Chapter One
Introduction
1.1: Introduction
Bacterial cell membranes form a selectively permeable barrier that function to protect
the cell from the external environment. Essential nutrients required for metabolism are
consequently occluded. High affinity dedicated membrane transport systems have
evolved to facilitate the uptake of these essential nutrients. Recognition of
environmental solutes by bacterial cells can occur by means of membrane sensory
components that elicit signals to induce expression of the genes encoding transport
systems. Internally, global regulatory proteins sense solute levels and mediate the
appropriate response. Thus, only dedicated membrane transport systems are expressed
when the corresponding solute is present. Transport protein activities are typically
coupled to metabolic energy in order to drive transport against the electrochemical ion
gradient of the cell.
Bacteria mainly contain iron in the redox center of redox enzymes owing to the large
potential ranging from – 300 to + 700 mV (Andrews et al., 2003). Thus iron is an
exellent prosthetic component for many enzymes and proteins, which are in turn
involved in essential roles within the cell. DNA synthesis, cellular metabolism, oxygen
transport, photosynthesis and nitrogen fixation are some of the crucial cellular processes
that rely on iron. Free iron concentrations of approximately 10-7M are required to
support bacterial growth, equating to 105 ions per bacterial cell. In soil and in aqueous
solution iron is not available above 10-9M, a level far below that required for
multiplication (Braun and Hantke, 2002). Iron scarcity is due to the property of ferrous
iron (Fe2+) becoming rapidly oxidised to its ferric form (Fe3+) in the presence of oxygen
and precipitating as insoluble Fe(OH)3.
To overcome conditions of iron limitation, many bacteria synthesise high-affinity iron
chelators, Kaff > 1030 M-1 (Rudolph et al., 2006), termed siderophores that efficiently
deliver iron to the bacterial cell. High affinity membrane transport systems facilitate the
internalisation of Fe3+-siderophores. Siderophores and their affinity uptake systems have
been identified in numerous microorganisms. Detailed genetic, biochemical and
structural analysis has lead to the identification and characterisation of the genes and
proteins involved in these systems. Bacterial siderophore membrane transport systems
generally consist of highly specific outer membrane receptors and less specific
2
periplasmic binding protein dependent ABC type transport systems (Koster, 2001;
Miethke and Marahiel, 2007), although recently, novel MFS type inner membrane
bacterial siderophore transport systems have been identified and characterised (Ó Cuív
et al., 2004; Ó Cuív et al., 2007).
Many pathogenic bacteria possess the ability to sequester iron directly from host
haemoproteins, although this ability is not strictly limited to pathogens. In human
extracellular fluid the concentration of free iron is approximately 10-24M.
Haemoproteins and host storage proteins such as ferritins limit the availability of 78%
and 15% of the iron in human blood, respectively (Braun and Hanke, 2002). With
similarity to siderophore acquisition, some bacteria have evolved an ability to acquire
iron bound to haem by synthesising, excreting and internalising molecules termed
hemophores which bind directly to haem. Alternatively, haem can in some cases bind
directly to outer membrane receptors on the bacterial cell surface. Haem transport
across the inner membrane is similar to that of Fe3+-siderophores in that it consists of
highly specific outer membrane receptors and less specific periplasmic binding protein
dependent ABC type transport systems. To date, the transport of haem compounds and
Fe3+-siderophores have been determined to be mediated by distinct and separate
transport systems.
3
1.2: Membrane Transport Superfamilies
Transport proteins are integral membrane proteins that facilitate the translocation of
solutes through the impermeable lipid bilayer. The membrane transport protein
complement of any bacterium can be extensive, representing 20-30% of the bacterial
proteome (Krogh et al., 2001), and as such a number of membrane transport protein
families have been classified. Bacterial energy-coupled transport systems can be divided
into three broad families based on the mechanism of energy-coupling.
1.2.1: The ATP-Binding Cassette (ABC) Superfamily
The ATP-binding cassette is highly conserved across eukaryotic, archaebacterial and
eubacterial species (Linton and Higgins, 1998). The ABC superfamily consists of 22
families of uptake permeases, 21 families of prokaryotic efflux and 11 families of
eukaryotic efflux systems (Saier and Paulsen, 2001). They represent the largest
paralogous family of proteins in Escherichia coli (Labedan and Riley, 1995), with
almost 5% of the entire genome encoding ABC transporters (Higgins, 2001). They are
capable of transporting both small molecules and macromolecules (Pao et al., 1998).
ABC transporters generally consist of four core domains. These domains can be
encoded separately on two polypeptides, although they can be fused into multidomain
polypeptides. ABC proteins are generally localised to the cytoplasmic membrane and
transmembrane domains (TMD) span the membrane multiple times. Generally, twelve
TMD α-helices are predicted per transporter, six per domain. The TMDs form a
gateway for the translocation of the solute across the membrane. Residues within the
TMDs determine the specificity of the substrate by substrate-binding sites (Higgins,
2001). The energy for the translocation of solutes is provided by ATP hydrolysis and is
created by the remaining two domains. These domains are hydrophilic and are
associated with the cytoplasmic side of the membrane. Sequence analysis lead to the
identification of conserved sequence motifs termed the Walker A and B motifs which
were found to be involved in the binding and hydrolysis of ATP (Linton and Higgins,
1998). Auxillary domains are used by ABC transporters for specific functions.
Periplasmic binding proteins associate with ABC transporters by binding specific
proteins with high affinity on the exterior of the cytoplasmic membrane and delivering
the substrate to the transporter. The periplasmic binding protein also determines the
4
direction of translocation. The binding of the periplasmic binding protein triggers ATP
hydrolysis and therefore transport. The absence of a periplasmic binding protein from
an ABC transporter generally implies solute export (Higgins, 2001). ABC transporters
are termed primary transporters because the mechanism of energy-coupling is provided
by ATP hydrolysis (Kelly and Thomas, 2001).
1.2.2: The Major Facilitator Superfamily (MFS)
The transport of solutes by ABC transporters in a single direction can cause
electrochemical potentials that MFS transporters then utilise to facilitate solute transport
(Pao et al., 1998). MFS transporters are therefore termed secondary transporters because
they take advantage of these electrochemical ion gradients. The ABC and MFS families
account for almost half of the solute transporters encoded on the genomes of
microorganisms (Pao et al., 1998).
MFS transporters are single protein transporters localised to the cytoplasmic membrane.
Their topology characteristically exhibits 12 TMD where the first six TMD display
sequence similarity to the last six (Saier, 2000). The MFS consists of 28 subfamilies
that each has specificity for a general class of substrates. The MFS is essentially
ubiquitous since it contains the largest and most diverse number of transporters.
Classification is an essential requirement when studying systems on such a scale. The
Transport Classification (TC) system is based on both function and phylogeny and it
defines the sugar MFS transporters (SP) family for example as TC 2.1.1, while the
siderophore-iron transport (SIT) family is TC 2.1.16 (Saier and Paulsen, 2001; Saier,
2002). The SP family TC 2.1.1 is the largest subfamily within the MFS consisting of
over 200 sequenced members in bacteria, archaea and eukarya (Saier, 2000).
Depending on the direction of transport and whether a coupling ion is used, the MFS
systems are termed symporters, antiporters and uniporters. Symporters work against a
concentration gradient by cotransport in the same direction with another solute to
transport a substrate into a cell. Antiporters use the coupling ion itself to transport the
molecules in the opposite direction. Uniporters transport single solutes along the
concentration gradient (Veenhoff et al., 2002). A schematic of electrochemical
potentials, symporters and antiporters is shown in figure 1.1.
5
Figure 1.1: Secondary Active Transport Systems in Bacteria. The large circle represents the
cytoplasmic membrane. A transmembrane electrochemical proton gradient is generated by hydrolysis of
ATP, shown on the left. The gradient may be used to drive ATP synthesis and the proton-nutrient symport
and proton-substrate antiport secondary active transport systems shown around the circumference (Ward
et al., 2001).
1.2.3: The Tripartite ATP-Independent Periplasmic (TRAP) Transporter Family
TRAP transport systems (TC 2.A.56) utilise extracytoplasmic solute receptor (ESR)
components for solute uptake; however unlike ABC transporters, translocation is not
driven by ATP hydrolysis. In a similar manner to that of MFS transporters, energy is
provided by an electrochemical ion gradient and thus TRAP transport systems are
termed secondary transporters (Kelly and Thomas, 2001). A comparison of the ABC
superfamily, MFS and TRAP transporters is illustrated in figure 1.2.
The C4-dicarboxylate transporter of Rhodobacter capsulatus is the best studied example
of TRAP transporters. This system is encoded within the dctPQM operon where DctP is
the ESR component with affinity for the C4-dicarboxylates malate, succinate and
fumarate. DctQ is a small integral membrane protein predicted to have 4 TMD and
DctM is a large integral membrane protein predicted to have 12 TMD. Two regulatory
genes, dctS and dctR, were identified adjacent to dctP but transcribed in the opposite
orientation. These encode a two-component sensory regulatory system that controls the
expression of dctPQM (Forward et al., 1997). Insertional mutagenesis of the dctPQM
operon of R. capsulatus resulted in the abolition of malate, succinate and fumarate
transport. While complementation analysis confirmed the presence of a C4dicarboxylate transport system, it did not reveal the mechanism of energy-coupling. A
6
Sinorhizobium meliloti strain with an insertional mutation in its ABC type transport
system for the C4-dicarboxylates was used as a heterologous host for the expression of
the R. capsulatus dctPQMSR locus. The ability to utilise malate, succinate and fumarate
was conferred on the S. meliloti strain. Taken together with the results of membrane
potential experiments, it was determined that the DctPQM transport system was driven
independently of ATP hydrolysis and represented a novel type of bacterial solute
transport systems (Forward et al., 1997).
Given that the first TRAP transporter to have been molecularly and functionally
characterised was the R. capsulatus DctPQM system, detailed in silico analysis was
performed using this system as a model to determine the ancestry of the TRAP transport
family. Phylogenetic data suggested that TRAP transporters are ancient and all share
common ancestry. Homologues of DctPQM have been identified in all major bacterial
subdivisions including the archaea. DctM homologues displayed greater percentage
identity than DctP or DctQ homologues, with DctQ being poorly conserved across
species (Rabus et al., 1999; Winnen et al., 2003). Many binding protein-dependent
secondary transporters have been identified subsequently, including a tricarboxylate
transporter (Tct) in Salmonella typhimurium (Winnen et al., 2003). The protein
constituents of this system were found to exhibit limited sequence similarity to the
DctPQM system, yet their topological and functional characteristics remained similar.
This system was therefore the archetype for a novel family termed the tripartite
tricarboxylate transporter (TTT) family (TC 2.A.80) (Winnen et al., 2003).
7
Figure 1.2: Model for ABC, TRAP and MFS Transport Systems. In an ABC uptake system, the
periplasmic binding protein (red circle with the solute depicted as a small black-filled circle) interacts
with two membrane proteins or domains (green rectangles) and solute translocation is coupled to the
hydrolysis of ATP by the ABC protein subunits (orange circles). The only common feature between
TRAP transporters and ABC systems is the possession of the extracytoplasmic solute binding protein (red
circle). Mechanistically, TRAP transporters are secondary transporters in their mode of action of energy
coupling to a transmembrane electrochemical gradient (depicted here by the nH+ arrow, where n is the
stoichiometry of proton translocation, although other coupling cations are also possible), and also possess
a 12 TMD helix protein (light blue rectangle labeled M) which is distantly related to some types of
secondary transporters in the ‘ion-transporter’ superfamily. Another protein of unknown function (dark
blue rectangle labeled Q) is present and is unique to TRAP transporters. MFS transporters are secondary
transporters and generally consist of a 12 TMD helix protein (light blue rectangle) (Kelly and Thomas,
2001).
8
1.3: Microbial Siderophores: Structure and Classification
A plethora of siderophores have been isolated and characterised from a variety of
microbial species. Most aerobic and facultative anaerobic microorganisms have the
capacity to synthesise siderophores. Siderophores have a high affinity for ferric iron
(formation constant in the range of 1030) but have a low affinity for ferrous iron,
indicating that the release of iron from siderophores may involve reduction. A
considerable amount of structural variety can be seen in siderophores, however they can
be classified into two major groups: hydroxamates and phenolates/catecholates.
1.3.1: Phenolates/Catecholates
Phenolate/Catecholate
siderophores
are
synthesised
only
by
prokaryotic
microorganisms. They can be either cyclic or linear molecules consisting of repeating
units of 2,3-dihydroxybenzoic acid (DHBA) or salicylic acid linked by amino acids or
amino alkane residues.
Cyclic Catechols
Enterobactin is classed as the prototypical siderophore of the catecholates (figure 1.3). It
was isolated from S. typhimurium (Pollack and Neilands, 1970) and also from E. coli
(O'Brien and Gibson, 1970) where it was termed enterochelin. At slightly acidic pH, the
affinity of enterobactin for ferric iron is reduced due to hydrolysis of its triester
backbone. The formation constant for enterobactin (log Kf of 52) was found to be the
highest compared to any know iron ligand (Neilands, 1981).
Figure 1.3: Structure of Enterobactin (Roosenberg et al., 2000)
9
Linear Catechols
Agrobactin is a linear catechol produced by the bacterial phytopathogen Agrobacterium
tumefaciens (Ong et al., 1979). It was characterised as a threonyl peptide of spermidine
with three residues of 2,3-dihydroxybenzoic acid and an oxazoline ring. Paracoccus
denitrificans produces a linear catechol termed parabactin, a N1,N8-bis-2,3dihydoxybenzoylspermidine which is acylated on the secondary amide N with a residue
of salicylthreonine (Peterson and Neilands, 1979). The structure of parabactin was
solved and designated parabactin A (Tait, 1975).
1.3.2: Hydroxamates
Hydroxamate siderophores can be synthesised by both prokaryotic and eukaryotic
microorganisms. They contain multiple bidentate coordinating groups with two donor
oxygen atoms forming a bidentate ligand with ferric iron.
Citrate Hydroxamates
Citrate hydroxamates are derivatives of citric acid. The distal carboxyl groups of the
molecule have been substituted by hydroxamate groups which have a stronger affinity
for ferric iron.
Aerobactin (figure 1.4) was first isolated from Aerobacter aerogenes (Gibson and
Magrath, 1969) and has since been detected in many enterobacteriaceae including E.
coli, Enterobacter cloacae, Enterobacter aerogens, Shigella spp and Salmonella spp
(Gibson and Magrath, 1969; Neilands, 1995). The distal carboxyl groups of the citrate
molecule have been substituted by residues of N6-hydroxyacetyl lysine. Salmonella
species have been shown to produce high yields of aerobactin when cultured on Trissuccinate medium. In addition, there appears to be a correlation between aerobactin
production and enterobacterial virulence (Cox, 1982). The formation constant for
aerobactin is log Kf of 22.9, (Neilands, 1981).
10
Figure 1.4: Structure of Aerobactin (Butler, 2005)
Rhizobactin 1021 (figure 1.5) was isolated and characterised from S. meliloti 1021 and
found to be a citrate-based dihydroxamate siderophore (Persmark et al., 1993). The
distal carboxyl groups of the citrate are amide linked to two different sidechains: 1amino-3-(N-hydroxy-N-acetylamino)propane
and
1-amino-3-(N-hydroxy-N-(E)-2-
decenylamino)propane. Rhizobactin 1021 is structurally similar to other citrate based
dihydroxamates, namely, aerobactin, arthrobactin and schizokinen. However, it
primarily differs in that it contains an additional fatty acid residue. This may account for
its amphiphilic properties. The genes encoding the biosynthesis and regulation of this
siderophore in S. meliloti 2011 have been identified and characterised (Lynch et al.,
2001).
Figure 1.5: Structure of Rhizobactin 1021 (Butler, 2005)
Ferrioxamines
The ferrioxamines are low molecular weight, water soluble, trihydroxamate type
siderophores. They were first isolated and characterised from a Streptomyces species in
1960 (Baumler and Hantke, 1992). There are many different types of ferrioxamines,
classified based on their chemical structure. Streptomyces pilosus produces and excretes
desferrioxamines A1, A2, B, C, D1, D2, E, F, G, and H. A characteristic structural feature
of the ferrioxamines is repeating units of α-amino-ω-hydroxyaminoalkane and succinic
11
or acetic acid, so that a thermodynamically stable octahedral ferric complex may be
formed with three hydroxamate groups (Muller and Raymond, 1984). The ferrioxamines
are also antagonistic to the structurally related ferrimycins, which are potent antibiotics.
For example, ferrioxamine B is capable of reversing ferrimycin A antibiotic activity
against organisms such as Bacillus subtilis (Muller et al., 1984).
Figure 1.6: Structure of Desferrioxamine B and Desferrioxamine E (Yamanaka et al., 2005)
As evident in figure 1.6, the ferrioxamines can be divided structurally into linear and
cyclic molecules. Ferrioxamine B is a linear siderophore whereas ferrioxamine E is a
cyclic siderophore. Ferrioxamine B and D1 differ only in one side chain: in ferrioxamine
B this side chain is H while in D1 it is COCH3. Ferrioxamine B has a positive charge at
physiological pH while its derivative D1 has a negative charge due to the acetylation of
its side chain. The charge of ferrioxamine B is a property which has been shown to
enable higher uptake rates than D1 in studies done in 1984 on S. pilosus (Muller et al.,
1984).
A methane-sulfonate derivative of iron-free ferrioxamine B has been used commercially
for the treatment in humans of hemochromatosis (Braun, 2001). The drug, approved by
the FDA and known as Desferal, sequesters excess iron in the blood and the
ferrisiderophore complex is later excreted from the body by the kidneys.
12
1.3.3: Fungal Siderophores
The number of fungal siderophores identified displaying varying structural detail has
reached approximately 100 to 150. Ferrichromes, coprogens, fusarinines and
polycarboxylates are the main classes (Winkelmann, 2007).
