Selectivity of ferric enterobactin binding and cooperativity of transport in gram-negative bacteria

Abstract

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd approximately 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (Kd </= 100 nM) and transported it at comparable rates (>/=50 pmol/min/10(9) cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.

FIG. 1

Structures of natural and synthetic catecholate siderophores. (A) Enterobactin; (B) TRENCAM; (C) agrobactin A; (D) TRENCAM-3,2-HOPO; (E) TREN-Me-3,2-HOPO; (F) MeME-Me-3,2-HOPO; (G) K₃MECAMS; (H) MeMEEtTAM.

FIG. 2

Binding (A) and transport (B) of FeEnt (○), FeEnEnt (•), and FeTRENCAM (□) by E. coli BN1071. Bacteria were cultured in iron-deficient MOPS medium and exposed to ⁵⁹Fe siderophores at the indicated concentrations.

FIG. 3

Binding and transport of FeEnt by FepA homologs. The concentration dependence of FeEnt binding (A) and transport (C) was measured for chromosomally expressed FepA of E. coli (•) and S. typhimurium (▴) grown in MOPS medium, and P. aeruginosa (■) and B. bronchisepticus (▾) cultured in MOPS medium with enterobactin (2 μM). Binding (B) and transport (D) of FeEnt was also measured for the individual proteins EcoFepA (○), StyFepA (▵), StyIroN (◊), PaeFepA (□), and BpeFepA (▿), expressed from plasmids in E. coli. Insets show transport of FeEnt by E. coli FepA at concentrations near the K_m. The plotted data represent mean values (with standard deviations) normalized to V_max from seven experiments. These curves gave Hill coefficients of 2.98 and 3.19 for chromosome- and plasmid-expressed FepA, respectively, with standard errors less than 5%.

FIG. 4

Chromosome- and plasmid-expression of FepA and its homologs. Outer membranes were prepared by Sarkosyl extraction of cell envelopes from bacteria grown in MOPS minimal medium, subjected to SDS-PAGE, and either stained with Coomassie blue (B) or transferred to nitrocellulose and stained with anti-FepA monoclonal antibodies 2, 27, 45, (30), goat anti-mouse immunoglobulin-alkaline phosphatase, and nitroblue tetrazolium-bromochloroindolyl phosphate (7) (A). E. coli KDF541 (lane 1) and BN1071 (lane 2), S. typhimurium Enb7 (lane 3), P. aeruginosa K407 (lane 4) and K201 (lane 5), and B. bronchisepticus 19387 (lane 7) and 19385 (lane 8) were cultured in iron-deficient MOPS medium. Strains K201 and 19385 were also grown in MOPS medium containing enterobactin (2 μM; lanes 6 and 9, respectively). Outer membranes from KDF541, grown in MOPS medium and harboring either pITS449 (EcoFepA; lane 10), pENB5 (StyFepA; lane 11), pTY994 (StyIroN; lane 12), pKP1 (BpeFepA; lane 13), or pCD3 (PaeFepA; lane 14) were also analyzed. Immunoreactive bands in the immunoblot correspond to the indicated stained bands in the SDS-PAGE gel.

FIG. 5

Siderophore nutrition tests. KDF541 (A) grown in Luria-Bertani broth and harboring plasmids pITS449 (EcoFepA) (C), pENB5 (StyFepA) (D), pTY994 (StyIroN) (E), or pCD3 (PaeFepA) (F) was plated in nutrient agar containing ampicillin (10 μg/ml), and 10 μl of 50 μM FeEnt was applied to a sterile paper disc on the agar surface. BN1071 (chromosomal EcoFepA) (B) was tested under the same conditions. A 1-cm ruler was embedded in the photograph.

FIG. 6

Transport of FeEnt by Salmonella proteins expressed in E. coli. Uptake of ⁵⁹FeEnt by EcoFepA (○), StyFepA (▵), and StyIroN (◊) was compared by two procedures, 5-min assays (filled symbols) and 60-min assays (open symbols).