• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Dec 1999; 181(24): 7588–7596.
PMCID: PMC94218

A Multifunctional ATP-Binding Cassette Transporter System from Vibrio cholerae Transports Vibriobactin and Enterobactin

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.

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. (Fig.1).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. (Fig.1).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.

FIG. 1
Structure of the catechol siderophores enterobactin and vibriobactin (17).

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. (Fig.1),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.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media.

Bacterial strains and plasmids used in this study are listed in Table Table1.1. 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).

TABLE 1
Bacterial strains and plasmids used in this study

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.

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. (Fig.2).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. (Fig.22 and unpublished data).

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 ...

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 (Table2),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. (Fig.3A).3A). The ViuP amino-terminal sequence is a better match to the consensus sequence for the prolipoprotein signal peptidase (Fig. (Fig.3D)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. (Fig.3D).3D).

TABLE 2
Selected homologies of vibriobactin transport proteins to other transport proteins
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 ...

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. (Fig.4),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. (Fig.3D).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.

FIG. 4
Visualization of cellular lipoproteins. E. coli DH5α 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% ...

The second ORF, viuD, encodes a protein with homology to FepD and other integral membrane proteins of cytoplasmic membrane permeases (Table (Table2).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 (Table2).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 (Table22).

The fourth ORF in the gene cluster, viuC, encodes a protein with homology to FepC and other ATPase proteins of ABC transporters (Table (Table2).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. (Fig.33C).

Upstream of the viuP translation start is a potential promoter sequence (Fig. (Fig.3A).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. (Fig.3B).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 106 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. (Fig.2).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. (Fig.2).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. (Fig.2),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 Tn5 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. (Fig.2).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 (Table3).3). Strains carrying pViuAB together with the viuPDGC genes encoded on pJSV90 formed large zones of growth around both Lou15 and DH5α, 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. (Fig.1)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).

TABLE 3
ViuPDGC, but not FepBDGC, transports vibriobactin in E. coli

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 DH5α, but growth around Lou15 was not observed (Table (Table3).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 (Table3).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. (Fig.2).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 (Table4).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 (Table4).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).

TABLE 4
Vibriobactin transport at high EDDA concentrations requires the Viu proteins in V. choleraea

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 (Table5).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 (Table5).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.

TABLE 5
Evidence for an additional catechol transport system in V. choleraea

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::TnphoA) and its classical biotype parental strain, O395, were tested for transport of vibriobactin and enterobactin (Table (Table4).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 (Table2).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. Fig.5.5. 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. Fig.5,5, 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. Fig.5,5, 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).

FIG. 5
Model for catechol siderophore transport in V. cholerae.

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.

