MUTATIONS IN TOXR AND TOXS THAT SEPARATE TRANSCRIPTIONAL ACTIVATION FROM DNA-BINDING AT THE CHOLERA-TOXIN GENE PROMOTER

ToxR and ToxS are integral membrane proteins that activate the transcr iption of virulence genes in Vibrio cholerae, ToxR can be separated in to three different domains: an N-terminal cytoplasmic DNA binding doma in, a central transmembrane domain, and a C-terminal periplasmic domai n. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection f or DNA binding, in combination with a screen for cholera toxin gene tr anscription, 12 toxR and toxS positive control mutants producing varia nt ToxR proteins from the toxRS operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mu tation in toxR specifies an E82K change in the predicted helix-loop-he lix DNA binding domain and destroys ToxR-mediated activation. Seven to xR mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce pro teolytically processed shorter forms of ToxR, suggesting that even sho rt periplasmic deletions alter the folding of ToxR in the periplasm, D eletion of toxS did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S vari ant rendered ToxR susceptible to proteolysis, suggesting that the natu ral function of ToxS is to complex with ToxR. Therefore, certain alter ations that map to the ToxR cytoplasmic DNA binding domain, to the per iplasmic domain, or to ToxS separate DNA binding activity from activat or function. These data support a model where proper assembly or stabi lity of the periplasmic domain of ToxR is enhanced by ToxS, This chape rone-like activity of ToxS may be required for the formation of the tr anscriptional activation complex but not the ToxR-DNA complex.