Figure 7.

A dual fluorescence reporter for measuring iron limitation. STM harboring p5008 for constitutive expression of DsRed, and sfGFP under control of P_sitA was cultured in LB media. Induction of P_sitA::sfGFP in vitro was determined by FC. (A) STM WT [p5008] was grown o/n in LB, diluted 1:31 in fresh LB and subcultured. Samples were collected after various timepoints of subculture as indicated. (B) WT [p5008] was grown o/n in LB, diluted 1:31 in fresh LB without or with addition of 100 µM FeCI_2, and/or MnCl_2, and 200 µM 2.2-dipyridyl, and subcultured for 8 h. (C, D) STM WT [p5008] was grown o/n in LB, or LB supplemented by various concentrations of FeCI₂ and MnCl₂ as indicated. Samples were collected after 8 h of culture (C) and X-means of sfGFP intensity of P_sitA-induced bacteria was determined by FC (D). sfGFP intensities above 4,000 RFI indicate FeCI₂ + MnCl₂ concentrations lower than 100 nM. sfGFP intensities of P_sitA-positive bacteria of a representative experiment are shown. Concentrations for induction of the reporter were derived from at least three independent experiments.