Ferrous Iron Efflux Systems in Bacteria

Abstract

Bacteria require iron for growth, with only a few reported exceptions. In many environments, iron is a limiting nutrient for growth and high affinity uptake systems play a central role in iron homeostasis. However, iron can also be detrimental to cells when it is present in excess, particularly under aerobic conditions where its participation in Fenton chemistry generates highly reactive hydroxyl radicals. Recent results have revealed a critical role for iron efflux transporters in protecting bacteria from iron intoxication. Systems that efflux iron are widely distributed amongst bacteria and fall into several categories: P1B-type ATPases, cation diffusion facilitator (CDF) proteins, major facilitator superfamily (MFS) proteins, and membrane bound ferritin-like proteins. Here, we review the emerging role of iron export in both iron homeostasis and as part of the adaptive response to oxidative stress.

Full text

Ferrous Iron Efflux Systems in Bacteria
Hualiang Pi and John D. Helmann*
Abstract
Bacteria require iron for growth, with only a few reported exceptions. In many environments, iron
is a limiting nutrient for growth and high affinity uptake systems play a central role in iron
homeostasis. However, iron can also be detrimental to cells when it is present in excess,
particularly under aerobic conditions where its participation in Fenton chemistry generates highly
reactive hydroxyl radicals. Recent results have revealed a critical role for iron efflux transporters in
protecting bacteria from iron intoxication. Systems that efflux iron are widely distributed amongst
bacteria and fall into several categories: P1B-type ATPases, cation diffusion facilitator (CDF)
proteins, major facilitator superfamily (MFS) proteins, and membrane bound ferritin-like proteins.
Here, we review the emerging role of iron export in both iron homeostasis and as part of the
adaptive response to oxidative stress.
Graphical abstract
Introduction
Iron is critical for cell growth and survival. However, when present in excess, it is also
detrimental to cells. Under aerobic conditions, iron toxicity is closely related to oxidative
stress through Fenton chemistry1. Hydrogen peroxide (H2O2) reacts with ferrous iron (Fe2+)
to generate highly reactive hydroxyl radicals that damage macromolecules such as DNA,
proteins and fatty acids, resulting in disruption of cell metabolism and ultimately cell death2.
Therefore, the toxicity of reactive oxygen species (ROS) is generally thought to be
exacerbated by conditions that elevate the intracellular iron pool. Conversely, high levels of
intracellular iron may also be toxic independent of ROS, presumably due to the ability of
*Corresponding author: John D. Helmann, Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA, Phone:
607-255-1517, Fax: 607-255-3904, jdh9@cornell.edu.
HHS Public Access
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Metallomics
. 2017 July 19; 9(7): 840–851. doi:10.1039/c7mt00112f.
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iron to compete with other transition metals, such as manganese, for binding to metal-
dependent enzymes or regulators, resulting in mismetallation and inactivation of these
proteins3, 4. ROS such as H2O2 and superoxide radical can disrupt iron-sulfur clusters and
mononuclear iron centers of iron-enzymes, thereby leading to iron release5, 6. Therefore,
iron intoxication may also be exacerbated by an elevation in ROS. Clearly, the toxicity of
iron and ROS are closely intertwined, with each potentially increasing the toxicity of the
other.
Bacteria adapt to environmental stresses by activation of specific transcriptional programs.
In the case of iron homeostasis, bacteria monitor intracellular iron levels using metal-sensing
(metalloregulatory) proteins7, 8. The ferric uptake regulator (Fur) protein is the most
widespread bacterial iron sensor9, but it can be replaced by functionally analogous proteins
such as IdeR (in actinomycetes)10, 11 and Irr (in alpha-proteobacteria)12–14. Fur helps to
maintain iron homeostasis by regulating genes implicated in iron uptake, storage, and
efflux15. Typically, Fur is considered to function as an Fe2+-activated transcriptional
repressor for most of its targets, but there are increasing examples where Fur functions as a
transcriptional activator or where it binds DNA in the absence of bound iron16–18.
Iron-sensing regulators such as Fur play a central role in the control of iron homeostasis19.
The
Escherichia coli
Fur regulon illustrates the diverse roles that Fur may play.
E. coli
Fur
(FurEC) binds to DNA when associated with Fe2+ and serves to repress the expression of
target operons20. This repression is relieved under iron-limited conditions, and this results in
the derepression of iron uptake systems, including the synthesis of the high-affinity iron-
chelating compound siderophore known as enterobactin and its cognate import system21.
Fur also helps bacteria to remodel their proteomes to prioritize the utilization of iron, in a
process known as "iron-sparing" (Fig. 1)22–24. In
E. coli
, the loss of FurEC DNA-binding
activity (under low iron conditions) results in expression of the RyhB small RNA (sRNA)
that represses translation of non-essential iron-enzymes22–24. Fur also participates in the
regulation of gene expression under conditions of iron excess. For example, FurEC positively
regulates expression of the iron storage protein ferritin by occluding the binding of the H-NS
transcriptional repressor25. In general, adaptation to iron excess often involves expression of
iron storage functions (including heme-containing bacterioferritins, ferritins, and Dps-family
mini-ferritins) but may additionally require iron efflux systems (Fig. 1). In light of the
central role of Fur in coordinating iron homeostasis, it is not surprising that some iron efflux
systems are induced by Fur in response to iron excess26, 27.
