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The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT.
- Source :
-
Molecular microbiology [Mol Microbiol] 2006 Mar; Vol. 59 (6), pp. 1779-89. - Publication Year :
- 2006
-
Abstract
- The Gram-negative, curved rod Vibrio cholerae causes the severe diarrhoeal disease cholera. The two major virulence factors produced by V. cholerae during infection are the cholera toxin (CT) and the toxin-coregulated pilus (TCP). Transcription of the genes encoding both CT and the components of the TCP is directly activated by ToxT, a transcription factor in the AraC/XylS family. ToxT binds upstream of the ctxAB genes, encoding CT, and upstream of tcpA, the first gene in a large operon encoding the components of the TCP. The DNA sequences upstream of ctxAB and tcpA that contain ToxT binding sites do not have any significant similarity other than being AT-rich. Extensive site-directed mutagenesis was performed on the region upstream of tcpA previously shown to be protected by ToxT, and we identified specific base pairs important for activation of tcpA transcription by ToxT. This genetic approach was complemented by copper-phenanthroline footprinting experiments that showed protection by ToxT of the base pairs identified as most important for transcription activation in the mutagenesis experiments. Based on this new information and on previous work, we propose the presence of a ToxT-binding motif - the 'toxbox'- in promoters regulated by ToxT. At tcpA, two toxbox elements are present in a direct repeat configuration and both are required for activation of transcription by ToxT. The identity of only a few of the base pairs within the toxbox is important for activation by ToxT, and we term these the core toxbox elements. Lastly, we examined ToxT binding to a mutant having 5 bp inserted between the two toxboxes at tcpA and found that occupancy of both binding sites is retained regardless of the positions of the binding sites relative to each other on the face of the DNA. This suggests that ToxT binds independently as a monomer to each toxbox in the tcpA direct repeat, in accordance with what we observed previously with the inverted repeat ToxT sites between acfA and acfD.
- Subjects :
- Base Sequence
Binding Sites
DNA Footprinting
DNA, Bacterial genetics
DNA, Bacterial metabolism
Genes, Bacterial genetics
Molecular Sequence Data
Point Mutation
Transcriptional Activation
Virulence
Bacterial Proteins metabolism
Fimbriae Proteins genetics
Gene Expression Regulation, Bacterial
Promoter Regions, Genetic genetics
Transcription Factors metabolism
Vibrio cholerae genetics
Vibrio cholerae pathogenicity
Subjects
Details
- Language :
- English
- ISSN :
- 0950-382X
- Volume :
- 59
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Molecular microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 16553883
- Full Text :
- https://doi.org/10.1111/j.1365-2958.2006.05053.x