PROTON MOTIVE FORCE-DRIVEN AND ATP-DEPENDENT DRUG EXTRUSION SYSTEMS IN MULTIDRUG-RESISTANT LACTOCOCCUS-LACTIS

Three mutants of Lactococcus lactis subsp. lactis MG1363, termed Eth(R ), Dau(R), and Rho(R), were selected for resistance to high concentrat ions of ethidium bromide, daunomycin, and rhodamine 6G, respectively. These mutants were found to be cross resistant to a number of structur ally and functionally unrelated drugs, among which were typical substr ates of the mammalian multidrug transporter (P-glycoprotein) such as d aunomycin, quinine, actinomycin D, gramicidin D, and rhodamine 6G. The three multidrug-resistant strains showed an increased rate of energy- dependent ethidium and daunomycin efflux compared with that of the wil d-type strain. This suggests that resistance to these toxic compounds is at least partly due to active efflux. Efflux of ethidium from the E th(R) strain could occur against a 37-fold inwardly directed concentra tion gradient. In all strains, ethidium efflux was inhibited by reserp ine, a well-known inhibitor of P-glycoprotein. Ionophores which select ively dissipate the-membrane potential or the pH gradient across the m embrane inhibited ethidium and daunomycin efflux in the wild-type stra in, corresponding with a proton motive force driven efflux system. The ethidium efflux system in the Eth(R) strain, on the other hand, was i nhibited by ortho-vanadate and not upon dissipation of the proton moti ve force, which suggests the involvement of ATP in the energization of transport. The partial inhibition of ethidium efflux by ortho-vanadat e and nigericin in the Dau(R) and Rho(R) strains suggest. that a proto n motive force-dependent and an ATP-dependent system are expressed sim ultaneously. This is the first report of an ATP-dependent transport sy stem in prokaryotes which confers multidrug resistance to the organism .