FIG. 5.
Increased σE activity in V. cholerae lacking functional T2S machinery. (A) The expression of the rpoEP2::lux fusion gene was determined with wild-type, PBAD::eps, ΔepsL, and ΔepsL+pEpsL strains of V. cholerae TRH7000 grown in LB broth at 37°C to mid-log and stationary phase (OD600 = 0.5 and OD600 = 4, respectively). The increased σE activities observed in PBAD::eps grown in the absence of arabinose (−) and TRHΔepsL strains could be complemented by the addition of 0.01% arabinose (+) to the growth medium and the expression of plasmid-encoded EpsL, respectively. The results presented are the means and corresponding SEM from three independent experiments. The difference in the induction of the σE response between the wild-type and PBAD::eps strains grown in the absence of arabinose and that of the TRHΔepsL mutant was statistically significant (P < 0.004) in mid-log-phase cultures, while the difference between stationary-phase cultures was not significant (P > 0.05). (B) Wild-type and rpoE mutant strains of V. cholerae O395 containing plasmid pMS43 were grown in LB broth at 37°C to late stationary phase (16 h) in the absence (−) or presence (+) of 10 μM IPTG to induce the expression of the dominant-negative mutant protein EpsE-K270A. Culture supernatants were separated from cells by centrifugation, matched by equivalent OD600 units, and analyzed by SDS-PAGE and silver staining.