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Direct Evidence of Iron Uptake by the Gram-Positive Siderophore-

Shuttle Mechanism without Iron Reduction
Tatsuya Fukushima,† Benjamin E. Allred,† and Kenneth N. Raymond*
Department of Chemistry, University of California, Berkeley, California 94720-1460, United States
*
S Supporting Information

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.

All organisms including animals, plants, and pathogenic recognition and import. In Gram-positive bacteria Fe-side-
microorganisms need iron as a cofactor for essential biological rophores are imported to the cytoplasm using a complex of a
processes including oxygen binding, electron transfer, and SBP, permease(s), and an ATPase. Several SBPs bind not only
catalysis.1 In nature iron is abundant, but the biologically Fe-siderophores but also apo-siderophores.7−9 The binding
available amount of iron is limited since Fe(III) is insoluble in affinity of Bacillus cereus FpuA, FatB, or FeuA for the target apo-
aqueous solution (10−10 M soluble Fe3+ at physiological pH).2 siderophore is similar to the affinity for the Fe-siderophore.8
Bacteria require 10−6 M intracellular iron,3 indicating that Two questions about the binding of apo-siderophores to
efficient iron acquisition systems are essential. Some micro- SBPs have arisen. The first question is what is the utility of
organisms have import machineries that import Fe(III) by binding an apo-siderophore? The answer is still not known.
oxidizing Fe(II), including YwbLMN Bacillus subtilis4 and One possible answer is that an apo-siderophore bound to a SBP
Ftr1p/Fet3p Saccharomyces cerevisiae,5,6 but many microorgan- can catch iron from a variety of ferric species. The second
isms cannot uptake Fe(III) without a chelator. Instead, many question is how is a Fe-siderophore imported when an apo-
microorganisms have transporters for iron-chelating small siderophore is initially bound to the SBP? When an SBP is
molecules, called siderophores, to efficiently import iron. bound to an apo-siderophore, the SBP can uptake a Fe-
Any siderophore transport system in Gram-positive bacteria
siderophore with two different mechanisms. One mechanism is
must differ from systems in Gram-negative bacteria because
the Gram-positive siderophore-shuttle where the SBPs
Gram-positive bacteria have one membrane (cytoplasmic
exchange Fe(III) from a Fe-siderophore to the initially bound
membrane) and a thick cell wall, while Gram-negative bacteria
have two membranes (outer and cytoplasmic membranes) and apo-siderophore followed by uptake. The other mechanism is
a thin cell wall in the periplasmic space. Gram-negative bacteria the displacement mechanism in which the apo-siderophore
use transmembrane outer membrane transporters (OMTs) to initially bound to the SBP is released and a Fe-siderophore is
selectively bind target Fe-siderophores on the cell surface. On then bound to the protein followed by uptake (Figure 1).7 We
the other hand, Gram-positive bacteria use lipoprotein
siderophore-binding proteins (SBPs) anchored on the mem- Received: April 29, 2014
brane to selectively bind the target Fe-siderophores. Thus, the Accepted: July 9, 2014
OMTs and SBPs are a key factor for Fe-siderophore Published: July 9, 2014

© 2014 American Chemical Society 2092 dx.doi.org/10.1021/cb500319n | ACS Chem. Biol. 2014, 9, 2092−2100
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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.

demonstrated that a B. cereus SBP, YxeB, binds the apo- and Fe- ment mechanism, and we successfully demonstrate that iron
siderophores DFO/DFch (desferrichrome) and FO/Fch exchanges from a Fe-siderophore to an apo-siderophore bound
(ferrichrome), respectively (chemical structures shown in to the protein without intermediate metal ion reduction.
Supporting Information Figure 1). When YxeB is initially
bound to an apo-siderophore, it facilitates exchange of iron
from a Fe-siderophore to the bound apo-siderophore, thus
■ RESULTS AND DISCUSSION
YxeB Binds Ga-DFO. Previously, the yxeB gene in B. cereus
explaining the efficacy of binding apo-siderophore. Metal ATCC 14579 in the laboratory stock was sequenced and the
exchange is a fundamental step in a siderophore-shuttle import nucleotides contained two variations, TT425A (residue 142 is
mechanism, and YxeB is the first protein identified to function Leu) and TC425A (residue 142 is Ser) (the number is with
in a Gram-positive siderophore-shuttle mechanism.7 respect to the first nucleotide of the yxeB translational start
Questions for YxeB and the Gram-positive shuttle mecha- codon).7 Although many B. cereus strains possess TT425A
nism remain. Does YxeB need to reduce Fe(III) of the Fe- (residue 142 is Leu) in yxeB, both variants were used for
siderophore, FO, during the iron exchange process? Does the studying the Gram-positive siderophore-shuttle mechanism.
shuttle provide any advantage over the displacement mecha- The natural YxeB variants YxeB-L142-6×His (residue 142 is
nism? Can YxeB facilitate exchange of iron from other Leu) and YxeB-S142-6×His (residue 142 is Ser) bind DFO,
siderophores such as Fe-enterobactin to the apo-siderophore FO, Fch, DFch, and Cr-DFO.7 To further study the metal
bound to the protein? To address these questions, the FO/Fch exchange by YxeB, a new substrate, Ga-DFO, was prepared
import system, YxeB (SBP) and BC_0382 and BC_0381 (chemical structure of DFO is shown in Supporting
(permeases renamed FhuB and FhuG) (Supporting Informa- Information Figure 1A). Gallium(III) cannot be reduced,
tion Figure 2A), have been further studied. The characteristics while iron(III) can be reduced to iron(II), an intermediate in
of YxeB and the Gram-positive siderophore-shuttle have broad many biological iron transport processes. Hence, Ga-DFO can
application since many Gram-positive bacteria such as B. be used as an irreducible FO analog. The binding ability of the
subtilis,4 Staphylococcus aureus,10 Listeria monocytogenes,11 and YxeB proteins for Ga-DFO was assessed by a fluorescence
Strepotococcus pneumoniae12 possess the FO/Fch import quenching assay. The fluorescence of YxeB-L142-6×His was
systems. We report that YxeB uses the Gram-positive increased instead of quenched by addition of the substrate
siderophore-shuttle mechanism in preference to the displace- (Figure 2A). The result is very similar to the fluorescence
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YxeB-FhuBG Machinery Can Import Cr- and Ga-DFO.

