Fig. 3.

Real-time inactivation of AcrB double-Cys mutant by cross-linking with MTS reagent. Cellular accumulation of ethidium by the ΔacrB dsbA host AG100YBD cells expressing each AcrB protein was monitored continuously by measuring the fluorescence of the ethidium-nucleic acid complex. After 2 min of incubation with ethidium bromide (arrow), solvent alone (curve 1) or MTS reagents (curve 2, 5-MTS; curve 3, MTS-2-MTS) was added to cell suspension. The AcrB with double-Cys mutation in the cleft, CL-F666C/Q830C, was active in the dsbA host in the efflux of ethidium, so that only very slow entry of ethidium was seen initially (bottom right panel). However, upon addition of MTS-2-MTS, cross-linking apparently occurred, so that AcrB became inactivated, inducing a rapid ethidium influx and increased fluorescence (curve 3, bottom right panel). In contrast, MTS-2-MTS had little effect on the ethidium entry rate in cells expressing no-Cys (CL-AcrB^His) or a single-Cys (CL-F666C and CL-Q830C) AcrB. A control reagent, 5-MTS (a non-cross-linker), produced no AcrB inactivation in any of the mutants (curves 2). As a positive control, a proton conductor (CCCP), was added to the cells expressing CL-AcrB^His, resulting in a rapid influx of ethidium because of inactivation of AcrB due to the loss of the proton-motive force (top left panel). (Reproduced from ref. 12.)