Fig. 12.

Real-time inhibition of pump function through closure of the periplasmic cleft. After the conversion of AcrB protein into the cysteineless (CL) form by substituting native Cys with Ser, two residues close to each other in the cleft, Phe666 and Gln830 were converted into cysteine by site-directed mutagenesis, generating CL-F666C/Q830C. A plasmid construct expressing this mutant AcrB was introduced into an E. coli ΔacrB::spc strain also containing dsbA::kan mutation, which prevents the formation of disulfide bonds in the periplasm. The AcrB efflux pump was active as it was able to prevent the rapid entry of the ethidium dye (curve 1, where only the solvent was added at the arrow). It remained active also when both Cys residues were modified by a methanethiosulfonate SH reagent 5-MTS (curve 2). However, the pump stopped functioning when the two Cys residues were cross-linked by the addition of a cross-linking methanethiosulfonate agent MTS-2-MTS (curve 3). For other control experiments see [60].