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-ethlypyridinium cation (DMP+) with cells of Escherichia coli AN120 (uncA) and AS-1 (acrA) was studied to elucidate the role of the envelope and 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 divalent cations. By contract, 50% of the DMP+ was displaced by micromolar concentrations of Mg2+ from resting cells of AN120. Energization of the 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 suspension. With AS-1, energization caused more DMP+ to be taken up from the medium. This was associated with an increase in fluorescence in the cell suspension. The extent of the quenching by addition of Mg2+ was not increased. Right-side out vesicles from AN120, like those of AS-1, showed DMP+ fluorescence behaviour which resembled that of intact cells of AS-1. Transformation of AS-1 with plasmids encoding the E. coli Mvr and EmrAB efflux systems resulted in the DMP+ fluorescence response of this strain becoming like that of AN120. It is suggested that with strain AN120 the changes in binding of DMP+ and fluorescence intensity were associated with activation of efflux systems on cell energization. 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 facilitated. Energization of dye update and release is driven by an electrochemical gradient of protons. ATP is not directly involved in energizing the movement of the dye.
Sedgwick
E G
EG
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Bragg
P D
PD
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D011726:Pyridinium Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; C025042:2-(dimethylaminostyryl)-1-ethylpyridinium; D000255:Adenosine Triphosphate; D008274:Magnesium