Figure 4.

(a) Covalent modification of FepA with CPM in the absence and presence of ferric enterobactin. Purified wild-type FepA (○) or FepAE280C (◊) was incubated with the thiol-specific probe CPM, and the time course of fluorescence was recorded. FepAE280C was also incubated with CPM in the presence of 10 μM ferric enterobactin (×). (b) Effect of PG modification on ferric enterobactin binding by FepA. Bacteria were grown and tested for siderophore binding as described in Fig. 2, either with (filled symbols) or without (open symbols) prior modification by PG. The binding of ⁵⁹Fe-enterobactin to RWB18-60 (fepA, □) and RWB18-60/pITS449 (fepA+), preincubated with PG (•), 10 μM ferric enterobactin and PG (▴), or 10 μM ferric enterobactin alone (▵), was measured. PG modification blocked ferric enterobactin binding, but preincubation with ferric enterobactin inhibited the PG-induced inactivation of FepA, protecting 70% of its binding capacity. The graph depicts mean values from two independent experiments.