Fig. 4.

Either ATP or GTP hydrolysis activity is required for in vivo ferrous iron transport. (A) For colony size assays, colony diameters of EPV6 carrying either empty vector (pWKS30) or a plasmid encoding Feo (pFeo) or its mutants were measured using ImageJ after 24 h of incubation at 37 °C on unsupplemented LB agar. The strains carrying empty vector (pWKS30) or the negative control construct (pT36K) did not grow, and their growth was indicated as zero. (B) The EPV6 strains were grown to midexponential phase in LB and resuspended in M9 medium containing 50 µg/ml of ethylene diamine diorthohydroxyphenyl acetic acid. The assay was begun with the addition of ⁵⁵Fe in the presence of 5 mM sodium ascorbate, and samples were removed after 10 min and filtered to determine the amounts of cell-associated radioactivity. The data are the averages from three replicates. The error bars represent SEM. Asterisks (***) represent statistical significance by one-way ANOVA with Dunnett’s multiple comparison test at the level of 0.001.