THE ROLE OF EFFLUX SYSTEMS AND THE CELL-ENVELOPE IN FLUORESCENCE CHANGES OF THE LIPOPHILIC CATION 2-(4-DIMETHYLAMINOSTYRYL)-1-ETHYLPYRIDINIUM IN ESCHERICHIA-COLI

The interaction of the fluorescent dye 2-(4-dimethylaminostyryl)-1-eth ylpyridinium cation (DMP(+)) with cells of Escherichia coli AN120 (unc A) and AS-1 (acrA) was studied to elucidate the role of the envelope a nd of efflux systems in the uptake of lipophilic cations, DMP(+) bound to the two strains in a different manner. With AS-1 the bound dye was displaced only to a small extent by addition of Mg2+ or other divalen t cations. By contrast, 50% of the DMP(+) was displaced by micromolar concentrations of Mg2+ from resting cells of AN120. Energization of th e cells by substrate oxidation resulted in the loss in AN120 of 50% of the bound dye and a decrease of the fluorescence in the cell suspensi on. With AS-1, energization caused more DMP(+) to be taken up from the medium, This was associated with an increase in fluorescence in the c ell suspension. The extent of the quenching by addition of Mg2+ was no t increased. Right-side out vesicles from AN120, like those of AS-1, s howed DMP(+) fluorescence behaviour which resembled that of intact cel ls of AS-1. Transformation of AS-1 with plasmids encoding the E. coli Mvr and EmrAB efflux systems resulted in the DMP(+) fluorescence respo nse of this strain becoming like that of AN120. It is suggested that w ith strain AN120 the changes in binding of DMP(+) and fluorescence int ensity were associated with activation of efflux systems on cell energ ization. With AS-1, it is suggested that the observed fluorescence and binding changes are due to inactivation of the AcrAB efflux system by the acrA mutation. Thus, the net entry of lipophilic cations is facil itated. Energization of dye uptake and release is driven by an electro chemical gradient of protons. ATP is not directly involved in energizi ng the movement of the dye.