Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter

The cholate-resistant Lactococcus lactis strain C41-2, derived from mild-ty pe L. lactis MG1363 through selection for growth on cholate-containing medi um, displayed a reduced accumulation of cholate due to an enhanced active e fflux, However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substr ates of the multidrug transporters LmrP and LmrA in L. lactis MG1363, The c holate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadat e, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Be sides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artifici al pH probe 2',7'-bis-(2 carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glut amate (FTUG). Surprisingly, the efflux: rate of BCECF, but not that of FTUG , was significantly enhanced in L. lactis C41-2. Further experiments with L , lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.