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Biometals 2002: Third International Biometals Symposium
10
I
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12
I3
14
Lu.
Z.
H.,
Dameron. C.
T.
and Solioz. M.
(2002)
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M.
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Biol. Chem.
273, 1277-
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H. H.
and Merchant,
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(I
995)
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Biol. Chem.
270,
Willett,
W.
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Brinen,
L.
S.,
Fletterick,
R.
J.
and Craik
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S.
(I
996)
Biochemistry
35,5992-5998
Odermatt, A,, Krapf,
R.
and Solioz, M.
(
1994)
Biochem.
Biophys. Res. Cornmun.
202, 4448
in the
press
23504-23510
Received
I0
March
2002
Metal
Transport
Microbial siderophore-mediated transport
G.
Winkelmann'
Microbiology
&
Biotechnology, University
of
Tuebingen,
Auf
der Morgenstelle 28, 72076 Tuebingen, Germany
Abstract
Microbial iron chelates, called siderophores, are
synthesized by bacteria and fungi in response to
low iron availability in the environment. The
present review summarizes structural details of
siderophore ligands with respect to their transport
properties. This presentation is largely centred on
the occurrence and function of siderophores in the
various bacterial and fungal genera.
Introduction
Siderophores are defined as low-molecular-mass
microbial compounds with a very high affinity for
iron. Their function is to mediate iron uptake by
microbial cells. For a more comprehensive review
of the structures and functions of siderophores the
reader is directed to reviews on iron transport
[l]
and microbial transport systems
[2].
Although
most siderophores are water soluble and are
excreted into the environment, there are some
siderophores that are not excreted at all, such as
the mycobactins, synthesized by mycobacteria,
that are located within the cell envelope
[3,4].
This
is in contrast to the carboxymycobactins and
exochelins that represent the real extracellular
siderophores of the mycobacteria. Fungal sidero-
phores may also be divided into extracellular and
intracellular siderophores, as found in spores
and mycelia of
Neurospora
and
Aspergillus
[S].
Key
words:
biosynthesrs. ecology, stereochemistry
'E-mail
Winkelmann@uni-tuebingen.de
Also, extremely lipophilic siderophores have been
found in marine bacteria that do not readily diffuse
into the surrounding medium, but which form
vesicles
[6].
Thus the environmental distribu-
tion of siderophores may vary to some extent.
However, their general iron-transport function
is
obvious and has been documented by radioactive
labelling experiments in a variety
of
microbial
organisms. Although a number of transport mech-
anisms are based on non-destructive shuttle sys-
tems, some of the ligands may be degraded by
esterases after iron delivery to the cells. In
general, most siderophore transport systems are
highly specific for certain siderophores, although
some broad-range siderophore-recognition sys-
tems have been described. Several novel sidero-
phore-transport systems have recently been
proposed based on ligand-exchange mechanisms
~7~81.
Functions
of
siderophores
Although their main function is to acquire iron
from insoluble hydroxides or from iron adsorbed
to solid surfaces, siderophores can also extract
iron from various other soluble and insoluble iron
compounds, such as ferric citrate, ferric phos-
phate, Fe-transferrin, ferritin or iron bound to
sugars, plant flavone pigments and glycosides or
even from artificial chelators like EDTA and
nitrilotriacetate by Fe(
I I
I)/ligand-exchange reac-
tions. Thus, even
if
siderophores are not directly
involved in iron solubilization, they are required
as carriers mediating exchange between extra
cellular iron stores and membrane-located
siderophore-transport systems.
69
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2002 Biochemical Society
Biochemical Society Transactions
(2002)
Volume
30,
part
4
The efficiency of siderophores in microbial
metabolism is based mainly on three facts.
(1) Siderophores contain the most efficient
iron-binding ligand types in Nature, consist-
ing of hydroxamate, catecholate or a-hydroxy-
carboxylate ligands that form hexadentate Fe(
111)
complexes, satisfying the six co-ordination sites
on ferric ions. Moreover, siderophores possessing
three bidentates in one molecule (iron-to-ligand
ratio
=
1
:
1)
show increased stability due to the
chelate effect. (2) Regulation of siderophore bio-
synthesis is an economic means of spending meta-
bolic energy, but it also allows for the production
of high local concentrations of siderophores in the
vicinity of microbial cells during iron limitation.
