Binding site requirements of the virulence gene regulator AphB: differential affinities for the Vibrio cholerae...

The differential expression of virulence genes be-tween the two disease-causing biotypes of Vibrio cholerae, classical and El Tor, is primarily due to a single basepair change in the tcpPH promoter, which strongly influences the ability of the LysR regulator AphB to activate transcription in response to environmental conditions. We show here that this single basepair change influences virulence gene expression by dramatically altering the affinity of AphB for its recognition site in the tcpPH promoter. AphB binds greater than 10-fold more efficiently to a wild-type classical tcpPH promoter fragment with an A at -65 relative to a wild-type El Tor fragment that has a G at this position. As this single basepair change is located within the left arm of the LysR recognition motif (5'-TGCAA-N7-TTGCA), which extends from -69 to -53, a systematic mutagenesis of the other positions within this site was carried out to assess their influence on AphB binding in vitro and transcriptional activation in vivo. This analysis revealed that the left and right arms of the interrupted dyad display a high degree of symmetry with respect to their role in AphB binding. The right promoter proximal arm also plays a role in transcriptional activation that is distinct from its role in AphB binding. A second AphB binding site (5'-TGCAA-N7-TGTCA) was identified upstream of the aphB gene itself, which extends from +17 to +33 relative to the start of transcription and functions in autorepression. Although the sequences of the AphB binding sites at the tcpPH and aphB promoters are highly conserved, important differences exist in the way that AphB functions at each of these sites.