Rhodotorulic acid, Dimerumic acid and Coprogen
Rhodotorula pilimanae and related species produce and excrete large quantities of
rhodotorulic acid to overcome adverse local conditions of iron depletion. The
rhodotorulic acid moiety may also exist in the related siderophores coprogen and
dimerumic acid (figures 1.7, 1.8 and 1.9). Dimerumic acid is produced by the fungus
Verticillum dahliae and coprogens are known to be produced by Penicillium species and
Neurospora crassa.
Figure 1.7: Structure of Rhodotorulic acid (Crichton and Charloteaux-Wauters, 1987)
Figure 1.8: Structure of Dimerumic acid (Crichton and Charloteaux-Wauters, 1987)
13
Figure 1.9: Structure of Coprogen (Crichton and Charloteaux-Wauters, 1987)
Ferrichromes
The ferrichrome family are fungal siderophores produced in response to iron limitation.
Ferrichrome was the first ferric trihydroxamate siderophore identified in nature, initially
isolated from Ustilago sphaerogena (Crichton and Charloteaux-Wauters, 1987). Many
bacteria are capable of utilising ferrichrome, although synthesis of the siderophore is
seemingly limited to fungi. Ferrichromes display variation in the amino acid sequence
of the cyclohexapeptide moiety, with glycine, serine or alanine as components. They
can also show variation in the hydroxamic acid residues. Here a variety of small
carboxylic acids can be substituted thereby creating a number of structural variants.
Figure 1.10 illustrates the structure of a number of ferrichrome class siderophores.
14
Figure 1.10: Ferrichrome Siderophores (Winkelmann, 2002)
15
1.4: Fe3+-Siderophore Active Transport in Gram Negative Bacteria
Gram negative bacteria have outer and inner membranes that function to protect the
internal contents of the cell from the external environment. These membranes serve as a
selective barrier to the passage of molecules, many of which are essential nutrients, into
the cystol. Peptidoglycan located between the inner and outer membranes provides
important structural support for the cell. Protein channels called porins are present in the
outer membrane to allow the diffusion of molecules into the periplasmic space between
the outer and inner membrane. The inner membrane is primarily composed of a lipid
bilayer. Only small uncharged molecules such as O2, CO2 and H2O can diffuse freely
through these phospholipids bilayers. However, larger molecules such as glucose or iron
must be actively transported into the cytoplasm by means of specific transmembrane
proteins. These transporters determine the selective permeability of the cell membrane.
Active transport occurs by the process of adenosine triphosphate (ATP) hydrolysis or by
the delivery of an electrochemical potential across the inner membrane.
Under conditions of iron deprivation, bacteria secrete low molecular weight (500 to
1000 Da) molecules called siderophores that scavenge this essential nutrient (section
1.3). When associated with iron, the Fe3+-siderophores bind to specific cognate outer
membrane receptors on the outer membrane cell surface. Commonly, bacteria possess a
battery of outer membrane Fe3+-siderophore receptors while often significantly fewer
inner membrane uptake systems are present that recognise the same Fe3+-sideophore
type. The proton motive force of the cytoplasmic membrane is transduced to the
receptor by means of an energy transducing complex termed the TonB-ExbB-ExbD
complex. This energy facilitates translocation of the Fe3+-siderophore complex to the
periplasm. Diffusion across the phospholipids bilayer of the inner membrane is not
possible due to the large size of the Fe3+-siderophore. Therefore the complex is actively
transported by inner membrane transport proteins. The model system for inner
membrane transport of hydroxamate siderophores in gram negative bacteria is the E.
coli FhuCDB system. The genetic arrangement of the genes encoding this system are
illustrated in figure 1.11 with a comparison of the genes encoding various Fe3+siderophore, Fe2+, transferrin and lactoferrin uptake systems. The following sections
describe in detail the transport of Fe3+-siderophore complexes across the outer and inner
membranes and the release of iron by reductase enzymes.
16
Figure 1.11: Genetic Arrangement of Genes Encoding fhuACDB of E. coli and Orthologues.
Examples of the genetic arrangement of Fe3+-siderophore, Fe2+, transferrin and lactoferrin systems are
given. In several systems not all participants have been identified and mechanisms of uptake been not all
been fully elucidated (Braun and Hantke, 2002).
1.4.1: Outer Membrane Fe3+-Siderophore Receptors
The crystal structures of five Fe3+-siderophore outer membrane receptors have been
solved to date, FhuA (Locher et al., 1998), FepA (Buchanan et al., 1999) and FecA
(Yue et al., 2003) from E. coli (figure 1.12), with FptA (Cobessi et al., 2005b) and
FpvA (Cobessi et al., 2005a) from Pseudomonas aeruginosa (figures 1.25, 1.24 and in
sections 1.9.1 and 1.9.2). Four of these crystal structures were solved with bound ligand
giving a valuable insight into the residues involved in ligand binding. All five structures
17
are similar and display similar domains. They consist of a 22 β-strand barrel composed
of 600 amino acids and a 150 amino acid N-terminal plug/cork domain that resides in
the lumen of the barrel. The plug domain consists of a four stranded β-sheet between
residues 47 and 154. The dimensions across the top of the barrel for FhuA, FepA and
FecA are 38 – 47 Å, 37 – 48 Å and 35 – 47 Å respectively and all were approximately
70 Å in height. The lengths of the external loops are greater than those of the
periplasmic side, ranging from 4 to 50 residues compared to the short periplasmic loops
(Chakraborty et al., 2007). The larger loops at the cell surface can extend 45 Å from the
cell surface and a large proportion of the receptor, greater than 50%, can be located
above the cell surface (Braun and Braun, 2002a). The -barrel has an elliptical shape,
with dimensions of approximately 35 Å by 47 Å. This is caused by the right-handed
twists of the -strands. The angle of the -strands to the axis is approximately 45.
Protein porins also possess such a structure but the Fe3+-siderophore receptors differ in
that their N-terminal region forms a globular domain which serves as a plug for the βbarrel. This completely closes the periplasmic side of the β-barrel. The external residues
together with the apices of the plug domain form a binding pocket for the cognate
ligand. The residues lining this pocket impart specific electrostatic charges that are
tailored to their cognate siderophores.
The plug domain is held in place by 50 – 60 hydrogen bonds between the plug and walls
of the β-barrel. This leaves a pore size of at least 10 Å in the case of FhuA which is too
small for passage of the ligand. The plug domain is composed of three loops termed
apices A, B and C which are involved in the interaction with the ligand. Many studies
have been involved in determining the structural mechanism of the final energy
dependent transport stage of the Fe3+-siderophore through the lumen of the β-barrel
(Ferguson et al., 1998; Locher et al., 1998; Buchanan et al., 1999). These studies
focused on two proposed models. The first proposal is where the plug domain with
bound siderophore was ejected into the periplasm following the induction of the plug
and/or barrel by energised TonB, the ‘ball and chain mechanism’, while the second is
where conformational rearrangement of the plug domain occurs and allows passage of
the siderophore substrate to the periplasm, the ‘transient pore model’. Many recent
studies involving site directed mutagenesis of plug domains and transport studies in
combination with disulphide tethering suggest that conformational changes within the
18
plug domain are necessary for ligand translocation. (Chakraborty et al., 2003;
Eisenhauer et al., 2005; Chakraborty et al., 2007). In silico analysis using multiple
sequence alignments of various combinations of receptor sequences revealed conserved
residues located below the binding site on the barrel and plug domains. This analysis
suggested the residues were involved in a common mechanism of transport from the
binding site through to the periplasm. There was a notable absence of conserved
residues in the extracellular loops or the apices of the plug domains (Chakraborty et al.,
2003; Chimento et al., 2005). Arginine and glutamate residues within a particular
conserved region termed the ‘Lock Region’ were mutated and time dependent transport
studies performed (1 nM labelled substrate). This analysis revealed that specific
individual mutations of FhuA (R93A in particular) resulted in an initial transport cycle
followed by several diminished rates of transport. This would suggest that the mutations
hindered structural reconfiguration of the region. This region was termed the ‘Lock
Region’ (Chakraborty et al., 2003). Double cysteine mutations were performed on
residues Tyr72/Ser615 and Pro74/Ser587 to tether the plug domain within the barrel.
Effectively normal transport rates of substrate passage through the receptor are observed
in receptors containing double cysteine mutations between the plug and barrel. In the
case of FhuA this suggested that transport does not require ejection of the plug domain
from the β-barrel and rather a conformational rearrangement of the plug domain occurs
(Eisenhauer et al., 2005; Chakraborty et al., 2007).
Ma et al. (2007) demonstrated that the interaction between an energised TonB complex
with a siderophore loaded outer membrane receptor results in a conformational change
within the receptor resulting in the displacement of the N-terminal plug domain and the
concomitant transfer of the siderophore to the periplasm. Fluorescein maleimide
labeling of specific FepA residues during transport occurred on the cell surface and also
on residues within the periplasm. Labelling of certain N-terminal residues could only
occur when the plug domain was expelled into the periplasm. A G54C FepA mutation
in particular indicated that fluorescein maleimide was translocated through the lumen of
FepA and interacted with residue G54C from the periplasm. Transport activity was
abolished and labeling of G54C reduced by disulphide bond formation between the Nterminal domain and the β-barrel. Thus the plug domain was tethered and unable to
eject from the lumen, thereby hindering ligand transport. This data supports a “ball and
19
chain” mechanism of membrane transport whereby the plug domain controls the
binding and movement of the ligand through the lumen of the receptor protein.
The FhuA receptor of E. coli is the cognate receptor for the hydroxamate type
siderophore ferrichrome which is produced by the fungus U. sphaerogena (Crichton and
Charloteaux-Wauters, 1987). Under conditions of iron limitation, 104 – 105 FhuA
molecules are synthesised per cell (Braun and Braun, 2002a). In addition to Fe3+siderophores, many bacteriophages, colicins and antibiotics such as albomycin and
rifamycin CGP 4832 utilise these receptors to bind and/or gain entry to the cell.
Consequently, the synthesis of Fe3+-siderophore receptors is restricted to conditions of
iron limitation. In FhuA ferrichrome binding occurs via interaction with ten amino
acids: R81, G99, Q100, Y116, Y244, W246, Y313, Y315, F391 and F693, six of which
are locate on the β-barrel while four are on the plug domain. Upon binding to
ferrichrome, FhuA undergoes major structural changes but the channel does not fully
open. Short α-helix residues 24 – 29 unwind and form an extended flexible
conformation in the periplasm. This results in the transition of residues Glu19, Ser20
and Trp22 by 17 Å (Braun and Braun, 2002a). The extension of this N-terminal region
to the periplasm is thought to form an interaction with the 160 amino acid region of the
energised TonB protein. The N-terminal regions of the Fe3+-siderophore receptors are
not seen in the crystal structures indicating the presence of a flexible structure (figure
1.12). Early studies on FhuA concentrated on the interaction with phage T1. The fhuA
gene was originally termed tonA to signify phage T one. Mutations in the protein
conferred resistance to phage T1. However, when it was discovered that it was involved
in ferrichrome uptake the gene was renamed FhuA (ferric hydroxamate uptake).
20
Figure 1.12: Crystal Structures of FhuA, FepA and FecA. Top and side views are displayed. The view
from outside the cell (top view) illustrates the closure of the β-barrel channel by the cork domain. The Nterminal segments of the protein, FhuA1-18, FepA1-10, FecA1-79 are not seen in the crystal structure
which suggests a flexible structure (Braun and Braun, 2002a; Chakraborty et al., 2006).
Bacteria often encode a battery of Fe3+-siderophore receptor proteins which can confer
upon the bacterium the ability to utilise xenosiderophores. In silico analysis of the
genome of P. aeruginosa PAO1 has revealed the presence of up to 34 putative outer
membrane receptors (Stover et al., 2000), while in contrast E. coli K12 posesses only
around 6 (Guerinot, 1994). In E. coli the FhuA receptor displays specificity for the
hydroxamate siderophore ferrichrome, FhuE for the fungal siderophore coprogen, FepA
for enterobactin (an endogenous siderophore of E. coli), FecA for ferric dicitrate, while
FoxA of Yersinia enterocolitica is specific for ferrioxamine B. This extends the range of
iron sources available to the organism without the energy expense of producing a
plethora of siderophores and may enable the organism to survive in extended ecological
niches.
21
1.4.2: Energy Coupled Active Transport: The TonB Complex
An energy source within either the outer membrane or the adjacent periplasmic space
that functions to drive the translocation of siderophores into the periplasm has not been
identified. The open porin channels of the outer membrane and the lack of any highenergy metabolites such as ATP restrict the development of an electrochemical potential
in the outer membrane. To facilitate energy coupled active transport of Fe3+siderophores, a cytoplasmic membrane anchored complex termed the TonB complex,
transduces the proton motive force of the cytoplasmic membrane to the outer membrane
(Postle, 1993; Braun, 1995).
The best characterised and most intensely studied of all TonB homologues is the 26 kDa
TonB protein of E. coli. While ferrichrome binding to FhuA occurs independently of the
TonB complex, translocation through the lumen of the FhuA requires energy mediated
by the TonB complex. In addition, energy is required to facilitate removal of the tightly
bound siderophore from ~10 amino acid side chains (Braun, 2006). TonB is a
cytoplasmic membrane anchored protein yet it can span the periplasmic space. TonB
interacts with two additional proteins termed ExbB and ExbD with a stoichiometry of
1(TonB):7(ExbB):2(ExbD) via its N-terminal domain (Higgs et al., 2002). Both ExbB
and ExbD are integral membrane proteins which utilise the proton motive force to
energise a conformational change in TonB that subsequently transduces this energy to
the outer membrane receptors. Ferrichrome binding to FhuA causes conformational
changes in the plug domain. FhuA residues 98 to 100 are shifted by 17 Å towards the
siderophore resulting in a counteracting shift of 17 Å by residue Glu19 away from its
original α-carbon position. This entire region (residues 24 to 29) is termed the ‘switch
helix’. It is understood that this motion may facilitate binding of TonB to FhuA
although the entire β-barrel channel is not opened by this movement. For this to happen,
energy must be transduced from the proton motive force of the cytoplasmic membrane
(Killmann et al., 2002).
There is currently no entire crystal structure for TonB, however NMR and X-ray
crystallographic structures of TonB domains indicate a structural plasticity of the
protein (Wiener, 2005). The TonB protein can be divided into three functional domains.
Hydrophobic residues (1 – 33) at the N-terminus function as an uncleaved export signal
sequence and anchors TonB to the cytoplasmic membrane (Chakraborty et al., 2006).
22
Within this domain, residues 12 to 33 interact with the ExbB and ExbD proteins and
form the energy transducing complex (Larsen et al., 2003). The second domain
(residues 34 to 154) consists of alternating Pro-Glu and Pro-Lys repeats from residues
66 to 100. Analysis of this second domain by NMR studies has suggested that it has an
extended rod shaped structure of 10nm length. It has been demonstrated in E. coli and
Vibrio cholerae that this region spans the periplasmic space (Seliger et al., 2001; Larsen
et al., 2003). NMR studies indicate that this domain may achieve an extended
conformation of 100 Å in length, which is approximately half the distance between the
cytoplasm and outer membrane (Postle and Larsen, 2007). This extension is probably
sufficient to enable the entire TonB protein to reach across the periplasmic space. The
third domain from 155 to 239 is located in the periplasm and can contact the N-terminus
of the outer membrane receptor. The crystal structure of the C-terminal domain in
contact with FhuA (figure 1.13) has been determined at 3.3 Å (Pawelek et al., 2006).
Additional crystal structures for the C-terminal domain (Chang et al., 2001; Kodding et
al., 2005; Sean Peacock et al., 2005) indicate that it is composed of two α-helices
positioned on the same face of a four stranded antiparallel β-sheet.
An N-terminal consensus sequence (8TITVTA13) termed the ‘Ton Box’ is a
characteristic of all outer membrane TonB-dependent receptors (Pawelek et al., 2006).
FhuA possesses such a consensus sequence (residues 7 to 11) and furthermore, binding
of TonB to five out of six of these residues has been shown (Pawelek et al., 2006).
Crystal structures reveal a mode of strand exchange whereby the Ton Box of FhuA
forms a parallel β interaction with the β3 sheet of the TonB C-terminal domain.
Residues Ile9, Thr10, Val11 and Ala13 of FhuA interact with the β3 sheet of TonB at
residues Val225, Val226, Leu229 and Lys231. This causes a conformational extension of the
β-sheet (β1 to β3). This study did not reveal the interaction of residue Gln160 of TonB
with the Ton Box; however a hydrogen bond interaction of Gln160 of TonB with Thr12 of
FhuA was indicated by sampling the preferred side chain rotamers at this position.
Gunter and Braun (1990) had previously demonstrated that TonB Gln160 substitution
mutations could abolish FhuA activity. I9P and V11D within the Ton Box abolished all
activity except phage T5 infection which is TonB-independent. Conversely, TonB
mutations Q160L, Q160K and R158L all partially restored the activity of the I9P
mutant. Evidence for interactions between these residues is corroborated by in vivo
disulphide formation between cysteine residues in the TonB Box of the receptor BtuB
23
and TonB (Cadieux and Kadner, 1999; Cadieux et al., 2000). Killmann et al. (2002) had
also demonstrated by genetic analysis that the Ton Box of FhuA interacts with the
region around residue Gln160 of TonB. Synthetic peptides identical to the region around
Gln160 of TonB reduced transport of ferrichrome in a FhuA mutant strain where the plug
domain (5 to 160) had been deleted. This indicated that TonB could also interact with
the -barrel of FhuA. Vakharia-Roa et al. (2007) created deletion mutants centring
around residue Gln160 of TonB. Deletion of up to five residues in this region did not
inactivate TonB or prevent it from associating with the outer membrane. Furthermore, a
TonB mutant lacking residues S157 to Y163 could still form disulphide bridges with
cysteine substitutions at W213 and F202 and the mutations could not prevent energy
dependent conformational changes in TonB. Thus, the TonB Gln160 region is probably
part of a periplasmic spanning region required to contact the outer membrane receptor
protein.
