FIG. 3.

Uptake of ⁶⁵Zn(II) by cells of E. coli GR362 (ΔznuABC ΔzupT ΔzntB::cam ΔzitB zntA::kan) expressing zupT. Cells were grown overnight in LB medium, diluted 50-fold into fresh prewarmed LB medium, and grown to an OD₆₀₀ of 0.5, and expression of zupT was induced with 200 or 20 ng of AHT/ml for 10 min or not induced. The cells were washed with buffer A (10 mM Tris-HCl [pH 7.0], 2 g of glucose/liter, 10 mM Na₂HPO₄) and resuspended in the same buffer. ⁶⁵ZnSO₄ was added to a final concentration of 10 μM. The cells were incubated at 37°C, and 0.1-ml aliquots were filtered through nitrocellulose membranes (pore size, 0.45 μm) at various times and immediately washed with 10 ml of buffer B (10 mM Tris-HCl [pH 7.0], 10 mM MgCl₂). The membranes were dried, and radioactivity was measured using a liquid scintillation counter. The protein concentration was determined using the method of Lowry et al. (11), and the amount of Zn(II) per milligram of protein was calculated. •, E. coli GR362 (ΔznuABC ΔzupT ΔzntB::cam ΔzitB zntA::kan)(pZUPT) (no inducer); (○), E. coli GR362 (ΔznuABC ΔzupT ΔzntB::cam ΔzitB zntA::kan)(pZUPT) (20-ng/ml AHT); ▾, E. coli GR362 (ΔznuABC ΔzupT ΔzntB::camΔzitB zntA::kan)(pZUPT) (200 ng/ml); ▿, E. coli GR362 (ΔznuABC ΔzupT ΔzntB::cam ΔzitB zntA::kan)(pASK-IBA3).