FIGURE 5.

TcpP mutants activity and homodimerization assay. (A) V. cholerae ΔtcpP containing P_BAD-controlled plasmids harboring TcpP or its cysteine mutant derivatives were grown in LB with 0.01% arabinose in the presence or absence of 1 mM TC at 37°C until OD600 ≈ 0.2. Luminescence was measured and reported as light units/OD₆₀₀. Data are the means ± SD (n = 3). (B) V. cholerae ΔtcpP containing P_BAD-controlled plasmids harboring TcpP or its cysteine mutant derivatives were grown in LB with 0.01% arabinose in the presence of 1 mM TC for 6 h. Then, 0.1-mg cell lysates were applied to SDS-PAGE and subjected to Western blotting using an anti-TcpA antibody. (C) Full-length TcpP wild type or cysteine mutants were fused with the T25 and T18 domains of adenylate cyclase (CyaA) from B. pertussis, respectively, and the T25, T18 fusion pairs were introduced into E. coli cyaA mutants (Karimova et al., 1998). Cultures were grown at 30°C for 8 h without shaking and β-galactosidase activity was measured and reported as Miller Units (Miller, 1972). Data are means ± SD (n = 3). (D) P_BAD-controlled plasmids harboring TcpP or its cysteine mutant derivatives and a P_toxT-lux transcriptional fusion plasmid in V. cholerae ΔtoxR/ΔtcpP (D) or E. coli DH5α (E) strains were grown in LB with 0.01% arabinose in the presence or absence of 1 mM TC at 37°C until OD600 ≈ 0.2. Luminescence was measured and reported as light units/OD₆₀₀. Data are the means ± SD (n = 3).