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To proliferate in the host environment during colonization or infection, bacteria acquire iron from eukaryotic tissues, fluids, cells and proteins. To do so, they secrete siderophores that chelate iron, and they express membrane transporters that recognize and internalize ferric siderophores. The production of siderophores and the acquisition of ferric siderophores are determinants of microbial pathogenesis. For example, the E. coli outer membrane protein FepA actively transports ferric enterobactin, a prototypic catecholate iron complex of the Enterobacteriaceae, whose utilization promotes colonization of the mouse gut. We genetically engineered several fluorescent sensors to detect, discriminate and quantify ferric siderophores, in purified form or in complex mixtures of metabolites and other biochemicals. By substituting single Cys residues in TonB-dependent outer membrane transporters, and modifying them with …