Suppression of hypersensitivity of Escherichia coli acrB mutant to organicsolvents by integrational activation of the acrEF operon with the IS1 of IS2 element

The AcrAB-ToIC efflux pump plays an intrinsic role in resistance to hydroph obic solvents in Escherichia coli. E. coli OST5500 is hypersensitive to sol vents due to inactivation of the acrB gene by insertion of IS30. Suppressor mutants showing high solvent resistance were isolated from OST5500. These mutants produced high levels of AcrE and AcrF proteins, which were not prod uced in OST5500, and in each mutant an insertion sequence (IS1 or IS2) was found integrated upstream of the acrEF operon, coding for the two proteins. The suppressor mutants lost solvent resistance on inactivation of the acrE F operon. The solvent hypersensitivity of OST5500 was suppressed by introdu ction of the acrEF operon with IS2 or IS2 integrated upstream but not by in troduction of the operon lacking the integrated IS. It was concluded that I S integration activated acrEF, resulting in functional complementation of t he acrB mutation. The acrB mutation was also complemented by a plasmid cont aining acrF or acrEF under the control of Pine. The wild-type tolC gene was found to be essential for complementation of the acrB mutation by acrEF. T hus, it is concluded that in these cells a combination of the proteins AcrA , AcrF, and ToIC or the proteins AcrE, AcrF, and ToIC is functional in solv ent efflux instead of the AcrAB-ToIC efflux pump.