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Journal of Bacteriology, December 1999, p. 7588-7596, Vol. 181, No. 24
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Multifunctional ATP-Binding Cassette Transporter
System from Vibrio cholerae Transports Vibriobactin
and Enterobactin
Elizabeth E.
Wyckoff,*
Ana-Maria
Valle,
Stacey L.
Smith, and
Shelley M.
Payne
Department of Molecular Genetics and
Microbiology, and Institute for Cellular and Molecular Biology,
University of Texas, Austin, Texas 78712-1095
Received 14 July 1999/Accepted 5 October 1999
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ABSTRACT |
Vibrio cholerae uses the catechol siderophore
vibriobactin for iron transport under iron-limiting conditions. We have
identified genes for vibriobactin transport and mapped them within the
vibriobactin biosynthetic gene cluster. Within this genetic region we
have identified four genes, viuP, viuD,
viuG and viuC, whose protein products have
homology to the periplasmic binding protein, the two integral
cytoplasmic membrane proteins, and the ATPase component, respectively,
of other iron transport systems. The amino-terminal region of ViuP has
homology to a lipoprotein signal sequence, and ViuP could be labeled
with [3H]palmitic acid. This suggests that ViuP is a
membrane lipoprotein. The ViuPDGC system transports both vibriobactin
and enterobactin in Escherichia coli. In the same assay,
the E. coli enterobactin transport system, FepBDGC, allowed
the utilization of enterobactin but not vibriobactin. Although the
entire viuPDGC system could complement mutations in
fepB, fepD, fepG, or
fepC, only viuC was able to independently
complement the corresponding fep mutation. This indicates
that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in
viuG were constructed by marker exchange. These mutations
reduced, but did not completely eliminate, vibriobactin utilization.
This suggests that V. cholerae contains genes in addition
to viuPDGC that function in the transport of catechol siderophores.
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INTRODUCTION |
Pathogenic bacteria require iron for
growth and survival. In most environments, however, iron is not readily
available. In the mammalian host, the vast majority of iron is present
in intracellular iron proteins, such as hemoproteins and ferritin. Most
of the extracellular iron is complexed with proteins, such as
transferrin, lactoferrin, hemopexin, or haptoglobin. The supply of free
iron also is limited in many environments outside the human host. To obtain iron, bacteria have evolved high-affinity iron acquisition systems, and many bacteria have several of these systems
(6). Some of these iron acquisition systems support direct
utilization of iron from specific host iron compounds. Others involve
the production and transport of siderophores, which are small molecules that bind iron with high affinity and are then transported back into
the cell. The expression of iron acquisition genes is repressed by
iron, usually by the negative regulatory transcription factor Fur
(4).
The gram-negative pathogen Vibrio cholerae, the causative
agent of cholera (13, 25), has multiple iron transport
systems. V. cholerae transports free heme and
hemoglobin-associated heme through the action of the HutABCD system
(21, 22, 31). V. cholerae strains also transport
siderophores, including the hydroxamate siderophore ferrichrome
(17), although the production of hydroxamate siderophores
has not been observed in V. cholerae (42). The siderophore produced and used by most V. cholerae strains is
the catechol vibriobactin (17) (Fig.
1). Vibriobactin contains three 2,3-dihydroxybenzoyl residues linked to a backbone of norspermidine, an
abundant polyamine in most members of the Vibrionaceae
(55, 56). Two of these dihydroxybenzoyl residues are joined
to the backbone via L-threonine, whereas the third is
linked directly to the norspermidine moiety (17) (Fig. 1).
Dihydroxybenzoate is synthesized from chorismate by VibABC (17,
53). The late steps in vibriobactin biosynthesis, in which
dihydroxybenzoate, threonine, and norspermidine are joined to each
other, are not well understood, but at least four proteins, VibD, VibE,
VibF, and VibH, are required for this process.