Figure 1.13: Overall Structure of the TonB-FhuA Complex. (A) Cartoon representation of TonB
residues 158 to 235 complexed to FhuA. β-strands are indicated as flat arrows; helices are indicated as
flat coils. View is along a plane parallel to the OM. Horizontal bars delineate approximate OM
boundaries. Arrow indicates direction towards the periplasm. TonB is bound at the periplacmic face to
FhuA. The FhuA plug domain (residues 19 to 160) is coloured green; remaining residues (8 to 18; 161 to
725) are coloured blue. TonB residues are coloured yellow. (B) View of the TonB-FhuA complex along
24
the longitudinal axis of the FhuA barrel, looking down on the periplasm-exposed surface of the complex.
TonB secondary-structure elements (α1, α2, β1, β2, β3) are labelled. FhuA periplasmic turns 1, and 7 to
10 (T1, T7, T8, T9, T10), are also labelled for reference. (C) Electron density (blue) from a simulatedannealing composite omit 2Fobs – Fcalc electron density map contoured at 1σ showing the extension of
electron density from FhuA Ile9 to Gln18. FhuA residues between 8 and 18 are shown as sticks and
coloured by atom (carbon, white; nitrogen, blue; oxygen, red). FhuA cork domain residues (19 to 160) are
shown as a green coil. FhuA barrel domain residues (161 to 725) are shown as a blue coil. TonB is shown
as a yellow coil. TonB helices α1 and α2 are lebelled for reference. (Pawelek et al., 2006)
The cytoplasmic membrane proteins ExbB and ExbD assist energy coupled active
transport mediated by TonB. The two proteins also contribute to the stability of TonB
and it is thought that ExbB may also function to chaperone newly synthesised TonB
into the cytoplasmic membrane (Karlsson et al., 1993). The majority of the ExbB
protein resides in the cytoplasm, however the N-terminus is located in the periplasmic
space and there are two additional membrane spanning segments. ExbD conversely
mainly occupies the periplasmic space with its N-terminus anchored in the cytoplasmic
membrane (Kampfenkel and Braun, 1992; Karlsson et al., 1993). The transduction of
the cytoplasmic membrane proton motive force to the outer membrane is thought to
involve the ExbBD complex in cooperative transition with TonB. The ExbBD complex
is probably a proton translocation apparatus that couples the chemiosmotic potential
with the conformational changes in TonB. A conformational change that can occur in
TonB is dependent upon the ability of ExbB to interact with the TonB N-terminal signal
anchor (Larsen et al., 1999).
E. coli ExbBD mutants result in a 90% reduction in all TonB-dependent activities.
Analogues of ExbB and ExbD, termed TolQ and TolR respectively, can functionally
substitute for their activity to which results 10% of normal TonB activity being restored
(Postle and Kadner, 2003). Double mutations made in exbB/tolQ and exbD/tolR resulted
in an abolition of Tol-dependent and Ton-dependent activity. However, TolA and TonB
cannot functionally substitute for one another, reflecting the significant differences in
amino acid sequence with the exception of the N-terminal membrane anchored segment
(Koebnik, 1993).
25
1.4.3: Delivery of Fe3+-Siderophores to the Cytoplasm by ABC-Transporters
The energy for the translocation of Fe3+-siderophores across the cytoplasmic membrane
is provided by the hydrolysis of ATP by cytoplasmic membrane associated-proteins.
The components of this type of transport system are typical of uptake systems for amino
acids, sugars and other nutrients (Clarke et al., 2001). While the outer membrane Fe3+siderophore receptors impart a high degree of specificity to the uptake system, a
common inner membrane periplasmic binding protein-dependent transport (PBT)
system can facilitate the transport of a variety of hydroxamate type siderophores to the
cytoplasm (Koster, 2001; Miethke and Marahiel, 2007). PBT systems are a subclass of
the ABC superfamily (section 1.2.1). The best characterised PBT system is the fhuCDB
system of E. coli and it has served as the model system for siderophore transport in
Gram-negative bacteria. The fhu genes of E. coli are found in an iron regulated operon,
fhuACDB. These genes encode an outer membrane ferrichrome specific receptor, FhuA,
a cytoplasmic membrane associated ATPase, FhuC, a periplasmic binding protein,
FhuD and a hydrophobic inner membrane protein, FhuB. The organisation of
siderophore family-related genes in operons (figure 1.11), such as fhuACDB, is common
to most Gram-negative bacteria, particularly in the enterobacteriaceae (Koster, 2001).
The 32 kDa periplasmic binding protein FhuD can shuttle a variety of hydroxamate type
Fe3+-siderophores to the cytoplasmic-membrane associated proteins FhuB and FhuC.
The crystal structure of FhuD (figure 1.14) in complex with the ferrichrome analogue
gallichrome, coprogen, desferal (desferrioxamine), and albomycin have been solved
(Clarke et al., 2000; Clarke et al., 2002a). Periplasmic binding proteins consist of Nterminal and C-terminal domains connected by a linker region and a ligand binding cleft
between the two domains. FhuD has a bilobate, kidney bean shaped, structure with
approximate dimensions of 60 Å X 30 Å X 40 Å. The N-terminal domain consists of a
five stranded twisted parallel β-sheet and the C-terminal domain consists of a mixed
five stranded β-sheet. These two domains are connected by an unusual 23 amino acid
kinked α-helix between residues 142 and 165. A shallow cleft, 10 Å deep, is formed
between the N and C-terminal domains and serves as a binding cleft for the Fe3+siderophores. FhuD represents a novel class of periplasmic binding proteins owing to
the unusual nature of the kinked α-helix backbone. Periplasmic binding proteins can be
grouped into three distinct classes. Class І and ІІ are defined as having two or three
central β-strand linker domains respectively. Class І proteins have a linker domain that
26
undergoes large open and closed conformational motion in the process of ligand binding
and release. Apo and holo class ІІІ periplasmic binding proteins such as FhuD display
little change in their structure other than minor alterations in the binding cleft. The rigid
nature of this class of proteins is due to the kinked α-helix backbone. Other periplasmic
binding proteins involved in iron acquisition have been crystallised and shown to
belong to class ІІІ periplasmic binding proteins. The vitamin B12 periplasmic binding
protein BtuF and the haem periplasmic binding proteins ShuT and PhuT are also
composed of a kinked α-helix backbone (Clarke et al., 2000; Clarke et al., 2002b; Ho et
al., 2007).
The binding cleft of FhuD is lined with aromatic residues that embrace the ligand.
Hydrogen bonds tether specific parts of the various hydroxamate siderophores to side
chains in the binding cleft. The cleft is larger than that of other periplasmic binding
proteins reflecting the need to bind the large hydrophobic ornithyl linkers of the
siderophores (Clarke et al., 2002b). The disassociation constant of FhuD ranges from
0.3 to 5.4 μM for hydroxamate siderophores such as ferrichrome, coprogen, aerobactin,
ferrioxamine B, schizokinen, rhodotorulic acid and the antibiotic albomycin (Braun,
2001). Analysis of the crystal structures of FhuD bound to coprogen, desferal,
albomycin and gallichrome indicated that the protein bound specifically to the iron
centre of the siderophores. Residues Arg84 and Tyr106 in the binding cleft are
particularly important for interaction with the iron coordinating components of the
siderophore. In hydroxamate siderophores, six oxygen atoms of the hydroxamate
portion coordinate the ferric iron. Hydrogen bonds also form between FhuD residues
Asn215 and Ser219 through an intermediate water molecule. The hydrogen bonds
between the siderophore and the binding cleft provide a low enough binding affinity to
allow rapid dissociation of the ligand (Clarke et al., 2002b).
The capture of Fe3+-siderophores by periplasmic binding proteins in a passive diffusion
controlled process in the periplasmic space is improbably due to the modest KD values
for these ligands and their low abundance in the periplasm. Analysis of E. coli
periplasmic extracts failed to detect FhuD in silver stained SDS polyacrylamide gels
(Koster and Braun, 1990). Carter et al. (2006) demonstrated that TonB specifically
interacts with FhuD by phage display mapping of protein-protein interactions that
contribute to ferrichrome uptake in E. coli and by use of dynamic light scattering,
27
surface plasmon resonance and fluorescence spectroscopy techniques. Direct transfer of
Fe3+-siderophore from the lumen of the outer membrane receptor to periplasmic binding
proteins is possible. Four FhuD-TonB binding sites were identified on FhuD at loop 2
(region І), helix 2 (region ІІ), loop 8 (region ІІІ) and loop 23 (region ІV). Regions І, ІІ
and ІV overlap with the binding cleft and comprise a binding surface of 17 X 17 Å.
Fe3+-siderophore interaction with the binding cleft may not be possible when FhuD is
bound to TonB. Binding sites were found to cluster at three distinct regions on TonB,
the N-terminal domain (region І), the intermediate domain (region ІІ) and the Cterminal domain (region ІІІ). Since crystal structures are available only for the Cterminal domain of TonB (Chang et al., 2001; Kodding et al., 2005; Pawelek et al.,
2006), only region ІІІ could be mapped to the crystal structure. Region ІІІ is adjacent to
the FhuA-TonB binding residues on TonB. Interaction of FhuD with TonB occurred
with a stoichiometry of 1:1. TonB functions as a scaffold for the delivery of FhuD to the
entry point of Fe3+-siderophores to the periplasmic space (Carter et al., 2006).
Figure 1.14: Crystal Structure of FhuD. The PDB accession code for FhuD is 1K7S. The bilobate
structure of FhuD is illustrated with the single long α-helix connecting the two mixed α/β domains
(Clarke et al., 2002a; Ho et al., 2007).
Following capture of Fe3+-siderophore by FhuD, the protein shuttles its ligand directly
to the hydrophobic 70 kDa cytoplasmic membrane protein FhuB. The transmembrane
28
topology of FhuB differs from that of all known integral membrane ABC permease
transporters. However it remains the model for siderophore permeases. FhuB consists
of two halves that are similar in structure and function [FhuB(N) residues 1 to 133 and
FhuB(C) residues 334 to 660]. Each half of FhuB consists of 10 membrane spanning
regions with both the N and C termini located in the cytoplasm. Both regions are
connected by a small linker segment of peptide. Each region of FhuB is essential for its
function, either one being deleted results in a complete loss of function. The greatest
sequence variability occurs in the periplasmic loops, indicating possible recognition and
binding sites for specific hydroxamate Fe3+-siderophores (Groeger and Koster, 1998).
Physical interactions between FhuD and FhuB were detected by cross-linking
experiments. Degradation of FhuB by trypsin and proteinase K was prevented by the
addition of His-tagged-FhuD (Rohrbach et al., 1995). Protection from protein
degradation occurred even in the absence of bound ligand. The α-helix backbone of
FhuD restricts the twisting motion common in other periplasmic binding proteins. It is
not know how FhuB can distinguish between ferrichrome-bound and ferrichrome-free
FhuD. However, FhuD is thought to attach to FhuB via interaction of negatively
charged Asp and Glu on each of the lobes of FhuD and via positively charged Arg
pockets on FhuB (Krewulak et al., 2005). Studies on the interaction of FhuD with FhuB
suggest that FhuD may insert significantly far into a predicted FhuB channel. FhuD
binding sites are located on exposed periplasmic loops of FhuB in addition to
transmembrane regions of FhuB and regions exposed to the periplasm. It is thought that
FhuD may even come close to the FhuC binding site. This binding may cause FhuB to
fold in on itself, thereby creating a channel allowing the translocation of Fe3+siderophores. The close proximity and/or direct contact of FhuD with FhuC may initiate
ATP hydrolysis without a need for transmembrane signalling (Braun, 2001; Koster,
2001; Braun and Braun, 2002b; Krewulak et al., 2005).
FhuC is a cytoplasmic membrane associated ATP hydrolase and functions to energise
the transport of the Fe3+-siderophore complex into the cytoplasm. It is localised to the
inner face of the cytoplasmic membrane. Conserved regions located 90 amino acids
from the C-terminus on each half of FhuB are located in the cytoplasm. These
conserved regions seem to be important in the function of PBT systems and may be
involved in the interaction of FhuB with FhuC (Groeger and Koster, 1998). Direct
interaction of FhuB with FhuC is probable since ATP hydrolysis needs to be transmitted
29
to facilitate Fe3+-siderophore translocation. Consensus motifs termed Walker A and
Walker B have been shown to be highly conserved in FhuC. These motifs are typical of
these types of ATPases. Mutations in the consensus motifs results in the inability of
FhuC to transport ferrichrome and albomycin (Becker et al., 1990). A model for
hydroxamate siderophore utilisation is illustrated in figure 1.15.
Figure 1.15: A Model for Siderophore Utilisation. Model of the FhuA-catalysed transport of
ferrichrome, albomycin, rifamycin CGP 4832, colicin M and microcin 25 across the outer membrane of
Escherichia coli, of the FhuA-mediated infection by the phages T5, T1, (80, and UC-1, and of
ferrichrome and albomycin transport across the cytoplasmic membrane. The crystal structure of FhuA is
shown (Ferguson et al., 1998; Locher et al., 1998) in which β-sheets on the front side were removed to
better visualise the cork domain. The binding sites of ferrichrome and albomycin (Ferguson et al., 2000)
are indicated. The N-terminus of FhuA (marked N) contains the TonB box, which is thought to interact
with the Gln-160 region of TonB (marked 160) in the periplasm. The crystal structure of FhuD with
bound ferrichrome is shown (Clarke et al., 2000). The regions of FhuB that bind to FhuD, as revealed
using synthetic FhuB peptides, are marked by dots in hydrophilic loops that may fold back into the
predicted FhuB channel, and by shaded transmembrane segments. Two copies of FhuC, the ATPase,
might bind to the indicated FhuB regions, as revealed by mutation analysis (Braun et al., 1998; Braun,
2001).
30
1.4.4: Iron Release by Fe3+-Siderophore Reductase Activity
The liberation of iron from Fe3+-siderophores following internalisation by high affinity
siderophore uptake systems occurs by means of ferric iron reduction. Siderophores have
an extremely high affinity for ferric iron while the affinity for ferrous iron is relatively
weak. This property enables the release of iron bound to siderophores by means of
reduction of the bound ferric iron to its ferrous state. Assimilation of iron by this means
is advantageous for the cell due to the fact that iron is metabolically active in the cell
when in its ferrous form.
Many bacterial reductases have been identified. For the most part these are flavin
reductases where the reactions strictly require NADH or NADHP as the reductant and
therefore depend strongly on the presence of flavin reductases. Most flavin reductases
are cytosolic proteins that consist of one 10 to 40 kDa polypeptide chain. They can be
categorised into three groups according to their specificity. The first being specific for
NADH, the second for NADPH and the third for both NADH and NADPH (Fontecave
et al., 1994). Flavin reductases can therefore catalyse the transfer of electrons from
these electron donors to ferric complexes. Previously, reductases have been identified in
E. coli that facilitate the release of iron from ferric citrate, Fe3+-siderophores and
ferritins (Coves et al., 1993) and in P. aeruginosa that facilitate the release of iron from
Fe3+-pyoverdine, ferrioxamine B and E, ferricrocin, ferrichrome A and Fe3+enterobactin (Halle and Meyer, 1992). However, the function of these reductases was
dependent on the involvement of NADH and they were thus classed as flavin
reductases. Flavin reductases are not regulated by iron and can also facilitate the
reduction of substrates other than ferric iron.
The only specific bacterial ferric siderophore iron reductase genetically and
biochemically identified and characterised is FhuF of E. coli (Stojiljkovic et al., 1994;
Muller et al., 1998; Matzanke et al., 2004). The gene encoding FhuF is not located
within an operon. A putative promoter was identified upstream with a -35 and -10
region overlapping Fur box consensus sequence (GATAATGATAACCAATATC). A
λplacMu insertion in fhuF resulted in reduced growth when supplemented with
ferrioxamine B as the sole iron source. Transcription of fhuF has been shown to be
derepressed by iron limiting conditions via the iron regulator Fur and also repressed by
an oxidative response regulator, OxyR. β-lactamase-fhuF transcriptional fusions
31
indictated that FhuF was localised to the cytoplasm. β-lactamase confers ampicillin
resistance when expressed in the periplasmic space and ampicillin sensitivity when in
the cytoplasm. In addition, fractionation of the cells into outer membrane, inner
membrane and cytoplasmic fractions resulted in the identification of labelled FhuF
primarily in the cytoplasm. A portion was identified in the inner membrane fractions
suggesting that FhuF is associated with the cytoplasmic membrane to some extent. FhuF
was not detected in outer membrane protein fractions of E. coli cells.
Mutational analysis of cysteine residues indicated that FhuF contains an unusual [2Fe2S] cluster (Cys-Cys-Xaa10-C-Xaa2-C). This is in contrast to other [2Fe-2S] proteins
(Cya-Xaa2-Cya-Xaa9-15-Cys-Xaa2-Cys). Cys244, Cys245, Cys256 and Cys259 form the
[2Fe-2S] cluster in FhuF. EPR and Mossbauer spectroscopic analysis indicated that
FhuF has unusual structural properties. Sequence homology analysis also revealed that
FhuF does not show significant similarities to any known [2Fe-2S] protein. The reaction
of FhuF with ferrioxamine B (FoxB) has been proposed as follows:
[2Fe-2S]FhuF1+ + [Fe]FoxB → [2Fe-2S]FhuF2+ + [Fe2+] + FoxB3-
32
1.5: Bacterial Haem Acquisition
Bacteria are capable of utilising haem-bound iron from sources such as haemoglobin,
haemopexin, and haptoglobin. This ability is common amongst pathogenic bacteria but
it is not limited to pathogens (Noya et al., 1997). Haem represents a large iron source in
the human body but its availability is limited due to low concentrations and insolubility
in aqueous solution (Braun and Hantke, 2002). In the human body 99.9% of iron is
exclusively available intracellularly, while in serum haemoglobin is available at
concentrations ranging from 80 to 800 nM and haptoglobin is available from 5 to 20
μM. Bacteria have evolved haem assimilation systems to take advantage of these iron
sources (Wandersman and Stojiljkovic, 2000; Braun, 2001).
