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Cholera, a severe diarrheal disease, is caused by ingestion of the gram-negative bacterium Vibrio cholerae. Expression of V. cholerae virulence factors is highly regulated at the transcriptional and posttranscriptional levels by a complex network of proteins and small noncoding RNAs. The direct activator of transcription of most V. cholerae virulence genes is the ToxT protein. ToxT binds to a 13-bp sequence, the toxbox, located upstream of genes in its regulon. However, the organization of toxboxes relative to each other and to the core promoter elements at different genes varies dramatically. At different ToxT-activated genes a single toxbox may be necessary and sufficient for full activation, or pairs of toxboxes organized as either inverted or direct repeats may be required for full activation. Although all toxboxes are located at positions consistent with a class I promoter architecture, the locations of toxboxes relative to the transcription start site also vary from gene to gene. To further assess the ability of ToxT to activate transcription from different configurations relative to the core promoter elements, we constructed promoter-lacZ fusions having altered spacing both between toxbox pairs and between the promoter-proximal toxbox and the -35 box at five different ToxT-activated promoters. Our results suggest that that ToxT has remarkable flexibility in its positioning as a transcription activator and that different interactions between ToxT and RNA polymerase occur during transcription activation of promoters having different toxbox configurations.

The Vibrio cholerae ToxT regulon includes the genes encoding cholera toxin (CT) and the toxin-coregulated pilus (TCP), which are the major virulence factors required for causing cholera disease and colonizing the upper small intestine of the host, respectively. The genes encoding CT, ctxAB, and the genes encoding the components of the TCP, tcpA to tcpJ, are organized within operons, upstream of which are DNA binding sites for the transcriptional activator ToxT. ToxT is a member of the large AraC/XylS family of transcriptional regulators and also activates transcription of five other genes whose roles in V. cholerae pathogenesis, if any, are poorly understood, acfA and acfD are divergently transcribed genes required for efficient colonization of the intestine. Transcriptional activation of acfA and acfD requires a pair of central ToxT binding sites in an inverted-repeat configuration for Tox[T.sup.-]directed transcription of both genes, tcpl has an unknown role in pathogenesis, aldA and tagA are divergently transcribed genes that also have unknown roles in pathogenesis. In this study, we map the aldA and tagA promoters and identify the ToxT binding sites upstream of each gene. Our results suggest that two ToxT binding sites in an inverted-repeat configuration are required for Tox[T.sup.-]directed transcription of tagA and that a single ToxT binding site is required for Tox[T.sup.-]directed transcription of aldA. Furthermore, to direct transcription of tagA and aldA, ToxT uses independent binding regions upstream of each gene, in contrast to what we previously found for the divergently transcribed acfA and acfD genes, which share ToxT binding sites between the two genes.

The Gram-negative bacteriumVibrio choleraeis the infectious agent responsible for the disease Asiatic cholera. The genes required forV. choleraevirulence, 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 ofV. choleraevirulence 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 thectxoperon, encoding CT, and thetcpAoperon, 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 thatctxandtcpAeach has a pair of ToxT binding sites arranged in a direct repeat orientation upstream of the core promoter elements. In this work, using both transcriptionallacZfusions andin vitrocopper-phenanthroline footprinting experiments, we have identified the ToxT binding sites between the divergently transcribedacfAandacfDgenes, which encode components of the accessory colonization factor required for efficient intestinal colonization byV. cholerae. Our results indicate that ToxT binds to a pair of DNA sites betweenacfAandacfDin 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 bothacfAandacfD. 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 betweenacfAandacfDto activate transcription of both genes. [ABSTRACT FROM AUTHOR]

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals. [ABSTRACT FROM AUTHOR]

The gram-negative curved bacillus Vibrio cholerae ( V. cholerae ) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how to measure linoleic acid entering V. cholerae when added exogenously and determine whether it is able to enter the cytoplasm. This protocol will quantify how much linoleic acid is able to enter the cell and then identify the amount of linoleic acid that stays in the membrane or ultimately enters the cytoplasm.