Less is known about the transport of vibriobactin. Because vibriobactin
is structurally similar to enterobactin, the catechol siderophore
produced by Escherichia coli and related species (Fig. 1),
it appeared likely that the transport systems would be similar. The
E. coli Fep system, which transports enterobactin, is
typical of high-affinity iron transport systems found in gram-negative bacteria. These consist of several components. The outer membrane receptors, such as FepA, bind their ligand with high affinity (6,
7, 11). Transport of the ligand through the outer membrane
requires the activity of TonB, ExbB, and ExbD, which are thought to
transduce the energy required for transport (30). Transport
of the siderophore through the periplasm and across the inner membrane
requires a periplasmic binding protein-dependent ATP-binding cassette
(ABC) transport system (5, 6). In this system, the
siderophore specifically binds its periplasmic binding protein, e.g.,
FepB, which then delivers the ligand to the corresponding inner
membrane permease complex. The permease usually consists of two
integral membrane proteins, which form a tight complex with each other
within the cytoplasmic membrane. Each of the integral membrane proteins
is bound to an additional protein that has ATPase activity. The
hydrolysis of ATP by the ATPase subunit is thought to generate the
energy for transport of the ligand across the inner membrane. In
E. coli, FepD and FepG form the integral membrane permease
complex (10, 41), and FepC is the ATPase (41). Following transport of enterobactin, a cytoplasmic protein, Fes, catalyzes the removal of iron from the ferri-siderophore complex (11).
In V. cholerae, the vibriobactin receptor, ViuA, has been
identified and characterized (9, 45). Like enterobactin,
vibriobactin transport is dependent upon a functional TonB system
(22, 31). Another vibriobactin utilization protein, ViuB, is
analogous to Fes and removes the iron from the iron-vibriobactin
complex in the cytoplasm (8). Prior to this work, however,
there was no information available about the transport of vibriobactin
through the periplasm and across the cytoplasmic membrane. In this
report we describe viu genes located within the vibriobactin
biosynthetic gene cluster which encode an ABC transporter system. The
system encoded by these genes is different from Fep in that it can
transport both vibriobactin and enterobactin. Further, we show that the Viu and Fep permeases each function as a complex, and, with the exception of the ATPase homologues, the individual Viu and Fep proteins
are not interchangeable. We also present evidence that V. cholerae has an additional system for the transport of catechol siderophores.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
Bacterial strains
and plasmids used in this study are listed in Table
1. The iron chelator ethylenediamine
di(ortho-hydroxyphenylacetic acid) (EDDA) was deferrated by
the method of Rogers (36). When added, the antibiotic
concentrations used were 250 µg of carbenicillin per ml, 50 µg of
kanamycin per ml, and 50 µg of chloramphenicol per ml (for E. coli) or 5 µg of chloramphenicol per ml (for V. cholerae).
DNA sequencing.
DNA was sequenced by using an Applied
Biosystems Prism 377 DNA sequencer (Perkin-Elmer Corp.). Routine DNA
sequence analysis was performed by using the program DNA Strider
(27). Amino acid sequence alignments were performed by using
the gap program of the Genetics Computer Group DNA sequence analysis
package. The BLAST program (2) was used to search the
National Center for Biotechnology Information protein database.
Detection of siderophore production and utilization.
The
colorimetric test for the production of catechols was performed by the
method of Arnow (3). The bioassay for siderophore production
and utilization was performed as previously described (53).
Construction of chromosomal mutations in V. cholerae.
To construct a mutation in viuP, a SmaI fragment
containing the chloramphenicol cassette from pMTLcam (54)
was inserted into the MscI site in viuP in the
plasmid pVIB119 to give the plasmid pVIB148. The PstI
fragment containing the disrupted viuP gene was then
subcloned into the PstI site of pWSc1 (31), a plasmid carrying the sacB gene, which confers sensitivity to
sucrose (15). This plasmid was introduced into the V. cholerae wild-type strain Lou15 by electroporation, and the marker
exchange mutant EWV103 was obtained by selecting colonies resistant to
both sucrose and chloramphenicol as previously described
(53). To construct SSV121, containing a mutation in
viuG, the chloramphenicol gene from pMA9 was inserted into
the StuI site within viuG in the plasmid pVIB121.
