A specific amount of RamA must be reached to trigger increased expression of AcrAB, enhance efflux, and confer multidrug resistance.

Using transcriptional GFP reporters, we previously found that most antibiotics tested induced acrAB via RamA, whilst other AraC/XylS family transcriptional activators, MarA, SoxS, or Rob, induced fewer signals. Surprisingly, we found that some antibiotics induced ramA with no subsequent acrAB induction. We postulated that expression of RamA, and subsequently AcrAB, must increase above basal levels to induce acrAB transcription. Furthermore, we hypothesized that a certain level of RamA is required to induce a measurable amount of acrAB, and likewise that a certain level of AcrAB is required to give a multidrug resistant (MDR) phenotype. The transcript levels of ramA and acrAB were measured in the presence of a range of concentrations of the ramA inducer, chlorpromazine. In parallel, the levels of RamA, AcrB, and antibiotic susceptibility were determined. Here, we show that a specific level of RamA, and subsequently AcrAB, must be reached before MDR is observed, and up to a maximum amount of RamA, there was enhanced production of AcrAB and MDR; higher RamA concentrations did not increase production of AcrAB or MDR. We postulate that this was due to saturation of the maximum number of RamA binding sites in acrB.