Overexpression of toxT in the ihfA mutant background. Strains were grown overnight in LB medium with a starting pH of 6.5 at 30°C with shaking and with the addition of 0.1% arabinose where indicated. Western blot analysis with anti-ToxT (α-ToxT) and α-TcpA antibodies was performed with 16 μg of total protein extract. WT, wild type.

Influence of IHF on ctx-lacZ and tcpA-lacZ expression. (A) Wild-type (WT) (tcpA-lacZ or ctx-lacZ), ihfA (ΔihfA::kan), or ihfB (ΔihfB::gen) mutants. (B) WT, ihfA, or ihfA mutants with the empty vector pBAD-TOPO or the IHFα expression vector pIHFA (pKAS161). Cultures were grown overnight in LB medium with a starting pH of 6.5 at 30°C with shaking (those with plasmids contained 0.1% arabinose) and assayed for β-galactosidase production. The values are the averages for at least two independent experiments.

Gel mobility shift assay for IHF binding to ctx and tcpA promoter DNA. The DIG-labeled promoter fragments of ctx from position −194 to +118 (C) and tcpA from position −226 to +78 (A1), −226 to −50 (A2), and −100 to +78 (A3) were incubated with purified IHF and analyzed by a gel mobility shift assay. The first lane in each set has no protein added, the second lane contains 180 nM IHF, and the third land contains 360 nM IHF. The boxes labeled −162 and −35 represent putative IHF consensus sites on the tcpA promoter fragments.

TcpA, CT, and ToxT production in O395 and the ihfA mutant. Strains were grown overnight in LB medium with a starting pH of 6.5 at 30°C with shaking. Western blot analysis with the anti-TcpA (α-TcpA) antibody (A) or α-ToxT polyclonal antibody (B) was performed with 16 μg of total protein extract. CT represents the amount of CT detected in the culture supernatant as measured by a GM₁-ganglioside enzyme-linked immunosorbent CT assay (A) (expressed in ng/mg of protein). WT, wild type.

Schematic of the transcriptional regulation of V. cholerae tcpA, toxT, and ctx expression. (A) OFF/INITIAL: H-NS represses gene expression at multiple promoters within the ToxR regulon, including tcpA to -F, toxT, and ctx. H-NS binding in the tcpA promoter region overlaps the IHF consensus sequence at position −162 as well as the ToxT binding site spanning from position −84 to −41. (B) ON: After a transition to virulence-inducing conditions, ToxR/S and TcpP/H in the inner membrane cooperate at the toxT promoter to activate toxT expression (TcpP/ToxR dependent). ToxT-mediated activation of tcpA-toxT is facilitated by IHF binding, bending of the DNA, and displacement of H-NS, allowing for greater accessibility of ToxT to its binding site. As ToxT levels accumulate, full activation of tcpA to -F, toxT, and ctx expression occurs.

Mutations in the tcpA IHF consensus site alter IHF and H-NS binding. Mutations in the tcpA promoter were constructed to disrupt IHF binding. The wild-type (WT) and mutant promoter fragments (Δconsensus and 4PM) from position −226 to +78 were incubated in the absence of protein or with 180 nM IHF (A) or 80, 160, 250, or 320 nM H-NS (B) and analyzed by a gel mobility shift assay. (C) Competitive gel mobility shift assay between H-NS and IHF on the wild-type promoter fragment. Lane 1, no protein added. Lane 2, 320 nM H-NS; lane 3, 420 nM IHF. Lanes 4 to 6, 320 nM H-NS and 120 nM (lane 4), 240 nM (lane 5), and 420 nM (lane 6) IHF. Lanes 7 to 9, 420 nM IHF and 160 nM (lane 7), 250 nM (lane 8), and 320 nM (lane 9) H-NS.

Bending of the tcpA promoter DNA by IHF as determined by a circular permutation assay. (A) Diagram of 190-bp linear probes produced by digestion of plasmid pEAS7 (pBEND5-tcpA) with five restriction enzymes. The probes contain the IHF consensus site (hatched box) within a 67-bp tcpA promoter fragment (−193 to −127) (black box) at various positions relative to the ends of the fragments. (B) Circular permutation analysis was performed on the tcpA IHF binding site with purified IHF. Each fragment was incubated without protein or with 180 nM IHF and analyzed by a gel mobility shift assay. The bending angle, α, was determined to be 120° using the equation (μ_M/μ_E) = cos(α/2), where μ_M is the relative migration of the fragment with the bend center in the middle and μ_E is the relative migration of the fragment with the bend center at the end (61).

DNase I protection by IHF and H-NS at the tcpA promoter. The top and bottom strands of the tcpA promoter fragments were incubated with increasing amounts of IHF (1.2 and 2.4 μM) (A) or H-NS (1, 2, and 4 μM) (B) and treated with DNase I. (A) IHF protection from DNase I on a promoter fragment from position −282 to −50 is shown with hatched bars. Arrows designate sites that were hypersensitive to DNase I treatment. NP, no protein. (B) H-NS protection on a promoter fragment from position −282 to +78 is shown with black bars to the right. On the left, the hatched bars designate the IHF consensus site and the gray boxes designate ToxT binding sites. (C) tcpA promoter sequence from position −200 to −125. Black bars designate the IHF protection on the top and bottom strands, and the IHF consensus site within the protected region is in bold and aligned with the E. coli consensus site below. W, A/T; R, A/G; N, any nucleotide. The 21 bp that were deleted in the Δconsensus mutation are marked above the sequence. The four base pair mutations that were incorporated into the consensus site are designated 4PM below the consensus alignment. Boxes indicate putative H-NS nucleation sites with seven matches to the 10-bp H-NS consensus site (29).