Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin.

Abstract

The effects of a set of conditions on aminoglycoside uptake were determined. Membrane vesicles either with a membrane potential (delta psi) of -125 mV (adequate to drive lysine uptake) or with succinate, lactate, or phenazine methosulfate did not accumulate gentamicin unless components of protein synthesis were included. Ribosomally resistant (rpsL) Escherichia coli cells demonstrated energy-dependent phase II uptake similar to that of a streptomycin-susceptible strain of E. coli when treated with 100 micrograms of puromycin per ml. Puromycin (100 micrograms/ml) also increased the uptake of the cationic compounds polyamine and arginine. These studies support a role of protein synthesis in aminoglycoside uptake and in the development of energy-dependent phase II. delta psi of cells did not increase either at the initiation of or during energy-dependent phase II, showing that energy-dependent phase II is not due to an elevation of delta psi. In a Bacillus subtilis system, significant streptomycin uptake requires a threshold value of delta psi which varies depending upon the concentration of streptomycin used. At 25 micrograms/ml, the uptake of streptomycin reached maximal levels after exceeding the threshold value, whereas at 100 micrograms/ml there was a gradual increase of the uptake to the maximal after the threshold value was exceeded. Several studies supported the view that electron transport has a specific role other than its requirement to produce the cellular delta psi. The uptake of gentamicin was stimulated to a greater extent by phenazine methosulfate-ascorbate than by the ionophore nigericin in strains of E. coli, although nigericin stimulated delta psi to a greater degree. Cells with 25% of the normal quinone concentration had delta psi values identical to cells with the normal quinone concentration, but the quinone-deficient cells had a significantly lower rate of gentamicin uptake. KCN prevented gentamicin uptake but did not prevent the development of delta psi. The effects of ubiquinone depletion in an E. coli strain were more evident on gentamicin uptake than on ATP-driven glutamine transport or proton motive force-driven proline transport, consistent with a specific requirement for quinones in aminoglycoside uptake. A detailed explanation of the mechanism of accumulation of streptomycin and gentamicin and a proposed mechanism for killing bacterial cells by these agents have been provided.