The term haem refers to ferrous iron protoporphyrin IX while haemin refers to the ferric
form of the molecule. However, typically, the term haem is used to indicate iron
protoporphyrin IX in either of the oxidation states (Genco and Dixon, 2001). The iron
chelated in the porphyrin ring can act as an iron source for the growth of bacteria
whereas the haem molecule itself is capable of functioning as a prosthetic group for
several proteins and acts as a cofactor to mediate oxygen transport.
Similarly to
ferrisiderophore complexes, the size of haem molecules (> 600 Da) impedes its
diffusion across the bacterial cell membranes. The structure of haem is illustrated in
figure 1.16.
Figure 1.16: Structure of Haem (Wilks and Burkhard, 2007)
1.5.1: Haem Utilisation Systems: Inner and Outer Membrane Transport
Bacterial haem assimilation systems share similarity with bacterial Fe3+-siderophore
assimilation systems in that transport is dependent on TonB-dependent outer membrane
33
haem receptors and high affinity inner membrane ABC transporters, figure 1.17.
Expression of these proteins is regulated by iron via the Fur regulatory protein. The
best-characterised bacterial haem acquisition systems involve direct binding of haem or
haem proteins to specific outer membrane receptor proteins. However as yet there are
no crystal structures for haem outer membrane receptor proteins available (Wilks and
Burkhard, 2007). The cytoplasmic proton motive force drives the translocation of these
compounds mediated via interaction of the TonB-ExbB-ExbD complex with the outer
membrane haem receptors. TonB complexes are highly conserved in all Gram-negative
bacteria and have been shown to be essential for haem transport in many species. Haem
transport systems from Y. enterocolitica (Stojiljkovic and Hantke, 1992, 1994), Yersinia
pestis (Thompson et al., 1999), V. cholerae and V. anguillarum (Stork et al., 2007),
Serratia marcescens (Izadi et al., 1997), Rhizobium leguminosarum (Wexler et al.,
2001) and Bradyrhizobium japonicum (Nienaber et al., 2001) have been identified and
characterised.
Figure 1.17: Mechanism of Haem Uptake in Gram Negative Bacteria (Wilks and Burkhard, 2007).
The first and best characterised haem transport system is encoded within the
hemPRSTUV regulon of Y. enterocolitica (Stojiljkovic and Hantke, 1992). The HemR
outer membrane protein was expressed in E. coli and endowed the organism with the
ability to utilise haem. This indicated that the HemR receptor functioned in haem
utilisation, but also indicated that E. coli itself encoded an unidentified haem specific
34
transport system. In addition, analysis of the amino acid sequence of HemR revealed the
presence of a conserved TonB box at position 16-22 of the mature protein (Stojiljkovic
and Hantke, 1992). Analysis of transcriptional hemR-phoA fusion constructs expressed
in Y. enterocolitica wild type and a Y. enterocolitica fur mutant strain indicated that the
Fur protein regulates transcription. By mutational analysis HemT was identified as a
haem specific periplasmic binding protein, HemU as a haem permease protein and
HemV as a haem ATPase. These proteins function together as a PBP-dependent
transport system. A haem degrading protein encoded by hemS was shown to be essential
for iron supply but not for haem supply. This indicated that the function of the HemS
protein was to release iron bound to haem (Stojiljkovic and Hantke, 1994).
In Y. pestis orthologues of Y. enterocolitica hemTUV were identified in a haem uptake
regulon, hmuRSTUV, and were identified as being required for the utilisation of haem
and haemoproteins as iron sources. HmuR was shown to be a TonB-dependent haem
outer membrane receptor with homology to TonB-dependent receptors of other Gramnegative bacteria. HmuTUV were identified as the ABC transporter component of the
system. Reconstitution of the hmu locus in an E. coli haem and enterobactin
biosynthesis deficient strain restored its ability to utilise haem. This transport system
was also shown to exhibit strong homology with haem transport systems of Y.
enterocolitica and Shigella dysenteriae (Thompson et al., 1999).
In R. leguminosarum a TonB protein was shown to be an absolute requirement for the
uptake of ferrisiderophore complexes and haem as iron sources. However, hmu mutants
with a fully functional TonB protein showed only slightly reduced growth when haem
was the sole iron source, indicating possible redundancy and the presence of a second
haem transport system. The TonB protein is located upstream of an operon termed
hmuPSTUV the products of which show homology to ABC haem transporters (Wexler
et al., 2001).
In Bradyrhizobium japonicum HmuR was identified as a TonB-dependent outer
membrane receptor. An inner membrane ABC haem transport system was also
identified consisting of HmuT, a periplasmic haem-binding protein, HmuU, an inner
membrane permease, and HmuV, an ATPase protein. Homologues of HmuR and
HmuTUV were shown by in silico analysis to be present in other members of the
35
rhizobia. Mutants in the HmuR, HmuTUV and an ExbBD-TonB complex showed
abolition in haem uptake, differing from the phenotype of R. leguminosarum mutants
which had implied the presence of a redundant inner membrane haem uptake system
(Nienaber et al., 2001).
1.5.2: Haemophores
Although direct binding of haem to outer membrane receptors is the major mechanism
of haem acquisition, a number of Gram-negative bacteria are capable of synthesising
and excreting haem binding proteins termed haemophores. Haemophores are highly
conserved and show little similarity to other haem-binding proteins (Wilks and
Burkhard, 2007). They are secreted via a type 1 secretion system and once associated
with haem deliver it to specific TonB-dependent haem outer membrane receptors.
Haemophores have very high affinities for haem and are thus capable of extracting
haem from haemoproteins. The S. marcescens HasA haemophore protein has been
genetically and biochemically analysed. It is a 19 kDa protein with a binding constant of
10-11 M and binds haem with a stochiometry of 1:1 (Wilks and Burkhard, 2007). The
cognate outer membrane haem receptor HasR is capable of direct binding to haem in the
presence of HasA (Letoffe et al., 1999). Homologues of this protein have been found in
P. aeruginosa and P. fluorescens (Letoffe et al., 1998; Letoffe et al., 1999; Letoffe et
al., 2000). The crystal structure of S. marcescens HasA has been solved (figure 1.18)
(Arnoux et al., 1999). Haem binds to residues His32 and Tyr75 and is held between two
loops at the interface of the α and β fold regions. Nonetheless, the precise mechanism
by which HasA extracts haem from haemoproteins remains elusive (Wandersman and
Stojiljkovic, 2000).
36
Figure 1.18: Structure of the Serratia marcescens HasA Haemophore. (a) ribbon diagram with helices
coloured in red and strands in blue. The ligands of the haem and the haem are shown in ball-and-stick
representation. The surface occupied by the haem is transparent. (b) same representation as (a) but rotated
by 90o. (Arnoux et al., 1999)
Another haemophore mechanism by which bacteria acquire haem is exemplified by
Haemophilus influenzae (Cope et al., 1995; Cope et al., 1998). HuxA (a 100 kDa
protein) is secreted by a signal peptide sequence involving a helper outer membrane
protein. HuxA has a strong affinity for serum haemopexin and once in complex, this is
shuttled to a specific outer membrane receptor protein (HuxB) for internalisation. HuxA
does not show amino acid homology with the HasA haemophore of S. marcescens.
1.5.3: Haem Oxygenase Facilitated Iron Release
The final step in haem utilisation for many bacteria is the delivery of haem to a haem
oxygenase. Homologues of human haem oxygenases have been identified in many
bacterial species. Oxidative cleavage disrupts the haem molecule, liberates ferrous iron
and produces carbon monoxide and biliverdin as by-products. Iron regulated haem
oxygenases have been identified in P. aeruginosa by screening for iron regulated genes
and by gene knockout mutants in Corynebacterium diphtheriae (hmuO) and Neisseriae
meningitidis (hemO), figure 1.19. Indeed, novel haem oxygenases have been identified
that share little homology to the classical enzymes. Complementation of a C. ulcerans
haem oxygenase deficient strain resulted in the identification of isdG and isdI of
Staphlylococcus aureus (figure 1.19) and isdG of Bacillus anthracis as novel haem
oxygenases (Wandersman and Delepelaire, 2004; Wilks and Burkhard, 2007).
37
Figure 1.19: Overall Structure of Haem Oxygenase (A) HemO from N. meningitides and (B) IsdG
from S. aureus (Wilks and Burkhard, 2007)
1.5.4: Haem Transport via Dipeptide Transport Systems
As alluded to previously, many bacteria harbour homologues of the Y. enterocolitica
hemTUV haem specific PBP-dependent transport system. Insertional mutagenesis of
these homologues in many species commonly results in a reduction in the haem uptake
ability of the organism but not abolition. This suggests that a redundant system may be
in existence in these species. In addition to these findings, certain bacterial species can
possess genes coding for outer membrane haem/haemophore receptors and utilise haem
as an iron source without any homologues of hemTUV in existence in their genomes.
Neisseria and Haemophilus species display these characteristics. Indeed, expression of
the Y. enterocolitica HemR outer membrane haem specific receptor in an E. coli host
endowed the organism with haem utilisation ability. This is despite the fact that E. coli
does not possess homologues of hemTUV.
Létoffé et al., (2006) demonstrated that an E. coli dipeptide transport system,
DppBCDF, functioned as the inner membrane permease complex for haem transport.
Indeed this system functioned in conjunction with the dipeptide periplasmic binding
protein DppA or the L-alanyl--D-glutamyl-meso-diaminopimelate periplasmic binding
protein MppA to mediate utilisation of haem and its precursor -aminolevulinic acid.
38
The dipeptide Ala-Ala competes with haem for binding to the periplasmic binding
protein DppA, while the tripeptide Pro-Phe-Lys competes with haem for the binding to
the periplasmic binding protein MppA. Both binding proteins subsequently shuttle their
substrate to the inner membrane transporters DppBCDF. Simultaneous competition of
both peptides against haem results in inhibition of haem utilisation by the Dpp permease
complex. The Dpp permease in E. coli, as in other Gram-negative bacteria, constitutes a
periplasmic binding protein dependent transport (PBT) system but uses two optional
binding proteins. E. coli therefore contains a heam/peptide inner membrane permease
despite being unable to utilse exogenous haem. It seems likely that haem transport by
this means is involved in endogenous haem recycling.
More recently, Létoffé et al., (2008) suggested that the HemTUV system of S.
marcescens forms a class of haem specific permeases on the basis of their restricted
substrate specificity as determined by comparison with the Dpp system of E. coli.
Expression of the S. marcescens hemTUV and hasR genes in E. coli FB827dppF::Km
restored haem utilisation. Inhibition assays demonstrated that peptides do not inhibit
haem utilisation by HemTUV and furthermore, competition assays demonstrated that
peptides do not compete with haem for binding to HemT. This suggests that haem
periplasmic binding proteins belong to two classes. One being composed of binding
proteins like DppA and its homologues which have broad substrate specificity and
another composed of binding proteins like HemT and its homologues which are highly
specific for haem.
The identification and characterisation of the Dpp system of E. coli may explain the
redundancy observed in mutants of hemTUV and their homologues in other species,
such as those observed in Y. enterocolitica (Stojiljkovic and Hantke, 1992, 1994), Y.
pestis (Thompson et al., 1999) and B. japonicum (Nienaber et al., 2001).
39
1.6: Iron-Regulated Gene Expression
Free cellular ferrous iron is potentially toxic due to its propensity to oxidise under
aerobic conditions. The hydroxyl radicals formed due to the Fenton reaction damage
essential cellular components. This coupled with extreme changes in iron availability,
due to dramatic environmental changes such as a switch from free-living to a symbiotic
or infectious state, necessitates regulation of supply. Bacteria have therefore developed
universal regulatory systems to control ferrous iron availability. Iron-responsive
regulators mediate this control.
1.6.1: Fur
The ferric uptake regulator (Fur) of E. coli is an iron-sensing repressor protein that
regulates expression of genes involved in iron acquisition. A working model for Fur
proposes that one ferrous iron atom per monomer coordinates with a 19 bp inverted
repeat DNA sequence termed the “Fur Box” (GATAATGAT[A/T]ATCATTATC)
which is located within the promoter region of iron regulated genes (Lee and Helmann,
2007). The N-terminus of Fur contains a helix-turn-helix motif which is needed for
binding to the Fur Box (Hantke, 2002). The affinity of Fur for ferrous iron enables it to
act as a sensor of intracellular iron levels whereby it allows the uptake of iron to a
sufficient level to maintain cellular growth and yet can repress further uptake once these
levels are exceeded. The binding of Fur to the Fur Boxes sterically blocks RNA
polymerase from transcribing the iron regulated genes.
The C-terminus of Fur is the dimerisation domain and contains a histidine cluster and
four cystine residues. Mutagenesis of the cystine residues indicated that those residues
located in the C92XXC95 motif were essential for the binding of Zn2+ while those
residues located in the C132XXXXC137 motif were dispensable. Zn2+ ions have been
shown to be tightly bound to Fur proteins and enable correct folding of the protein (Lee
and Helmann, 2007). The crystal structure of P. aeruginosa Fur has been determined
(figure 1.20) (Pohl et al., 2003). The DNA-binding domain consists of four α-helices
and a two-stranded antiparallel β-sheets. Helix four is thought to be the DNA-binding
helix. The dimerisation domain consists of three antiparallel β-sheets covering an αhelix. Both domains are necessary to produce a functional protein. The structure also
identifies two metal-binding sites. One of these sites (site one) coordinates the Zn2+ ion
40
via the side chains of His86, Asp88, Glu107, His124 and by a water molecule. The
other metal-binding site (site two) connects the DNA binding domain to the
dimerisation domain and is composed of the side chains His32, Glu80, His89 and
Glu100. Site one has been shown to be capable of exchanging the Zn2+ ion for Fe2+ and
has thus been assigned as the regulatory metal binding site (Lee and Helmann, 2007).
Fur regulators have been identified by homology searches in both Gram negative and
Gram positive bacterial species. These proteins are likely to act as metal-dependent
DNA-binding repressors; however within this Fur superfamily a considerable amount of
variability in metal specificity exists. Fur homologues have been shown to respond to
other metals such as zinc, manganese and nickel. Furthermore, in the Rhizobiales the
gene with the most homology to Fur is in actuality a manganese responsive repressor. In
these species the predominant iron responsive repressor is RirA, a member of the Rrf2
protein family (Johnston et al., 2007).
Figure 1.20: Crystal Structure of P. aeruginosa Fur. Ribbon diagram of the crystal structure of the P.
aeruginosa Fur dimer with secondary structure elements annotated. The view shown is approximately
perpendicular to the crystallographic twofold axis. The DNA-binding domains are depicted in blue and
the dimerisation domain is green (Pohl et al., 2003).
1.6.2: RirA
The Rhizobial iron regulator (RirA) was first identified in R. leguminosarum bv. viciae
(Todd et al., 2002; Wexler et al., 2003) and subsequently shown to be the global iron
regulator in S. meliloti 2011 (Chao et al., 2005; Viguier et al., 2005). RirA displays little
41
sequence similarity to Fur and has been shown to be a member of a protein family
called Rrf2. Only three members of the Rrf2 family of metallo-regulators Rrf2, NsrR
and IscR have been studied in detail. Rrf2 regulates the genes coding for a cytochrome
in Desulfovibrio, NsrR regulates genes involved in nitrogen oxide metabolism in
Nitrosomonas, E. coli and other proteobacteria, while IscR regulates the expression of
genes involved in the assembly of FeS clusters in proteins (Johnston et al., 2007).
Instead of recognising and binding to Fur boxes, RirA responds to regions in the
promoters of RirA-repressed genes termed IRO boxes. IRO motifs are conserved across
many species in the α-proteobacteria. RirA-regulated genes have been identified in
Agrobacterium, Brucella, Bartonella, Mesorhizobium, Rhizobium and Sinorhizobium. In
these species RirA is the global iron regulator however, it has been shown to be capable
of exerting a more parochial effect. In R. leguminosarum RirA represses rpoІ, which
codes for a σ factor that transcribes some of the vicibactin siderophore biosynthesis
genes (Todd et al., 2002). Also, in S. meliloti 2011 RirA has been shown to repress
rhrA, which codes for an AraC-type regulator of the rhizobactin 1021 biosynthesis
genes (Viguier et al., 2005). These regulatory cascades which RirA is involved in
demonstrate that in addition to having a global effect on the iron status of the cell, RirA
can have a more accurate and local effect on the cells iron status.
1.6.3: Mur
Fur homologues in Rhizobium and Sinorhizobium have been studied and shown to act as
transcriptional regulators in response to manganese instead of iron. The sitABCD operon
codes for ABC-type Mn2+ transporters. Under conditions of high manganese this operon
is repressed by a Fur homologue which was renamed Mur to indicate manganese uptake
regulator. Manganese responsive motifs have been found in the promoter regions of
genes involved in manganese uptake. These motifs are different from the Fur Boxes
from E. coli. However it has been shown that Mur can use Fe2+, Co2+ or Zn2+ as active
metals and once activated Mur can bind to manganese responsive motifs and Fur Boxes.
So, the DNA-binding specificity of Mur may in fact overlap with that of Fur (Johnston
et al., 2007; Lee and Helmann, 2007).
42
1.6.4: Irr
Another iron responsive regulator termed Irr was first identified in B. japonicum but is
also present in most α-proteobacteria including Rhizobium and Brucella. In B.
japonicum Irr regulates the gene (hemB) coding for δ-aminolevulanic acid dehydratase,
a haem biosynthesis enzyme, iron transport genes and genes involved in iron
metabolism. Irr functions only in iron limited conditions and the mechanism by which
Irr functions is different to other members of the Fur superfamily: an enzyme called
ferrochelatase delivers haem to Irr which then forms a complex and results in an
increase in the haem biosynthesis enzyme. Irr is subsequently destroyed by proteolitic
degradation (Yang et al., 2006; Johnston et al., 2007; Lee and Helmann, 2007).