The SalI-XbaI fragment of the resulting plasmid
was cloned into SalI-XbaI-digested pHM5, to give
pUNK143. Following transfer of pUNK143 by conjugation into Lou15,
sucrose-resistant, chloramphenicol-resistant colonies were selected.
The presence of the cam cassette within the chromosomal
viuP gene in strain EWV103 and within viuG in SSV121 was confirmed by Southern hybridization (data not shown).
Construction of pViuAB.
pViuAB was constructed by PCR
amplification of the viuA and viuB genes from
V. cholerae O395. The PCR was performed as follows: 1 ml of
an overnight culture of O395 was pelleted by centrifugation and was
resuspended in 100 µl of sterile water. One microliter of this cell
suspension was mixed with 100 pmol of each deoxynucleoside triphosphate, 50 pmol of each primer, Qiagen PCR buffer (1× final concentration), 1.25 U of Taq polymerase (Qiagen), and 1.25 U of Pfu polymerase (Stratagene) in a total volume of 100 µl. The reaction was 95°C for 5 min, followed by 30 cycles of
94°C for 30 s, 45°C for 60 s, and 72°C for 20 min in a
GeneAmp PCR system 2400 (Perkin-Elmer). The primer sequences were
5'-AAGCTTTGTAGGAAGGGAA and
5'-TCTAGATAAGCAATGTGCTCATAAA. The 3.6-kbp product was made blunt with Klenow and ligated in the SmaI site of pWSK29
(50).
Labeling of lipoproteins with [3H]palmitic
acid.
Overnight cultures were diluted into 1 ml of L broth
containing 20 µCi of [3H]palmitic acid per ml and 50 µg of carbenicillin per ml. Cultures were grown for 8 h with
shaking at 37°C. Cells were harvested by centrifugation, resuspended
in 50 µl of sodium dodecyl sulfate (SDS) gel sample buffer and lysed
by boiling for 15 min. Proteins were separated by SDS-polyacrylamide
gel electrophoresis (PAGE). The gel was treated with
EN3HANCE (NEN Life Science Products, Inc., Boston, Mass.),
and [3H]-labeled proteins were visualized by fluorography.
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RESULTS AND DISCUSSION |
Identification of genes encoding potential iron transport
proteins.
As a continuation of our studies of V. cholerae iron transport systems, we characterized genes for the
transport of vibriobactin across the cytoplasmic membrane. Because
siderophore synthesis and transport genes are often clustered, we
tested previously isolated cosmid clones containing vibriobactin
biosynthetic genes (53) for the presence of iron transport
functions. Preliminary experiments showed that one of the cosmid clones
complemented a mutation in the E. coli enterobactin
transport gene, fepC (data not shown). Further subcloning
and complementation experiments indicated that the V. cholerae iron transport protein genes reside in a 4,100-bp region
which is within the vibriobactin synthetic gene cluster (Fig.
2). The nucleotide sequence of this
region was obtained on both strands (GenBank accession no. U52150). This region contained four open reading frames (ORFs), which have been
named viuPDGC. They are flanked by the vibriobactin
biosynthetic genes vibH and vibD (Fig. 2 and
unpublished data).
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FIG. 2.
Organization of the vibriobactin transport genes and
complementation of E. coli fep mutations. The horizontal
arrows indicate the direction of transcription of the viu
genes, and selected restriction sites are indicated below the line. The
vertical arrowheads above the lines indicate the position of the
cam cassette insertion in the marker exchange mutations in
EWV103 and SSV121. The arrow in pVIB154 indicates the position of
insertion of a cam cassette into the MscI site in
viuP. The ability of plasmids to complement E. coli
fep mutants in a bioassay is shown to the right. +, growth on
enterobactin;  , no growth on enterobactin. Strains used for
complementation studies were as follows: fepB, AB1515.414;
fepD, AB1515.718; fepG, AB1515.764;
fepC, AB1515.199.