The gene encoding the DFO/Fch-binding protein, YxeB,
makes an operon with the predicted permease genes, f huB and
f huG (Supporting Information Figure 2A). The f huB and f huG
genes were disrupted (strain TC137) and the iron uptake was
assessed by growth assay and disc diffusion assay as described in
the Methods. The B. cereus ATCC 14579 host strain does not
produce DFO and DFch,13 indicating that the concentrations
of DFO/FO and the DFO analogs, Cr-DFO and Ga-DFO, can
be quantitatively controlled by adding the compounds to the
culture. The growth assay showed that the growth of the
TC137 strain is delayed with DFO or DFch compared to the
Figure 2. Fluorescence quenching assays of YxeB-L142-6×His (panel growth of wild-type strains, TC129 and TC128 (Supporting
A) and YxeB-S142-6×His (panel B). The dissociation constants were Information Figure 2B and C). Moreover, the strain did not
calculated by Hyperquad22 using the assay data (see Table 1). Open grow around a disc containing DFO or DFch (Supporting
squares, AcDFO; closed squares, AcFO; closed triangle, Ga-DFO; Information Figure 2D), indicating that FhuB and/or FhuG are
open triangles, TBS buffer (control). The fluorescence quenching
essential for importing FO and Fch.
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 To assess whether Ga-DFO and Cr-DFO are actually
quenching curves (lines) by Hyperquad.22 imported into the cytoplasm, the amount of Ga or Cr in the
cells was measured by ICP. The wild-type strains, TC128 and
enhancement of the protein when binding DFO or DFch,7 TC129, incubated with the respective metal complex contained
suggesting that the protein may bind Ga-DFO. The dissociation Ga or Cr in the cells while TC111 (yxeB− f huB+ f huG+) and
constant (Kd) of the protein for Ga-DFO is 59.2 nM (Table 1), TC137 (yxeB+ f huB− f huG−) did not (Figure 3A and B). Since
the amount of Ga or Cr in TC137 (with YxeB) was the same as
Table 1. Kd and Substrate Binding Assay of YxeB-L/S142- the amount in TC111 (no YxeB) (Figure 3A and B), the Ga
6×His for Several Substrates and Cr levels of the wild-type strains represent imported metal-
siderophore, not metal-siderophore bound at the cell surface by
YxeB-L142-6×His YxeB-S142-6×His
Kd (nM) by binding assay Kd (nM) by binding assay
substrates FQ assay (HPLC) FQ assay (HPLC)
Ga-DFO 59.2 boundd 44.6 boundd
(0.0151a) (0.0058a)
AcDFO NCb boundd 30.7 boundd
(0.0068a)
AcFO 25.4 boundd 31.6 boundd
(0.0128a) (0.0047a)
DFch 103.8c boundd 23.0c boundd
(0.0189a) (0.0113a)
Fch 43.0c boundd 29.3c boundd
(0.0184a) (0.0096a)
a
Numbers with parentheses are the SDs. bNC is not calculated by
Hyperquad22 because the fluorescence curve of YxeB-L142-6×His for
AcDFO did not show saturation (Figure 2). cKd for DFch or Fch was
calculated using fluorescence quenching data previously described.7
d
Bound means that substrates are detected in the protein complex
analyzed by RP-HPLC (Supporting Information Figures 3 and 4).

