Figure 7.

A and B, FD HTS assay for Gram-negative bacterial TonB-dependent FeEnt (A) and Fc (B) transport. We used a ΔtonB host harboring either pEcoFepA-FM (A) or pEcoFhuA-FM (B), suspended in microtiter plates, to create universal FD sensors of FeEnt or Fc uptake, respectively. The inert sensors effectively monitored active iron transport in 96-well microtiter plates by both E. coli (blue) and K. pneumoniae (red). The proton ionophore CCCP (23) abrogated iron uptake, so fluorescence did not recover in the presence of these compounds. C and D, statistical comparisons of these positive and negative controls yielded Z′ factors approaching 1.0 within 10 min in the case of FeEnt (C) and 35 min in the case of Fc (D). The panels show the results of single representative experiments that were performed in triplicate, from which we calculated and plotted the mean values. Standard deviations of the mean measurements (data not shown) were 1.9–4.1%.