Abstract
We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology. The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein. FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C. These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods. Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer. These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport. We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons. Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion.
Highlights
Gram-negative bacteria recognize and transport a variety of ferric siderophores [1, 2] through outer membrane (OM)1 receptor proteins that function as ligand-gated porins (LGP) [3]
From the FepA crystal structure we designed and introduced 10 more individual, unpaired Cys residues at positions either on the cell surface or within the periplasm: W101C, S150C, S211C, Y260C, S271C, F329C, S397C, S423C, S575C, and S595C. Among these 10 target Cys residues, fluorescein maleimide (FM) labeled W101C, S271C, F329C, and S397C, which all reside in cell surface-exposed loops of FepA (Fig. 1A) above the level of the LPS core sugars; W101 lies in the second loop of the N-domain, whereas S271, F329, and S397 exist in loops 3, 4, and 5, respectively, of the C-domain
Western blots revealed specific covalent modification of some of the Cys mutants, FM nonspecifically adsorbed to deep rough strains, which was apparent in the yellow color of the cell pellets that was not diminished by repeated washing
Summary
Gram-negative bacteria recognize and transport a variety of ferric siderophores [1, 2] through outer membrane (OM) receptor proteins that function as ligand-gated porins (LGP) [3]. Specificity with which LGP select their iron-containing ligands (4 –7), the high affinity of the receptor-ligand interactions [8, 9], conformational changes in the receptors during ligand binding (10 –13) and transport [14],2 the requirement for the accessory proteins TonB [16], ExbB, and ExbD [17, 18], and the need for cellular energy to accomplish active transport of the metalcontaining ligands through the OM (19 –22). In this report we applied fluorescence methodologies to FeEnt transport in vivo; the sensitivity and specificity of the technique provided an explanation for the discrepancies in affinity that were observed in vivo and in vitro and more detailed information on the ligand internalization reaction through the OM, including its temperature, pH, and energy dependence
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