This kind of overproduction may also be operating
in host-adapted bacterial and fungal strains, lead-
ing to increased virulence.
(3)
Besides their ability
to solubilize iron and to function as external iron
carriers, siderophores exhibit structural and con-
formational specificities to fit into membrane
receptors and/or transporters. This has been
amply demonstrated by modifying siderophore
chemical structure, i.e. using derivatives, enanti-
omers, metal-replacement studies or by genetic
and mutational analysis of receptors and mem-
brane transporters
[%lo].
Biosynthetic and structural aspects
The biosynthetic pathways of siderophores are
tightly connected to aerobic metabolism involving
molecular oxygen activated by mono-, di- and N-
oxygenases and the use of acids originating from
the final oxidation of the citric acid cycle, such as
citrate, succinate and acetate. Moreover, all sidero-
phore peptides are synthesized by non-ribosomal
peptide synthetases and in the case of fungal
siderophores are mainly built up from ornithine, a
non-proteinogenic amino acid. Thus, siderophore
synthesis is largely independent from the primary
metabolism. Most siderophores contain one or
more of the following simple bidentate ligands as
building blocks: (1) a dihydroxybenzoic acid
(catecholate) coupled to an amino acid, (2)
hydroxamate groups containing N5-acyl-N5-
hydroxyornithine or
N'-acyl-N'-hydroxylysine
and
(3)
hydroxycarboxylates consisting of citric
acid or P-hydroxyaspartic acid.
Besides being precursors most of the mono-
meric bidentates may also act as functional sidero-
phores after excretion. The iron-binding affinity
of bidentate siderophores, however, remains low
compared with hexadentate siderophores. A phy-
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2002
Biochemical Society
logeny
of
siderophore structures is difficult to
delineate. However, starting from simple precur-
sors of each class, one can imagine that extended
siderophore structures have been favoured during
evolution, resulting in hexadentate siderophores
possessing higher stability constants (chelate
effect) compared with their monomeric pre-
cursors. Thus, higher denticity seems to have a
selective advantage in siderophore evolution.
A further aspect of siderophore evolution is
the optimization of chelate conformation. Al-
though linear di-, tetra- and hexadentate sidero-
phores have been found in all siderophore classes,
there is a tendency for cyclization in the final
biosynthetic end products (Figure 1). Examples
are enterobactin or corynebactin
[ll]
in the cate-
cholate class, fusigen, triacetylfusarinine and ferri-
oxamines
E
and
G,
as well as the ferrichromes and
asperchromes, in the hydroxamate class [12].
Cyclization enhances complex stability, chemical
Figure
I
Cyclic siderophores
(a) Ferrichrorne, (b) triacetylfusarinine
C
and (c) enterobactin.
04..
YH
on
692
Biometals
2002:
Third International Biometals Symposium
stability and improves resistance to degrading
enzymes. Cyclization is regarded as a common
feature of secondary metabolism and is found
in microbial peptides, polyketides, macrocyclic
antibiotics and other bioactive compounds.
Cyclization might also be advantageous for dif-
fusion-controlled transport processes across cellu-
lar membranes. Moreover, due to a reduction of
residual functional groups, the surface of the
siderophores becomes non-reactive or inaccessible
to modifying enzymes.
Ecological
aspects
If we consider siderophore production within
different microbial genera, we realize that
catecholate siderophores predominate in certain
Gram-negative genera, like the Enterobacteria and
the genus
Vibrio,
but also in the nitrogen-fixing
Azotobacteria and the plant-associated Agro-
bacteria. The reasons that these bacteria use
catecholates may be manifold. However, lipo-
philicity, complex stability, high environmental
pH and a weak nitrogen metabolism might favour
catecholates. The Gram-positive Streptomycetes
produce hydroxamate-type ferrioxamines and the
ascomycetous and basidiomycetous fungi synthe-
size ester- and peptide-containing hydroxamate
siderophores that are acid-stable and well suited
for environmental iron solubilization. Both the
Streptomycetes and fungi show a versatile nitro-
gen metabolism with active N-oxygenases.