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The first ORF,
viuP, encodes a protein with a predicted
molecular weight of 36,100 and predicted pI of 5.97. It has amino
acid
sequence homology with
E. coli FepB (Table
2), the periplasmic
binding protein for
the transport of enterobactin (
12). The
amino-terminal
region of ViuP is rich in hydrophobic amino acids
but lacks a good
match to the standard signal peptidase cleavage
site (
34)
(Fig.
3A). The ViuP amino-terminal
sequence is a better
match to the consensus sequence for the
prolipoprotein signal
peptidase (Fig.
3D) (
52), suggesting
that ViuP may be a lipoprotein.
In gram-positive bacteria, the binding
protein components of membrane
transport systems are membrane
lipoproteins (
46). In gram-negative
bacteria, a few of the
periplasmic binding protein homologues
also appear to be membrane
lipoproteins. Examples include the
FatB protein for anguibactin
transport in
Vibrio anguillarum (
1)
and the CeuE
protein for enterobactin transport in
Campylobacter jejuni
and
Campylobacter coli (
32,
35). In bacterial
lipoproteins,
the lipid is covalently attached to a cysteine residue
present
within a consensus sequence located near the amino-terminal end
of the protein (
34,
46,
52). The cysteine residue in the
amino-terminal region of ViuP aligns with the cysteines in FatB
and in
CeuE that are believed to be the modified residues (Fig.
3D).
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FIG. 3.
Portions of the nucleotide sequence and the predicted
amino acid sequences of ViuP and related proteins. (A) The nucleotide
sequence and predicted translation of the 5' end of viuP and
the sequence of the vibH-viuP intergenic region. Possible
fur boxes for vibH and viuP are
underlined. The potential cleavage site for lipoprotein modification is
indicated by an arrowhead. (B) The viuG-viuC intergenic
region. An inverted repeat that may function as a transcription
terminator is indicated by arrows, and a possible fur box
for the viuC transcript is underlined. (C) The
viuC-vibD intergenic region. The end of viuC and
predicted start of vibD are shown. Three potential ATG start
codons are present at the 5' end of the vibD ORF. (D) The
predicted amino terminal sequence of FatB (1), C. coli CeuE (35), HutB (31), and ViuP. The
consensus lipoprotein cleavage site is also shown; the asterisk below
the alignments indicates the conserved cysteine.
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To determine whether ViuP is a membrane lipoprotein,
E. coli
carrying either pBluescript encoding
viuP or the pBluescript
vector as a control were labeled with [
3H]palmitic acid.
SDS-PAGE of tritium-labeled proteins showed the
presence of a labeled
protein with a molecular weight of approximately
35,000, the predicted
size of ViuP. This labeled protein was present
in cells carrying
viuP on a plasmid, but not in cells carrying
the pBluescript
vector (Fig.
4), suggesting that ViuP is
a lipoprotein.
Interestingly, a cysteine in HutB, the putative
periplasmic binding
protein for heme transport in
V. cholerae, also can be aligned
with the cysteine in FatB and ViuP
as shown by a ClustalW alignment
of the amino-terminal regions of these
three proteins (Fig.
3D).
These data raise the possibility that
attachment of the binding
protein component of iron transport systems
to the inner membrane
may be a common feature in
Vibrio spp.
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FIG. 4.
Visualization of cellular lipoproteins. E. coli DH5 carrying either pBluescript (lane 1) or pVIB119
encoding viuP (lane 2) were grown in the presence of
[3H]palmitic acid. Proteins were then separated by
SDS-12.5% PAGE and visualized by fluorography. The arrow indicates
the position of ViuP. The position of the 30-kDa marker is shown at the
left.