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-
and the value is similar to the Kd for FO (38.8 nM).7 The S142, wild-type), TC111 (yxeB−), and TC137 (f huBG−). 2 μM Cr-
fluorescence of YxeB-S142-6×His for Ga-DFO was quenched DFO or Ga-DFO was added in the culture and the amount of Cr or
(Figure 2B), and the dissociation constant (Kd) is 44.6 nM Ga in the cells was measured by ICP. The optical density (OD) of the
(Table 1), indicating that the protein binds Ga-DFO like FO, cultures at 600 nm after 0 and 120 min incubation with Cr-DFO were
for which the Kd is 29.1 nM.7 0.9−1.3 and 2.0−2.1, respectively. The OD at 600 nm after 0 and 80
To confirm binding of Ga-DFO to the YxeB proteins, after min incubation with Ga-DFO were 0.8−1.1 and 1.6−1.8, respectively.
the proteins and Ni-agarose beads had been mixed in order to Data are the average of two independent experiments. Bars are the
bind the proteins to the beads, Ga-DFO was added in the standard errors. (C and D) In vitro substrate binding (exchange or
mixture. The mixture was centrifuged to separate the protein displacement) assays for the DFO:YxeB-L/S142-6×His complex. After
complex binding to the beads (pellet) and unbound solution 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
(supernatant). The bound ligand, Ga-DFO, was separated from was collected as described in the Methods and the amount of Fe, Ga
the protein complex as described in the Methods and was then or Cr in the complex was then measured by ICP. Fe amount, closed
quantified by RP-HPLC. As shown in Supporting Information squares; Ga amount, open triangles; Cr amount, closed circles. Data
Figure 3B and C, both proteins, YxeB-L/S142-6×His, are the average of three independent experiments. Bars indicate the
contained Ga-DFO. Thus, this result by RP-HPLC confirmed standard errors. The amount of Fe, Ga, or Cr in the complex after 30
that the YxeB proteins bind Ga-DFO. min incubation is shown in Table 2.

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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.

