Altering expression of targets impacts triclosan sensitivity. The coding sequences of selected genes were cloned into the pBAD30 expression vector under transcriptional control of the arabinose-inducible promoter. This was to modify their expression and test the predicted impact on triclosan sensitivity. Genes fabI, infB, marAB, and fabZ were cloned in the forward orientation, allowing their up-regulation after induction (under the green bar). In contrast, fabA, rpoN, pstA, and lacA were cloned in the reverse direction, allowing inducible production of an antisense transcript and consequent repression of expression (under the red bar). The lacA and pBAD30 vector alone constructs were included as negative controls. Duplicate recombinants (Rec1, Rec2) for each gene were tested by spotting cultures onto LB-agar (top), LB agar supplemented with 0.04 µg/mL triclosan (middle), or LB agar supplemented with 0.04 µg/mL triclosan and 0.5% arabinose. All recombinants grew without selection, and none grew in the presence of triclosan without arabinose. With triclosan and arabinose induction, the fabI, infB, and marAB recombinants consistently grew better than the controls.
