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Vibrio cholerae is the causative agent of cholera, a severe diarrhoeal illness. V. cholerae produces two major virulence factors: the cholera toxin, which directly causes diarrhoea, and the toxin-coregulated pilus, which is required for intestinal colonization. Production of these virulence factors is dependent on the major virulence regulator, ToxT. Under virulence-inducing growth conditions, transcription factors ToxR and TcpP initially activate transcription of toxT. However, once ToxT has been expressed, it produces more of itself independent of ToxR and TcpP by activating transcription of the long tcpA operon, within which toxT is located. It is known that V. cholerae terminates virulence gene expression prior to escape from the host, but it is unknown how this ToxT-positive feedback loop is broken, an essential step in terminating virulence gene expression. To better understand how ToxT protein activity is regulated, we monitored ToxT accumulation and activity under virulence-inducing and -repressing growth conditions. Our results suggest that ToxT protein undergoes proteolytic degradation to terminate virulence gene expression. This directed degradation of ToxT supports a model for terminating V. cholerae virulence gene expression late in infection, with both ToxT and TcpP undergoing proteolysis prior to escape from the host., (© 2011 Blackwell Publishing Ltd.)

Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post-transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae. Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT-activated gene tcpI. TarA regulates ptsG, encoding a major glucose transporter in V. cholerae. Cells overexpressing TarA exhibit decreased steady-state levels of ptsG mRNA and grow poorly in glucose-minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base-pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae. Although somewhat functionally analogous to SgrS of Escherichia coli, TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates., (© 2010 Blackwell Publishing Ltd.)

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.

The Gram-negative bacterium Vibrio cholerae is the infectious agent responsible for the disease Asiatic cholera. The genes required for V. cholerae virulence, such as those encoding the cholera toxin (CT) and toxin-coregulated pilus (TCP), are controlled by a cascade of transcriptional activators. Ultimately, the direct transcriptional activator of the majority of V. cholerae virulence genes is the AraC/XylS family member ToxT protein, the expression of which is activated by the ToxR and TcpP proteins. Previous studies have identified the DNA sites to which ToxT binds upstream of the ctx operon, encoding CT, and the tcpA operon, encoding, among other products, the major subunit of the TCP. These known ToxT binding sites are seemingly dissimilar in sequence other than being A/T rich. Further results suggested that ctx and tcpA each has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptional lacZ fusions and in vitro copper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribed acfA and acfD genes, which encode components of the accessory colonization factor required for efficient intestinal colonization by V. cholerae. Our results indicate that ToxT binds to a pair of DNA sites between acfA and acfD in an inverted repeat orientation. Moreover, a mutational analysis of the ToxT binding sites indicates that both binding sites are required by ToxT for transcriptional activation of both acfA and acfD. Using copper-phenanthroline footprinting to assess the occupancy of ToxT on DNA having mutations in one of these binding sites, we found that protection by ToxT of the unaltered binding site was not affected, whereas protection by ToxT of the mutant binding site was significantly reduced in the region of the mutations. The results of further footprinting experiments using DNA templates having +5 bp and +10 bp insertions between the two ToxT binding sites indicate that both binding sites are occupied by ToxT regardless of their positions relative to each other. Based on these results, we propose that ToxT binds independently to two DNA sites between acfA and acfD to activate transcription of both genes.