YxeB. Thus, the results of the in vivo metal uptake studies Fe and Ga from FO and Ga-DFO are Transferred to
(Figure 3A and B) show that Ga-DFO and Cr-DFO can be DFO Bound to YxeB. Ga-DFO is bound to YxeB and
imported via the YxeB-FhuBG machinery. imported by B. cereus (Figures 2 and 3, Supporting Information
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Figure 3, and Table 1). Thus, the substrate can be used as an Kd is 25.4 nM (Table 1), which is similar to the Kd for FO (38.8
irreducible FO analog. The substrate binding (exchange or nM).7 Binding by AcDFO increased the fluorescence of YxeB-
displacement) experiment in vitro was performed using FO, Ga- L142-6×His (Figure 2A). Analysis of a solution of protein and
DFO, and Cr-DFO. First the DFO:YxeB complex was formed siderophore by RP-HPLC shows that the protein contains
and confirmed by RP-HPLC (see Figure 4F), and then Ga- AcDFO and AcFO (Supporting Information Figure 4 and
DFO, FO, or Cr-DFO was added to the sample. As shown in Table 1), indicating that both ligands bind to the protein. YxeB-
Figure 3C and D and Table 2, the YxeB complexes contained S142-6×His also binds AcDFO and AcFO such as DFO and
FO, respectively, since the fluorescence was quenched by
Table 2. Substrate Amounts Bound to YxeB by Exchange or addition of AcDFO or AcFO (Figure 2B). The calculated Kd
Displacement with YxeB:DFO values for AcDFO and AcFO were 30.7 and 31.6 nM,
respectively (Table 1), similar to the Kd values for DFO
metal−substrate added to YxeB:DFO substrates bound to YxeB except
[0.2 nmol/mL sample] DFO [nmol/mL sample] (35.9 nM) and FO (29.1 nM) calculated previously.7
Additionally, the complex analysis by RP-HPLC demonstrates
ICP analysis for DFO:YxeB-L142-6×His complexa (Figure 3C)
that the protein−substrate complex contained AcDFO and
Ga-DFO Ga-DFO, 0.155 (78%b)
AcFO (Supporting Information Figure 4 and Table 1). Thus,
FO FO, 0.172 (86%b)
Cr-DFO Cr-DFO, 0.014 (7%b)
AcDFO and AcFO can be used as DFO and FO analogs.
ICP Analysis for DFO:YxeB-S142-6×His Complexa (Figure 3D)
Iron Is Transferred from AcFO to DFO by YxeB
Ga-DFO Ga-DFO, 0.165 (83%b)
(“Direct Evidence” Of Iron Transfer by YxeB). To study
FO FO, 0.166 (83%b)
the iron exchange by YxeB, after the DFO:YxeB complexes had
Cr-DFO Cr-DFO, 0.022 (11%b)
been created, synthesized AcFO was added to the sample. After
RP-HPLC Analysis for DFO:YxeB-L142-6×His Complex
the addition, both YxeB proteins, YxeB-L142-6×His and -S142-
(Figure 5 and Supporting Information Figure 6) 6×His, contained FO (Figures 4G and H, 5B [YxeB-L142-
AcFO FO, 0.153 (77%b); AcDFO, 0.110 6×His] and Supporting Information Figure 5 [YxeB-S142-
(55%c) 6×His]). Since AcFO was the only iron source added to the
Fe-Ent FO, 0.011 (≤5%b) solution of DFO:YxeB complex, the increased amount of iron
Fe-EDTA FO, 0.003 (≤1%b) observed in the protein-siderophore complex comes from
Fch FO, 0 (0%b); Fch, 0.037 (19%b) AcFO. Thus, this result is strong evidence that the iron is
Fe-Cit FO, 0.089 (45%b) transferred from AcFO to DFO:YxeB. Remarkably, the protein
hematin FO, 0.003 (≤1%b) complexes also contained AcDFO, the iron-depleted substrate,
RP-HPLC Analysis for DFO:YxeB-S142-6×His Complex clearly indicating that the iron-released substrate after iron
(Supporting Information Figure 5)
exchange remains bound to YxeB.
AcFO FO, 0.144 (72%b); AcDFO, 0.089
(45%c) Iron exchange between AcFO and DFO did not occur
a
Substrate amounts bound to YxeB after 30 min incubation of without YxeB even after 24 h (Figure 4C, D, and I). On the
YxeB;DFO with metal−substrate are shown. bPercentages are metal other hand, the iron exchange with YxeB was complete after
exchange rates or displaced substrate rates. cPercentages indicate rates only 2 min incubation (Figure 4G), and the exchange amount
of iron-released substrate remaining bound to YxeB. after 8 min incubation was approximately 0.2 μM (0.2 nmol/
mL reaction sample) FO (Figure 4J). Since the AcFO added to
Ga and Fe (>0.15 nmol/mL reaction solution) but not Cr the sample gave a final concentration of 0.2 μM, Figure 4J
(≤0.02 nmol/mL reaction solution). If the protein uses the clearly shows that almost all iron (0.2 μM, final concentration)
displacement mechanism over the Gram-positive siderophore- from AcFO is immediately transferred to DFO bound to YxeB.
shuttle mechanism, the YxeB complexes should contain similar Iron Cannot Be Transferred from Several Iron-
amounts of Ga, Fe, or Cr since the binding affinities of YxeB Chelators to DFO Bound to YxeB. YxeB transfers iron
(especially YxeB-S142-6×His) for Ga-DFO, FO, and Cr-DFO from FO or AcFO to DFO bound to the protein. However, it is
are similar (Table 1). The Cr(III) of Cr-DFO (an inert, not known whether the protein can obtain iron from the other
nonexchangeable metal complex14,15) was not contained in the iron-chelators. Several iron-chelators including Fe-enterobactin
DFO:YxeB complexes and the exchangeable metals, Ga(III) (Fe-Ent, pFeIII = 34.3),16 AcFO (pFeIII for DFO = 26.6),17 Fch
and Fe(III), were contained by the complex. The Ga(III) and (pFeIII = 25.2),18 Fe-EDTA (pFeIII = 23.4),19 hematin, and Fe/
Fe(III) from Ga-DFO and FO, respectively, are transferred to citrate (the ligand should be FeCit2 as the predominant species
YxeB-bound DFO by the Gram-positive siderophore-shuttle [Supporting Information Figure 6C]) were used for the iron-
mechanism. Significantly, the amounts of metal transferred exchange assays. As shown in Figure 5, Supporting Information
from Ga-DFO and FO after 5 min incubation are 0.14−0.18 Figure 6 and Table 2 the iron of AcFO or FeCit2 (weak iron-
nmol/mL reaction solution. Since the final concentration of chelator) was transferred to the DFO:YxeB complex, while the
Ga-DFO or FO added in the assay was 0.2 μM (see Methods), iron of the other ligands, Fe-Ent, Fe-EDTA, or hematin, was
>70% of Ga and Fe are transferred to the DFO:YxeB complex not. Thus, the iron exchange does not occur between DFO and
within the 5 min incubation time (Figure 3C and D). the other chelators except FeCit2. Iron transfer does not
Synthetic AcDFO and AcFO Can Be Used as DFO and depend solely on the stability of the potential iron donor
FO Analogs. To study the iron exchange by YxeB, acetylated because the iron from Fe-EDTA is not transferred to the
DFO and FO, AcDFO and AcFO, respectively, were complex. Iron exchange to the DFO:YxeB complex may also
synthesized. Since the four compounds, AcDFO, AcFO, depend on the ability of the Fe-chelators to fit in a binding
DFO, and FO, are separated by RP-HPLC (see Figure 4), pocket of YxeB.
they are suitable probes to examine the iron exchange by YxeB. The analysis of YxeB and DFch or Fch by RP-HPLC shows
The fluorescence of YxeB-L142-6×His was quenched by that YxeB binds DFch and Fch (Supporting Information Figure
binding AcFO similar to FO (Figure 2A), and the calculated 3D−I). The iron of Fch was not transferred; however, a small
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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.

