FIG 6.
FeEnt uptake by fluoresceinated AbaFepA-Cys mutants. (A) FM labeling. A. baumannii 17978 ΔfepA harboring plasmids expressing AbaFepA Cys mutants were labeled with 5 μM FM and subjected to SDS-PAGE, followed by fluorescent imaging. We included E. coli OKN3/pITS23-S271C as a positive control. The imaged gel was representative of three experiments. (B) FeEnt transport Vmax screening. All unlabeled mutants normally utilized FeEnt in siderophore nutrition tests (data not shown), but quantitative measurements of [59Fe]Ent uptake showed that certain Cys substitutions (T223C, A326C, T383C, and S482C) impaired iron transport, especially when fluoresceinated. The graph depicts the transport of [59Fe]Ent by the mutant strains before and after FM labeling, relative to wild-type A. baumannii that was untreated but harboring the empty plasmid vector. S279C, T562C, and T665C mutants transported [59Fe]Ent with 38 to 75% of the efficiency of wild-type AbaFepA. Each bar derives from the average of 2 or 3 experiments, performed in triplicate.