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The second ORF,
viuD, encodes a protein with homology to
FepD and other integral membrane proteins of cytoplasmic membrane
permeases (Table
2).
viuD has two possible translation
starts,
one overlapping the
viuP stop codon and a second,
in-frame ATG
four codons downstream of the first one. The tight linkage
of
viuP and
viuD suggests that they are
cotranscribed. The calculated
molecular weight for the larger possible
ViuD protein is 37,033,
and the calculated pI is 9.40. Highly basic pIs
are typical of
cytoplasmic membrane permease proteins. ViuD is
extremely hydrophobic,
as would be expected for an integral membrane
protein. Staudenmaier
et al. (
44) found that the inner
membrane permease proteins
of iron transport systems share a high level
of sequence conservation
throughout their entire length and also
identified seven regions
of these proteins with especially high amino
acid sequence conservation.
Each of these seven sequence motifs is
present within the ViuD
sequence (data not
shown).
The third ORF,
viuG, encodes a protein with homology to FepG
and other cytoplasmic membrane permease proteins (Table
2).
The
translational start site of
viuG has not been identified,
but we believe that the most likely start is an ATG 20 nucleotides
upstream of the
viuD stop codon. The predicted protein has a
molecular
weight of 37,470 and a pI of 11.36. Like ViuD, ViuG has the
amino
acid sequence features characteristic of cytoplasmic membrane
permease proteins. As has been reported for other inner membrane
protein pairs, ViuD and ViuG have homology with each other (Table
2).
The fourth ORF in the gene cluster,
viuC, encodes a protein
with homology to FepC and other ATPase proteins of ABC transporters
(Table
2). ViuC contains both Walker motif A, GPNGCGKS (amino
acids 52 to 60), and motif B, YLLLDEPT (amino acids 177 to 184)
(
49),
indicating that it is likely to be the ATPase component
of the complex.
It has a predicted molecular weight of 30,914
and a predicted pI of
7.56. ViuC is more hydrophilic than either
ViuD or ViuG, consistent
with it being more loosely associated
with the cytoplasmic membrane.
The vibriobactin biosynthetic gene
vibD is located
immediately downstream of
viuC (Fig.
3C).
Upstream of the
viuP translation start is a potential
promoter sequence (Fig.
3A). This region contains a sequence with
homology
to the
E. coli Fur box, suggesting that the
expression of these
genes is regulated by iron. Approximately 200 bp
upstream of
viuP is the potential promoter region for the
vibriobactin biosynthetic
gene
vibH, which also contains a
potential Fur binding site. The
vibH promoter does not
appear to overlap the promoter for
viuP.
In
E. coli,
fepB and the divergently transcribed gene
entC are
separated by a complex region that contains direct
repeats, possible
stem-loop structures, and an ORF of unknown function
(
12). The
function of these features is not well understood,
but they appear
to influence
fepB expression
(
40). No direct or inverted repeat
sequences are located
between the
viuP and
vibH genes, and no
ORFs of
significant size are present, indicating that the regulation
of
viuP is likely to be different from that of
fepB.
viuG and
viuC are separated by 116 nucleotides
(Fig.
3B). This region contains a small, inverted repeat sequence,
which might
function as a rho-independent transcription terminator. The
region
also contains a sequence with homology to the consensus Fur box.
These data suggest that
viuC is transcribed separately from
viuPDG.
The presence of a promoter in this region is
supported by the
complementation data shown
below.
Frequently, genes for inner membrane permease proteins are linked to
the gene for the outer membrane receptor for the same
ligand
(
11). Our analysis of this region did not reveal the
presence of the vibriobactin receptor gene,
viuA, or any
other
potential outer membrane receptor protein gene. Data from the
unfinished TIGR (The Institute for Genomic Research)
V. cholerae database also failed to show a closely linked potential
receptor
protein gene. Recent physical mapping studies indicate that
the
V. cholerae genome consists of two large replicons.
viuA and the
vibriobactin gene cluster described here are
both located on replicon
I but are separated by about 10
6
bp (
48).
Complementation of E. coli fep mutations with
viu region clones.
Preliminary complementation data,
together with the sequence data presented above, suggest that the
ViuPDGC proteins function as a periplasmic binding protein-dependent
transport system. In initial experiments to determine the functions of
these genes, segments of the viu gene region were subcloned
into low-copy-number vectors, and these plasmids were tested for the
ability to complement mutations in E. coli fep genes (Fig.
