Cooperative uptake of microcin E492 by receptors FepA, Fiu, and Cir and inhibition by the siderophore enterochelin and its dimeric and trimeric hydrolysis products

Abstract

Microcin E492 uptake by FepA, Fiu, and Cir is cooperative, with FepA being the main receptor. No TonB-mediated interaction with the ferric catecholate receptors is needed for microcin to exert action at the cytoplasmic membrane. Microcin E492 uptake by the receptors is inhibited by the dimer and trimer of dihydroxybenzoylserine.

FIG. 1.

Contribution of receptors FepA, Fiu, and Cir to microcin E492 uptake and its dispensability in outer membrane permeabilized cells. (A and B) Bacterial strains were grown in minimal medium to 5 × 10⁸ cells/ml and treated with increasing concentrations of a MccE492 preparation with a specific activity of 180 AU/μg. The percentage of cell death was calculated relative to the viability of the cells without MccE492 treatment. E. coli H1443 is the parental strain with the three receptors (•), and E. coli H1876 was used as the triple-mutant strain (FepA⁻ Cir⁻ Fiu⁻) (○). (A) E. coli mutant strains in one receptor: H873 (FepA⁻) (▿), H1594 (Fiu⁻) (□), and H800 (Cir⁻) (⋄). (B) E. coli mutant strains in two receptors: H1728 (Fiu⁻ Cir⁻) (▾), H1875 (FepA⁻ Cir⁻) (▪), and H1877 (FepA⁻ Fiu⁻) (♦). (C) The specific activity of MccE492 used was 39 AU/μg, and the concentration of spheroplasts used was 2 × 10⁶ cells/ml. Symbols: E. coli H1876 (FepA⁻ Cir⁻ Fiu⁻) with (▵) or without (○) treatment to form spheroplasts; E. coli H1443 (wild type) with (▴) or without (•) treatment to form spheroplasts. Mcc, microcin E492. Error bars represent the standard deviations from three determinations.

FIG. 2.

Effect of increasing concentrations of the dimer and trimer of DHBS on microcin E492 bactericidal activity. DHBS₂ (DHBS dimer), DHBS₃ (trimer), and Ent (enterochelin) were incubated for 15 min at 37°C with MccE492 (0.15 μM; specific activity, 49 AU/μg), further incubated with 10⁷ cells of E. coli AN260 (FepC⁻) at 37°C for 15 min, and plated for survivors. The indicated symbols are used for DHBS₂ (▴), DHBS₃ (□), and enterochelin (○). The data were fit by using the program “standard curve, four-parameter logistic curve” equation (Sigma Plot 8.0). Error bars represent the standard deviation from three determinations.