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Journal of Bacteriology, April 1999, p. 2584-2592, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analysis of an Autoregulatory Loop Controlling ToxT, Cholera Toxin, and Toxin-Coregulated Pilus Production in Vibrio cholerae

Rosa R. Yu1 and Victor J. DiRita1,2,*

Department of Microbiology and Immunology1 and Unit for Laboratory Animal Medicine,2 University of Michigan Medical School, Ann Arbor, Michigan 48109

Received 15 October 1998/Accepted 10 February 1999

Coordinate expression of many virulence genes in the human pathogen Vibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activate toxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of toxT transcription. We tested a model whereby activation of toxT by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the tcpA promoter reads through a proposed terminator between the tcpF and toxT genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxTDelta pro) resulted in the abolishment of toxT transcription, as predicted. Deletion of the tcpA promoter (tcpADelta pro) had no effect on subsequent detection of the smaller toxT primer extension product, but the larger toxT product was not detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. The toxTDelta pro strain produced little detectable CTX, while the tcpADelta pro strain produced CTX levels intermediate between those of the wild-type and toxTDelta pro strains. Dependence of toxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectable toxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest that toxT transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109. Phone: (734) 936-3804. Fax: (734) 936-3235. E-mail: vdirita{at}umich.edu.

Journal of Bacteriology, April 1999, p. 2584-2592, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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