amount of Fch was bound to YxeB (Figure 5E). The bound Table 3. Kinetics of Cr-DFO Import by TC129a
Fch is due to the displacement mechanism, and the amount of
Vmax
Fch bound to the protein was 0.037 μM (Table 2). Since 0.2 ligand Km (μM) (pmol Cr mL−1 min−1) Vmax/Km
μM Fch was used for the iron exchange experiment (see
Cr-DFO only 0.74 (0.19) 3.11 (0.36) 4.20
Methods), less than 20% of Fch was bound to the protein after
Cr-DFO + 2 μM DFO 1.59 (0.17) 1.82 (0.28) 1.14
40 min incubation. Thus, YxeB uses the displacement
(DFO:YxeB-L142
mechanism for the Fch binding, although this mechanism is complex initially
less efficient than the Gram-positive siderophore-shuttle formed)
mechanism. a
Numbers with parentheses are the standard errors.
YxeB Uses the Gram-Positive Siderophore-Shuttle
Mechanism in Preference to the Displacement Mecha-
nism When Apo-Siderophore Is Present. When DFO is value, Vmax/Km, with DFO is four times lower than the Vmax/Km
initially bound to YxeB, very little Cr-DFO binds to the protein without DFO (Table 3), indicating that the Cr-DFO is less
by displacing the apo-siderophore (the displacement mecha- efficiently imported when the DFO:YxeB complex is initially
nism) (Figure 3C and D and Table 2) and only a small amount formed. Thus, the presence of apo-siderophore inhibits metal
of the DFO is displaced by Fch (Figure 5E and Table 2). On uptake when the shuttle mechanism is blocked by an inert
the other hand, the iron from FO or AcFO is immediately metal. The displacement mechanism is an inefficient metal
transferred to DFO bound to YxeB within 5 min by iron uptake mechanism when apo-siderophore is initially bound.
exchange (Figures 3 and 4) (the Gram-positive siderophore- The kinetic observations taken together with the metal
shuttle mechanism). These in vitro experiments clearly show exchange experiments show the advantage of the shuttle
that the DFO:YxeB-L/S142 complex accumulates metal- mechanism over the displacement mechanism. Siderophores
siderophore by metal exchange, diagnostic of the Gram-positive are secreted from the cell surface during growth in iron-limited
siderophore-shuttle mechanism, over the displacement mech- conditions, and apo-siderophore concentration is highest near
anism. the cell surface. It is probable that SBPs with affinity for apo-
The in vivo Cr-DFO import experiment shows that the Km of siderophores, such as YxeB, are occupied, which would inhibit
Cr-DFO import by the TC129 strain (wild-type) without DFO iron uptake if metal exchange to the bound apo-siderophore did
is two times smaller than the Km with 2 μM DFO (to make the not take place. In this situation, ferric siderophore is more
DFO:YxeB complex), and the Vmax without DFO is two times efficiently acquired via the shuttle mechanism, facilitated by the
higher than the Vmax with 2 μM DFO (Table 3). The kinetic YxeB, than by the displacement mechanism.
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We suggest that the FO/Fch-binding proteins in many Since the Cr-DFO imported by the YxeB-FhuBG in the cytoplasm
Gram-positive bacteria might use the Gram-positive side- is the product (P), the added Cr-DFO in the culture is the substrate
rophore-shuttle and the displacement mechanisms described (S) and the YxeB-FhuBG system is like enzyme (E), the Cr-DFO
here if they can bind apo- and Fe-siderophores. Having SBPs import rate by the YxeB-FhuBG system can be considered as an
enzymatic reaction (E + S ↔ ES → E + P).8,23 For calculating the
with the two mechanisms enables the bacteria to obtain iron kinetics parameters, Vmax and Km, of the Cr-DFO import, Cr-DFO
not only from the target Fe-siderophores but also from free (several concentrations) was added to the culture after 0 or 2 μM
iron and weak iron-chelators. Finally, the iron acquisition DFO had been added in the culture and the sample had been
mechanism of YxeB differs from the siderophore-shuttle incubated for 15 min at 37 °C. The values of imported Cr amounts for
mechanism in Gram-negative bacterium, Aeromonas hydrophila, 20 min incubation at 37 °C were used for determining the initial rates
in that the A. hydrophila siderophore-shuttle protein uses only since the import amounts are constant within 20 min.7 The Vmax and
the iron-exchange mechanism.20,21 Km were calculated with Excel.

