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J Bacteriol. Sep 2008; 190(17): 5953–5962.
Published online Jun 27, 2008. doi:  10.1128/JB.00569-08
PMCID: PMC2519515

Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters[down-pointing small open triangle]


Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

Most organisms have an absolute requirement for iron; however, despite being relatively abundant in nature, iron is often growth limiting due to its insolubility in aerobic environments at neutral pH. Iron acquisition poses particular challenges for microbial pathogens, because their hosts severely limit the availability of iron by keeping it tightly bound to protein carriers. In response to iron limitation, most pathogens express high-affinity iron transport systems (14). Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, encodes a large number of iron transport systems (68), reflecting the importance of iron for this pathogen. V. cholerae secretes vibriobactin, a low-molecular-weight iron chelator, or siderophore, which binds iron in the extracellular environment (17). Iron-loaded vibriobactin is transported back into the cell via a specific outer membrane receptor, ViuA (9, 57). In addition, V. cholerae expresses receptors for the exogenous siderophores enterobactin and ferrichrome (17, 37, 47). This ability to take advantage of siderophores produced by other microbial species may allow V. cholerae to compete more effectively for limited iron sources when growing in mixed microbial communities. V. cholerae also encodes three outer membrane receptors dedicated to heme transport (20, 35) and grows well in medium containing heme as the sole source of iron (21, 35, 41, 58, 69). All these receptor-mediated transport systems require the TonB/ExbB/ExbD complex to provide energy for transport across the outer membrane. V. cholerae encodes two distinct TonB/ExbB/ExbD complexes with overlapping as well as specific roles in iron acquisition (34, 41, 53). Transport of siderophores and heme across the inner membrane is accomplished via ligand-specific periplasmic binding protein-dependent permeases belonging to the ATP binding cassette (ABC) transporter family.

In addition to the TonB-dependent systems, V. cholerae encodes cytoplasmic membrane transport systems for the uptake of both ferrous and ferric inorganic iron (67). The Feo system belongs to a well-conserved family of ferrous iron transporters with a characteristic GTPase component essential for transport function (10, 18, 29). The Fbp system for ferric iron uptake is a periplasmic binding protein-dependent ABC transporter similar to those used to shunt siderophores and other iron complexes across the inner membrane. How free inorganic iron crosses the outer membrane and is made available in the periplasmic space is not currently known.

Like most bacteria, V. cholerae coordinates iron uptake with its use and storage in the cell to maintain an appropriate concentration of iron. This avoids toxicity due to oxidative damage that occurs in the presence of excess cellular iron, while ensuring sufficient levels of iron for metabolism and growth. Regulation of the expression of iron transport systems is mediated by the iron-binding repressor protein Fur (19). When iron is abundant, iron-bound Fur binds to specific promoter sequences and represses transcription of a large number of V. cholerae genes, including those encoding iron transport systems (36). The importance of iron sensing and iron regulation of gene expression can be seen in the reduced virulence of a fur mutant (36).

Although most of the iron uptake systems in V. cholerae have now been identified, it is clear from studies of mutants in the known transport systems that additional, uncharacterized systems are present in this pathogen. In this report we show that V. cholerae encodes a protein, VciB, which greatly enhances growth during iron stress by stimulating iron uptake via a cytoplasmic ferrous iron transport system such as Feo.


Bacterial strains and plasmids and growth conditions.

Bacterial strains and plasmids used in this study are listed in Table Table1.1. All strains were maintained at −80°C in tryptic soy broth plus 20% glycerol. Strains were routinely grown at 37°C in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, 1% NaCl) (38) or on LB agar. Iron-depleted medium was prepared by the addition of ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA), deferrated by the method of Rogers (46), at the concentrations indicated in the legend to each table or figure. For growth of Shigella flexneri strains in minimal medium, MM9 (52) without added iron and containing 2 μg nicotinic acid per ml and 0.2% glucose was used. The aerobactin for supplementing S. flexneri iron transport mutants was sterile culture supernatant of S. flexneri SA101 prepared as previously described (49). Antibiotics were used at the following concentrations for Escherichia coli strains: 250 μg of carbenicillin per ml, 50 μg of kanamycin per ml, and 30 μg of chloramphenicol per ml. For V. cholerae strains, the concentrations used were 125 μg of carbenicillin per ml, 25 μg of kanamycin per ml, 7.5 μg of chloramphenicol per ml, and 75 μg of streptomycin per ml. Electroporation of V. cholerae strains was carried out as described previously (41).

Bacterial strains and plasmids used in this study

Utilization of iron sources.

The ability of V. cholerae and E. coli strains to use various iron sources was tested in halo assays and in liquid culture growth assays as described previously (34, 41, 53). Iron utilization assays were carried out with strains defective in siderophore production in order to reduce background growth. All assays were repeated at least three times.


The oligonucleotide primers for PCR were purchased from IDT Inc. (Coralville, IA) and from Invitrogen (Carlsbad, CA). PCR was performed using Taq polymerase (Qiagen, Valencia, CA) or Platinum Pfx polymerase (Invitrogen) according to the manufacturers' instructions. Bacterial cultures grown overnight were used as the template. All clones derived from PCR products were verified by sequencing.

Sequence analysis.