Bacteria also adapt to oxidative stress by the induction of specific defensive genes. For
example, H2O2 induces a specific peroxide-stress response that is regulated by the OxyR
repressor in
E. coli
28 and by the PerR repressor in
Bacillus subtilis
29. In both model
organisms, a rise in intracellular H2O2 triggers the induction of defensive enzymes such as
catalase and alkyl hydroperoxide reductase, which can directly detoxify H2O2. In addition,
cells scavenge excess iron from the cytosol by sequestration into mini-ferritin proteins,
including Dps in
E. coli
30 and the Dps ortholog MrgA in
B. subtilis
31. The co-regulation of
H2O2 degradation enzymes and iron-sequestering proteins further highlights the central role
of iron in peroxide intoxication. In addition to scavenging iron, peroxide stress also
frequently modulates metal uptake and efflux systems32. In
E. coli
, H2O2 induces an OxyR-
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activated Mn2+ uptake system (MntH)33, 34, and in
B. subtilis
H2O2 induces a PerR-
regulated iron efflux system, PfeT35, 36. PfeT is a member of the P1B4-type ATPases, and
recent results indicate that several close homologs also function as Fe2+ efflux
pumps27, 37–39. Fe2+ efflux pumps have now been documented in a wide variety of bacteria,
and include P1B-type ATPases, cation diffusion facilitator (CDF) proteins, major facilitator
superfamily (MFS) proteins, and membrane bound ferritin-like proteins (Table 1 & Fig. 2).
Here, we summarize the emerging role of these ferrous iron efflux pumps in helping
ameliorate the deleterious effects of excess iron and peroxide.
P-type ATPases
The P-type ATPases are a large group of transmembrane proteins that transport ions and
lipids across cellular membranes, energetically driven by ATP hydrolysis40. Five subgroups
of P-type ATPases have been defined based on sequence homology and substrate
specificity41. These are the P1-type (K+ and transition metal transporters), P2-type (Ca2+,
Na+/K+, and H+/K+ pumps), P3-type (H+ pumps), P4-type (phospholipid transporters), and
P5-type ATPases (unknown substrate). The P2-type ATPases have been well studied and are
more prevalent in eukaryotes than in prokaryotes. The majority of P3-type ATPases are H+
pumps found in plants and fungi. Some of the P4-type ATPases have been revealed to be
phospholipid transporters42, 43. No specific substrate has yet been identified for the P5-type
ATPases that are only found in eukaryotes.
The P1-type ATPases exist predominately in prokaryotes but are omnipresent across all
domains of life44: P1A-ATPases are involved in K+ transport whereas P1B-ATPases are
important for maintaining transition metal homeostasis. P1B-ATPases are known to transport
Cu+ 45, 46, Ag+ 47, Zn2+ 48, Cd2+ 49, Cu2+ 50, Co2+ 51 and Fe2+ 27, 36, 37. The structure of a
typical P1B-ATPase includes a transmembrane domain with 6–8 helices, a soluble actuator
domain, and an ATP-binding domain52 (Fig. 2). The P1B-ATPases can be further divided
into seven subclasses based on sequence similarity and metal substrate specificity52. The
P1B4-type ATPases were originally assigned a role in Co2+ export, based on the properties of
some of the first characterized members53. However, P1B4-type ATPases have recently been
found to function instead, or in addition, as Fe2+ efflux transporters including
Bacillus
subtilis
PfeT36,
Listeria monocytogenes
FrvA27,
Mycobacterium tuberculosis
CtpD37, and
group A
Streptococcus
PmtA38, 39.
PfeT in Bacillus subtilis
B. subtilis
is a Gram-positive soil microorganism and encodes two transcriptional regulators
critical for iron homeostasis, FurBs and PerR. FurBs is a global transcriptional regulator of
iron homeostasis analogous to FurEC54 and PerR mediates the adaptive response to peroxide
stress by regulating genes involved in iron storage and peroxide detoxification29. The
regulons for both FurBs and PerR have been well defined55, 56. FurBs senses intracellular
iron sufficiency and represses genes that are involved in siderophore synthesis and
uptake54, 57. FurBs also regulates an iron sparing response mediated by the small non-coding
RNA FsrA (Fig. 1) and its coregulators FbpA, FbpB and FbpC58–60. This system, analogous
to RyhB in
E. coli
, blocks the translation of non-essential iron-containing enzymes such as
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aconitase and succinate dehydrogenase58–60. PerR regulates peroxide detoxification
enzymes (catalase, alkyl hydroperoxide reductase), iron sequestration (MrgA) and the P1B4-
type ATPase (PfeT). Although the Fur and PerR regulons are largely non-overlapping,
pfeT
is the exception and is regulated by both proteins26. The result is that
pfeT
is induced by
either peroxide stress or by iron excess (unpublished data, Pinochet-Barros A & Helmann
JD).