■
Fe-/Ga-/Cr-DFO Substrate Binding (Exchange or Displace-
ment) Assay In Vitro Using ICP. The exchanged or displaced
METHODS amounts of Fe, Ga and Cr ions bound to the YxeB proteins were
Information for all plasmids and strains used in the study is shown in measured using ICP (inductively coupled plasma). DFO (4 μM, final
Supporting Information Table 1 and the method of construction of a concentration), 2 μM YxeB-L142-6×His or YxeB-S142-6×His (final
B. cereus f huBG markerless mutant is shown in the Supporting concentration), and 200 μL Ni Sepharose 6 Fast Flow agarose beads
Information. (Ni-agarose beads, GE Healthcare) were mixed in 26 mL of TBS
Fluorescence Quenching Assays of YxeB-L/S142-6×His for buffer (25 mM Tris-HCl, 3.2 g/L NaCl, 0.08 g/L KCl, [pH 7.4]) and
Synthesized Ga-DFO, AcDFO, and AcFO. Fluorescence quenching the mixture was gently shaken for 2 h at RT to create the DFO:YxeB
assays of YxeB-L/S142-6×His for the Ga-DFO, AcDFO, and AcFO complex. After 5 mL of the mixture had been collected and
were performed as described previously.7 The dissociation constants centrifuged, the pellet was collected (0 min sample). Purified 0.2
were calculated by Hyperquad.22 μM FO, Ga-DFO, or Cr-DFO (final concentration) was added in the
YxeB-L/S142-6×His Binding Assay for Ga-DFO, DFch, Fch, sample and the mixture was incubated at RT. After 5, 10, 20, and 30
AcDFO, and AcFO Using RP-HPLC (Reverse-Phase High min incubation, 5 mL of the mixture was collected and centrifuged,
Performance Liquid Chromatography). The binding assay was followed by collection of the pellet (5, 10, 20, and 30 min samples).
described previously.7 The YxeB-L/S142-6×His protein (2 μM, final Nitric acid (2.5 mL of 3.5% (v/v)) was added into the samples and the
concentration) and 50 μL of Ni Sepharose 6 Fast Flow agarose beads mixtures were kept overnight. After 0.1 ppm Eu had been added as an
(Ni-agarose beads) (GE Healthcare) were mixed in 5 mL of TBS internal control and the samples had been filtered, the amount of Fe,
buffer and the mixture was then gently shaken for 2 h at RT to make Ga or Cr was measured by ICP.
the YxeB-L/S142-6×His:Ni-agarose beads complex. The substrate, 10 Iron Exchange Experiment with or without the YxeB Protein
μM Ga-DFO, Fch or AcFO, or 20 μM DFch or AcDFO, was added in In Vitro. DFO (4 μM, final concentration), 2 μM YxeB-L/S142-6×His
the sample and the mixture was then gently shaken for 10 min at RT, (final concentration), and 200 μL of Ni-agarose beads were mixed in
followed by centrifuging the sample. The pellet containing the 15 mL of TBS buffer for 2 h at RT. After 0.2 μM AcFO (final
complex was washed by TBS buffer twice. concentration) had been added in the sample, 2 mL of the sample was
For the Ga-DFO samples, AcCN (400 μL of 20% (v/v)) was added collected and then centrifuged. The pellet containing the protein
in the pellet containing YxeB, and the sample was then kept at RT for complex was washed with TBS buffer twice and the YxeB-L/S142-
20 min, followed by centrifuge of the samples. After the supernatant 6×His and its substrates were eluted by addition of 2 mL of 0.01% (v/
had been collected, 1600 μL of Milli-Q was added in the samples and v) TFA for 20 min at RT. The elution was filtered and the sample was
the samples were filtered. The samples were analyzed by RP-HPLC then analyzed by RP-HPLC using a Luna 5 μ C18 column (150 × 4.60
with a Luna 5 μ C18 column (150 × 4.60 mm 5 μm, Phenomenex) to mm 5 μm, Phenomenex; flow rate, 1 mL/min; monitoring wavelength
determine whether the protein binds the substrates or not (flow rate, 1 220 nm). The elution buffer A of RP-HPLC is 0.1% (v/v) TFA and
mL/min; monitoring wavelength 220 nm). Elution buffer A and B for the buffer B is 100% (v/v) AcCN. The elution was performed for 50
the RP-HPLC are 20 mM ammonium acetate (pH 5.5) and 100% (v/ min with a linear gradient of 0% to 25% buffer B.
v) CH3CN, respectively. The elution was performed for 50 min with a For the iron exchange experiment without the YxeB protein, 4 μM
linear gradient of 0% to 25% buffer B. DFO (final concentration) and 0.2 μM AcFO (final concentration)
For the DFch, Fch, AcDFO, and AcFO samples, 1 mL of 0.1% (v/ were mixed in 15 mL of TBS buffer and 2 mL of the mixture was then
v) TFA was added in the samples and the samples were kept at RT for collected at 0 min, 5 min, 1 h, 3 and 24 h incubation at RT. The
20 min, followed by filtration of the samples. The samples were sample was immediately analyzed by RP-HPLC, as described above.
analyzed by RP-HPLC with a Luna 5 μ C18 column (150 × 4.60 mm Iron Exchange Experiment with Several Iron-Chelators or
5 μm, Phenomenex; flow rate, 1 mL/min; monitoring wavelength 220 Fe-Siderophores In Vitro. DFO (4 μM, final concentration), 2 μM
nm). Elution buffer A and B for the RP-HPLC are 0.1% (v/v) TFA YxeB-L142-6×His (final concentration), and 300 μL of Ni-agarose
and 100% (v/v) CH3CN, respectively. The elution was performed for beads were mixed in 11 mL of TBS buffer, and the mixture was gently
50 min with a linear gradient of 0% to 25% buffer B. shaken for 2 h at RT. After 5 mL of the mixture had been collected and
Growth Assays of TC111 and TC137. Growth assays were centrifuged, the pellet was collected (”No substrate added” sample).
performed as described previously.7 In the experiment 10 or 100 nM Equal amounts (20 μM) of FeCl3 and iron-chelator/apo-siderophores
DFO or DFch was added in iron-limited minimum medium (5 g/L (DFch, EDTA or apo-Ent) were mixed in TBS buffer [pH 7.4] and the
glucose, 3 g/L Difco bacto casamino acid, 1 g/L [NH4]2HPO4, 2.5 g/L sample was then kept for 2 h to form the ferric complexes Fch, Fe-
K2HPO4, 2.5 g/L KH2PO4, 40 μM nicotinic acid, 100 μM thiamine, 36 EDTA, and Fe-Ent. For ferric citrate solution after 20 μM FeCl3 and 4
μM MnSO4, 0.3 μM ZnSO4, 830 μM MgSO4, and 0.05 g/L mM citrate (Fe/citrate = 1:200) had been mixed and the pH of the
tryptophan).23 solution was adjusted at pH 7, the sample was kept for 2 h.
Disc Diffusion Assays of TC111 and TC137. Disc diffusion Equilibrated iron-substrate (0.2 μM Fch, Fe-EDTA, or Fe-Ent, final
assays were performed as described previously.7,9 In the experiment 10 concentration), AcFO (0.2 μM, final concentration), or hematin
nmol of DFO, DFch, or bacillibactin (BB) or 6 μL of DMSO as the (Sigma-Aldrich) (0.2 μM, final concentration) was added in the
negative control was infused in a sterilized filter. sample. Equilibrated ferric citrate was also added in the sample (Fe/
Measurement of Cr-DFO and Ga-DFO Import in Wild-Type, citrate = 0.2 μM: 40 μM, final concentration). In the final
TC111, and TC137. Cr-DFO and Ga-DFO import assays were concentration FeCit2 is a main component (Supporting Information
performed, as described previously.7 In the experiment, 2 μM Cr-DFO Figure 6C) and the speciation diagram of ferric citrate shown in
or Ga-DFO was added in the culture. Supporting Information Figure 6C was generated by HySS,24 as

