Flexibility of Vibrio cholerae ToxT in Transcription Activation of Genes Having Altered Promoter Spacing

Abstract

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 gram-negative bacterium Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, continues to be a serious public health problem despite over 100 years of research on its pathogenesis. The current cholera pandemic, which began in 1961, continues unabated, and an estimated 5 million cases of cholera occur worldwide each year. Although over 200 known serogroups of V. cholerae have been identified, only the O1 and O139 serogroups are capable of causing pandemic cholera (34,35).Pandemic V. cholerae strains require two major virulence factors for colonization and pathogenesis. The first factor, cholera toxin, is an A 1 B 5 -type ADP-ribosylating toxin that is responsible for producing the voluminous watery diarrhea characteristic of cholera (8,26). The genes encoding cholera toxin, ctxAB, are carried in the genome of a lysogenic bacteriophage, CTX⌽ (40). The second major virulence factor is the toxin-coregulated pilus (TCP) (38), a type IV pilus that initiates microcolony formation and is required for intestinal colonization by V. cholerae (39). Genes encoding the TCP, which are in a long operon beginning with the tcpA pilin gene, are located in the Vibrio pathogenicity island (VPI) (22). In addition to its role as a colonization factor, the TCP is also the receptor for CTX⌽ and thus allows horizontal transfer of ctxAB to nontoxigenic V. cholerae carrying the VPI (40). In addition to the TCP-encoding genes, several other genes thought to...

Identification and Characterization of the Functional Toxboxes in the Vibrio cholerae Cholera Toxin Promoter

“…The DNA sequences of the identified toxboxes are consistent with the consensus toxbox in that they are degenerate but contain the 5= poly(T) tracts common among all known ToxT DNA binding sites (42)(43)(44). The toxboxes in PctxAB are also consistent with other ToxT-activated promoters in their positioning relative to the transcriptional start site (2,(42)(43)(44). Although ToxT is a flexible transcription activator in regard to sequence requirements, configuration, and the number of binding sites, it has specific requirements for the activation of PctxAB, and a single mutation within one the two toxboxes is enough to severely decrease transcription activation by ToxT.…”

“…Individual point mutations within a region spanning positions Ϫ72 to Ϫ59 caused severe defects in ToxTdependent activity, with the exception of positions Ϫ65 and Ϫ64. This 13-bp sequence, which we designated toxbox 1, is consistent with previously characterized ToxT binding sites in both sequence and proximity to the transcriptional start site (2,(42)(43)(44). Interestingly, there are no single point mutations within toxbox 1 that significantly increased ToxT-independent activity, unlike the double point mutation within heptad repeat 5.…”

“…Toxboxes are characterized by a well-conserved 5= portion containing a poly(T) tract and a degenerate 3= portion that is generally A/T rich. In addition to having somewhat degenerate sequences, toxboxes also vary in configuration and location relative to the transcriptional start site (2,(42)(43)(44). However, toxboxes are invariably located upstream of the Ϫ35 sequence recognized by the RNA polymerase (RNAP) 70 , suggesting that ToxT interacts with RNA polymerase ␣-subunit CTDs (␣-CTDs) to activate transcription (3).…”

Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

“…ToxT is thought to have optimal activity when acting as a dimer (27)(28)(29)(30)(31). In contrast, it also has been shown that ToxT can bind DNA as a monomer (18,19,26). The sigmoidal ToxT binding curve at two toxbox promoters (Fig.…”

“…All ToxT-dependent virulence genes have two toxboxes upstream of their transcriptional start site in either direct or inverted repeat configurations, with the exception of aldA, which has only a single toxbox (19). ToxT can bind toxboxes as a monomer (18,19,26) but is presumed to dimerize to fully activate at least some virulence genes (27)(28)(29)(30)(31). The activation of the ToxT-dependent virulence genes can be altered in the presence of the positive and negative ToxT effectors.…”