PfeT is one of three P1B ATPases encoded by
B. subtilis
. CopA is a P1B1-ATPase that
functions as a Cu+ efflux transporter and, appropriate to its function, is regulated by the
CsoR Cu+ sensor61. CadA is a P1B2-ATPase that confers resistance to Cd2+, Zn2+, and Co2+
and is regulated by the divalent cation sensor CzrA62. PfeT (formerly named as ZosA) is a
P1B4-type ATPase and was discovered as a transporter induced by H2O2 that plays a role in
protecting cells against oxidative stress35. Initial results indicated that deletion of
pfeT
enhanced Zn2+ tolerance, as monitored in cells lacking the CadA efflux system35. This led to
the proposal that PfeT might function as a Zn2+ importer under oxidative stress conditions,
consistent with the idea that Zn2+ has a role in protecting cells against oxidative damage35.
As a result, PfeT was originally named for this proposed role as ZosA (Zn2+ uptake under
oxidative stress)35.
Contrary to this model, most P1B-type ATPases function in metal export rather than import,
which motivated a reinvestigation of the role of PfeT. Further study revealed that a
pfeT
null
mutant is sensitive to Fe2+ and Fe3+, particularly under acidic media conditions, but not to
Zn2+ or Co2+. Moreover, a
pfeT
null mutant accumulates elevated levels of intracellular
Fe2+, as judged by sensitivity to the Fe2+-activated antibiotic streptonigrin and by direct
chemical measurement36. Biochemical studies confirmed that the ATPase activity of PfeT is
induced the most by Fe2+, with modest induction by Co2+ but not with other metals,
including Zn2+. In addition to H2O2,
pfeT
is strongly and specifically induced by iron, but
not by other metals. Together, these findings indicate that PfeT function as a peroxide- and
iron induced ferrous efflux transporter36. The ability of PfeT to protect against H2O2 is
secondary to that of the detoxification enzymes catalase and alkyl hydroperoxide reductase.
However, PfeT plays a dominant role in protecting cells from iron overload with the MrgA
miniferritin playing a secondary role36. The revelation that PfeT functions in Fe2+ efflux, in
turn, prompted a re-evaluation of the roles of several other P1B4-type ATPases in bacterial
iron homeostasis.
FrvA in Listeria monocytogenes
L. monocytogenes
is the causative agent of the foodborne disease listeriosis, which is
associated with central nervous system infections and bacteraemia. FrvA (Lmo0641) is a
P1B4-ATPase originally described as a Fur-regulated virulence factor63. FrvA was proposed
to function as a heme exporter that was suggested to be induced by iron deficiency and to be
under negative regulation of both Fur and PerR63, 64. However, a different transcriptome
study showed a downregulation of
frvA
in a
fur
null mutant65, indicating a positive
regulatory role of Fur in
frvA
expression.
To resolve these contradictory reports of iron regulation, and to test if FrvA might function
in Fe2+ efflux, the mutant phenotype was reinvestigated and the FrvA protein was purified
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for biochemical studies27. As predicted based on studies of
pfeT
, a
frvA
null mutant was
sensitive to iron intoxication, but not to other metals or heme. Like
B. subtilis pfeT, frvA
is
positively regulated by Fur in response to high Fe2+ levels and is repressed by PerR27, 64.
Biochemical studies indicate the FrvA ATPase activity is stimulated most strongly by Fe2+
with weaker stimulation in the presence of Co2+ or Zn2+. Based on the Fe2+ concentration
dependence of ATPase activity, FrvA seems to have a higher affinity for Fe2+ than
B. subtilis
PfeT. Consistent with this, not only does FrvA complement the iron-sensitive phenotype of a
B. subtilis pfeT
null mutant, its expression depletes the cytosol of iron (even under iron-rich
conditions) thereby leading to derepression of the Fur regulon27. These results support the
hypothesis that FrvA functions as a Fe2+ efflux transporter that protects cells from Fe2+
intoxication27.
FrvA is required for virulence in murine and insect (
Galleria mellonella
) infection models63.
The
frvA
null mutant strain shows strong attenuation in virulence, but is still able to invade
and propagate inside antigen-presenting cells66, suggesting an important link between iron
homeostasis and virulence, but it is not clear at which stage(s) of the
L. monocytogenes
life
cycle FrvA is important. The phagocytic vacuole is generally considered to be an iron-
limited environment. One possibility is that the expression of high affinity iron uptake
systems by iron limitation during infection or in the phagocytic vacuole can contribute to
iron overload upon escape of cells into the relatively iron-rich cytosol. Alternatively, the
imposition of oxidative damage from host immune cells may trigger iron release from
listerial iron enzymes and this may lead to iron overload. The points in the infection cycle
where FrvA plays a critical role are not yet clearly defined and provide an interesting avenue
for future research.
CtpD in Mycobacterium tuberculosis
M. tuberculosis
is an obligate pathogen and the causative agent of human tuberculosis.
Nearly one-third of the world's population is infected with
M. tuberculosis
, which can persist
in a latent state for decades and then later emerge (in ~10% of cases) as an active lung
infection.
