Fig. 2.

A, ⁵⁵Fe uptake by ΔpvdFΔpchA cells in the presence of various exosiderophores. ΔpvdFΔpchA cells were grown in CAA medium supplemented with one exosiderophore to induce the expression of its corresponding transporters (13). Bacteria were then washed with 50 mm Tris-HCl (pH 8.0) and transport assays initiated by adding the corresponding exosiderophore loaded with ⁵⁵Fe to a concentration of 200 nm, as described in Materials and Methods. After 30 min incubation, samples were centrifuged, and the radioactivity retained in the cells monitored. Uptake experiments were also carried out for each exosiderophore in the presence of the protonophore CCCP to evaluate the radioactivity because of binding of ⁵⁵Fe-exosiderophore complexes to the bacterial cell surface or to precipitation. These values were subtracted from those obtained in the absence of CCCP to consider only the radioactivity because of ⁵⁵Fe uptake. The results are expressed as pmol of ⁵⁵Fe transported per ml/OD_{600 nm}. The data represent 3 independent experiments. B, ⁵⁵Fe uptake by PVD or PCH in ΔpvdFΔpchAΔpfeA cells in the presence of ENT acting as a competitor for iron chelation. ⁵⁵Fe uptake assays were carried out with strain ΔpvdFΔpchAΔpfeA, which is unable to transport ⁵⁵Fe-ENT. Siderophore complexes were prepared by either mixing 500 nm ⁵⁵Fe with 10 μm ENT (red dots), 10 μm PVD (dark blue dots), 10 μm PVD and 10 μm ENT (light blue dots), 20 μm PCH (dark green dots), or 20 μm PCH and 10 μm ENT (light green dots). ΔpvdFΔpchAΔpfeA cells were incubated for 30 min in the presence of the various siderophore-⁵⁵Fe mixtures at 500 nm. Bacteria were then harvested by centrifugation and the radioactivity counted. As in panel A, each uptake assay was repeated in the presence of 200 μm CCCP to specifically evaluate the radioactivity because of ⁵⁵Fe uptake from that because of binding of the siderophore-⁵⁵Fe complexes to the cell surface and these values were subtracted from those obtained in the absence of CCCP. The data represent 3 independent experiments with 3 technical replicates. C, ⁵⁵Fe uptake by PVD or PCH in ΔpvdFΔpchAΔfiuAΔfoxA cells in the presence of FERRI acting as a competitor for iron chelation. ⁵⁵Fe uptake assays were carried out with the ΔpvdFΔpchAΔfiuAΔfoxA strain, which is unable to efficiently transport ⁵⁵Fe-FERRI. Siderophore complexes were prepared by either mixing 500 nm ⁵⁵Fe with 10 μm FERRI (red dots), 10 μm PVD (dark blue dots), 10 μm PVD and 10 μm FERRI (light blue dots), 20 μm PCH (dark green dots), or 20 μm PCH and 10 μm FERRI (light green dots). The uptake assays were carried out as in graph B, with or without 200 μm CCCP. The data represent 3 independent experiments with 3 technical replicates.