2). E. coli strains with a Tn5 insertion in one
of each of the fep genes were transformed with a plasmid
containing the entire viu region (pVIB147). Each of these
transformed strains was able to use the siderophore enterobactin as an
iron source, as measured in a bioassay (Fig. 2). This indicates that
the four Viu proteins can functionally substitute for the Fep proteins
in the transport of enterobactin.
In the above experiments, all four of the
viu genes were
present on the plasmids. Additional plasmids were constructed to
determine whether the Viu proteins must assemble together as a
Viu
complex, or whether the individual Viu protein ViuP, ViuD,
ViuG, or
ViuC can replace the homologous Fep protein, FepB, FepD,
FepG, or FepC,
respectively, to form an active transport complex.
The plasmid pVIB154,
in which a polar chloramphenicol cassette
was inserted into the
MscI site in
viuP (Fig.
2), complemented
a
mutation in
fepC but not in
fepB,
fepD, or
fepG. The cassette
inserted into
viuP should be polar on
viuD and
viuG
but not on
viuC, which appears to have its own promoter.
Thus,
viuC is the
only gene likely to be expressed from this
plasmid, suggesting
that ViuC can substitute for FepC. This is
supported by the observation
that the plasmid pVIB109, containing
viuG and
viuC also complemented
the
fepC mutation. pVIB109 did not complement the
fepG mutation.
The plasmid pVIB159, containing
viuP, failed to complement a mutation
in the
viuP
homologue,
fepB. Although the
E. coli fepB
mutation
is a Tn
5 insertion and is polar on downstream
genes, the
fepDGC genes are transcribed from a separate
promoter (
11), and thus
the transport defect is specific for
fepB. When the plasmids pVIB109
and pVIB159 were carried
together in the same cell, complementation
of each of the
fep mutations was observed (Fig.
2). This indicates
that the
lack of
fepB or
fepG complementation observed
with a
single plasmid is not due to poor expression of the
viuP and
viuG genes from these
plasmids.
The observation that pVIB109 and pVIB159 did not individually
complement the
fepBDG mutations, but did complement these
mutations
when present together, suggests that the proteins encoded by
these
two plasmids may interact. We propose that ViuPDGC function
together
as a unit and are unable to form the proper protein-protein
contacts
with
E. coli Fep proteins. Thus, with the exception
of ViuC, the
individual Viu proteins are unable to assemble with Fep
proteins
to form an active permease complex. The ability of the ATPase
homologue, ViuC, to functionally substitute for FepC is consistent
with
observations in other ABC transport systems, where some ATPase
subunits
can function with two different inner membrane permease
complexes
(
20,
39,
51).
Transport of vibriobactin by the ViuPDGC proteins.
The ability
of the viuPDGC genes to complement E. coli fep
mutations indicates that they encode a transport system capable of
transporting enterobactin across the cytoplasmic membrane. The presence
of these genes within a vibriobactin biosynthetic cluster, however,
suggested that their usual ligand in V. cholerae is
vibriobactin. E. coli strains carrying only the
viuPDGC genes do not transport vibriobactin. These E. coli strains, however, lack both ViuA, the vibriobactin outer
membrane receptor, and ViuB, which catalyzes the removal of iron from
the ferri-vibriobactin complex. To test for vibriobactin transport in
E. coli, we first constructed the plasmid, pViuAB, which
contains the viuA and viuB genes. The plasmids
pViuAB and pVIB147 are incompatible, so E. coli fep mutants
carrying pViuAB were transformed with the compatible plasmid pJSV90,
which encodes ViuPDGC (53). These strains were then tested
for the ability to transport vibriobactin and enterobactin (Table
3). Strains carrying pViuAB together with
the viuPDGC genes encoded on pJSV90 formed large zones of
growth around both Lou15 and DH5, indicating that the ViuPDGC
proteins are capable of transporting vibriobactin as well as
enterobactin. This ability of ViuPDGC to transport these two rather
dissimilar catechols (Fig. 1) is reminiscent of hydroxamate transport
in E. coli. In this system, each hydroxamate has a specific
outer membrane receptor, but are all transported across the cytoplasmic
membrane by the same periplasmic binding protein and permease complex
(FhuBCD) (6). In our experiments, FepA and ViuA were
specific for their ligands, in that FepA transported enterobactin but
not vibriobactin and ViuA transported vibriobactin but not enterobactin
(data not shown).