Conserved motifs termed ICE motifs (Iron-Control Elements) are the location of Irr
DNA-binding in the promoters of regulated genes. In B. japonicum Irr has been shown
to recognise ICE motifs which are close to or overlap the promoter regions and acts as a
repressor of these genes. However, Irr can act as an activator of genes depending on the
proximity of the ICE motif to the promoter region which would cause up-regulation of
genes in iron deplete cells (Johnston et al., 2007).
B. japonicum does not encode a homologue of RirA but does encode a Fur homologue
in its genome. Fur homologues are not found in all rhizobial species and when present
are commonly found to mediate manganese-dependent control of manganese associated
genes. These Fur homologues are termed Mur. However, B. japonicum Fur has been
shown to be capable of complementing an E. coli fur mutant. The Fur box which B.
japonicum Fur binds to is not identical to that of E. coli. In addition to this, residues in
the dimerisation domain that are involved in metal binding by Fur of P. aeruginosa are
not essential for DNA binding or repression activity (Yang et al., 2006). Dispite these
differences to the model Fur proteins, B. japonicum Fur is capable of iron-dependent
gene regulation.
1.6.5: AraC-type Transcriptional Regulators
The expression of many genes involved in iron acquisition has been shown to be
regulated by AraC-type regulators. The model for AraC regulation is the arabinoseinduced binding of the AraC protein in E. coli to araI2 which activates the expression of
the araBAD operon (Lee et al., 1987). Homologues of the E. coli AraC are present in
43
many species and are associated with the regulation of many genes involved in iron
acquisition. In P. aeruginosa a 31 kDa AraC-type transcriptional regulator, PchR, has
been identified and characterised (Heinrichs and Poole, 1996). This regulator is required
for the production of the FptA outer membrane receptor in response to iron limitation
and in response to the presence of its cognate siderophore pyochelin. Fur-mediated
repression of iron acquisition genes is abated during conditions of iron limitation.
However, full expression of iron acquisition genes often requires the presence of the
cognate siderophore (Michel et al., 2007). PchR deficient strains resulted in an inability
to produce FptA under both iron limited conditions and in the presence of pyochelin.
This indicated that the PchR AraC-type transcriptional regulator mediated the
expression of FptA. β-Galactosidase assays on P. aeruginosa strains harbouring lacZ
transcriptional fusion constructs indicated that activation of fptA by PchR was however
dependent on the presence of pyochelin. More recently Michel et al., (2007)
demonstrated that pyochelin signaling also functions through PchR to activate the
expression of the two pyochelin biosynthesis operons pchDCBA and pchEFGHI in
addition to the fptA gene. In Y. pestis an AraC-type homologue encoded by ybtA directly
upstream of the ybtSXQP cluster is thought to use the siderophore yersiniabactin as an
inducer to regulate the expression of the YbtA receptor and biosynthesis genes
(Fetherston et al., 1996; Fetherston et al., 1999). In S. meliloti 2011 an AraC-type
transcriptional regulator termed RhrA has been identified and characterised (Lynch et
al., 2001; Viguier et al., 2005), which functions to regulate the transcription of
rhizobactin 1021 biosynthesis and transport, discussed in detail in section 1.8.1.
1.6.6: Two-Component Signal Transduction
Bacteria have evolved sophisticated signaling systems termed “two-component”
systems that allow them to respond to environmental stimuli. The model system consists
of a histidine protein kinase, containing a conserved kinase core, and a response
regulator protein, containing a conserved regulatory domain. The environmental
stimulus is detected by the histidine kinase which subsequently becomes altered by this
interaction. The histidine kinase donates a phosphoryl group to the response regulator
and results in the activation of a downstream effector domain that elicits the bacterium’s
response. In E. coli 30 histidine kinases and 32 response regulators have been identified.
These two-component signaling systems have been adapted and integrated into a wide
variety of cellular signaling cascades (Stock et al., 2000).
44
Proteins of the histidine kinase family range in size from 40 to 200 kDa and contain a
diverse sensing domain and a highly conserved kinase core. The kinase core is
approximately 350 amino acids in length and contains a conserved five amino acid
motif termed the ‘H box’. The sensing domain is located at the N-terminus of the
protein and possesses little sequence homology. Thus, ligand-stimuli interactions are
detected by the sensing domain and cause an ATP-dependent autophosphorylation of a
conserved His residue in the H box of the kinase core. A phosphoryl group is then
transferred to a conserved Asp residue in the response regulators regulatory domain.
Most response regulators are transcription factors and they consist of two domains, an
N-terminal regulatory domain and a C-terminal DNA-binding effector domain. The
response regulator normally functions to activate and/or repress transcription of target
genes (Stock et al., 2000).
In E. coli the transport of ferric citrate is regulated by a novel signal transduction
mechanism which can be considered an adaptation of the prototypical histidine kinase
two-component signaling system. Ferric citrate is transported through the outer
membrane by the TonB-dependent FecA receptor protein and then passed to the
periplasmic binding protein FecB. Transport proceeds to the cytoplasmic membrane
where an ATP-binding cassette transport system FecCDE facilitates translocation to the
cytoplasm. In addition to its transport function, FecA can independently function as a
signaling protein, as illustrated in figure 1.21 (Braun et al., 2006). Binding of the
inducer ferric citrate causes a signaling cascade from FecA at the outer membrane to the
cytoplasm where gene transcription is regulated.
45
Figure 1.21: The Ferric Citrate Transport and Regulatory Systems. The signaling pathway from
FecA to FecI; the involvement of TonB, ExbB and ExbD in signaling and transport; and transport of iron
through the periplasmic FecB protein and the ABC transporter FecCDE proteins are shown. Fe 2+-loaded
Fur repressor binds to the promoter upstream of fecI and fecA and dissociates from the promoter under
low iron conditions. Interactions between the FecA TonB box and TonB and between the FecA signaling
domain and FecR are indicated. N indicates the N-terminal end, C the C-terminal end of the proteins. σ2
and σ4 indicate FecI domains involved in binding to FecR and DNA, respectively (Braun et al., 2006).
Located N-terminally to the TonB box of the mature FecA protein is a 79 amino acid
peptide that functions as the signaling domain. Deletion of this domain retains ferric
citrate transport but abolishes the induction of fec gene transcription. Crystal structures
of FecA (Braun and Braun, 2002a; Chakraborty et al., 2006) do not reveal the signaling
domain indicating that the structure may be flexible. Upon binding of ferric citrate,
loops 7 and 8 move by 11 and 15 Å respectively however citrate binding does not elicit
this response. These loops have been shown to be essential for ferric citrate transport
and their movement causes the protein gate to close (Braun et al., 2006).
46
The C-terminal region of the cytoplasmic transmembrane protein FecR interacts with
the signaling domain of the FecA outer membrane protein. This interaction was
revealed in vitro by binding of FecA to FecR which had been bound to a Ni-agarose
column via a recombinant N-terminal His10 tag. Removal of the FecA signaling domain
resulted in the protein losing the ability to interact with bound FecR. In addition, in vivo
binding studies utilising a bacterial two-hybrid system identified residues 237 to 317 at
the C-terminal region of FecR as being sufficient for binding to the FecA signaling
domain (Enz et al., 2000).
The signal cascade is delivered to the cytoplasm by binding of the N-terminal region of
FecR to an extracytoplasmic sigma factor (ECF) termed FecI. Sigma factors function by
enabling specific binding of the RNA polymerase to the -10 and -35 promoter regions.
Bacterial two-hybrid binding assays identified interactions between residues 1 to 85 of
FecR and 1 to 173 of FecI as forming a direct interaction. In addition, Ni-agarose
column binding assays complemented this finding (Enz et al., 2000).
The sophistication of this signal transduction system is brought to light when fecIR
regulation is understood. The fecIR genes are not induced by ferric citrate but rather are
repressed by Fe2+ Fur. Therefore under conditions of iron limitation Fur repression
ceases and the fecIR genes are transcribed. Similarly, fecABCDE are regulated by Fe2+
Fur and are thus only transcribed under iron limiting conditions. The FecIR proteins
remain inactive until a signal from FecA indicates the availability of ferric citrate. FecI
enhances transcriptional activation by recruiting RNA polymerase and directing it to
transcription of fecABCDE. The bacterium therefore first recognises the internal iron
starvation and will only synthesise the Fec system when ferric citrate becomes available
(Braun et al., 2006).
47
1.7: Rhizobium-Legume Symbiosis
The rhizobia are a group of bacteria that are capable of forming a nitrogen fixing
symbiotic relationship with legumes. These bacteria must change from a free-living
state and enter the cells of the host plant where they differentiate into nitrogen-fixing
bacteriods. Iron is a critical requirement for the symbiosis between the rhizobia and
their host plants. Proteins involved in the nitrogen fixation process such as nitrogenase
use iron as a key co-factor. This, in addition to the presence of enormous quantities of
iron proteins such as leghaemoglobin underlies the importance of this element within
the nodule. While the plant benefits from fixed nitrogen in the form of ammonium
provided by the bacteriod, the bacteriods in return receive sugars for respiration in the
form of C-4 dicarboxylates such as succinate, fumarate and malate.
Rhizobia often have large and multipartite genomes reflecting the extensive genetic
information necessary to maintain a free-living and symbiotic state. The tripartite
genome of S. meliloti harbours two symbiotic megaplasmids with a combined size of
3.1 Mb. The pSyma megaplasmid contains genes coding for nitrogen fixation (fix) and
nodule formation (nod), while the pSymb megaplasmid contains genes coding for
exopolysaccharide (EPS) biosynthesis and dicarboxylate transport and utilisation. For a
successful symbiotic infection the plant/bacterium signalling and gene induction
choreography must be perfect. Furthermore, the signals determine which plant and
bacterium will engage in symbiosis. S. meliloti forms nodules on the plant species in the
genera Medicago, Melilotus and Trigonella.
Plant signalling molecules such as flavonoids and betaines act as chemoattractants to
draw the rhizobia to the plant roots and induce bacterial gene expression. Many of the
expressed bacterial genes encode lipochitooligosaccharide response signalling
molecules called Nod factors. These are members of the LysR family of transcriptional
activators and act as plant morphogens by stimulating plant root meristem growth that
will eventually form the root nodule (figure 1.22). The Nod factors affect the epidermal
cells by depolarising the plasma membrane, calcium spiking and deformation of polar
tip growth in root hairs. The inner cortical cells are affected by the induction of mitotic
activation which leads to the development of the nodule primordium. S. meliloti
contains three genes encoding Nod factors nodABC and in addition it carries three
48
homologues of regulatory genes, nodD1, nodD2 and nodD3. The plant flavonoid
inducer luteolin targets the activation of these nod genes in S. meliloti.
Figure 1.22: Early Signal Exchange between Alfalfa (M. sativa) and S. meliloti (Barnett and Fisher
2006).
Nod factors cause curling of the epidermal root hairs to which the bacteria are attached
and subsequently become snared in the curling root hairs. Here the bacteria multiply and
infect the outer cells of the root hair. An infection thread is synthesised from plant tissue
and harbours the invading bacteria. Plant plasma membrane extends and surrounds the
infection thread which extends inwards to the root cortex cells. The bacteria are released
from the tip of the infection thread and enter the cytoplasm of plant cells which is then
termed the symbiosome (figure 1.23). The bacteria then differentiate to their nitrogenfixing bacteriod form. Bacteriods appear larger and more club shaped. Nitrogenase, the
enzyme that catalyses the conversion of dinitrogen to ammonium functions under
microoxic conditions. These microoxic conditions are provided by the action of the
oxygen sequestering plant protein, leghaemoglobin. Indeed, the genes required for
nitrogen fixation, nif and fix, are induced under low oxygen conditions.
49
Figure 1.23: The Infection Thread Leading to the Development of the Symbiosome (Barnett and
Fisher 2006).
The transformation to a nitrogen-fixing state is an extreme change for the invading
bacterium. Bacteriod development leads to a reduction in metabolism as the genes
required for microoxic respiration are induced. The FixL/FixJ two-component
regulatory system of S. meliloti senses the microoxic conditions and responds by
inducing the genes required for microoxic respiration. Interestingly there are more genes
repressed in bacteriods than induced, however genes required for DNA repair are
induced in bacteriods. This would imply that maintenance of the prokaryotic genome is
still a priority at this stage.
50
1.8: Iron Acquisition in The Rhizobia
The α-proteobacteria is a taxonomic group of bacteria which includes a number of
important genera, including some which form a relationship with higher eukaryotes in
either a symbiotic or pathogenic lifestyle. Rhizobia are members of the α-proteobacteria
and can be found free-living in the soil or in a symbiotic nitrogen-fixing relationship
with a specific leguminous host. Other members of the α-proteobacteria include the
plant pathogen Agrobacterium and mammalian pathogens Brucella and Bartonella.
Within the Rhizobials, iron acquisition has been studied in greatest detail in three
species, namely S. meliloti, R. leguminosarum and B. japonicum.
1.8.1: Sinorhizobium meliloti
S. meliloti is a gram negative soil microorganism that can be found free-living in the soil
or in symbiosis with specific legumes. It can form nitrogen fixing root nodules on
Medicago, Melilotus and Trigonella. The genome of S. meliloti 1021 consists of three
replicons, a 3.7 Mb chromosome, and 1.4 Mb and 1.7 Mb megaplasmids called pSyma
and pSymb. The entire genome of S. meliloti 1021 has been sequenced (Barnett et al.,
2001; Finan et al., 2001; Galibert et al., 2001).
Rhizobactin 1021 is a citrate based hydroxamate siderophore produced by S. meliloti
2011, the parent of the sequenced strain S. meliloti 1021. This siderophore is structurally
similar to other citrate based hydroxamate siderophores, namely schizokinen, aerobactin
and arthrobactin. Rhizobactin 1021 is distinguished by the presence of a lipid tail, (E)-2decenoic acid residue (Persmark et al, 1993), figure 1.5. The genes involved in the
biosynthesis of this siderophore have been identified as rhbABCDEF and are arranged
in an operon located on the pSyma megaplasmid. A 72 kDa iron-regulated outer
membrane receptor protein, termed RhtA, is encoded downstream of the rhbABCDEF
operon. RhtA displays 61 % sequence similarity with the aerobactin receptor IutA of E.
coli and mutagenesis of the gene encoding RhtA indicated that the receptor was
necessary for transport of the rhizobactin 1021 siderophore. RT-PCR analysis of RNA
isolated from mature alfalfa nodules did not yield products for rhtA or rhbF despite nifH
being strongly expressed. This indicated that the biosynthesis and transport genes for
rhizobactin 1021 were not strongly expressed in bacteriods when the nitrogenase genes
are expressed (Lynch et al., 2001).
51
A novel single unit siderophore transporter termed RhtX was identified in S. meliloti
2011 which functioned to facilitate transport of rhizobactin 1021 and schizokinen across
the cytoplasmic membrane. The gene rhtX encoding the permease was located at the
beginning of the operon extending through to the rhizobactin 1021 biosynthetic genes.
An E. coli mutant carrying a polar Tn10 insertion in fhuC and expressing the iutA gene
was used to heterologously express rhtX. RhtX is capable of substituting for FhuCDB in
the transport of rhizobactin 1021 and schizokinen but not aerobactin. This indicated that
the lipid moiety of rhizobactin 1021 is not involved in utilisation by means of RhtX.
Schizokinen and rhizobactin 1021 are structurally identical with the exception of the
lipid moiety, in contrast to aerobactin which displays structural differences (Ó Cuív et
al., 2004).
An AraC-type transcriptional activator is encoded between the rhbABCDEF operon and
rhtA and is transcribed in the opposite orientation. Mutagenesis studies and RNase
protection assays demonstrated that RhrA functioned to activate expression of the
rhizobactin 1021 biosynthesis and transport genes (Lynch et al., 2001). Transcripts of
rhbABCDEF and rhtA were not detected in an rhrA mutant culture grown under iron
limiting conditions, confirming the role of RhrA as a transcriptional activator of the
biosynthesis and rhtA genes. An iron-responsive regulator termed RirA was identified
by in silico analysis and mutagenesis of S. meliloti (Viguier et al., 2005). Real-time RT
PCR analysis was used to assess gene expression in a rirA mutant compared to wild
type. In addition to their regulation by iron, the expression of rhtA and the rhbABCDF
operon was determined to be regulated by RirA. Iron nutrition bioassay analysis
confirmed that rhizobactin 1021 production was occurring in the presence of iron in a
rirA mutant. RirA had also been shown to repress rhrA, the gene coding for the AraCtype regulator of the rhizobactin 1021 biosynthesis genes. Furthermore, the ShmR
(smc02726) receptor was shown by real-time RT PCR analysis to be regulated by RirA.
This indicated that RirA functioned independently from RhrA as a global iron
responsive regulator. Mutations in the rhizobactin 1021 regulon in these studies did not
affect symbiotic nitrogen fixation.
1.8.2: Rhizobium leguminosarum
R. leguminosarum has biovars that form nitrogen fixing nodules on peas, clovers and
various beans. R. leguminosarum biovar viciae 8401pRL1JI nodulates peas and
52
produces a cyclic trihydroxamate type siderophore called vicibactin. Vicibactin
comprises three moieties each of D-3-hydroxybutyric acid and N2-acetyl-N2-hydroxy-Dornithine linked by alternating amide and ester bonds (Carter et al., 2002).
R.
leguminosarum biovar viciae contains a sequenced 7.75 Mb genome which is composed
of a chromosome and six large plasmids.
A fhu system homologous to that of E. coli is encoded on the genome of R.
leguminosarum biovar viciae 3841. The genes, fhuDBC, were found to be involved in
the transport of vicibactin across the inner membrane (Stevens et al., 1999). A gene
coding for a TonB homologue was found to be essential for the transportation of
vicibactin and haem compounds (Wexler et al., 2001). A mutant in fhuA of R.
leguminosarum biovar viciae 3841 was found to be incapable of vicibactin transport. A
fhuA::gus fusion demonstrated that this receptor was expressed only in the meristematic
zone of pea nodules and not in the mature bacteriods where nitrogen fixation occurs
(Yeoman et al., 2000).
