ToxR of Vibrio cholerae binds multiple sites along the toxT promoter based on structural analysis

Alhabeil, Jamal, Jonathan Zora, Joshua Thomson, Albert Canals, Pieretti Simone, Rosa PĂ©rez, Miquel Coll, and Eric Krukonis

Cholera, an acute diarrheal disease caused by Vibrio cholerae, is estimated to cause over 100,000 deaths each year. Two key virulence factors, cholera toxin and toxin co-regulated pilus, are directly regulated by ToxT. ToxT synthesis requires activation of the toxT promoter by ToxR in conjunction with TcpP, which stimulates RNA polymerase (RNAP). We previously defined two ToxR-binding sites within the toxT promoter, but our recent crystallographic studies of ToxR bound to the toxT promoter identified three other ToxR-binding sites. Based on these structural findings, ToxR mutant proteins predicted to impact DNA binding were generated. ToxR residues W64, D72, T77 and R84 when mutated to alanine (or proline for D72) resulted in ToxR molecules unable to activate either of two ToxR-dependent promoters, toxT or ompU. To assess DNA-binding activity, the DNA-binding and transactivation domains of these ToxR mutants were purified and used in electrophoretic mobility shift assays (EMSAs). While all four mutants were strongly defective for promoter activation, only ToxR-R84A was completely defective for DNA binding. ToxR-W64A, and ToxR-T77A showed intermediate levels of DNA binding and ToxR-D72P bound DNA like wild-type ToxR. T77 and R84 lie within the DNA recognition helix of ToxR while W64 lies upstream of the transactivation loop of ToxR and D72 lies within the transactivation loop. The fact that ToxR-D72P maintains DNA-binding, but fails to activate both the toxT and ompU promoters suggests this substitution may alter presentation of the transactivation loop for interaction with RNAP. This finding implies ToxR and TcpP may both interact with RNAP on the toxT promoter or ToxR interacts with TcpP via its transactivation loop. In addition, EMSA analysis supports the structurally-based identification of additional promoter-proximal ToxR-binding sites on the toxT promoter. Future studies will more specifically define the DNA sequences required for ToxR binding and regulation of V. cholerae virulence.