M. tuberculosis
encodes a total of 11 P-type ATPases, which have been suggested
to be possible targets for therapeutic intervention67. Of these, two encode P1B4-ATPases:
CtpD (Rv1469) and CtpJ (Rv3743)37. CtpD, but not CtpJ, was found to be important for
survival in macrophages and the mouse lung37. Biochemical studies had previously
highlighted the activity of these two P1B4-ATPases with Co2+, but it was not clear why
M.
tuberculosis
would encode two such proteins, nor was it understood why Co2+ efflux would
be important for survival in the host.
In light of the finding that PfeT functions as an Fe2+ efflux transporter, the roles of CtpD and
CtpJ were reinvestigated. Biochemical studies indicated that the ATPase activity of CtpD is
most strongly activated by Fe2+. Although Co2+ also activates ATPase activity, the maximal
activity (Vmax) is 10-fold lower than with ferrous iron37. CtpD also binds Fe2+ with 3-fold
higher affinity than Co2+. In contrast, the CtpJ ATPase activity is activated by both Fe2+ and
Co2+, and has a slightly higher affinity for Co2+ than Fe2+. To better understand their roles
in vivo, metal accumulation and sensitivity was monitored for strains lacking either
ctpD
or
ctpJ
. The
ctpD
mutant strain did not accumulate Co2+ and was impaired in growth in iron-
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amended medium, consistent with a primary role in resistance to iron intoxication37.
Mutation of
ctpJ
led to a significant increase in Co2+ accumulation and expression was
induced by Co2+, consistent with a primary role in Co2+ resistance37. However, the
ctpJ
mutant was also growth impaired in the presence of excess iron. Thus, these two paralogous
transporters seem to have overlapping metal selectivity, but largely distinct physiological
roles. Further studies are needed to understand the molecular mechanism of substrate
specificity, but based on X-ray absorption spectroscopy (XAS) analysis, it is likely that
distinct metal coordination geometry plays an important role37.
During infection,
M. tuberculosis
propagates in the host macrophages, which are considered
iron-poor environments. Just as noted for
L. monocytogenes
, it is not yet clear where in the
infection process cells experience iron intoxication. Further studies are needed to better
understand the conditions that lead to induction of
ctpD
. In prior work
ctpD
was not induced
by metals such as Co2+, Zn2+, and Ni2+, but its cognate substrate Fe2+ was not tested68. It
might be induced by Fe2+ and, by analogy with its orthologs, this might involve an iron-
sensing transcription factor. IdeR, a member of DtxR family, is the major iron-dependent
transcriptional regulator in
M. tuberculosis
10, 11. IdeR represses transcription of genes
involved in iron uptake and siderophore biosynthesis and activates expression of genes
encoding iron-storage proteins such as bacterioferritin and a ferritin-like protein10, 11. Since
M. tuberculosis
is primarily a pathogen of the mammalian respiratory system it might
frequently encounter oxidative stress. Thus, it is also possible that
ctpD
might be induced in
response to H2O2 stress. Future work to monitor the expression of
ctpD
in vitro in response
to specific stresses and in vivo during the course of infection will be needed to elucidate the
physiological role of CtpD during the infection process.
PmtA in group A Streptococcus
Group A
Streptococcus
(GAS), a human pathogen, is the causative agent of a wide range of
diseases, from mild skin infection to life-threatening diseases such as necrotizing fasciitis69.
GAS encodes a P1B4-type ATPase under regulation of PerR, and was therefore named a
PerR-regulated metal transporter (PmtA). In a
perR
null mutant, high level expression of
pmtA
is associated with derepression of genes normally responsive to cellular zinc status
due to repression by AdcR70, a Zn2+-dependent repressor. This simplest interpretation of
this result is that PmtA may function as a Zn2+ efflux transporter. Consistent with this
notion, a
perR
null mutant has an increased resistance to Zn2+, and this depends on PmtA70.
However, it is unclear why cells would efflux Zn2+ in response to H2O2 stress, nor is there
any evidence that PmtA is important for Zn2+ resistance in wild-type cells, which
presumably relies on the Zn2+-inducible CzcD efflux pump to ameliorate Zn2+-toxicity.
By analogy with PfeT and its orthologs, an alternative interpretation is that the primary role
of PmtA is as a H2O2-inducible Fe2+-efflux pump and that activity with Zn2+ may only be
revealed when it is constitutively overexpressed in a
perR
null mutant. Two recent studies
have confirmed the primary role of PmtA as an Fe2+-efflux pump38, 39. PmtA is important
for resistance to iron intoxication, and a
pmtA
null mutant accumulates elevated levels of
intracellular iron. As expected, expression of
pmtA
is strongly induced by Fe2+. Although a
pmtA
null mutant shows similar sensitivity to peroxide stress as a wild type strain in the
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absence of excess Fe2+, it exhibits significantly increased susceptibility to peroxide stress
when treated with Fe2+. Since GAS is catalase negative, PmtA might be a frontline defense
against peroxide stress. PmtA is also a critical virulence factor and is required for survival
during infection in both intramuscular and subcutaneous mouse models38, which again links
iron efflux and peroxide resistance to pathogen virulence.