The biosynthesis of vicibactin is orchestrated by a cluster of eight genes, vbsGSO,
vbsADL, vbsC and vbsP. An ECF σ factor of RNA polymerase, rpoІ, was required for
the transcription of vbsGSO and vbsADL but not vbsC and vbsP. Fixation assays on pea
plants demonstrated that these vicibactin biosynthesis genes were not essential for
symbiotic nitrogen fixation (Carter et al., 2002).
1.8.3: Bradyrhizobium japonicum
B. japonicum is the nitrogen-fixing endosymbiont of soybean. B. japonicum 61A152
secretes citric acid as a siderophore under iron deplete conditions. It is also capable of
utilising xenosiderophores including rhodoturulate and ferrichrome as well as utilising
haem compounds. A gene, fegA, coding for an 80 kDa iron-regulated TonB-dependent
outer membrane receptor was identified (LeVier and Guerinot, 1996). A haem uptake
system was also identified and shown to consist of nine genes. The products of these
genes includes a TonB-dependent outer membrane haem receptor, inner membrane
ABC transporters composed of HmuT, a periplasmic binding protein, HmuU, a
permease and HmuV, and ATPase. Also, homologues of TonB and ExbBD were
identified. HmuR and HmuTUV were shown to function together in haem utilisation,
although redundancy at the inner membrane was present. Mutations in the TonB53
ExbBD system demonstrated that these were essential for haem utilisation but not for
siderophore utilisation. This indicated that there must be a second energy transducing
system present. Also, all mutants were shown to form effective nodules on their host
plant and were capable of nitrogen fixation (Nienaber et al., 2001).
54
1.9: Siderophore Mediated Iron Acquisition in Pseudomonas aeruginosa
P. aeruginosa is a Gram-negative opportunistic human pathogen that can cause severe
and often fatal infections that are a particular risk for immuno-compromised patients.
Iron is acquired by this organism by the production and utilisation of two endogenous
siderophores termed pyoverdine (Cornelis and Matthijs, 2002) and pyochelin (Poole and
McKay, 2003), the utilisation of numerous xenosiderophores ( Poole and McKay, 2003;
Ó Cuív et al., 2007) and the utilisation of host haemoproteins (Ochsner et al., 2000). P.
aeruginosa encodes a multitude of ferrisiderophore outer membrane receptors (up to 34
putative TonB-dependent receptors), yet its genome sequence exhibits few periplasmic
and cytoplasmic ferrisiderophore transport system components (Koster, 2001; Poole and
McKay, 2003). The production of endogenous siderophores by P. aeruginosa has been
shown to contribute to the virulence of the organism. Furthermore, the presence of
siderophores has been shown to regulate the production of other virulence factors in
Pseudomonas (Buckling et al., 2007).
1.9.1: Pyoverdine Utilisation
Pyoverdine is the primary siderophore produced by P. aeruginosa in response to iron
limitation. The molecule is composed of a conserved dihydroyquinoline chromophore, a
variable peptide chain and a dicarboxylic acid or a dicarboxylic acid amide side chain
(Cornelis and Matthijs, 2002). The fluorescence of pyoverdine is a characteristic that is
conferred upon the molecule by the dihyroxyquinoline chromophore. The pvd locus on
the P. aeruginosa chromosome encodes the genes required for the biosynthesis of
pyoverdine (Tsuda et al., 1995). The genes required for the synthesis of the
dihyroxyquinoline chromophore have been identified in the pvcABCD operon (Stintzi et
al., 1996; Stintzi et al., 1999).
The transport of pyoverdine across the outer membrane is facilitated by the 90 kDa
receptor FpvA. Recently, Wirth et al (2007) solved the crystal structure of FpvA bound
to ferripyoverdine at a 2.7Å resolution (figure 1.24). With similarity to the ferric
dicitrate receptor, FecA of E. coli (section 1.6.6), an N-terminal sequence that is
involved in cell surface signalling was identified in FpvA between residues Asn44 and
Ala118. NMR spectroscopy of this region resulted in the identification of a novel
protein fold involved in ECF signalling (Garcia-Herrero and Vogel, 2005). Many
55
putative signalling systems appear to be present in P. aeruginosa, most of which have a
role in metal uptake and with the putative ECF sigma factors PA0149, PA1912,
PA2050, PA2093 and PA4896 having a predicted role in iron acquisition (Llamas et al.,
2008). The binding of ferripyoverdine to FpvA initiates a signal transduction cascade to
the cytoplasmic membrane spanning antisigmafactor FpvR. The signal can then
transduce to PvdS, whose activity is regulated by FpvR, which releases the sigma factor
PvdS to subsequently stimulate the expression of the pvd biosynthesis genes and also
the virulence factors exotoxin A and PrpL (Lamont et al., 2002). Ferripyoverdine
binding to FpvA also stimulates the expression of fpvA through the involvement of FpvI
and FpvR (Redly and Poole, 2003).
Figure 1.24: The FpvA-Pvd-Fe Crystal Structure. Signalling domain (residues 44 to 118) in blue, the
plug (residues 139 to 276) in red and the β-barrel (residues 277 to 815) in green (Wirth et al., 2007).
1.9.2: Pyochelin Utilisation
Pyochelin binds ferric iron with a stoichiometry of 2:1 and has a lower affinity for ferric
iron than that of pyoverdine (Cox, 1980; Poole and McKay, 2003). The siderophore is
nevertheless capable of effectively promoting iron acquisition in P. aeruginosa and
interestingly also has the capability of binding other transition metals. Zn(II), Cu(II),
Co(II), Mo(VI) and Ni(II) can be bound with appreciable affinity, while pyochelin
bound Co(II) and Mo(IV) have been shown to be transported by P. aeruginosa (Visca et
al., 1992). The pchDCBA and pchEF genes coding for the biosynthesis of pyochelin and
its precursors salicylate and dihydroaeruginoate are localised on the chromosome of P.
56
aeruginosa clustered with the pchR regulatory gene (section 1.6.5). Three additional
genes, pchGHI, are found downstream and adjacent to pchEF and are thought to form
an operon. PchG has been attributed a siderophore reductase function while PchH and
PchI are predicted to have an ATP binding cassette export function (Crosa and Walsh,
2002).
Transport of ferripyochelin across the outer membrane is mediated by the 75 kDa
receptor FptA, whose corresponding gene is located directly downstream of the
pchEFGHI operon, and is transcribed from a separate promoter (Heinrichs and Poole,
1996). The activity and regulation of FptA has been described in section 1.6.6. The
crystal structure of the FptA receptor bound to ferripyochelin has been solved at a 2.0 Å
resolution (Cobessi et al., 2005b). This was the first report of a structure for pyochelin
also and indicated that the molecule provides a tetra-dentate coordination for iron.
Figure 1.25 illustrates a two-dimensional topology representation of FptA and indicates
the residues involved in interaction with ferripyochelin.
Figure 1.25: Two-Dimensional Topology of FptA. Squares represent residues in the β-strands and
circles represent residues in the loops. The residues involved in interaction with ferripyochelin are shaded
57
in red. Residues Glu524 and Glu578 involved in conserved interaction between the plug domain and the
β-barrel domain are highlighted in green (Cobessi et al., 2005b).
Ó Cuív et al. (2004) identified and characterised the first inner membrane siderophore
transporter in P. aeruginosa. FptX, a novel single unit siderophore transporter, functions
to facilitate the transport of ferripyochelin across the inner membrane. This MFS type
transporter was initially identified based on sequence homology to the RhtX
siderophore transporter of S. meliloti. Insertional mutagenesis of fptX in a pyoverdine
biosynthesis defective P. aeruginosa strain resulted in an abrogation in ferripyochelin
utilisation. Recently, Michel et al. (2007) demonstrated that fptX is encoded with an
operon composed of four genes, fptABCX, transcribed from the fptA promoter. Thus, the
genes are PchR and Fur dependent. The fptA and fptX genes were found to be necessary
for ferripyochelin utilisation, signalling and pyochelin production. Conversely fptB and
fptC were not necessary for these functions. Pyochelin mediated signalling requires the
function of both FptA and FptX although FptX appears to be less important in
signalling as an fptX mutation delayed utilisation of ferripyochelin rather than
abolishing it.
1.9.3: Xenosiderophore Utilisation
The occurrence of a battery of cell surface signalling systems encoded on the genome
of P. aeruginosa that have putative roles in iron acquisition (Llamas et al., 2008) makes
it tempting to speculate that these systems induce gene expression by sensing the
presence of xenosiderophores.
There are 34 putative TonB dependent receptors
encoded on the genome (Koster, 2001) and P. aeruginosa has been shown to utilise
many xenosiderophores including enterobactin, ferric citrate, ferrichrome, ferrioxamine
B, coprogen, aerobactin, rhizobactin 1021 and schizokinen (Dean et al., 1996; Vasil
and Ochsner, 1999; Poole and McKay, 2003; Ó Cuív et al., 2006; Ó Cuív et al., 2007).
Enterobactin, a catecholate siderophore of E. coli, translocates through the outer
membrane of P. aeruginosa by the ferric enterobactin receptor PfeA. The expression of
pfeA under iron limited conditions requires the products of the pfeRS operon, encoding a
response regulator, PfeR, and a histidine kinase component, PfeS (Poole and McKay,
2003). The mechanism of regulation is similar to that described for histidine kinase
signal transduction systems (section 1.6.6). An inner membrane system for enterobactin
58
utilisation has yet to be discovered although putative components have been identified
by in silico analysis.
A 78 kDa P. aeruginosa outer membrane receptor termed ChtA was identified based on
significant sequence homology to the IutA aerobactin specific receptor of E. coli (Ó
Cuív et al., 2006). Insertional mutagenesis of chtA resulted in an inability to utilise the
siderophores aerobactin, rhizobactin 1021 and schizokinen, indicating that there was no
redundancy in the utilisation of these xenosiderophores. A motif with similarity to the
TonB box consensus sequence was identified at residues 24 – 31 of the mature protein.
A P. aeruginosa TonB1 mutant strain was tested and found to be defective in its ability
to utilise aerobactin, rhizobactin 1021 and schizokinen implying that ChtA is TonB1
dependent. Analysis using chtA-gfp transcriptional fusions indicated that chtA
expression was iron regulated. Furthermore, a Fur box was identified upstream of chtA
and in vivo Fur titration assays demonstrated the region was capable of binding Fur.
The two P. aeruginosa outer membrane receptor proteins FoxA and FiuA were
identified and characterised as having ferrioxamine B and ferrichrome specificity
respectively (Banin et al., 2005; Llamas et al., 2006). More recently, Ó Cuív et al.
(2007) identified and characterised a novel single unit inner membrane siderophore
transporter, FoxB, which functions to facilitate transport of the xenosiderophores
ferrichrome, ferrioxamine B and schizokinen across the cytoplasmic membrane of P.
aeruginosa. FoxB is a 45 kDa protein that is encoded directly downstream of foxA on
the chromosome of P. aeruginosa. Insertional mutagenesis of foxB did not result in any
discernable siderophore utilisation deficient phenotype in bioassays or growth
promotion assays suggesting redundancy at the inner membrane. A siderophore
utilisation mutant of S. meliloti was used as a heterologous host to examine the
siderophore utilisation ability of FoxB. FoxB conferred the ability to utilise the
xenosiderophores ferrichrome, ferrioxamine B and schizokinen. The siderophore
utilisation ability of FoxB was also examined in E. coli. The ferrioxamine B cognate
receptor FoxA from Y. enterocolitica (Baumler and Hantke, 1992) and the receptor
IutA from E. coli, which has been shown to facilitate the transport of schizokinen and
rhizobactin 1021 in addition to aerobactin (Ó Cuív et al., 2004), were both expressed in
an E. coli fhuB mutant expressing foxB. However when the ability of foxB to
complement the E. coli fhuB mutant was assessed it was found to be incapable of
59
restoring siderophore utilisation. This suggests that to function, FoxB possibly requires
additional transport proteins or that it possesses a limited ability to respond to
chemiosmotic gradients. FoxB represents a novel family of single unit siderophore
transport proteins.
60
1.10: Iron Acquisition in Fungi
Fungi are generally immobile organisms and consequently the available nutrients in
their surrounding environment can rapidly become limited. Fungal mycelia must
develop a branching system to explore and exploit environmental resources. Since ferric
iron insolubility creates challenges for fungal growth and development, two major
mechanisms have been developed to mediate iron acquisition: ferric reduction and
siderophore utilisation (Howard, 2004; Winkelmann, 2007). Saccharomyces cerevisiae
was the first eukaryotic genome to have been completely sequenced and as such is a
model organism for the study of eukaryotic molecular cell biology. Siderophore
mediated iron acquisition and reductive uptake evolved as vital systems for S. cerevisiae
to satisfy its absolute nutritional requirement for iron. S. cerevisiae does not synthesise
any endogenous siderophores yet retains the ability to utilise many xenosiderophores
(Winkelmann, 2002).
Reductive uptake occurs when ferric iron is reduced to ferrous iron at the cell surface
and high affinity ferrous uptake systems transport the reduced iron to the cystol. The
metalloreductase Fre1p encoded by the FRE1 gene catalyses the reduction of ferric iron
to ferrous iron. Significant homologues of FRE1 are present in yeast and therefore
redundancy in the ferric iron reduction ability is exhibited. The FRE genes are
transcriptionally regulated by iron. The reduced ferrous iron can then be transported by
uptake systems. A high affinity ferrous uptake system utilises the FET3 and FTR1
proteins. FET3 contains a single transmembrane domain localised to the cytoplasm.
This protein surprisingly oxidises the ferrous ion back to its ferric form prior to
transport via FTR1. It is possible that this reaction may impart specificity to the system.
FET3 is partly dependent on copper since it is an essential co-factor for the oxidase
activity. The iron permease FTR1 then facilitates translocation through the cytoplasmic
membrane. An iron binding motif, REGLE, present in FTR1 has been shown to be
essential for iron uptake activity. FET3 and FTR1 must be co-localised at the cell
surface to facilitate iron transport under iron deplete conditions (Philpott and
Protchenko, 2008). Under iron replete conditions, a low affinity iron transporter termed
FET4 is upregulated to facilitate the uptake of ferrous iron. FET4 displays a Km of
approximately 30 µM for ferrous iron (Winkelmann, 2002).
61
Siderophore mediated iron uptake in yeast can occur by means of two mechanisms:
transport of iron-loaded siderophores followed by intracellular ferric reduction or cell
surface siderophore reduction followed by iron uptake via the ferrous uptake system
(Johnson, 2008). Uptake of iron-loaded siderophores is designated the ‘shuttle
mechanism’. The Siderophore Iron Transport (SIT) family are a group of MFS type
transporters that have been identified in yeast and function to transport iron-loaded
siderophores. The SIT family were previously designated the UMF family standing for
unknown major facilitator family. Four membrane transporters found in S. cerevisiae
encoded by sit1, taf1, arn1 and enb1 are members of the SIT family, figure 1.26.
Figure 1.26: Specificity of Transporters of Siderophore-Iron Transporters in S. cerevisiae
(Winkelmann, 2002).
The Sit1 transporter displays broad substrate specificity by being capable of utilising
ferrioxamine, ferrichrome and coprogen type hydroxamate siderophores. Arn1 also
displays broad substrate specificity but cannot utilise the ferrioxamines. These two
transporters have been shown to normally be localised to intracellular vesicles and are
only present in the cytoplasmic membrane when the cognate siderophore is available
(Philpott and Protchenko, 2008). The Enb1 transporter is an enterobactin specific
transporter and displays the most significant divergence from the Sit1 transporter
compared to the other members of the SIT family. This divergence reflects the structural
differences between catecholate and hydroxamate siderophores. Taf1 is a transporter
with tight specificity for the fungal siderophore triacetylfusarinine C, which is
commonly produced by Aspergillus and Penicillium species (Winkelmann, 2002).
62
Other fungal species harbour homologues of the SIT family transporters. Candida
albicans for instance harbours a single broad substrate range transporter designated
SIT1p/ARN1p which facilitates the transport of ferrichrome-type siderophores. An
Arn1 S. cerevisiae homologue termed fgSIT1 is present in Fusarium graminearum
which has substrate specificity for ferrichrome. A. nidulans contains paralogues of SIT
transporters termed MirA, MirB and MirC. The genes encoding these transporters have
been shown to be iron regulated. MirA displays specificity for the xenosiderophore
enterobactin,
MirB
displays
specificity
for
the
endogenous
siderophore
triacetylfusarinine C, and no siderophore specificity has been determined for MirC as
yet (Johnson, 2008).
63
1.11: Summary
Bacteria have evolved sophisticated mechanisms to overcome the impermeability of the
cell membrane to essential nutrients and the severely restricted availability of iron.
These acquisition strategies take advantage of high affinity iron chelators, dedicated
specific receptor proteins and high affinity uptake mechanisms. The presence of iron
can be detected by cell surface signalling receptors and internally by global iron
regulators. These facilitate the regulation of the genes encoding the iron acquisition
machinery.
This thesis explores the utilisation of haem and xenosiderophores in S. meliloti 2011,
the endosymbiont of M. sativa. A novel dual ABC transport system is described, where
components of haem and hydroxamate siderophore utilisation systems have evolved
into a single consolidated uptake system. Furthermore, evidence is provided which
indicates that this dual ABC system may be more widespread within the bacterial
kingdom. The realease of iron internally from the siderophore ferrioxamine B is shown
to be mediated by a ferric iron reductase protein termed FhuF (Smc01658). This protein
is the first ferric iron siderophore reductase protein to have been identified and
characterised in the α-proteobacteria and displays significant similarity to the
prototypical ferric iron reductase, FhuF of E. coli. The presence of additional haem
utilisation systems is also investigated. In particular, putative dipeptide transport
systems are identified displaying strong homology to the E. coli dpp pearmease
complex which displays specificity for haem and dipeptide utilistation. The most
significant homologues were mutated and their phenotype analysed. The effect of
mutation of these haem and xenosiderophore utilisation systems on plant nodulation and
nitrogen fixation was analysed.