2098 dx.doi.org/10.1021/cb500319n | ACS Chem. Biol. 2014, 9, 2092−2100

ACS Chemical Biology Articles

described previously25 using values of stability constants (log β) for (2) Boukhalfa, H., and Crumbliss, A. L. (2002) Chemical aspects of
ferric citrate,26 pKa values of citrate27,28 and iron hydroxide formation siderophore mediated iron transport. Biometals 15, 325−339.
constants.26,29 Reaction sample (5 mL) was collected and centrifuged (3) Braun, V., Hantke, K., and Koster, W. (1998) Metal Ions in
after 40 min incubation and the pellet was then collected (“Incubation Biological Systems (Sigel, A., and Sigel, H., Eds.) Vol. 35, pp 67−145,
with substrate” sample). The samples were analyzed by RP-HPLC as Marcel Dekker, Inc., New York.
described above (see “Iron exchange experiment with or without the (4) Ollinger, J., Song, K. B., Antelmann, H., Hecker, M., and
YxeB protein in vitro”, the section in Methods). Helmann, J. D. (2006) Role of the fur regulon in iron transport in
Synthesis and Purification of FO, Cr-DFO, Ga-DFO, AcDFO, Bacillus subtilis. J. Bacteriol. 188, 3664−3673.
and AcFO. FO was synthesized and purified, as described previously.7 (5) Singh, A., Severance, S., Kaur, N., Wiltsie, W., and Kosman, D. J.
Cr-DFO was synthesized and purified using the procedure of Leong (2006) Assembly, activation, and trafficking of the Fet3p.Ftr1p high
and Raymond.30 affinity iron permease complex in Saccharomyces cerevisiae. J. Biol.
Ga-DFO was synthesized as follows: Ga(acac)3 (0.18 g, 0.5 mmol), Chem. 281, 13355−13364.
desferrioxamine methanosulfonate (0.30 g, 0.45 mmol), and KOH (6) Kwok, E. Y., Severance, S., and Kosman, D. J. (2006) Evidence
(0.11 g, 1.83 mmol) were stirred in methanol overnight. Water was for iron channeling in the Fet3p-Ftr1p high-affinity iron uptake
added and the solution was acidified with HCl(aq). The solvent was complex in the yeast plasma membrane. Biochemistry 45, 6317−6327.
removed and the residue was dissolved in MeOH/EtOH. A white (7) Fukushima, T., Allred, B. E., Sia, A. K., Nichiporuk, R., Andersen,
precipitate formed which was removed by filtration. The precipitation U. N., and Raymond, K. N. (2013) Gram-positive siderophore-shuttle
and filtration were repeated to give the title compound (0.219 g, 76% with iron-exchange from Fe-siderophore to apo-siderophore by
yield): ESI-MS (positive mode) m/z calcd for (M+H) C25H46N6O8Ga Bacillus cereus YxeB. Proc. Natl. Acad. Sci. U.S.A. 110, 13821−13826.
627.2627, found 627.2625; Anal. Calcd (Found) for C25H45N6O8Ga· (8) Zawadzka, A. M., Abergel, R. J., Nichiporuk, R., Andersen, U. N.,
HCl·KCl·2H2O·2MeOH: C, 38.67 (38.42); H, 6.97 (6.91); N, 10.02
and Raymond, K. N. (2009) Siderophore-mediated iron acquisition
(10.10).
systems in Bacillus cereus: Identification of receptors for anthrax
AcDFO was synthesized following the procedure of Ihnat et al.31
virulence-associated petrobactin. Biochemistry 48, 3645−3657.
The 1H NMR spectrum matches the literature characterization. ESI-
(9) Zawadzka, A. M., Kim, Y., Maltseva, N., Nichiporuk, R., Fan, Y.,
MS (pos. mode) for C27H51O9N6 (M+H) calc’d for 603.3712, found
603.3719; anal. calcd (found) for C27H50N6O9: C, 53.80 (53.86); H, Joachimiak, A., and Raymond, K. N. (2009) Characterization of a
8.36 (8.35); N, 13.94 (13.97). Bacillus subtilis transporter for petrobactin, an anthrax stealth
AcFO was synthesized as follows: AcDFO (0.15 g, 0.25 mmol) was siderophore. Proc. Natl. Acad. Sci. U.S.A. 106, 21854−21859.
dissolved in H2O and KOH (0.08 g, 1.5 mmol). FeCl3 (0.04 g, 0.27 (10) Sebulsky, M. T., and Heinrichs, D. E. (2001) Identification and
mmol) was added to the solution, and the dark red solution was stirred characterization of f huD1 and f huD2, two genes involved in iron-
at RT for 36 h. Washed two times with ethyl acetate and one time with hydroxamate uptake in Staphylococcus aureus. J. Bacteriol. 183, 4994−
CH3Cl. Filtered the aqueous layer to break an emulsion. Washed the 5000.
aqueous layer two more times with CH3Cl. Removed water and (11) Xiao, Q., Jiang, X., Moore, K. J., Shao, Y., Pi, H., Dubail, I.,
dissolved the residue in methanol. Cooled the solution and filtered off Charbit, A., Newton, S. M., and Klebba, P. E. (2011) Sortase
colorless salt. Purified the red solution with columns of Na+ exchange independent and dependent systems for acquisition of haem and
resin, BioGel, and cellulose powder. Removed solvent to give a dark haemoglobin in Listeria monocytogenes. Mol. Microbiol. 80, 1581−1597.
red solid (0.1 g, 0.15 mmol, 61% yield): ESI-MS (pos. mode) for (12) Pramanik, A., and Braun, V. (2006) Albomycin uptake via a
C27H48O9N6 (M+H) calc’d for 656.2832, found 656.2820; anal. calcd ferric hydroxamate transport system of Streptococcus pneumoniae R6. J.
(found) for C27H47N6O9Fe·2CH3OH·H2O: C, 47.22 (47.34); H, 7.79 Bacteriol. 188, 3878−3886.
(7.65); N, 11.39 (11.40). (13) Hotta, K., Kim, C. Y., Fox, D. T., and Koppisch, A. T. (2010)

