Stages of polymyxin B interaction with the Escherichia coli cell envelope

The effects of polymyxin B (PMB) on the Escherichia coli outer (OM) and cyt oplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K+ and H+ ions. At concentrations between 2 and 20 mug/ml, PMB increased the OM permeabilit y to lipophilic compounds and induced a leakage of K+ from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolariz ed the CM, forming ion-permeable pores in the cell envelope. The permeabili ty characteristics of PMB-induced pores mimic those of bacteriophage- and/o r bacteriocin-induced channels. However, the bactericidal effect of PMB too k place at concentrations below 20 mug/ml, indicating that this effect is n ot caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K+ grad ient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg2+. The majo r new findings of this work are as follows: (i) the OM-permeabilizing actio n of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB -induced ion-permeable pores in bacterial envelope were registered, and (ii i) the pore formation and depolarization of the CM are not obligatory for t he bactericidal action of PMB and dissipation of the K+ gradient on the CM.