Characterization of ferric and ferrous iron transport systems in Vibrio cholerae

Abstract

Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

FIG. 1.

Growth of S. flexneri strain SM193w. SM193w carrying genes for the indicated iron transport systems was streaked on either (A) LB agar or (B) LB agar supplemented with an aerobactin-containing culture supernatant. Genes were carried on the following plasmids: pWKS30 (vector), pFbp1 (fbpABC), pVfu4 (VCA0685-0687), pVtu100 (VCA0601-0603), pFeo101 (V. cholerae feoABC), and pUH18E (E. coli feoABC).

FIG. 2.

Iron transport assays. SM193w carrying a plasmid encoding the indicated iron transport system was grown to late exponential phase and resuspended in the transport buffer. The assay was begun with the addition of ⁵⁵Fe, and samples were removed at the times shown (A and B) or after 5 min (C) and filtered to determine the amounts of cell-associated radioactivity. SM193w carrying plasmids with (A) feoABC (pFeo101) or (B) fbpABC (pFbp1) was compared to strains carrying the vector (pWKS30). (B) The transport reaction mixture contained 5 μM sodium ascorbate. (C) Uptake of iron by SM193w containing the indicated system genes was carried out in buffer with (+) or without (−) supplementation with 5 μM sodium ascorbate. The data are the averages from three experiments. The error bars represent 1 standard deviation.

FIG. 3.

Growth of an E. coli tonB mutant containing the V. cholerae fbpABC genes. E. coli tonB entF mutant strain ARM100 carrying either the vector plasmid pWKS30 or the fbp genes on the plasmid pFbp1 was spread at low density on LB agar containing either 1 μg EDDA per ml or 20 μM FeSO₄. Colony diameters were measured after 24 h at 37°C. The data are the mean diameters of 10 colonies from one representative experiment. The error bars indicate 1 standard deviation.

FIG. 4.

Growth of V. cholerae iron transport mutants. Bacteria were spread on LB agar or on LB agar containing either 40 μM FeSO₄ or 5 μg EDDA per ml. The sizes of the colonies formed were measured after incubation at 37°C for 24 h. The strains used in this assay were O395 (wild type), ALV101 (vib), ALV102 (vib feo), ARM591 (vib fbp), CFV1 (vib fbp feo), and EWV125 (vib feo fbp VCA0685-0687). The data are the mean diameters of 10 colonies from one representative experiment. The error bars indicate 1 standard deviation.