Nia in Sinorhizobium meliloti
In addition to the P1B4-ATPases featured above, it is possible that P1B-ATPases of other
groups may also have physiologically relevant activity with iron. One example is Nia, a
P1B5-ATPase with a C-terminal hemerythrin domain. Since hemerythrin domains bind O2
via a diiron active site, this suggests a possible role in O2-sensing71, 72. Nia is encoded by
the symbiotic plasmid A of
Sinorhizobium meliloti
, a nitrogen fixing microbe in the
Rhizobiales
lineage that has a symbiotic relationship with legumes in which it establishes
nodules associated with roots.
Consistent with a possible role in Fe2+ efflux, a
nia
null mutant accumulates Fe2+ under
excess metal conditions73. However, Nia also functions with Ni2+ and a
nia
null mutant
accumulates Ni2+ when in excess. The precise physiological role of Nia is not yet resolved.
Biochemical assays suggest that Nia interacts with both Fe2+ and Ni2+ (but not Co2+).
However, a
nia
null mutant showed moderate sensitivity to Ni2+, but not to Fe2+, under the
conditions tested73. Expression of
nia
was moderately induced by Fe2+ (3-fold), Ni2+ (3-
fold), and Co2+ (2-fold), but not by other metals. Interestingly,
nia
was most strongly
induced (20-fold) in root nodules, thought to be a microaerobic, iron-rich environment74.
These results lead to a model in which Nia is expressed in nitrogen-fixing root nodules, in
response to either iron excess or microaerobic conditions. The C-terminal hemerythrin
domain may also participate in or regulate transport activity, perhaps in response to O273.
More work needs to be done to characterize the details of
nia
gene regulation and to more
clearly define the physiological role of Nia during the
S. meliloti
-plant symbiosis.
Cation diffusion facilitator (CDF) proteins
Cation diffusion facilitators (CDFs) are a family of membrane-bound proteins that export
and thereby confer tolerance to heavy metal ions75, 76. CDF proteins are ubiquitous in
bacteria, archaea, and eukaryotes77. Collectively, bacterial CDF proteins have been
implicated in transport of a wide range of metal ions (Zn2+, Cd2+, Co2+, Ni2+, Fe2+ and
Mn2+) with some transporters able to transport multiple metals78–82. Phylogenetic analysis
of the CDF transporters defines three major groups corresponding to substrate specificity: 1)
manganese efflux, 2) iron/zinc efflux, 3) zinc and other metals (but not manganese or iron)
efflux83.
A typical bacterial CDF contains an N-terminal domain (NTD), 6 transmembrane helices
(TM), a histidine-rich interconnecting loop (IL) between TM4 and TM5, and a C-terminal
cytoplasmic domain (CTD)75 (Fig 2). However, the detailed mechanisms of metal selectivity
are unknown. Some studies suggest the cytoplasmic domain or the IL loop is important for
metal specificity84–86, but other studies highlight the role of residues in the TM3 helix on
metal selectivity87. For the
E. coli
FieF transporter, evidence supports a role for a tetrahedral
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metal-binding site formed between TM2 and TM5 in metal selectivity88. So far, there is no
unifying model that can account for metal selectivity of CDF proteins.
FieF in E. coli: Zn2+ vs. Fe2+ efflux
There are two CDF transporters in
E. coli
: ZitB and FieF (also named as YiiP). ZitB is the
secondary zinc efflux system that is critical for maintaining zinc homeostasis only when the
zinc efflux ATPase ZntA is absent89. FieF has been studied for more than a decade, but its
physiological function has been controversial. In 2004, the first two reports of its structural
analysis were built on the assumption that FieF acts as a zinc efflux protein90, 91. In fact,
prior studies had demonstrated that
fieF
is induced by either zinc or iron89. However, ectopic
expression of FieF does not restore zinc tolerance in a zinc-sensitive strain, suggesting it
might not play a role in zinc homeostasis89.
Physiological studies suggest that the major physiological role of FieF may be in iron
tolerance. Indeed, FieF is important for full resistance to iron intoxication in a
fur
null
mutant, where iron homeostasis is disrupted and iron uptake systems are constitutively
expressed92. Ectopic expression of FieF leads to reduced accumulation of iron in a
fieF
null
mutant. Moreover, reconstitution of FieF in proteoliposomes showed that it mediates iron
transport in vitro92. These results all support the assignment of FieF (ferrous iron efflux) as
an iron efflux transporter. However, this notion has been challenged by others. For example,
FieF was shown to selectively bind zinc and cadmium with high affinity, but not iron or
other metals tested93. Based on the site-directed fluorescence resonance energy transfer
(FRET) measurements, Lu
et al
. proposed an autoregulation model of transport activity in
response to intracellular zinc levels94. Currently, FieF (YiiP) is referred to as a Zn2+
transporter in most published papers.
