H-NS Binding and Repression of the ctx Promoter in Vibrio cholerae

Abstract

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the ؊35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the ␣-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.Vibrio cholerae is the etiological agent of the human diarrheal disease cholera. Two virulence factors that are produced by V. cholerae and are essential for disease are toxin-coregulated pilus (TCP) (62) and cholera toxin (CT) (28). TCP is a type IV pilus that is assembled by polymerization of the pilin subunit, TcpA, and forms long filaments that laterally associate into bundles. Expression of TCP in vitro results in autoagglutination of the bacterium, and TCP-mediated bacterium-bacterium interactions in vivo facilitate microcolony formation on the intestinal epithelium. Pilus biogenesis requires at least 9 other proteins in addition to TcpA, which are encoded in an operon located on the Vibrio pathogenicity island (VPI). The pilus biogenesis apparatus plays a second role in colonization by secreting the cotranscribed, soluble colonization factor TcpF, which is also essential for colonization but for which a mechanism remains unknown (29, 30). The second main virulence factor, CT, is a potent A 1 B 5 subunit, ADP-ribosylating toxin that is responsible for the severe watery diarrhea that is associated with cholera. CT is encoded by the ctxAB operon, which is located on the lysogenic bacteriophage CTX. The VPI and CTX were both acquired by horizontal gene transfer. TCP serves as the high-affinity receptor for CTX that links TCP production to ctx acq...

Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus

“…For example, nucleoid-associated protein (H-NS) binds DNA at lower temperatures blocking the transcription of multiple genes, whereas higher temperatures cause loosening in the DNA structure allowing transcription to occur. H-NS suppresses virulence-associated genes, such as RTX and CTX, in V. vulnificus (Liu et al, 2009) and V. cholerae (Stonehouse et al, 2011), respectively. The concurrent upregulation of H-NS (ZP_05887985.1) and RTX toxin (ZP_05887531.1) on CPI-1 in the Vc450 proteome at 27 1C could be the result of a conserved Vc450 H-NS unable to regulate a more recently acquired RTX toxin.…”

Interaction of the Histone-Like Nucleoid Structuring Protein and the General Stress Response Regulator RpoS at Vibrio cholerae Promoters That Regulate Motility and Hemagglutinin/Protease Expression

“…There are numerous evidences indicating that repression by H-NS can be relieved in response to environmental stimuli that activate the expression of other regulators whose binding site overlaps with that of H-NS. For instance, transcriptional silencing of V. cholerae tcpA and ctxA promoters by H-NS is antagonized by the AraC-like transcriptional regulator ToxT and IHF (51,52,59).…”

H-NS Is a Negative Regulator of the Two Hemolysin/Cytotoxin Gene Clusters in Vibrio anguillarum

“…It has been reported that H-NS function is based on self-oligomerization and binding to DNA motifs to create DNA-protein-DNA bridges that can impede the movement of RNA polymerase (8). H-NS has been shown to repress expression of several virulence genes, including the cholera toxin (ctx) (9,10) and exopolysaccharide biosynthesis (vps) genes in V. cholerae (10,11), the RTX toxin gene (rtxA1) in V. vulnificus (12), and T3SS1 genes in V. parahaemolyticus (13). In many bacterial species, repression by H-NS can be relieved by other regulators, and each bacterial system has developed specific approaches to attenuate the repressive action of H-NS (8).…”