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. 2000 Sep;182(18):5196-201.
doi: 10.1128/JB.182.18.5196-5201.2000.

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

A Yokota  1 , M Veenstra, P Kurdi, H W van Veen, W N Konings
Affiliations

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

A Yokota et al. J Bacteriol. 2000 Sep.

Abstract

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate 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-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (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.

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Figures

FIG. 1
FIG. 1
Sensitivity of wild-type L. lactis MG1363 and the cholate-resistant L. lactis C41-2 to cholate, deoxycholate, and taurocholate. Both strains were grown for 6 h in M17 medium containing various concentrations of cholate (●), deoxycholate (▴), and taurocholate (■). The solid lines and the dotted line represent L. lactis MG1363 and L. lactis C41-2, respectively.
FIG. 2
FIG. 2
Loading of L. lactis C41-2 cells with cholate. Incubations were done in the presence (●) and absence (■) of 10 mM glucose. Cholate was added at time zero to an external concentration of 0.116 mM.
FIG. 3
FIG. 3
Energy-dependent extrusion of cholate in the cholate-resistant L. lactis C41-2 and in wild-type L. lactis MG1363. After the cells were loaded with cholate (■), glucose was added to a final concentration of 10 mM at the time point indicated by the arrow. Subsequently, the amount of cell-associated cholate was measured over time in the absence (●) and presence (▴) of 2 μM valinomycin, which selectively dissipated the transmembrane potential. (A) Strain C41-2; (B) strain MG1363.
FIG. 4
FIG. 4
Effect of ortho-vanadate on the transport of cholate in L. lactis C41-2. The transport of cholate was measured in the presence (●) and absence (■) of 1 mM ortho-vanadate, using cells which obtained metabolic energy from the catabolism of l-arginine. To maximize the ΔpH-driven accumulation of cholate in the cells, measurements were performed at pH 6.5 in the presence of 2 μM valinomycin.
FIG. 5
FIG. 5
BCECF efflux in the wild-type L. lactis MG1363 and in L. lactis C41-2. Cells of L. lactis MG1363 (●) and L. lactis C41-2 (■) were loaded with BCECF. At time zero, BCECF efflux was initiated by the addition of glucose to a final concentration of 20 mM.
FIG. 6
FIG. 6
Effect of cholate on the efflux of BCECF and FTUG in L. lactis C41-2. Cells were loaded with BCECF (A) or FTUG (B) and equilibrated with 10 mM potassium cholate (■) or 10 mM potassium chloride (●) in the presence of 1 μM nigericin. At time zero, 20 mM glucose was added to the cells to initiate efflux.
FIG. 7
FIG. 7
Effect of BCECF on the efflux of cholate in L. lactis C41-2. Cells loaded with BCECF (■) or unloaded cells (●) were equilibrated in the presence of 2.7 mM [14C]cholate and 1 μM nigericin. Transport was initiated at time zero by the addition of glucose to a final concentration of 20 mM.
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