Ever since its structure was solved in 200779, FieF has been considered as a prototype for
bacterial CDF proteins, which makes it more frustrating that its physiological role has
remained controversial. The regulation of
fieF
expression has not been well defined, but it
does not appear to be regulated by Fur92. The physiological studies of FieF are certainly
supportive of a role in Fe(II) efflux. This inference is further supported by the observation
that the FieF homologs MamM and MamB form a heterodimeric CDF protein required for
Fe(II) import into vesicles in support of magnetosome formation in the magnetotatic
bacterium
Magnetospirillum gryphiswaldense
95, 96.
AitP in Pseudomonas aeruginosa
Pseudomonas aeruginosa
is Gram-negative, opportunistic pathogen that is highly antibiotic
resistant.
P. aeruginosa
encodes three paralogous CDF efflux systems: CzcD (PA0397), AitP
(PA1297), and YiiP (PA3963). Of these, the alternative iron transport protein (AitP) most
likely functions physiologically in Fe2+ efflux. Deletion of
aitP
leads to an increased
sensitivity to both Fe2+ and Co2+, increased intracellular accumulation of both ions, and
decreased survival in presence of H2O297. The observed sensitivity to H2O2 is most
consistent with a role in Fe2+ efflux, as noted above for P-type ATPases. In contrast with
AitP, the CzcD and YiiP proteins were inferred to function physiologically in Zn2+
resistance, although this role is largely masked in wild-type cells by the activity of the Zn2+
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efflux P-type ATPase, ZntA98. All the three transporters are critical for virulence in a plant
infection model97. However, it remains unclear why this organism requires multiple classes
of Zn2+ efflux proteins or under what conditions the three proteins are physiologically
important during the infection process.
FeoE in Shewanella oneidensis MR-1
Shewanella oneidensis
MR-1 is a facultative anaerobe in the γ-proteobacterium family that
is capable of respiration using metals (e.g. manganese, lead, uranium and ferric iron) as
electron acceptors99.
S. oneidensis
cells are usually pink or red, reflective of a high iron
content in hemoproteins and cytochromes100. When Fe3+ is used as a terminal electron
acceptor, cells generate a large amount of soluble Fe2+ which could potentially lead to iron
intoxication. FeoE, a CDF protein, is required for cell growth during anaerobic iron
respiration, and deletion of
feoE
increased susceptibility to Fe2+ intoxication, consistent
with a physiological role in Fe2+ efflux101. Further work is required to understand how
feoE
expression is regulated. It is unclear, for example, whether
feoE
is induced in response to
excess iron. Fur is the primary regulator that modulates iron acquisition in
S. oneidenis
102,
and is a candidate for an iron-responsive transcription factor that could be involved.
Major facilitator superfamily (MFS)
The major facilitator superfamily (MFS) of membrane transporters function with a wide
scope of small molecules such as ions, nucleosides, amino acids, small peptides, and
lipids103. They can be categorized into three groups: uniporters that transport a single
substrate, symporters that transport a substrate coupled with another ion (generally a
proton), and antiporters that transport two substrates in opposite directions104, 105. All the
MFS transporters share a canonical structural fold composed of two distinct domains [Fig.
2], each consisting of six transmembrane helices. The substrate binding site is located at the
interface between these two domains103.
The mechanism of transport by MFS proteins is not clear, but several related models have
been proposed. The first, an alternate-access model, was proposed more than five decades
ago106. This model speculates that the transporters undergo a conformational change that
alternates between a form where substrate can bind from one side of the membrane to one
where it can only bind from the other side. This has been validated by many structural
studies such as the xylose/H+ symporter XylE and for LacY107–109. The second, a rocker-
switch model, postulates that conformational changes are accomplished through rocker-
switch-type rotation between the N and C domain. This model is supported by some open-
conformation structures110 but not by the structures in occluded states111–114. A third,
clamp-and-switch model, provides a two-step transport mechanism: a clamping step that
mediates occlusion of the binding site and a switching step that mediates the exposure of the
binding site. This model postulates four conformational states: inward open, outward open,
inward-facing occlusion, and outward-facing occlusion105. This model is in a good
agreement with studies of some MFS transporters115, 116, but more structural analyses
combined with biochemical and computational analyses are needed to further understand the
transport mechanism of MFS transporters.
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IceT (iron and citrate efflux transporter) in Salmonella Typhimurium
Salmonella
Typhimurium is a Gram-negative pathogen commonly found in the
gastrointestinal tract. IceT (MdtD) is a member of the MFS superfamily in
S.
Typhimurium.
The mdtABCD baeSR operon encodes IceT and two other systems: a RND (resistance-
nodulation-division) drug efflux system MdtABC and a two-component regulatory system
BaeSR that regulates antibiotic resistance and efflux117–119. IceT is proposed to be an iron-
citrate efflux transporter and it can export either iron citrate or citrate alone120. The
iceT
null
mutant shows increased susceptibility to the antibiotic streptonigrin (SN), the activity of
which is modulated by the level of intracellular free iron121. This result suggests that the
mutation of
iceT
leads to an increase in intracellular labile iron pools. Consistent with this
result, induction of IceT expression leads to reduced levels of intracellular iron120.
