Direct evidence of iron uptake by the Gram-positive siderophore-shuttle mechanism without iron reduction

Abstract

Iron is an essential element for all organisms, and microorganisms produce small molecule iron-chelators, siderophores, to efficiently acquire Fe(III). Gram-positive bacteria possess lipoprotein siderophore-binding proteins (SBPs) on the membrane. Some of the SBPs bind both apo-siderophores (iron-free) and Fe-siderophore (iron-chelated) and only import Fe-siderophores. When the SBP initially binds an apo-siderophore, the SBP uses the Gram-positive siderophore-shuttle mechanism (the SBPs exchange Fe(III) from a Fe-siderophore to the apo-siderophore bound to the protein) and/or displacement mechanism (the apo-siderophore bound to the SBP is released and a Fe-siderophore is then bound to the protein) to import the Fe-siderophore. Previously, we reported that the Bacillus cereus SBP, YxeB, exchanges Fe(III) from a ferrioxamine B (FO) to a desferrioxamine B (DFO) bound to YxeB using the siderophore-shuttle mechanism although the iron exchange was indirectly elucidated. Synthetic Cr-DFO (inert metal FO analog) and Ga-DFO (nonreducible FO analog) are bound to YxeB and imported via YxeB and the corresponding permeases and ATPase. YxeB exchanges Fe(III) from FO and Ga(III) from Ga-DFO to DFO bound to the protein, indicating that the metal-exchange occurs without metal reduction. YxeB also binds DFO derivatives including acetylated DFO (apo-siderophore) and acetylated FO (AcFO, Fe-siderophore). The iron from AcFO is transferred to DFO when bound to YxeB, giving direct evidence of iron exchange. Moreover, YxeB also uses the displacement mechanism when ferrichrome (Fch) is added to the DFO:YxeB complex. Uptake by the displacement mechanism is a minor pathway compared to the shuttle mechanism.

Figure 1

Models of the Gram-positive siderophore-shuttle mechanism and displacement mechanism of YxeB. YxeB is initially bound to an apo-siderophore. (1) A Fe-siderophore approaches YxeB and rests near the binding pocket occupied by the apo-siderophore. At this step two pathways are possible. Steps 2–4 are the shuttle pathway. (2) Iron exchanges from the Fe-siderophore to the apo-siderophore in the binding pocket. The protein facilitates this step by increasing the local concentration of the entering ligand and the ferric complex. (3) The new Fe-siderophore (B) is transported and the created iron-released ligand (A) may remain to be bound the YxeB protein. (4) The receptor is bound to an apo-siderophore. Steps 5–7 are the displacement pathway. (5) The Fe-siderophore displaces the apo-siderophore and occupies the binding pocket. (6) The original Fe-siderophore (A) is transported. (7) The SBP is bound to an apo-siderophore. In the Gram-positive siderophore-shuttle both pathways operate but the shuttle pathway is preferred.

Figure 2

Fluorescence quenching assays of YxeB-L142-6×His (panel A) and YxeB-S142-6×His (panel B). The dissociation constants were calculated by Hyperquad using the assay data (see Table 1). Open squares, AcDFO; closed squares, AcFO; closed triangle, Ga-DFO; open triangles, TBS buffer (control). The fluorescence quenching curves of YxeB-L142-6×His for AcFO and Ga-DFO and of YxeB-S142-6×His for AcFO, AcDFO, and Ga-DFO were fit to the calculated quenching curves (lines) by Hyperquad.

Figure 3

(A and B) Imported Cr amounts (panel A) and Ga amounts (panel B) in cells of TC129 (YxeB-L142, wild-type), TC128 (YxeB-S142, wild-type), TC111 (yxeB^–), and TC137 (fhuBG^–). 2 μM Cr-DFO or Ga-DFO was added in the culture and the amount of Cr or Ga in the cells was measured by ICP. The optical density (OD) of the cultures at 600 nm after 0 and 120 min incubation with Cr-DFO were 0.9–1.3 and 2.0–2.1, respectively. The OD at 600 nm after 0 and 80 min incubation with Ga-DFO were 0.8–1.1 and 1.6–1.8, respectively. Data are the average of two independent experiments. Bars are the standard errors. (C and D) In vitro substrate binding (exchange or displacement) assays for the DFO:YxeB-L/S142-6×His complex. After the DFO:YxeB-L/S142-6×His complex had been created, 0.2 μM FO, Ga-DFO or Cr-DFO was added in the samples. The protein complex was collected as described in the Methods and the amount of Fe, Ga or Cr in the complex was then measured by ICP. Fe amount, closed squares; Ga amount, open triangles; Cr amount, closed circles. Data are the average of three independent experiments. Bars indicate the standard errors. The amount of Fe, Ga, or Cr in the complex after 30 min incubation is shown in Table 2.

Figure 4

Iron exchange from AcFO (0.2 μM, final concentration) to DFO (4 μM, final concentration) with or without YxeB-L142-6×His (2 μM, final concentration). The amounts of AcFO, AcDFO, FO, and DFO were assessed by RP-HPLC. (A, B, and E) AcFO (panel A), DFO (panel B), and AcDFO (panel E) standards analyzed by RP-HPLC. As shown in panel B there is a small amount of FO in the DFO standard solution arising from minor iron impurities. (C, D, and I) Amounts of ligands after 0 min and 24 h incubation with AcFO, DFO, and no YxeB were analyzed by RP-HPLC. The amounts of formed FO (iron-transferred DFO from AcFO) after 0, 1, 3, and 24 h incubation without the protein are shown in panel I. (F to H and J) Amount of substrates bound to the protein without AcFO (panel F) and after 2 and 8 min incubation with AcFO (panel G and H) as determined by RP-HPLC. The amount of formed FO that is bound to the protein after 2, 4, 6, and 8 min incubation with AcFO and the DFO:YxeB-L142-6×His complex are shown in panel J.

Figure 5

Iron exchange from Fe-siderophores or iron-chelators to the DFO:YxeB-L142-6×His complex. (A to E) After 4 μM DFO and 2 μM the YxeB protein had been mixed to create the DFO:YxeB complex, 0.2 μM AcFO (panel B), Fe-Ent (panel C), Fe-EDTA (panel D), or Fch (panel E) was added to the sample. The complex without substrate addition (blue chromatograph) and the complex after 40 min incubation with the substrate (red chromatograph) were collected and analyzed by RP-HPLC. Peaks with the thick and underlined letters are the increased products. The calculated amount of compounds bound to the protein is shown in Table 2. (F) RP-HPLC analysis of DFch and Fch standards.