Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter

The expression of the Vibrio cholerae virulence factors, toxin-co-regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co-operate with a second protein, AphA, to activate the expression of the membrane-bound transcription factors TcpP and TcpH. To gain insights into the mechanism by which AphA and AphB co-operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites. As shown by tcpP-lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between -101 and -71 from the start of transcription. AphB binds to and activates from a partially overlapping downstream site between -78 and -43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well-characterized LysR-binding motif. A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease-causing biotypes of V. cholerae, classical and El Tor. We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP-CRP. Purified CRP binds to a near-consensus sequence in the tcpPH promoter in a cAMP-dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB. These findings raise the possibility that the negative effect of cAMP-CRP on virulence gene expression is the result of its ability to influence AphA- and AphB-dependent transcriptional activation of tcpPH under various conditions.