Although the mdtABCD baeSR operon is not induced directly by high Fe2+ 122, it is induced
by disruption of iron homeostasis in a
fur
null mutant where iron uptake systems are
constitutively expressed, supportive of a physiological role for IceT in iron efflux. Although
IceT confers resistance to peroxide stress in a
fur
null mutant, the mdtABCD baeSR operon
is not induced by H2O2 or superoxide-generating reagents such as paraquat120. However, it
is induced by nitric oxide, which is also known to interact with the labile iron pool120. The
significance of the regulation of IceT, together with its co-transcribed ABC transporter, by
the BaeSR two-component system is not understood, nor is it yet clear whether or not IceT
is important for pathogenesis.
Membrane bound ferritin A (MbfA) in Agrobacterium tumefaciens and
Bradyrhizobium japonicum
Agrobacterium tumefaciens
belongs to the
Rhizobiales
lineage and is the causative agent of
the economically important plant disease, crown gall. MbfA was originally described as
membrane-bound ferritin A, and is a member of the erythrin-vacuolar iron transport (Er-
VIT1) ferritin-like superfamily. MbfA has two major domains: an N-terminal ferritin-like or
Er domain (Er) and a C-terminal membrane-embedded vacuolar iron transporter domain
(VIT1) (Fig. 2). The Er domain has a di-iron binding site and the VIT1 domain shows
sequence homology to
Arabidopsis
VIT1, which is responsible for transferring iron into
vacuoles123. Ferritin is a cytosolic iron storage protein ubiquitous in prokaryotes and
eukaryotes124, however, MbfA is not a
bona fide
ferritin and its physiological function was
not immediately apparent.
Plant hosts often produce reactive oxygen species as a defense mechanism in response to
microbial infection. Initial studies revealed that MbfA confers resistance to H2O2 stress,
suggesting that it may play an important role in plant-pathogen interaction125. Moreover,
mbfA
expression was induced in response to high iron conditions as sensed by the iron
response regulator protein, Irr125. However, these results could not distinguish between a
role for MbfA in sequestration of iron (through its ferritin domain) or iron efflux. A follow
up study revealed that MbfA is important for resistance to iron intoxication under acidic
conditions (pH 5.5), which enhances iron solubility thereby promoting toxicity126.
Compared to wild-type, an
mbfA
null mutant had a modest increase in intracellular total iron
Pi and Helmann Page 10
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as well as labile iron125. Since its expression is induced by high iron under acidic
conditions125, and leads to reduced intracellular iron levels, MbfA was postulated to
function as an iron efflux transporter125.
Bradyrhizobium japonicum
also encodes an MbfA protein implicated in iron efflux127.
B.
japonicum
is a nitrogen-fixing endosymbiotic microbe that, like
A. tumefaciens
, belongs to
the
Rhizobiales
lineage. As in
A. tumefaciens
, iron homeostasis in
B. japonicum
is also
under control of Irr128, which regulates iron uptake, storage, and utilization129. MbfA in
B.
japonicum
is specifically induced by high iron and confers resistance to iron intoxication
and H2O2 stress. Moreover, an
mbfA
null mutant accumulates significantly high levels of
iron.
Collectively, these data support the idea that MbfA functions physiologically as an iron
efflux transporter127. Interestingly, the N-terminal ferritin-like domain located on the
cytoplasmic side of inner membrane is required for iron transport activity and stress
resistance. The purified ferritin domain forms a dimer in solution, which suggests that MbfA
may dimerize to form a functional channel127. By mediating the efflux of Fe2+, MbfA
functions cooperatively with bacterioferritin (Bfr), which functions in iron sequestration, to
prevent iron intoxication130. Mutation of either
mbfA
or
bfr
increases Fe2+ sensitivity, but a
double
mbfA bfr
mutant is extremely sensitive to iron130.
Conclusions
Efflux systems play a central role in the resistance of bacteria to heavy metals, but their role
in iron homeostasis has been relatively slow to emerge. This is perhaps a reflection of the
fact that iron limitation is a far more prevalent challenge for bacteria than iron
intoxication131, due in part to the very low solubility of iron under aerobic conditions of near
neutral pH. Recent results, however, have greatly expanded our appreciation of the central
importance of iron efflux systems and their contribution to virulence in human
pathogens27, 37, 38. This implies that iron intoxication imposes a selective pressure during
infection, although how this arises is not yet clear. For example, iron intoxication may arise
from an uncontrolled influx of iron into the cell from the outside. Indeed, it is thought that
macrophages impose Zn2+ and Cu+ toxicity on engulfed bacteria by import of metals into
the phagolysosome132. However, iron is not known to be imported into the phagocytic
vacuole. Iron overload may also result when bacteria exposed to an iron limited
environment, and therefore expressing high affinity uptake systems, transition to an iron-rich
environment. The sudden influx of iron may then be best accommodated by storage or
efflux. Alternatively, or in addition, iron intoxication may arise from within the cell. For
example, oxidative stress may lead to the release of iron from abundant iron-sulfur and
mononuclear iron enzymes, thereby leading to an increase in cytosolic iron levels.