Siderophores are also involved in mycorrhizal
symbiosis, as found in all terrestrial plant com-
munities. One of the major types of mycorrhizum
are the ectomycorrhiza, typically formed by almost
all tree species in temperate forests.
So
far, only a
few siderophores have been described due to the
difficulties with cultivating the mycorrhizal fungi
in pure culture under iron limitation. However,
siderophores from three ericoid mycorrhizal fun-
gal species,
Hymenoscyphus ericae, Oidiodendron
griseum
and
Rhodothamnus chamaecistus,
and
an ectendomycorrhizal fungus
Wilcoxina
and an
ectomycorrhizal fungus
Cenococcusm geophilum,
have been isolated which all produce hydroxamate
siderophores of the ferrichrome and fusigen class
Zygomycetes produce solely aminocarboxyl-
ates based on citric acid and amines, which show
optimal iron-binding activity at a weakly acidic
pH
[14].
Although phenolate and catecholate
pigments have been detected in higher fungi,
defined structures of catecholate-based sidero-
~31.
693
phores have never been reported in fungi. The
concomitant production of organic acids by most
fungi probably prevents the use of ferric catecho-
lates that are unstable at acidic pH, while ferric
hydroxamates are generally stable down to pH
2.
Characterization of siderophore classes based on
microbial groups, however, is not always possible.
The phylogenetic distance between catechol- and
hydroxamate-producing genera can be very small
and occasionally both siderophore types have been
observed in the same genus, and indeed in at least
one case in a single siderophore
[
151.
We reported
earlier that both catecholate- and hydroxamate-
type siderophores have been isolated from the
ErwinialEnterobacterlHafnia
group, representing
closely related genera of the family of Entero-
bacteriaceae
[16,17].
Fluorescent Pseudomonads
and the related non-fluorescent
Burkholderia
group, which are well-known producers of linear
peptide siderophores
[18],
might profit from the
generally neutral environment of soil, where acid
stability is of minor importance. The alternating
D-
and L-configuration of peptidic amino acids also
makes these siderophores very resistant to micro-
bial proteases.
When we look more deeply into the large
group of marine
Vibrios,
we notice that a broad
range of structurally different siderophores is
produced
[
121.
Thus, catecholate siderophores
have been detected in
Vibrio cholerae, Vib-
rio vulni’cus
and
Vibrio jluvialis.
A mixed-type
catecholate-thiazoline-hydroxamate
siderophore,
named anguibactin, has been isolated from
Vibrio
anguillarun
and the citrate-based hydroxamate,
aerobactin, has been described in certain marine
Vibrios.
Also the occurrence of ferrioxamine G has
been reported in
Vibrio
species and we have
recently identified the structurally related di-
hydroxamate, bisucaberin, in the fish pathogen
Vibrio salmonicida
[
191.
This broad range of
structurally different siderophores in the family
Vibrionaceae may reflect the existence of a large
pool of siderophore biosynthetic genes and may
also indicate that the different genera of the family
are more heterogeneous than previously assumed.
Vibrios
are widespread in marine water, but this
does not necessarily mean that they are really free-
living bacteria. We may assume that most
Vibrios
are somehow associated with particles in marine
coastal water. Siderophores have also been isolated
from several other marine bacteria, like
Altero-
monas, Halomonas
and
Marinobacter,
indicating
that siderophore production in the marine en-
vironment is widespread
[6].
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2002
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Biochemical Society Transactions
(2002)
Volume
30,
part
4
The role
of
citric
acid
With respect to the diversity of siderophores, it is
interesting to note that citrate is the starting point
of a variety of siderophores. There is evidence that
citrate-containing siderophores are present in a
variety of bacterial and fungal genera. Some of
these siderophores, like aerobactin, arthrobactin,
schizokinen, acinetoferrin and nannochelin, con-
tain citrate linked to hydroxamate residues, while
others, like rhizoferrin, staphyloferrin and vibrio-
ferrin, represent aminocarboxylate siderophores
Citric acid may be linked amidically to the
a-amino group of ornithine or lysine, as found in
staphyloferrin A and aerobactin. On the other
hand citric acid may be bound to an amine
group, as found in rhizoferrin, or to the terminal
amine group in staphyloferrin A. Thus, carboxylic
groups may combine with both a-amino groups
and terminal amine groups. Condensation
of
succinic acid with amines is common among all
ferrioxamines, suggesting that decarboxylation
may occur prior to condensation. However, while
succinic acid forms both, amide and hydroxamate
groups, citric acid has never been shown to form
hydroxamic acyl residues. A functionally anal-
ogous hydroxycarboxylate donor is provided by
p-
hydroxyaspartic acid that is inserted in the peptide
backbone of some pyoverdines and ornibactins,
representing siderophores of the genus Pseudo-
monas and the non-fluorescent genus Burkholderia
respectively [20].