In addition, a representative of a novel family of inner membrane single unit
siderophore transporters in P. aeruginosa, an opportunistic human pathogen, has been
expressed and purified to levels necessary for structural analysis. Methodologies to
purify membrane proteins in large quantities and to overcome problems with protein
hydrophobicity and insolubility are described.
64
(Killmann et al., 2002)
(Gunter and Braun, 1990)
(Vakharia-Rao et al., 2007)
(Carter et al., 2006)
(Carter et al., 2006)
(Ma et al., 2007)
Andrews, S.C., Robinson, A.K., and Rodriguez-Quinones, F. (2003) Bacterial iron
homeostasis. FEMS Microbiol Rev 27: 215-237.
Arnoux, P., Haser, R., Izadi, N., Lecroisey, A., Delepierre, M., Wandersman, C., and
Czjzek, M. (1999) The crystal structure of HasA, a hemophore secreted by
Serratia marcescens. Nat Struct Biol 6: 516-520.
Banin, E., Vasil, M.L., and Greenberg, E.P. (2005) Iron and Pseudomonas aeruginosa
biofilm formation. Proc Natl Acad Sci U S A 102: 11076-11081.
Barnett, M.J., Fisher, R.F., Jones, T., Komp, C., Abola, A.P., Barloy-Hubler, F.,
Bowser, L., Capela, D., Galibert, F., Gouzy, J., Gurjal, M., Hong, A., Huizar, L.,
Hyman, R.W., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H., Palm, C.,
Peck, M.C., Surzycki, R., Wells, D.H., Yeh, K.C., Davis, R.W., Federspiel,
N.A., and Long, S.R. (2001) Nucleotide sequence and predicted functions of the
entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A
98: 9883-9888.
Baumler, A.J., and Hantke, K. (1992) Ferrioxamine uptake in Yersinia enterocolitica:
characterization of the receptor protein FoxA. Mol Microbiol 6: 1309-1321.
Becker, K., Koster, W., and Braun, V. (1990) Iron(III)hydroxamate transport of
Escherichia coli K12: single amino acid replacements at potential ATP-binding
sites inactivate the FhuC protein. Mol Gen Genet 223: 159-162.
Braun, V. (1995) Energy-coupled transport and signal transduction through the gramnegative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins.
FEMS Microbiol Rev 16: 295-307.
Braun, V., Hantke, K., and Koster, W. (1998) Bacterial iron transport: mechanisms,
genetics, and regulation. Met Ions Biol Syst 35: 67-145.
Braun, V. (2001) Iron uptake mechanisms and their regulation in pathogenic bacteria.
Int J Med Microbiol 291: 67-79.
Braun, V., and Braun, M. (2002a) Iron transport and signaling in Escherichia coli. FEBS
Lett 529: 78-85.
Braun, V., and Braun, M. (2002b) Active transport of iron and siderophore antibiotics.
Curr Opin Microbiol 5: 194-201.
Braun, V. (2006) Energy transfer between biological membranes. ACS Chem Biol 1:
352-354.
Braun, V., Mahren, S., and Sauter, A. (2006) Gene regulation by transmembrane
signaling. Biometals 19: 103-113.
Buchanan, S.K., Smith, B.S., Venkatramani, L., Xia, D., Esser, L., Palnitkar, M.,
Chakraborty, R., van der Helm, D., and Deisenhofer, J. (1999) Crystal structure
of the outer membrane active transporter FepA from Escherichia coli. Nat Struct
Biol 6: 56-63.
65
Buckling, A., Harrison, F., Vos, M., Brockhurst, M.A., Gardner, A., West, S.A., and
Griffin, A. (2007) Siderophore-mediated cooperation and virulence in
Pseudomonas aeruginosa. FEMS Microbiol Ecol 62: 135-141.
Butler, A. (2005) Marine siderophores and microbial iron mobilization. Biometals 18:
369-374.
Cadieux, N., and Kadner, R.J. (1999) Site-directed disulfide bonding reveals an
interaction site between energy-coupling protein TonB and BtuB, the outer
membrane cobalamin transporter. Proc Natl Acad Sci U S A 96: 10673-10678.
Cadieux, N., Bradbeer, C., and Kadner, R.J. (2000) Sequence changes in the ton box
region of BtuB affect its transport activities and interaction with TonB protein. J
Bacteriol 182: 5954-5961.
Carter, D.M., Miousse, I.R., Gagnon, J.N., Martinez, E., Clements, A., Lee, J., Hancock,
M.A., Gagnon, H., Pawelek, P.D., and Coulton, J.W. (2006) Interactions
between TonB from Escherichia coli and the periplasmic protein FhuD. J Biol
Chem 281: 35413-35424.
Carter, R.A., Worsley, P.S., Sawers, G., Challis, G.L., Dilworth, M.J., Carson, K.C.,
Lawrence, J.A., Wexler, M., Johnston, A.W., and Yeoman, K.H. (2002) The vbs
genes that direct synthesis of the siderophore vicibactin in Rhizobium
leguminosarum: their expression in other genera requires ECF sigma factor
RpoI. Mol Microbiol 44: 1153-1166.
Chakraborty, R., Lemke, E.A., Cao, Z., Klebba, P.E., and van der Helm, D. (2003)
Identification and mutational studies of conserved amino acids in the outer
membrane receptor protein, FepA, which affect transport but not binding of
ferric-enterobactin in Escherichia coli. Biometals 16: 507-518.
Chakraborty, R., Storey, E., and van der Helm, D. (2006) Molecular mechanism of
ferricsiderophore passage through the outer membrane receptor proteins of
Escherichia coli. Biometals.
Chakraborty, R., Storey, E., and van der Helm, D. (2007) Molecular mechanism of
ferricsiderophore passage through the outer membrane receptor proteins of
Escherichia coli. Biometals 20: 263-274.
Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) Crystal structure of the
dimeric C-terminal domain of TonB reveals a novel fold. J Biol Chem 276:
27535-27540.
Chao, T.C., Buhrmester, J., Hansmeier, N., Puhler, A., and Weidner, S. (2005) Role of
the regulatory gene rirA in the transcriptional response of Sinorhizobium
meliloti to iron limitation. Appl Environ Microbiol 71: 5969-5982.
Chimento, D.P., Kadner, R.J., and Wiener, M.C. (2005) Comparative structural analysis
of TonB-dependent outer membrane transporters: implications for the transport
cycle. Proteins 59: 240-251.
Clarke, T.E., Ku, S.Y., Dougan, D.R., Vogel, H.J., and Tari, L.W. (2000) The structure
of the ferric siderophore binding protein FhuD complexed with gallichrome. Nat
Struct Biol 7: 287-291.
Clarke, T.E., Tari, L.W., and Vogel, H.J. (2001) Structural biology of bacterial iron
uptake systems. Curr Top Med Chem 1: 7-30.
Clarke, T.E., Braun, V., Winkelmann, G., Tari, L.W., and Vogel, H.J. (2002a) X-ray
crystallographic structures of the Escherichia coli periplasmic protein FhuD
bound to hydroxamate-type siderophores and the antibiotic albomycin. J Biol
Chem 277: 13966-13972.
66
Clarke, T.E., Rohrbach, M.R., Tari, L.W., Vogel, H.J., and Koster, W. (2002b) Ferric
hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved
and non-conserved regions. Biometals 15: 121-131.
Cobessi, D., Celia, H., Folschweiller, N., Schalk, I.J., Abdallah, M.A., and Pattus, F.
(2005a) The crystal structure of the pyoverdine outer membrane receptor FpvA
from Pseudomonas aeruginosa at 3.6 angstroms resolution. J Mol Biol 347: 121134.
Cobessi, D., Celia, H., and Pattus, F. (2005b) Crystal structure at high resolution of
ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa.
J Mol Biol 352: 893-904.
Cope, L.D., Yogev, R., Muller-Eberhard, U., and Hansen, E.J. (1995) A gene cluster
involved in the utilization of both free heme and heme:hemopexin by
Haemophilus influenzae type b. J Bacteriol 177: 2644-2653.
Cope, L.D., Thomas, S.E., Hrkal, Z., and Hansen, E.J. (1998) Binding of hemehemopexin complexes by soluble HxuA protein allows utilization of this
complexed heme by Haemophilus influenzae. Infect Immun 66: 4511-4516.
Cornelis, P., and Matthijs, S. (2002) Diversity of siderophore-mediated iron uptake
systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol
4: 787-798.
Coves, J., Niviere, V., Eschenbrenner, M., and Fontecave, M. (1993) NADPH-sulfite
reductase from Escherichia coli. A flavin reductase participating in the
generation of the free radical of ribonucleotide reductase. J Biol Chem 268:
18604-18609.
Cox, C.D. (1980) Iron uptake with ferripyochelin and ferric citrate by Pseudomonas
aeruginosa. J Bacteriol 142: 581-587.
Cox, C.D. (1982) Effect of pyochelin on the virulence of Pseudomonas aeruginosa.
Infect Immun 36: 17-23.
Crichton, R.R., and Charloteaux-Wauters, M. (1987) Iron transport and storage. Eur J
Biochem 164: 485-506.
Crosa, J.H., and Walsh, C.T. (2002) Genetics and assembly line enzymology of
siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66: 223-249.
Cuiv, P.O., Clarke, P., Lynch, D., and O'Connell, M. (2004) Identification of rhtX and
fptX, novel genes encoding proteins that show homology and function in the
utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and
pyochelin by Pseudomonas aeruginosa, respectively. J Bacteriol 186: 29963005.
Cuiv, P.O., Clarke, P., and O'Connell, M. (2006) Identification and characterization of
an iron-regulated gene, chtA, required for the utilization of the xenosiderophores
aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa.
Microbiology 152: 945-954.
Cuiv, P.O., Keogh, D., Clarke, P., and O'Connell, M. (2007) FoxB of Pseudomonas
aeruginosa functions in the utilization of the xenosiderophores ferrichrome,
ferrioxamine B, and schizokinen: evidence for transport redundancy at the inner
membrane. J Bacteriol 189: 284-287.
Dean, C.R., Neshat, S., and Poole, K. (1996) PfeR, an enterobactin-responsive activator
of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa. J
Bacteriol 178: 5361-5369.
Eisenhauer, H.A., Shames, S., Pawelek, P.D., and Coulton, J.W. (2005) Siderophore
transport through Escherichia coli outer membrane receptor FhuA with
disulfide-tethered cork and barrel domains. J Biol Chem 280: 30574-30580.
67
Enz, S., Mahren, S., Stroeher, U.H., and Braun, V. (2000) Surface signaling in ferric
citrate transport gene induction: interaction of the FecA, FecR, and FecI
regulatory proteins. J Bacteriol 182: 637-646.
Ferguson, A.D., Hofmann, E., Coulton, J.W., Diederichs, K., and Welte, W. (1998)
Siderophore-mediated iron transport: crystal structure of FhuA with bound
lipopolysaccharide. Science 282: 2215-2220.
Ferguson, A.D., Braun, V., Fiedler, H.P., Coulton, J.W., Diederichs, K., and Welte, W.
(2000) Crystal structure of the antibiotic albomycin in complex with the outer
membrane transporter FhuA. Protein Sci 9: 956-963.
Fetherston, J.D., Bearden, S.W., and Perry, R.D. (1996) YbtA, an AraC-type regulator
of the Yersinia pestis pesticin/yersiniabactin receptor. Mol Microbiol 22: 315325.
Fetherston, J.D., Bertolino, V.J., and Perry, R.D. (1999) YbtP and YbtQ: two ABC
transporters required for iron uptake in Yersinia pestis. Mol Microbiol 32: 289299.
Finan, T.M., Weidner, S., Wong, K., Buhrmester, J., Chain, P., Vorholter, F.J.,
Hernandez-Lucas, I., Becker, A., Cowie, A., Gouzy, J., Golding, B., and Puhler,
A. (2001) The complete sequence of the 1,683-kb pSymB megaplasmid from the
N2-fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci U S A 98:
9889-9894.
Fontecave, M., Coves, J., and Pierre, J.L. (1994) Ferric reductases or flavin reductases?
Biometals 7: 3-8.
Forward, J.A., Behrendt, M.C., Wyborn, N.R., Cross, R., and Kelly, D.J. (1997) TRAP
transporters: a new family of periplasmic solute transport systems encoded by
the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gramnegative bacteria. J Bacteriol 179: 5482-5493.
Galibert, F., Finan, T.M., Long, S.R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler,
F., Barnett, M.J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L.,
Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R.W.,
Dreano, S., Federspiel, N.A., Fisher, R.F., Gloux, S., Godrie, T., Goffeau, A.,
Golding, B., Gouzy, J., Gurjal, M., Hernandez-Lucas, I., Hong, A., Huizar, L.,
Hyman, R.W., Jones, T., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H.,
Kiss, E., Komp, C., Lelaure, V., Masuy, D., Palm, C., Peck, M.C., Pohl, T.M.,
Portetelle, D., Purnelle, B., Ramsperger, U., Surzycki, R., Thebault, P.,
Vandenbol, M., Vorholter, F.J., Weidner, S., Wells, D.H., Wong, K., Yeh, K.C.,
and Batut, J. (2001) The composite genome of the legume symbiont
Sinorhizobium meliloti. Science 293: 668-672.
Garcia-Herrero, A., and Vogel, H.J. (2005) Nuclear magnetic resonance solution
structure of the periplasmic signalling domain of the TonB-dependent outer
membrane transporter FecA from Escherichia coli. Mol Microbiol 58: 12261237.
Genco, C.A., and Dixon, D.W. (2001) Emerging strategies in microbial haem capture.
Mol Microbiol 39: 1-11.
Gibson, F., and Magrath, D.I. (1969) The isolation and characterization of a hydroxamic
acid (aerobactin) formed by Aerobacter aerogenes 62-I. Biochim Biophys Acta
192: 175-184.
Groeger, W., and Koster, W. (1998) Transmembrane topology of the two FhuB domains
representing the hydrophobic components of bacterial ABC transporters
involved in the uptake of siderophores, haem and vitamin B12. Microbiology
144 (Pt 10): 2759-2769.
68
Guerinot, M.L. (1994) Microbial iron transport. Annu Rev Microbiol 48: 743-772.
Gunter, K., and Braun, V. (1990) In vivo evidence for FhuA outer membrane receptor
interaction with the TonB inner membrane protein of Escherichia coli. FEBS
Lett 274: 85-88.
Halle, F., and Meyer, J.M. (1992) Iron release from ferrisiderophores. A multi-step
mechanism involving a NADH/FMN oxidoreductase and a chemical reduction
by FMNH2. Eur J Biochem 209: 621-627.
Hantke, K. (2002) Members of the Fur protein family regulate iron and zinc transport in
E. coli and characteristics of the Fur-regulated fhuF protein. J Mol Microbiol
Biotechnol 4: 217-222.
Heinrichs, D.E., and Poole, K. (1996) PchR, a regulator of ferripyochelin receptor gene
(fptA) expression in Pseudomonas aeruginosa, functions both as an activator and
as a repressor. J Bacteriol 178: 2586-2592.
Higgins, C.F. (2001) ABC transporters: physiology, structure and mechanism--an
overview. Res Microbiol 152: 205-210.
Higgs, P.I., Larsen, R.A., and Postle, K. (2002) Quantification of known components of
the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and
FepA. Mol Microbiol 44: 271-281.
Ho, W.W., Li, H., Eakanunkul, S., Tong, Y., Wilks, A., Guo, M., and Poulos, T.L.
(2007) Holo- and apo-bound structures of bacterial periplasmic heme-binding
proteins. J Biol Chem 282: 35796-35802.
Howard, D.H. (2004) Iron gathering by zoopathogenic fungi. FEMS Immunol Med
Microbiol 40: 95-100.
Izadi, N., Henry, Y., Haladjian, J., Goldberg, M.E., Wandersman, C., Delepierre, M.,
and Lecroisey, A. (1997) Purification and characterization of an extracellular
heme-binding protein, HasA, involved in heme iron acquisition. Biochemistry
36: 7050-7057.
Johnson, L. (2008) Iron and siderophores in fungal-host interactions. Mycol Res 112:
170-183.
Johnston, A.W., Todd, J.D., Curson, A.R., Lei, S., Nikolaidou-Katsaridou, N., Gelfand,
M.S., and Rodionov, D.A. (2007) Living without Fur: the subtlety and
complexity of iron-responsive gene regulation in the symbiotic bacterium
Rhizobium and other alpha-proteobacteria. Biometals.
Kampfenkel, K., and Braun, V. (1992) Membrane topology of the Escherichia coli
ExbD protein. J Bacteriol 174: 5485-5487.
Karlsson, M., Hannavy, K., and Higgins, C.F. (1993) ExbB acts as a chaperone-like
protein to stabilize TonB in the cytoplasm. Mol Microbiol 8: 389-396.
Kelly, D.J., and Thomas, G.H. (2001) The tripartite ATP-independent periplasmic
(TRAP) transporters of bacteria and archaea. FEMS Microbiol Rev 25: 405-424.
Killmann, H., Herrmann, C., Torun, A., Jung, G., and Braun, V. (2002) TonB of
Escherichia coli activates FhuA through interaction with the beta-barrel.
Microbiology 148: 3497-3509.
Kodding, J., Killig, F., Polzer, P., Howard, S.P., Diederichs, K., and Welte, W. (2005)
Crystal structure of a 92-residue C-terminal fragment of TonB from Escherichia
coli reveals significant conformational changes compared to structures of
smaller TonB fragments. J Biol Chem 280: 3022-3028.
Koebnik, R. (1993) Structural organization of TonB-dependent receptors. Trends
Microbiol 1: 201.
69
Koster, W., and Braun, V. (1990) Iron (III) hydroxamate transport into Escherichia coli.