Iron intoxication may also be present in specific environments. For example, acidophilic
bacteria grow in low pH environments where iron concentrations may be 1018 times higher
than that found in pH neutral environments133. In the case of iron-respiring bacteria, high
local concentrations of Fe2+ may be produced by reduction of Fe3+-containing minerals101.
Pi and Helmann Page 11
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Further work is needed to better define the prevalence of iron intoxication in natural
environment settings and the role of iron efflux in these environments.
With the identification of the several families of iron efflux systems noted here, the stage is
now set for further structural, biochemical and genetic studies to address their mechanisms
of metal selectivity. It is presently unclear how these efflux transporters discriminate Fe2+
from competing substrates and how, at a structural level, efflux is coupled to substrate
binding and energy consumption. It is also unclear why some cells rely on ATP-dependent
P-type transporters and others utilize CDF proteins, which are coupled to the proton motive
force. It is notable that in several cases efflux pumps were initially assigned a role for
substrates others than Fe2+ (PfeT, FrvA, CtpD), and in other cases (FieF, Nia) the most
relevant physiological substrate is still unclear. This highlights the fact that metal selectivity
cannot be easily predicted from protein sequence alone, and biochemical assays need to be
interpreted in context of the physiology of the organisms. In several of the cases described,
the most compelling evidence to assign function has emerged from a careful analysis of
mutant phenotypes combined with detailed analysis of regulation to infer those conditions
that specifically induce expression.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Pete Chandrangsu for helpful comments. This work was supported by a grant from the NIH (GM059323)
to JDH.
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fig. 1. Iron homeostasis in bacteria
Under iron deficient conditions (left), high affinity iron uptake systems are induced to
scavenge iron from the surroundings to maintain the cell's labile iron pool. when iron is
limiting, it is selectively partitioned to the most essential functions and incorporation into
lower priority iron enzymes is translationally inhibited as part of an iron sparing response. in
many cases, iron-independent enzymes may be derepressed to replace functions that would
otherwise depend on iron. under iron excess conditions, the cell will have a full complement
of iron-requiring enzymes, and iron in excess of immediate needs will be either stored for
future use or exported by fe2+ efflux transporters to prevent iron overload.
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fig. 2. Ferrous iron efflux systems in bacteria
Four different groups of transporters can function as fe2+ efflux pumps. i. p1b-atpase; ii.
cation diffusion facilitator (cdf); iii. major facilitator superfamily (mfs); iv. membrane-bound
ferritin. a typical p1b-atpase consists of a transmembrane domain (tmd) that has 6–8 helices,
a soluble actuator domain (not shown), and an atp-binding domain (atp-bd)52. a cdf
transporter contains a n-terminal domain (ntd), a transmembrane domain (tmd) that has 6
helices, a histine-rich interconnecting loop (il) between tm4 and tm5 (not shown), and a c-
terminal cytoplasmic domain (ctd)75. the common structural fold (mfs fold) of a mfs
transporter is composed of two distinct domains, n domain and c domain. each domain has
six consecutive transmembrane helices103. a membrane-bound ferritin transporter has two
major domains, n-terminal ferritin-like or er domain (er) and c-terminal membrane-
embedded vacuolar iron transporter domain (vit1).
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Table 1
Fe2+ efflux transporters in bacteria.
Protein Organism Function Category Substrate
specificity*Transcription regulation References
PfeT
Bacillus subtilis
Fe2+ efflux P1B4-type ATPase Fe2+
a
,
b
, Co2+
a
PerR & Fur 36
FrvA
Listeria monocytogenes
Fe2+ efflux P1B4-type ATPase Fe2+
a
,
b
, Co2+
a
, Zn2+
a
PerR & Fur 27
CtpD
Mycobacterium tuberculosis
Fe2+ efflux P1B4-type ATPase Fe2+
a
,
b
, Co2+
a
Unknown 37
PmtA group A
Streptococcus
Fe2+ efflux P1B4-type ATPase Fe2+
b
PerR 38, 39
Nia
Sinorhizobium meliloti
Fe2+ or Ni2+ efflux P1B5-type ATPase Fe2+
a
,
b
, Ni2+
a
,
b
Unknown 73
FieF (YiiP)
Escherichia coli
Fe2+ or Zn2+ efflux CDF family Fe2+
b
, Zn2+
a
, Cd2+
a
Unknown 92–94
AitP
Pseudomonas aeruginosa
Fe2+ or Co2+ efflux CDF family Fe2+
b
Unknown 97
FeoE
Shewanella oneidensis
Fe2+ efflux CDF family Fe2+
b
Unknown 101
IceT
Salmonella typhimurium
Fe2+ citrate or citrate efflux MFS family Fe2+
b
BaeSR 120
MbfA
Agrobacterium tumefaciens
Fe2+ efflux Membrane bound ferritin Fe2+
b
Irr 125
MbfA
Bradyrhizobium japonicum
Fe2+ efflux Membrane bound ferritin Fe2+
b
Irr 127
*
Note: the substrate specificity of the transporters is either based on biochemical measurements (a), inferred from physiology studies (b), or both (a, b).
Metallomics
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