QstR drives the expression of comEA in the absence of competence induction. (A) qRT- PCR data showing the relative expression of the indicated genes in wild-type V. cholerae strain A1552 (WT) carrying an empty vector (p) as control or a plasmid encoding qstR (p_qstR; ara-inducible). Strains were grown to high cell density, and the growth medium was supplemented with arabinose to induce expression of qstR in the absence of competence-induction (e.g., independent of TfoX). Data are the average of three biological replicates. Error bars indicate standard deviation. Statistically significant differences were determined by Student’s t- tests. *P < 0.05, n.s. = not significant. (B) Detection of the ComEA protein (with and without the N-terminal signal sequence peptide; upper and lower band, respectively) by Western blot analysis using protein-specific antibodies. Total protein was extracted from the indicated strains after growth to high cell density. Lanes: 1, V. cholerae strain A1552-TntfoX, competence-non-induced; 2, A1552-TntfoX competence-induced; 3, wild-type strain A1552 carrying plasmid pBAD/Myc-HisA (vector control); 4, A1552 containing plasmid p_qstR in trans. Strains indicated in lanes 3 and 4 were grown in the presence of 0.02% arabinose to induce the PBAD promoter, which is located on the plasmid.