNA Footprinting , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Molecular Sequence Data , Porins/genetics , Porins/metabolism , Promoter Regions, Genetic , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism
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