Substrate binding to the periplasmic FhuD protein. J. Biol. Chem. 265: 2140721410.
Koster, W. (2001) ABC transporter-mediated uptake of iron, siderophores, heme and
vitamin B12. Res Microbiol 152: 291-301.
Krewulak, K.D., Shepherd, C.M., and Vogel, H.J. (2005) Molecular dynamics
simulations of the periplasmic ferric-hydroxamate binding protein FhuD.
Biometals 18: 375-386.
Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E.L. (2001) Predicting
transmembrane protein topology with a hidden Markov model: application to
complete genomes. J Mol Biol 305: 567-580.
Labedan, B., and Riley, M. (1995) Gene products of Escherichia coli: sequence
comparisons and common ancestries. Mol Biol Evol 12: 980-987.
Lamont, I.L., Beare, P.A., Ochsner, U., Vasil, A.I., and Vasil, M.L. (2002) Siderophoremediated signaling regulates virulence factor production in
Pseudomonasaeruginosa. Proc Natl Acad Sci U S A 99: 7072-7077.
Larsen, R.A., Thomas, M.G., and Postle, K. (1999) Protonmotive force, ExbB and
ligand-bound FepA drive conformational changes in TonB. Mol Microbiol 31:
1809-1824.
Larsen, R.A., Letain, T.E., and Postle, K. (2003) In vivo evidence of TonB shuttling
between the cytoplasmic and outer membrane in Escherichia coli. Mol Microbiol
49: 211-218.
Lee, J.W., and Helmann, J.D. (2007) Functional specialization within the Fur family of
metalloregulators. Biometals.
Lee, N., Francklyn, C., and Hamilton, E.P. (1987) Arabinose-induced binding of AraC
protein to araI2 activates the araBAD operon promoter. Proc Natl Acad Sci U S
A 84: 8814-8818.
Letoffe, S., Redeker, V., and Wandersman, C. (1998) Isolation and characterization of
an extracellular haem-binding protein from Pseudomonas aeruginosa that shares
function and sequence similarities with the Serratia marcescens HasA
haemophore. Mol Microbiol 28: 1223-1234.
Letoffe, S., Nato, F., Goldberg, M.E., and Wandersman, C. (1999) Interactions of HasA,
a bacterial haemophore, with haemoglobin and with its outer membrane receptor
HasR. Mol Microbiol 33: 546-555.
Letoffe, S., Omori, K., and Wandersman, C. (2000) Functional characterization of the
HasA(PF) hemophore and its truncated and chimeric variants: determination of a
region involved in binding to the hemophore receptor. J Bacteriol 182: 44014405.
LeVier, K., and Guerinot, M.L. (1996) The Bradyrhizobium japonicum fegA gene
encodes an iron-regulated outer membrane protein with similarity to
hydroxamate-type siderophore receptors. J Bacteriol 178: 7265-7275.
Linton, K.J., and Higgins, C.F. (1998) The Escherichia coli ATP-binding cassette
(ABC) proteins. Mol Microbiol 28: 5-13.
Llamas, M.A., Sparrius, M., Kloet, R., Jimenez, C.R., Vandenbroucke-Grauls, C., and
Bitter, W. (2006) The heterologous siderophores ferrioxamine B and
ferrichrome activate signaling pathways in Pseudomonas aeruginosa. J Bacteriol
188: 1882-1891.
Llamas, M.A., Mooij, M.J., Sparrius, M., Vandenbroucke-Grauls, C.M., Ratledge, C.,
and Bitter, W. (2008) Characterization of five novel Pseudomonas aeruginosa
cell-surface signalling systems. Mol Microbiol 67: 458-472.
70
Locher, K.P., Rees, B., Koebnik, R., Mitschler, A., Moulinier, L., Rosenbusch, J.P., and
Moras, D. (1998) Transmembrane signaling across the ligand-gated FhuA
receptor: crystal structures of free and ferrichrome-bound states reveal allosteric
changes. Cell 95: 771-778.
Lynch, D., O'Brien, J., Welch, T., Clarke, P., Cuiv, P.O., Crosa, J.H., and O'Connell, M.
(2001) Genetic organization of the region encoding regulation, biosynthesis, and
transport of rhizobactin 1021, a siderophore produced by Sinorhizobium
meliloti. J Bacteriol 183: 2576-2585.
Ma, L., Kaserer, W., Annamalai, R., Scott, D.C., Jin, B., Jiang, X., Xiao, Q., Maymani,
H., Massis, L.M., Ferreira, L.C., Newton, S.M., and Klebba, P.E. (2007)
Evidence of ball-and-chain transport of ferric enterobactin through FepA. J Biol
Chem 282: 397-406.
Matzanke, B.F., Anemuller, S., Schunemann, V., Trautwein, A.X., and Hantke, K.
(2004) FhuF, part of a siderophore-reductase system. Biochemistry 43: 13861392.
Michel, L., Bachelard, A., and Reimmann, C. (2007) Ferripyochelin uptake genes are
involved in pyochelin-mediated signalling in Pseudomonas aeruginosa.
Microbiology 153: 1508-1518.
Miethke, M., and Marahiel, M.A. (2007) Siderophore-based iron acquisition and
pathogen control. Microbiol Mol Biol Rev 71: 413-451.
Muller, G., Matzanke, B.F., and Raymond, K.N. (1984) Iron transport in Streptomyces
pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantiorhodotorulic acid. J Bacteriol 160: 313-318.
Muller, G., and Raymond, K.N. (1984) Specificity and mechanism of ferrioxaminemediated iron transport in Streptomyces pilosus. J Bacteriol 160: 304-312.
Muller, K., Matzanke, B.F., Schunemann, V., Trautwein, A.X., and Hantke, K. (1998)
FhuF, an iron-regulated protein of Escherichia coli with a new type of [2Fe-2S]
center. Eur J Biochem 258: 1001-1008.
Neilands, J.B. (1981) Microbial iron compounds. Annu Rev Biochem 50: 715-731.
Neilands, J.B. (1995) Siderophores: Structure and Function of Microbial Iron Transport
Compounds. J. Biol. Chem. 270: 26723-26726.
Nienaber, A., Hennecke, H., and Fischer, H.M. (2001) Discovery of a haem uptake
system in the soil bacterium Bradyrhizobium japonicum. Mol Microbiol 41:
787-800.
Noya, F., Arias, A., and Fabiano, E. (1997) Heme compounds as iron sources for
nonpathogenic Rhizobium bacteria. J Bacteriol 179: 3076-3078.
Ochsner, U.A., Johnson, Z., and Vasil, M.L. (2000) Genetics and regulation of two
distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa.
Microbiology 146 (Pt 1): 185-198.
Ong, S.A., Peterson, T., and Neilands, J.B. (1979) Agrobactin, a siderophore from
Agrobacterium tumefaciens. J Biol Chem 254: 1860-1865.
Pao, S.S., Paulsen, I.T., and Saier, M.H., Jr. (1998) Major facilitator superfamily.
Microbiol Mol Biol Rev 62: 1-34.
Pawelek, P.D., Croteau, N., Ng-Thow-Hing, C., Khursigara, C.M., Moiseeva, N.,
Allaire, M., and Coulton, J.W. (2006) Structure of TonB in complex with FhuA,
E. coli outer membrane receptor. Science 312: 1399-1402.
Philpott, C.C., and Protchenko, O. (2008) Response to iron deprivation in
Saccharomyces cerevisiae. Eukaryot Cell 7: 20-27.
Pohl, E., Haller, J.C., Mijovilovich, A., Meyer-Klaucke, W., Garman, E., and Vasil,
M.L. (2003) Architecture of a protein central to iron homeostasis: crystal
71
structure and spectroscopic analysis of the ferric uptake regulator. Mol
Microbiol 47: 903-915.
Pollack, J.R., and Neilands, J.B. (1970) Enterobactin, an iron transport compound from
Salmonella typhimurium. Biochem Biophys Res Commun 38: 989-992.
Poole, K., and McKay, G.A. (2003) Iron acquisition and its control in Pseudomonas
aeruginosa: many roads lead to Rome. Front Biosci 8: d661-686.
Postle, K. (1993) TonB protein and energy transduction between membranes. J
Bioenerg Biomembr 25: 591-601.
Postle, K., and Kadner, R.J. (2003) Touch and go: tying TonB to transport. Mol
Microbiol 49: 869-882.
Postle, K., and Larsen, R.A. (2007) TonB-dependent energy transduction between outer
and cytoplasmic membranes. Biometals.
Rabus, R., Jack, D.L., Kelly, D.J., and Saier, M.H., Jr. (1999) TRAP transporters: an
ancient family of extracytoplasmic solute-receptor-dependent secondary active
transporters. Microbiology 145 (Pt 12): 3431-3445.
Redly, G.A., and Poole, K. (2003) Pyoverdine-mediated regulation of FpvA synthesis in
Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function
sigma factor, FpvI. J Bacteriol 185: 1261-1265.
Rohrbach, M.R., Braun, V., and Koster, W. (1995) Ferrichrome transport in Escherichia
coli K-12: altered substrate specificity of mutated periplasmic FhuD and
interaction of FhuD with the integral membrane protein FhuB. J Bacteriol 177:
7186-7193.
Roosenberg, J.M., 2nd, Lin, Y.M., Lu, Y., and Miller, M.J. (2000) Studies and
syntheses of siderophores, microbial iron chelators, and analogs as potential
drug delivery agents. Curr Med Chem 7: 159-197.
Rudolph, G., Hennecke, H., and Fischer, H.M. (2006) Beyond the Fur paradigm: ironcontrolled gene expression in rhizobia. FEMS Microbiol Rev 30: 631-648.
Saier, M.H., Jr. (2000) Families of transmembrane sugar transport proteins. Mol
Microbiol 35: 699-710.
Saier, M.H., Jr., and Paulsen, I.T. (2001) Phylogeny of multidrug transporters. Semin
Cell Dev Biol 12: 205-213.
Sean Peacock, R., Weljie, A.M., Peter Howard, S., Price, F.D., and Vogel, H.J. (2005)
The solution structure of the C-terminal domain of TonB and interaction studies
with TonB box peptides. J Mol Biol 345: 1185-1197.
Seliger, S.S., Mey, A.R., Valle, A.M., and Payne, S.M. (2001) The two TonB systems
of Vibrio cholerae: redundant and specific functions. Mol Microbiol 39: 801812.
Stevens, J.B., Carter, R.A., Hussain, H., Carson, K.C., Dilworth, M.J., and Johnston,
A.W. (1999) The fhu genes of Rhizobium leguminosarum, specifying
siderophore uptake proteins: fhuDCB are adjacent to a pseudogene version of
fhuA. Microbiology 145 (Pt 3): 593-601.
Stintzi, A., Cornelis, P., Hohnadel, D., Meyer, J.M., Dean, C., Poole, K., Kourambas,
S., and Krishnapillai, V. (1996) Novel pyoverdine biosynthesis gene(s) of
Pseudomonas aeruginosa PAO. Microbiology 142 (Pt 5): 1181-1190.
Stintzi, A., Johnson, Z., Stonehouse, M., Ochsner, U., Meyer, J.M., Vasil, M.L., and
Poole, K. (1999) The pvc gene cluster of Pseudomonas aeruginosa: role in
synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS. J
Bacteriol 181: 4118-4124.
Stock, A.M., Robinson, V.L., and Goudreau, P.N. (2000) Two-component signal
transduction. Annu Rev Biochem 69: 183-215.
72
Stojiljkovic, I., and Hantke, K. (1992) Hemin uptake system of Yersinia enterocolitica:
similarities with other TonB-dependent systems in gram-negative bacteria.
Embo J 11: 4359-4367.
Stojiljkovic, I., Baumler, A.J., and Hantke, K. (1994) Fur regulon in gram-negative
bacteria. Identification and characterization of new iron-regulated Escherichia
coli genes by a fur titration assay. J Mol Biol 236: 531-545.
Stojiljkovic, I., and Hantke, K. (1994) Transport of haemin across the cytoplasmic
membrane through a haemin-specific periplasmic binding-protein-dependent
transport system in Yersinia enterocolitica. Mol Microbiol 13: 719-732.
Stork, M., Otto, B.R., and Crosa, J.H. (2007) A Novel Protein, TtpC, Is a Required
Component of the TonB2 Complex for Specific Iron Transport in the Pathogens
Vibrio anguillarum and Vibrio cholerae. J Bacteriol 189: 1803-1815.
Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J.,
Brinkman, F.S., Hufnagle, W.O., Kowalik, D.J., Lagrou, M., Garber, R.L.,
Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L.L.,
Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D.,
Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory,
S., and Olson, M.V. (2000) Complete genome sequence of Pseudomonas
aeruginosa PA01, an opportunistic pathogen. Nature 406: 959-964.
Tait, G.H. (1975) The identification and biosynthesis of siderochromes formed by
Micrococcus denitrificans. Biochem J 146: 191-204.
Thompson, J.M., Jones, H.A., and Perry, R.D. (1999) Molecular characterization of the
hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for
hemin and hemoprotein utilization. Infect Immun 67: 3879-3892.
Todd, J.D., Wexler, M., Sawers, G., Yeoman, K.H., Poole, P.S., and Johnston, A.W.
(2002) RirA, an iron-responsive regulator in the symbiotic bacterium Rhizobium
leguminosarum. Microbiology 148: 4059-4071.
Tsuda, M., Miyazaki, H., and Nakazawa, T. (1995) Genetic and physical mapping of
genes involved in pyoverdin production in Pseudomonas aeruginosa PAO. J
Bacteriol 177: 423-431.
Vakharia-Rao, H., Kastead, K.A., Savenkova, M.I., Bulathsinghala, C.M., and Postle,
K. (2007) Deletion and substitution analysis of the Escherichia coli TonB Q160
region. J Bacteriol 189: 4662-4670.
Vasil, M.L., and Ochsner, U.A. (1999) The response of Pseudomonas aeruginosa to
iron: genetics, biochemistry and virulence. Mol Microbiol 34: 399-413.
Veenhoff, L.M., Heuberger, E.H., and Poolman, B. (2002) Quaternary structure and
function of transport proteins. Trends Biochem Sci 27: 242-249.
Viguier, C., P, O.C., Clarke, P., and O'Connell, M. (2005) RirA is the iron response
regulator of the rhizobactin 1021 biosynthesis and transport genes in
Sinorhizobium meliloti 2011. FEMS Microbiol Lett 246: 235-242.
Visca, P., Colotti, G., Serino, L., Verzili, D., Orsi, N., and Chiancone, E. (1992) Metal
regulation of siderophore synthesis in Pseudomonas aeruginosa and functional
effects of siderophore-metal complexes. Appl Environ Microbiol 58: 2886-2893.
Wandersman, C., and Stojiljkovic, I. (2000) Bacterial heme sources: the role of heme,
hemoprotein receptors and hemophores. Curr Opin Microbiol 3: 215-220.
Wandersman, C., and Delepelaire, P. (2004) Bacterial iron sources: from siderophores
to hemophores. Annu Rev Microbiol 58: 611-647.
Ward, A., Hoyle, C., Palmer, S., O'Reilly, J., Griffith, J., Pos, M., Morrison, S.,
Poolman, B., Gwynne, M., and Henderson, P. (2001) Prokaryote multidrug
73
efflux proteins of the major facilitator superfamily: amplified expression,
purification and characterisation. J Mol Microbiol Biotechnol 3: 193-200.
Wexler, M., Yeoman, K.H., Stevens, J.B., de Luca, N.G., Sawers, G., and Johnston,
A.W. (2001) The Rhizobium leguminosarum tonB gene is required for the
uptake of siderophore and haem as sources of iron. Mol Microbiol 41: 801-816.
Wexler, M., Todd, J.D., Kolade, O., Bellini, D., Hemmings, A.M., Sawers, G., and
Johnston, A.W. (2003) Fur is not the global regulator of iron uptake genes in
Rhizobium leguminosarum. Microbiology 149: 1357-1365.
Wiener, M.C. (2005) TonB-dependent outer membrane transport: going for Baroque?
Curr Opin Struct Biol 15: 394-400.
Wilks, A., and Burkhard, K.A. (2007) Heme and virulence: how bacterial pathogens
regulate, transport and utilize heme. Nat Prod Rep 24: 511-522.
Winkelmann, G. (2007) Ecology of siderophores with special reference to the fungi.
Biometals.
Winnen, B., Hvorup, R.N., and Saier, M.H., Jr. (2003) The tripartite tricarboxylate
transporter (TTT) family. Res Microbiol 154: 457-465.
Wirth, C., Meyer-Klaucke, W., Pattus, F., and Cobessi, D. (2007) From the periplasmic
signaling domain to the extracellular face of an outer membrane signal
transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine
outer membrane receptor. J Mol Biol 368: 398-406.
Yamanaka, K., Oikawa, H., Ogawa, H.O., Hosono, K., Shinmachi, F., Takano, H.,
Sakuda, S., Beppu, T., and Ueda, K. (2005) Desferrioxamine E produced by
Streptomyces griseus stimulates growth and development of Streptomyces
tanashiensis. Microbiology 151: 2899-2905.
Yang, J., Sangwan, I., Lindemann, A., Hauser, F., Hennecke, H., Fischer, H.M., and
O'Brian, M.R. (2006) Bradyrhizobium japonicum senses iron through the status
of haem to regulate iron homeostasis and metabolism. Mol Microbiol 60: 427437.
Yeoman, K.H., Wisniewski-Dye, F., Timony, C., Stevens, J.B., deLuca, N.G., Downie,
J.A., and Johnston, A.W. (2000) Analysis of the Rhizobium leguminosarum
siderophore-uptake gene fhuA: differential expression in free-living bacteria and
nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different
strains. Microbiology 146 (Pt 4): 829-837.
Yue, W.W., Grizot, S., and Buchanan, S.K. (2003) Structural evidence for iron-free
citrate and ferric citrate binding to the TonB-dependent outer membrane
transporter FecA. J Mol Biol 332: 353-368.
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