REFERENCES

1. Actis L A, Tolmasky M, Crosa L M, Crosa J. Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid. Mol Microbiol. 1995;17:197–204. [PubMed]
2. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. [PubMed]
3. Arnow L E. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine tyrosine mixtures. J Biol Chem. 1937;118:531–537.
4. Bagg A, Neilands J B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987;26:5471–5477. [PubMed]
5. Boos W, Lucht J M. Periplasmic binding protein-dependent ABC transporters. In: Neidhardt F C, Curtiss III R, Ingraham J L, Lin E C C, Brooks Low K, Magasanik B, Reznikoff W S, Riley M, Schaechter M, Umbarger H E, editors. Escherichia coli and Salmonella: cellular and molecular biology. 2nd ed. Vol. 1. Washington, D.C.: ASM Press; 1996. pp. 1175–1209.
6. Braun V, Hantke K, Koster W. Bacterial iron transport: mechanisms, genetics, and regulation. In: Sigel A, Sigel H, editors. Metal ions in biological systems. Vol. 35. New York, N.Y: Marcel Dekker; 1998. pp. 67–145. [PubMed]
7. Buchanan S K, Smith B S, Venkatramani L, Xia D, Esser L, Palnitkar M, Chakraborty R, van der Helm D, Deisenhofer J. Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nat Struct Biol. 1999;6:56–63. [PubMed]
8. Butterton J R, Calderwood S B. Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae. J Bacteriol. 1994;176:5631–5638. [PMC free article] [PubMed]
9. Butterton J R, Stoebner J A, Payne S M, Calderwood S B. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992;174:3729–3738. [PMC free article] [PubMed]
10. Chenault S S, Earhart C F. Organization of genes encoding membrane proteins of the Escherichia coli ferrienterobactin permease. Mol Microbiol. 1991;5:1405–1413. [PubMed]
11. Earhart C F. Uptake and metabolism of iron and molybdenum. In: Neidhardt F C, Curtiss III R, Ingraham J L, Lin E C C, Brooks Low K, Magasanik B, Reznikoff W S, Riley M, Schaechter M, Umbarger H E, editors. Escherichia coli and Salmonella: cellular and molecular biology. 2nd ed. Vol. 1. Washington, D.C.: ASM Press; 1996. pp. 1075–1090.
12. Elkins M F, Earhart C F. Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. J Bacteriol. 1989;171:5443–5451. [PMC free article] [PubMed]
13. Faruque S M, Albert M J, Mekalanos J J. Epidemiology, genetics and ecology of toxigenic Vibrio cholerae. Microbiol Mol Biol Rev. 1998;62:1301–1314. [PMC free article] [PubMed]
14. Fiss E H, Yu S, Jacobs W R. Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways. Mol Microbiol. 1994;14:557–569. [PubMed]
15. Gay P, LeCoq D, Steinmetz M, Berkelman T, Kado C I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985;164:918–921. [PMC free article] [PubMed]
16. Goldberg M B, DiRita V J, Calderwood S B. Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis. Infect Immun. 1990;58:55–60. [PMC free article] [PubMed]
17. Griffiths G L, Sigel S P, Payne S M, Neilands J B. Vibriobactin, a siderophore from Vibrio cholerae. J Biol Chem. 1984;259:383–385. [PubMed]
18. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983;166:557–580. [PubMed]
19. Hantke K. Dihydroxybenzoylserine—a siderophore for E. coli. FEMS Microbiol Lett. 1990;67:5–8. [PubMed]
20. Hekstra D, Tommassen J. Functional exchangeability of the ABC proteins of the periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli. J Bacteriol. 1993;175:6546–6552. [PMC free article] [PubMed]
21. Henderson D P, Payne S M. Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins. J Bacteriol. 1994;176:3269–3277. [PMC free article] [PubMed]
22. Henderson D P, Payne S M. Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin. Mol Microbiol. 1993;7:461–469. [PubMed]
23. Henderson D P, Payne S M. Vibrio cholerae iron transport systems: role of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems. Infect Immun. 1994;62:5120–5125. [PMC free article] [PubMed]
24. Hong M, Payne S M. Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance. Mol Microbiol. 1997;24:779–791. [PubMed]
25. Kaper J B, Morris J G, Levine M M. Cholera. Clin Microbiol Rev. 1995;8:48–86. [PMC free article] [PubMed]
26. Mahe B, Masclaux C, Rauscher L, Enard C, Expert D. Differential expression of two siderophore-dependent iron-acquisition pathways in Erwinia chrysanthemi 3937: characterization of a novel ferrisiderophore permease of the ABC transporter family. Mol Microbiol. 1995;18:33–43. [PubMed]
27. Marck C. “DNA strider”: a “C” program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988;16:1829–1836. [PMC free article] [PubMed]
28. Martinez E, Bartolomé B, de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ α reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988;68:159–162. [PubMed]
29. Miller V L, Mekalanos J J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae required toxR. J Bacteriol. 1988;170:2575–2583. [PMC free article] [PubMed]
30. Moeck G S, Coulton J W. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol Microbiol. 1998;28:675–681. [PubMed]
