Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

Abstract

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

FIG. 1

Expression, purification, and functional reconstitution of hexa-histidine-tagged HorA. The HorA protein was overexpressed in L. lactis as a hexa-histidine-tagged protein using the NICE system. A silver-stained sodium dodecyl sulfate-polyacrylamide gel is shown. Lane 1, total membrane protein (20 ug) of L. lactis harboring pNZHHorA; lane 2, soluble fraction (20 μg of protein) of a lysate of HorA-expressing cells; lane 3, Western blot of the membrane fraction (20 μg of protein) of HorA-expressing cells, with anti-hexa-histidine antibody; lane 4, flowthrough fraction of membrane proteins (20 μl of the total fraction of 2 ml) eluted from the Ni²⁺-NTA resin; lanes 5, 6, and 7, histidine-tagged HorA eluted from the NTA resin (20 μl out of the total fraction of 2 ml) in three consecutive steps with buffer supplemented with 250 mM imidazole; lane 8, molecular mass markers; lane 9, HorA reconstituted into proteoliposomes. Lanes 3 and 9 are Western blots; the other lanes are silver-stained gels. The arrow indicates the position of hexa-histidine-tagged HorA protein.

FIG. 2

(A) Growth of control L. lactis harboring pNZ8048 (triangles) and of HorA-expressing L. lactis harboring pNZHHorA (squares) in the absence of iso-α-acids. (B) Inhibition of growth by iso-α-acids of control L. lactis (triangles) and of HorA-expressing L. lactis (squares). Cells were grown at 15°C in the absence or presence of a 50, 100, 200, or 300 μM concentration of iso-α-acids. The OD₆₉₀ was measured every 10 min. The growth rates were determined at the mid-exponential phase.

FIG. 3

Ethidium transport in HorA-expressing cells and nonexpressing cells of L. lactis. Panel (A) De-energized HorA-expressing and control cells (0.2 mg of protein/ml; OD₆₉₀, 0.5) were preequilibrated with 10 μM ethidium bromide at 30°C. The development of fluorescence of the DNA-ethidium complex in the cell suspension was monitored at 20°C over time. At the arrow, 25 mM glucose was added. (B) Effect of ortho-vanadate on the accumulation of ethidium bromide in control cells. Cells were energized with arginine and incubated for 7.5 min in the presence or absence of 0.5 mM ortho-vanadate prior to the addition of 10 μM ethidium bromide (at the arrow). (C) Effect of ortho-vanadate on the accumulation of ethidium bromide in HorA-expressing cells. Cells were treated as described for panel B.

FIG. 4

Hoechst 33342 transport in inside-out membrane vesicles of HorA-expressing cells and nonexpressing cells of L. lactis. Membrane vesicles prepared from HorA-expressing cells (H) and control cells (C) were diluted to a concentration of 0.5 mg of membrane protein/ml in buffer containing the ATP regenerating system (see Materials and Methods) and 0.4 μM of each of the ionophores valinomycin and nigericin to dissipate the membrane potential and transmembrane pH gradient, respectively. After incubation for 1 min at 20°C, 2.3 μM Hoechst 33342 was added to the assay mixture. At the arrow, 2 mM Mg-ATP or 2 mM Mg-ATPγS was added. Hoechst 33342 transport was measured at 20°C by fluorimetry.

FIG. 5

Transport of Hoechst 33342 in proteoliposomes. Liposomes without reconstituted HorA protein (A) and proteoliposomes containing reconstituted HorA protein (B) were diluted in buffer containing the ATP regenerating system. After incubation for 1 min at 20°C, 2.3 μM Hoechst 33342 was added to the assay mixture. At the arrow, 2 mM Mg-ATP or 2 mM Mg-ATPγS was added.

FIG. 6

HorA displays specificity for hop compounds. The ATP-dependent transport of Hoechst 33342 in HorA-containing inside-out membrane vesicles was measured as described in the legend to Fig. 4. Hop compounds at indicated concentrations were added to the assay mixture prior to the addition of Hoechst 33342. The hop compounds did not affect the fluorescence of Hoechst 33342 in control membrane vesicles without HorA (data not shown). To dissipate a proton motive force generated by F₁F₀-ATPase, the ionophores valinomycin (0.4 μM) and nigericin (0.4 μM) were included in the assay medium.