[121.
Stereochemistry and recognition
An interesting finding is the occurrence of rhizo-
ferrin in both fungal and bacterial genera. The
fungal rhizoferrin was isolated from Rhizopus
strains and other Mucorales of the Zygomycetes,
while the bacterial rhizoferrin was isolated from
Ralstonia pickettii [21]. However, as we have
reported earlier, the configuration
of
the chiral
centre of citric acid residues in the rhizoferrins is
different (Figure
2).
While the fungal rhizoferrin
has an R,R-configuration, the bacterial enantio-
rhizoferrin has an S,S-configuration, suggesting
different biosynthetic pathways [21], This also
indicates that the rhizoferrin molecule must have
arisen twice in Nature. The stereochemistry of
the corresponding iron complexes has also been
identified by
CD
spectroscopy, being
A
for R,R-
rhizoferrin and
A
for S,S-rhizoferrin. The bac-
terial staphyloferrin
A
is structurally very similar
to rhizoferrin, but possesses D-ornithine instead of
putrescine, which results in a third chiral centre at
the
C-a
atom [22]. Although the chirality of the
citryl residues in staphyloferrin A has never been
determined, there is evidence from analogous
synthetic compounds for an S,S-configuration
identical to enantio-rhizoferrin from
R.
pickettii
(H. Drechsel and
G.
Winkelmann, unpublished
work). A growth-promotion test with Staphylo-
coccus
strains confirmed this observation by show-
ing that staphyloferrin A and enantio-rhizoferrin
were both functionally active, while the fungal
rhizoferrin was inactive. We therefore suggest that
the two bacterial ferric carboxylate complexes
staphyloferrin A and enantio-rhizoferrin are
stereochemically equivalent and that their iron
complexes are recognized by the same transport
system in Staphylococcus.
We had previously shown that the chirality of
siderophores is an important structural feature for
recognition and transport in fungi [23,24]. The
first observation of the functionally inactive enan-
tio-ferrichrome in filamentous fungi like
Neuro-
spora, Penicillium and Aspergillus was recently
confirmed when studying siderophore trans-
porters of the major facilitator superfamily
(MFS)
in yeast [25,26]. Enantio-ferrichrome was not
recognized by the SIT1 and ARNl transporters in
the yeast Saccharomyces cerevisiae [26]. A function
Figure
2
Configuration
of
S,S-enantio-rhizoferrin (bacterial) and R,R-rhizoferrin (fungal)
bacterial Rhlroferrln fungal Rhlzoferrln
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2002
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694
Biometals
2002:
Third International Biometals Symposium
Figure
3
Siderophore iron transporters and routes
of
entry
of
siderophores
in
5.
cerevisiae
coprogen
Ferrioxamines
Tfla&ylfuuflnine
c
Ferrirubin Ferrichrysin
I
Ferrirhcdin Ferricrodn
I
Ferrichrome
A
Ferrichrcine
I
Enterr
J
of SIT1 in
ferrioxamine/ferrichrome
transport
had previously been proven by mutational analysis
of yeast cells
[27].
Using a functional genomics
strategy by starting from known genome sequence
data, we were able to disrupt six open reading
frames previously assigned to unknown
MFS
transporters in
S.
cerevisiae.
Of
the four sidero-
phore transporter proteins identified (Figure
3),
we found specificities for ferrioxamines/ferri-
chromes (Sitlp), triacetylfusarine
C
(Taflp)
[24],
anhydromevalone-ferrichromes
(Arnl p)
[26]
and
enterobactin (Enblp)
[28].
The expression of all
siderophore transporters is iron-regulated by the
transcription factor Aft1 p
[29].
