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Journal of Bacteriology, October 2002, p. 5533-5544, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5533-5544.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mechanism of ToxT-Dependent Transcriptional Activation at the Vibrio cholerae tcpA Promoter{dagger}

Robin R. Hulbert and Ronald K. Taylor*

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755

Received 17 January 2002/ Accepted 21 June 2002

The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae. ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others. In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from -95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to -63. A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation. Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer. Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit. A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at -72 and -51 and requires the alpha C-terminal domain of RNA polymerase.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, HB7550, Dartmouth Medical School, Hanover, NH 03755. Phone: (603) 650-1623. Fax: (603) 650-1318. E-mail: ronald.k.taylor{at}dartmouth.edu.

{dagger} For a commentary on this article, see p. 5529 in this issue.

Journal of Bacteriology, October 2002, p. 5533-5544, Vol. 184, No. 20
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.20.5533-5544.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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