To test whether the
E. coli FepBDGC proteins are also
capable of transporting both enterobactin and vibriobactin, the
fepBDGC genes were introduced into
fep mutant
strains carrying pViuAB.
These cells grew well around DH5
, but
growth around Lou15 was
not observed (Table
3). Thus, unlike
ViuPDGC, the
E. coli enterobactin
ABC transport system
transported only enterobactin in these experiments.
Thus, the ability
to transport both of these catechols does not
appear to be a universal
property of catechol transport systems.
The
E. coli FepBDGC
system is not absolutely specific for the
transport of enterobactin,
since it has been shown previously
to transport the catechol
dihydroxybenzoylserine (
19).
When neither pJSV90 (carrying
viuPDGC) nor pCP111 (carrying
fepBDGC) were present in
fep mutant strains
carrying pViuAB, very
weak growth was observed around Lou15 (Table
3).
The
fep mutant
strains used in this assay can synthesize,
but not use, enterobactin.
When the
fep genes are supplied
on a plasmid, a higher level of
background growth is observed, due to
the ability of the strains
to transport the enterobactin that they are
producing. This background
growth may obscure a small amount of growth
around an iron source.
In contrast, the level of background growth
observed with strains
unable to transport enterobactin is extremely
low, which allows
detection of very weak utilization of an iron source.
One of the
many systems that transports ligands across the inner
membrane
is likely also to transport vibriobactin at very low
efficiency.
Characterization of V. cholerae viu mutants.
To
determine the role of the viu genes in V. cholerae, chromosomal mutations were constructed in the wild-type
El Tor strain Lou15 using marker exchange. The strain EWV103 has a
chloramphenicol resistance cassette inserted in the MscI
site within viuP, and SSV121 has a chloramphenicol cassette
in the StuI site in viuG (Fig. 2). The ability of
the strains to use vibriobactin as an iron source was determined in a
bioassay using a high EDDA concentration (1 mg/ml) (Table
4). Under conditions of iron limitation,
the growth of the parental strain, Lou15, was stimulated by
vibriobactin, whereas both EWV103 and SSV121 failed to use vibriobactin
as an iron source. This indicates that ViuP and ViuG function in the transport of vibriobactin in V. cholerae. The mutants did
not have a general iron transport defect, since both EWV103 and SSV121 utilized the hydroxamate siderophore ferrichrome (Table 4). When the
mutant strains were transformed with plasmids carrying the viu genes, vibriobactin utilization was restored, indicating
that the observed defects were due to the viu mutation. Both
EWV103 and SSV121 stimulated the growth of V. cholerae
strains under conditions of iron limitation, indicating that neither
strain was defective in vibriobactin production (data not shown).
Evidence for additional genes for the transport of catechol
siderophores in V. cholerae.
The V. cholerae
strains were also tested for siderophore transport at a reduced EDDA
concentration of 500 µg/ml (Table 5). Transport of enterobactin has been previously observed in V. cholerae (38), and at this EDDA concentration, the
transport of enterobactin was observed in our assay. When assayed at
this EDDA concentration, the viu mutants used both
vibriobactin and enterobactin, but the zones were smaller and less
dense than those observed with the wild-type strain. In the mutant
strains, the zones around vibriobactin and enterobactin are of
approximately equal size, in contrast to the wild-type strain, which
had a larger zone around vibriobactin (Table 5). Although it was
necessary to reduce the EDDA concentration to observe growth of the
mutants on vibriobactin, the amount of EDDA used (500 µg/ml) is still
a relatively high concentration of the chelator. It is expected that a
high-affinity transport system would be required to observe iron
transport under these conditions. The ability of the viu
mutants to use vibriobactin under these conditions suggests the
presence of an additional system for the transport of catechol
siderophores in V. cholerae.