The relatively
large number of siderophore transporters in
S.
cerevisiae
is surprising, since this fungus is un-
able
to
synthesize its own siderophores. However,
iron-regulated siderophore transporters in yeast,
now collectively called siderophore iron trans-
porter (SIT), are of high environmental value,
since they enable yeast cells to survive in a low-
iron environment by using external siderophores
produced by various accompanying bacteria and
fungi.
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Siderophore production by
Fusarium
venenaturn
A3/5
M.
G.
Wiebe'
Sohngaardsholmsvej 49, Institute
of
Life Sciences, Aalborg University, DK-9000 Aalborg, Denmark
Abstract
Fusarium venenatum
A315 was grown in iron-
restricted batch cultures and iron-limited chemo-
stat cultures to determine how environmental
conditions affected siderophore production. The
specific growth rate in iron-restricted batch cul-
tures was 0.22 h-', which was reduced to 0.12 h-'
when no iron was added to the culture.
Dcrit
in iron-limited chemostat culture was 0.1 h-'.
Siderophore production was correlated with
specific growth rate, with the highest siderophore
production occurring at
D
=
0.08
h-' and the
lowest at
D
=
0.03 h-'. Siderophore production
was greatest at pH 4.7 and was significantly
reduced at pHs above 6.0. Siderophore production
could be enhanced by providing insoluble iron
instead of soluble iron in continuous flow cultures.
Introduction
Siderophores are produced by micro-organisms to
chelate and take up ferric iron from the environ-
ment, in which it is typically available in an
insoluble form [l]. The majority of filamentous
fungi produce hydroxamate-type siderophores,
derived from
N'-acyl-NS-hydroxyornithine,
with
the hydroxamic acid forming the functional group
[2]. Although it is possible to produce siderophores
by chemical synthesis, the processes are often slow
and expensive [3], and there is continued interest
in producing and studying siderophores from
biological systems. Therefore it is important to
establish conditions under which optimal sidero-
phore production will take place. This paper
Key words chernostat, iron limitation.
Abbreviation used.
DFO,
desfemoxamine B.
IE-mail rngw@bio auc dk
describes the effect of iron-restricted growth on
the specific growth rate of
Fusarium venenatum
A315 and the production
of
siderophores by
F. venenatum
A315 in iron-restricted cultures.
The effects of iron concentration, dilution rate
and pH were assessed.
Materials and methods
F. venenatum
A315 was obtained from Mr
T.
W.
Naylor (Marlow Foods, Billingham, Cleve-
land, U.K.). The defined medium contained
(per litre) 10 g
of
glucose, 3.3 g of (NH,),SO,,
0.3 g of KH,PO,, 0.2 g of MgSO,
.
7H,O, 0.1 g
of CaCI,. 2H,O,
5
mg of citric acid,
5
mg of
ZnSO,
.
7H,0,0.26 mgofCuSO,
.
5H,0,0.05 mg
of MnSO, .4H,O,
0.05
mg of H,BO,,
0.05
mg of
NaMoO,
.
2H,O and
0.05
mg of biotin. For con-
tinuous flow cultures, the same medium was
used at half-concentration. Iron was provided as
FeCl,
.
6H,O or as a suspension
of
FeO(0H). All
media were prepared using MilliQ-purified water.
Batch cultures were grown in 250 ml shake
flasks containing
50
ml of medium, buffered to
pH
5.8
with Mes and incubated on rotary shakers
at 200 rev./min. Chemostat cultures were grown
in either a Braun Biostat M or an Infors IFSlOO
(working volumes 2.1-2.3
1)
as described by Wiebe
and Trinci [4]. The pH was kept constant by
addition of
0.5
M NaOH and foaming was con-
trolled by addition of polypropylene glycol.
FeCl,
.
6H,O was added to the glucose/mineral
salts medium, but FeO(0H) was suspended in
0.15
yo
(w/v) sodium polyacrylic acid and supplied
to the cultures in a separate feed line (4 ml
.
h-') in
order to avoid problems with insoluble particles
settling in the medium reservoir.
Maximum specific growth rates were meas-
ured by increase in attenuance using a Cecil
Ce7200 spectrophotometer. For bioreactor cul-
0
2002
Biochemical Society
696
