FIG. 2.

⁶⁵Zn(II) uptake by cells of E. coli GG48 (ΔzitB::Cm zntA::Km)/pZITB expressing zitB. Cells were grown overnight in Luria-Bertani medium and diluted 100-fold into fresh prewarmed Luria-Bertani medium. The cells were grown to an optical density at 600 nm of 0.8 and induced with 200 μg of AHT per liter. After growth for 2.5 h, the cells were washed with buffer A (10 mM Tris-HCl [pH 7.0], 2 g of glucose per liter, 10 mM Na₂HPO₄) and concentrated fourfold in the same buffer. ⁶⁵ZnSO₄ was added to a final concentration of 5 μM. The cells were incubated at 37°C, and 0.1-ml aliquots were filtered through nitrocellulose membranes (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 bicinchoninic acid kit (Sigma), and the amount of Zn(II) per milligram of protein was calculated.