FIG 7.

Metal sequestration assay. (A) Schematic of the metal sequestration assay. Briefly, in this assay, a metal sequestering protein and acinetobactin were added to the LB medium sequentially in that order with a 1-h interval for effective complexation of the former with metal ions in the medium before competition with the latter. After an additional 2 h of incubation at 37°C, each mixture was filtered through a membrane with 10-kDa cutoff, and the filtrate was then analyzed by ICP-OES/MS to quantify metals not withheld by the protein. The size of each acinetobactin is appreciably smaller than 10 kDa; therefore, any metal ion scavenged by this siderophore would be detected in the filtrate. (B) Metal sequestration assay results. All ICP-OES/MS signals were normalized with respect to the metal contents in the intact LB medium (100%). Error bars represent the standard deviations from independent triplicate experiments. Statistical significance was assessed by one-way ANOVA tests (ns, not significance; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).