31. Occhino D A, Wyckoff E E, Henderson D P, Wrona T J, Payne S M. Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes. Mol Microbiol. 1998;29:1493–1507. [PubMed]
32. Park S F, Richardson P T. Molecular characterization of a Campylobacter jejuni lipoprotein with homology to periplasmic siderophore-binding protein. J Bacteriol. 1995;177:2259–2264. [PMC free article] [PubMed]
33. Pierce J R, Earhart C F. Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake. J Bacteriol. 1986;166:930–936. [PMC free article] [PubMed]
34. Pugsley A P. The complete general secretory pathway in gram-negative bacteriae. Microbiol Rev. 1993;57:50–108. [PMC free article] [PubMed]
35. Richardson P T, Park S F. Enterochelin acquisition in Campylobacter coli: characterization of components of a binding-protein-dependent transport system. Microbiology. 1995;141:3181–3191. [PubMed]
36. Rogers H J. Iron-binding catechols and virulence in Escherichia coli. Infect Immun. 1973;7:445–456. [PMC free article] [PubMed]
37. Runyen-Janecky L J, Hong M, Payne S M. Virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation. Infect Immun. 1999;67:1415–1423. [PMC free article] [PubMed]
38. Rutz J M, Abdullah T, Singh S P, Kalve V I, Klebba P E. Evolution of the ferric enterobactin receptor in gram-negative bacteria. J Bacteriol. 1991;173:5964–5974. [PMC free article] [PubMed]
39. Schlosser A, Kampers T, Schrempf H. The Streptomyces ATP-binding component of MsiK assists in cellobiose and maltose transport. J Bacteriol. 1997;179:2092–2095. [PMC free article] [PubMed]
40. Schmitt M P, Payne S M. Genetic analysis of the enterobactin gene cluster in Shigella flexneri. J Bacteriol. 1991;173:816–825. [PMC free article] [PubMed]
41. Shea C M, McIntosh M A. Nucleotide sequence and genetic organization of the ferric enterobactin transport system: homology to other periplasmic binding protein-dependent systems in Escherichia coli. Mol Microbiol. 1991;5:1415–1428. [PubMed]
42. Sigel S P, Payne S M. Effect of iron limitation on growth, siderophore production and expression of outer membrane proteins of Vibrio cholerae. J Bacteriol. 1982;150:148–155. [PMC free article] [PubMed]
43. Sigel S P, Stoebner J A, Payne S M. Iron-vibriobactin transport system is not required for virulence of Vibrio cholerae. Infect Immun. 1985;47:360–362. [PMC free article] [PubMed]
44. Staudenmaier H, Van Hove B, Yaraghi Z, Braun V. Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism of iron(III) dicitrate in Escherichia coli. J Bacteriol. 1989;171:2626–2633. [PMC free article] [PubMed]
45. Stoebner J A, Butterton J R, Calderwood S B, Payne S M. Identification of the vibriobactin receptor of Vibrio cholerae. J Bacteriol. 1992;174:3270–3274. [PMC free article] [PubMed]
46. Sutcliffe I C, Russell R R B. Lipoproteins of gram-positive bacteria. J Bacteriol. 1995;177:1123–1128. [PMC free article] [PubMed]
47. Tashima K T, Carroll P A, Rogers M B, Calderwood S B. Relative importance of three iron-regulated outer membrane proteins for in vivo growth of Vibrio cholerae. Infect Immun. 1996;64:1756–1761. [PMC free article] [PubMed]
48. Trucksis M, Michalski J, Deng Y K, Kaper J B. The Vibrio cholerae genome contains two unique circular chromosomes. Proc Natl Acad Sci USA. 1998;95:14464–14469. [PMC free article] [PubMed]
49. Walker J E, Saraste M, Runswick M J, Gay N J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1:945–951. [PMC free article] [PubMed]
50. Wang R F, Kushner S R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991;100:195–199. [PubMed]
51. Wilken S, Schmees G, Schneider E. A putative helical domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG. A study using the LacK protein of Agrobacterium radiobacter as a tool. Mol Microbiol. 1996;22:655–666. [PubMed]
52. Wu H C. Biosynthesis of lipoprotein. In: Neidhardt F C, Curtiss III R, Ingraham J L, Lin E C C, Brooks Low K, Magasanik B, Reznikoff W S, Riley M, Schaechter M, Umbarger H E, editors. Escherichia coli and Salmonella: cellular and molecular biology. 2nd ed. Vol. 1. Washington, D.C.: ASM Press; 1996. pp. 1005–1014.
53. Wyckoff E, Stoebner J A, Reed K E, Payne S M. Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis. J Bacteriol. 1997;179:7055–7062. [PMC free article] [PubMed]
54. Wyckoff E E, Duncan D, Torres A G, Mills M, Maase K, Payne S M. Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria. Mol Microbiol. 1998;28:1139–1152. [PubMed]
55. Yamamoto S, Chowdhury M A R, Kuroda M, Nakano T, Koumoto Y, Shinoda S. Further study on polyamine compositions in Vibrionaceae. Can J Microbiol. 1991;37:148–153. [PubMed]
56. Yamamoto S, Shinoda S, Kawaguchi M, Wakamatsu K, Makita M. Polyamine distribution in Vibrionaceae: norspermidine as a general constituent of Vibrio species. Can J Microbiol. 1983;29:724–728.

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Conserved Domains
    Conserved Domains
    Link to related CDD entry
  • Gene
    Gene
    Gene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...