TOBRAMYCIN UPTAKE IN ESCHERICHIA-COLI MEMBRANE-VESICLES

The uptake of tobramycin was measured in Escherichia coli membrane ves icles prepared in KMES [K+-2-(N-morpholino)ethanesulfonic acid] buffer at pH 6.6. Uptake occurred in vesicles energized with ascorbic acid a nd phenazine methosulfate, in which the electrical potential (Delta ps i) was -120 mV, but not in vesicles energized with D-lactate (Delta ps i = -95 mV). The addition of nigericin to vesicles energized with D-la ctate did not induce tobramycin uptake despite an increase in Delta ps i to -110 mV. However, when Delta psi was increased or decreased by th e addition of nigericin or valinomycin, respectively, uptake in vesicl es energized with ascorbic acid and phenazine methosulfate was stimula ted or inhibited, respectively, confirming studies,vith whole cells sh owing that uptake of aminoglycosides is gated by Delta psi rather than by proton motive force (Delta mu(H+)) or Delta pH. N-ethylmaleimide p revented uptake, suggesting that the aminoglycoside transporter is a c ytoplasmic membrane protein with accessible sulfhydryl groups. The obs ervation that uptake is gated in vesicles as well as in whole cells su ggested that diffusion occurs through a voltage-gated channel. In vesi cles preloaded with tobramycin, no efflux occurred after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone. In susce ptible cells, aminoglycosides themselves decreased the magnitude of De lta psi. We propose a mechanism of aminoglycoside-induced killing in w hich aminoglycosides themselves close the voltage-gated channel by dec reasing the magnitude of Delta psi. Channel closure causes aminoglycos ides accumulated prior to the fall in Delta psi to be trapped, which i n turn causes irreversible uptake and subsequent bactericidal effects.