Zinc uptake assay.

A nonradioactive assay was established as described by Bayle et al. (30) and Chimalapati et al. (31) by use of FluoZin-3AM (Thermo Fisher Scientific, Waltham, MA, USA) to quantify zinc uptake in different MSMEG strains. Precultures of MSwt, MSΔΔ4, MSΔΔ4::A::B, MSΔΔ4::C, MSΔΔ4::D, and MSΔΔ4::E were grown in MB to an OD₆₀₀ of 2.0, washed twice, and resuspended in SM. Main cultures in 10 ml SM were inoculated with the preculture at an OD₆₀₀ of 0.1 and incubated for 72 h in a shaking incubator at 37°C and 150 rpm to starve bacteria of zinc. Cells were then harvested, washed twice with 10 ml 1× PBS, resuspended in 1 to 2 ml 1× PBS (dependent on the pellet size), and adjusted to an OD₆₀₀ of 2.0. For labeling, FluoZin-3AM was added to a final concentration of 1 μM in dark reaction tubes to protect the FluoZin-3AM from light. To allow FluoZin-3AM uptake by the cells, samples were subsequently incubated for 30 min at room temperature on a rotator. Cells were then harvested by centrifugation, washed three times, and resuspended in 1 to 2 ml 1× PBS.

For complete deesterification of intracellular FluoZin-3 esters, cells were incubated for another 30 min at room temperature on a rotator and were subsequently diluted with 1× PBS to an OD₆₀₀ of 0.1 (input). Two hundred microliters of the samples and 1× PBS as a blank were transferred to a black 96-well plate (in triplicate), and the samples were incubated at 37°C in a TECAN microtiter plate reader. Excitation and emission (495 and 516 nm) were measured every 10 min. First, a 1-h measurement was performed to obtain the baseline. Next, 25 μM ZnSO₄ was added to each well, and measurement was performed for another 3 h to monitor zinc uptake. Then, 1 μM TPEN was added, and measurement was performed for 1 h to show zinc specificity. Results were normalized to the negative control (PBS) and adjusted to the precise OD₆₀₀ of the input.