■
Siderophore-mediated iron acquisition in Bacillus anthracis and related
ASSOCIATED CONTENT strains. Microbiology 156, 1918−1925.
(14) Cotton, F. A., and Wilkinson, G. (1988) Advanced Inorganic
*
S Supporting Information
Chemistry: A Comprehensive Text, pp 1283−1334, John Wiley and
Supplemental figures and methods. This material is available Sons, New York.
free of charge via the Internet at http://pubs.acs.org. (15) Leong, J., and Raymond, K. N. (1975) Coordination isomers of

■ AUTHOR INFORMATION
Corresponding Author
biological iron transport compounds, IV, Giometrical isomers of
chromic desferriferrioxamine B. J. Am. Chem. Soc. 97, 293−296.
(16) Loomis, L. D., and Raymond, K. N. (1991) Solution equilibria
of enterobactin and metal−enterobactin complexes. Inorg. Chem. 30,
*Phone: 510-642-7219. Fax: 510-486-5283. Email: raymond@ 906−911.
socrates.berkeley.edu. (17) Schwarzenbach, G., and Schwarzenbach, K. (1963) Hydrox-
Author Contributions amatkomplexe I. Die Stabilität der Eisen(III)-Komplexe einfacher
† hydroxamsäuren und des ferrioxamins B. Helv. Chim. Acta 46, 1390−
T.F. and B.E.A. contributed equally to this work.
1400.
Notes
(18) Wong, G. B., Kappel, M. J., Raymond, K. N., Matzanke, B., and
The authors declare no competing financial interest.

■
Winkelmann, G. (1983) Coordination chemistry of microbial iron
transport compounds. 24. Characterization of coprogen and
ACKNOWLEDGMENTS ferricrocin, two ferric hydroxamate siderophores. J. Am. Chem. Soc.
We thank Dr. J. Xu for setting up reverse-phase HPLC; Dr. E. 105, 810−815.
Kreimer for helping with inductively coupled plasma analysis; (19) Abergel, R. J., Zawadzka, A. M., and Raymond, K. N. (2008)
Prof. J. A. Hoch and Prof. M. Perego for gifting the pBKJ223 Petrobactin-mediated iron transport in pathogenic bacteria: Coordi-
nation chemistry of an unusual 3,4-catecholate/citrate siderophore. J.
plasmid. This work is supported by National Institutes of
Am. Chem. Soc. 130, 2124−2125.
Health Grants AI11744 (to K.N.R.).

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(20) Stintzi, A., Barnes, C., Xu, J., and Raymond, K. N. (2000)
Microbial iron transport via a siderophore shuttle: A membrane ion
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