To investigate this further, the transport phenotype of a strain
carrying a mutation in the vibriobactin outer membrane receptor
gene,
viuA, was further examined. The strain MBG14
(
viuA::Tn
phoA)
and its classical
biotype parental strain, O395, were tested for
transport of
vibriobactin and enterobactin (Table
4). MBG14 was
completely defective
in vibriobactin transport, consistent with
previous characterization of
this mutant (
45). Transport of
enterobactin, however, was
unaffected in the mutant, indicating
that enterobactin enters the cell
via a ViuA-independent transport
system. This indicates that additional
genes for the transport
of catechols are present in
V. cholerae.
In this paper, we have presented additional characterization of the
genes within a vibriobactin locus in
V. cholerae. Four
of
the genes within this locus encode proteins with sequence homology
to
periplasmic binding proteins and cytoplasmic permease proteins
(Table
2). These proteins transport both vibriobactin and enterobactin
in
E. coli. With the exception of ViuC, they appear to function
together as a complex rather than assembling with the
E. coli Fep proteins to form a mixed complex. In
V. cholerae, the Viu
proteins function in the transport of
vibriobactin, but additional
proteins that transport catechol
siderophores are also
present.
A model for the transport of catechols in
V. cholerae is
presented in Fig.
5. In this model,
vibriobactin crosses the outer
membrane through ViuA, and enterobactin
crosses through a separate
outer membrane protein, which is specific
for enterobactin. This
protein has been designated VctA for
Vibrio catechol transport.
The ViuPDGC system can transport
both vibriobactin and enterobactin
in
E. coli, and we
anticipate that it can also transport both
of these ligands in
V. cholerae. Because the
viuP and
viuG
mutations
reduced, but did not completely abolish, catechol transport,
we
also propose that
V. cholerae contains at least one
additional
system for the transport of catechol siderophores across the
inner
membrane (designated VctPDGC). In Fig.
5, we show a single,
additional
cytoplasmic membrane permease system that transports both
vibriobactin
and enterobactin. This is the simplest model consistent
with the
data, but more complex models involving multiple, additional
permease
systems are also possible. At this time, there is no
information
about the organization of these unidentified catechol
transport
protein genes. It is also not known whether the Vct system
transports
intact vibriobactin and enterobactin, or whether it
transports
breakdown products derived from these siderophores. As shown
in
Fig.
5, the final step in iron transport is the removal of iron
from
the iron-siderophore complex by ViuB. Previously published
data suggest
that ViuB can function in the utilization of both
ferri-vibriobactin
and ferri-enterobactin (
8).
It has been difficult to determine the relative contribution of the
different iron transport systems to the survival and growth
of
V. cholerae in mammalian hosts. Classical strains carrying
a mutation
in the gene for vibriobactin synthesis (
43), vibriobactin
transport (
23), or heme transport (
23,
47) are
only weakly
attenuated in animal models. The virulence of the
viuA-hutA double
mutant was more attenuated than any of the
single mutants, suggesting
that both of these transport systems
contribute to iron acquisition
in vivo (
23,
47). While it is
possible that an unidentified
system is responsible for most iron
acquisition in vivo, it is
more likely that multiple systems are
present, each contributing
to the acquisition of iron in different
environments or at different
times during the course of infection. The
presence of multiple
systems may reflect the overall importance of iron
acquisition
in this
organism.
| |
ACKNOWLEDGMENTS |
This work was supported by the Foundation for Research and by
grant AI16935 from the National Institutes of Health.
We thank Charles Earhart for providing strains and for helpful
discussions. We also thank Charles Earhart, Douglas Henderson, and
Laura Runyen-Janecky for comments on the manuscript and Chris Tinkle
for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712-1095. Phone: (512) 471-5204. Fax: (512) 471-7088. E-mail: ewyckoff{at}mail.utexas.edu.
| |
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Abstract
Introduction