DNA sequencing was performed by the University of Texas Institute for Cellular and Molecular Biology DNA Core Facility using an ABI Prism 3700 DNA sequencer. Analysis of DNA sequences was carried out using MacVector 7.2 and Clone Manager 7.04. BLAST searches and other bioinformatics analyses were done using the National Center for Biotechnology (NCBI) and the National Microbial Pathogen Data Resource (NMPDR) (31) databases. Pairwise alignments were carried out using ClustalW from within MacVector 7.2.

Construction of plasmids and chromosomal mutants.

pCosA was isolated from a V. cholerae CA401 genomic DNA library of partial Sau3AI fragments in pLAFR3 (21) during a selection for clones allowing growth of an E. coli siderophore mutant (W3110 entF) in low-iron medium (LB containing the iron chelator EDDA). A partial NcoI digest of pCosA yielded a fragment containing both vciA and vciB that was cloned into pACYC184 digested with NcoI to generate pAMH30. To create pAMR42, pAMH30 was digested with EcoRV and BamHI, and the fragment containing vciA was cloned into pWKS30 digested with EcoRV and BamHI. To construct pAMR43, the HpaI-MunI fragment containing vciA and vciB was excised from pAMH30 and cloned into pWKS30 digested with SmaI and EcoRI. To generate an in-frame deletion in vciA, pAMR43 was digested with NcoI and AgeI to remove approximately 1,200 bp. The overhangs produced by digestion were filled in using the Klenow fragment of DNA polymerase I (USB Corp., Cleveland, OH) and then ligated together to create pAMR45. pVciB was constructed by excising the vciB-containing EcoRV fragment from pAMR45 and cloning it into the ScaI site of pACYC184. To create pHit, pAH10 (1) was digested with EcoRI and BamHI and the fragment containing hitABC was cloned into pWKS30 digested with EcoRI and BamHI.

To mutate the vciA gene, the HpaI-SalI fragment of pAMH30 containing vciA was cloned into the λpir-dependent suicide vector pHM5 cut with SalI and EcoRV. The resulting clone was digested with NcoI and blunted with Klenow fragment, and the cam cassette from pMTLcam (66) was inserted as a SmaI fragment to yield pAMS24. The vciA::cam mutation is likely to be polar on vciB. To create a mutation in vciB alone, a fragment containing vciB was excised from pAMR43 by using EcoRV and XbaI and cloned into pHM5 digested with EcoRV and XbaI. The resulting plasmid was digested with NcoI and blunted with mung bean nuclease, and the kanamycin resistance cassette from pUC4K (Pharmacia) was inserted as a Klenow fragment-blunted SalI fragment to yield pAMS23. Allelic exchange in V. cholerae was carried out as described previously (33, 35). To create E. coli strain ARM110, pAMS7 (34) was transferred into W3110N and allelic exchange was performed as described previously (33, 35). Strains ARM113 and ARM114 were created by P1 transduction of the tonB::kan mutation from KP1032N (41) and the feoB::kan mutation from Keio strain JWK3372-1 (3), respectively, as described in reference 50. All mutations were verified by PCR.

Iron transport assays.

Cultures of S. flexneri strain SA167w or SM193w containing the indicated plasmid were grown overnight in LB broth supplemented with a 1/10 volume of sterile supernatant from S. flexneri strain SA101 as a source of aerobactin. Overnight cultures were diluted 1/25 into minimal medium with a 1/10 volume of SA101 culture supernatant and grown to late exponential phase with aeration at 37°C. Transport assays were performed in triplicate at room temperature, as previously described (67).

In vivo competition assays.

In vivo competition assays were performed using 5-day-old BALB/c mice as described by Taylor et al. (60), with modifications. Prestarved mice were inoculated intragastrically with 50 μl saline containing 0.5% sucrose, 0.02% Evan's blue dye, and approximately 5 × 105 CFU of each competing strain grown to mid-log phase at 30°C in Luria broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) at pH 6.5 to induce expression of virulence genes (39). The mice were sacrificed after 24 h, and the small intestines were removed and homogenized in sterile phosphate-buffered saline. Serial dilutions were plated on medium selective for V. cholerae and then replica plated on medium selective for the mutant to determine the viable counts for each competing strain. The output ratios were normalized to the input ratios to determine the competitive index [(mutant output/wild-type output)/(mutant input/wild-type input)].


Isolation of a novel V. cholerae iron utilization system.

Many, but not all, of the iron acquisition systems in V. cholerae have been identified (68). To screen for additional iron uptake systems, a siderophore synthesis mutant of E. coli was transformed with a cosmid library of V. cholerae classical strain CA401 (21), and clones were selected that allowed the siderophore mutant to grow in iron-restricted medium. Further subcloning of one of these cosmids, pCosA, identified a putative two-member operon, designated vciAB (VC0284 and VC0283), within the region conferring growth stimulation (data not shown). The presence of a predicted Fur-binding sequence within the promoter region of the vciAB operon (36) suggested regulation by iron and Fur and pointed to a role for these genes in iron acquisition or metabolism. Microarray analyses confirmed that these genes are repressed by iron in a Fur-dependent manner (36) (data not shown).

The vciA gene encodes a predicted TonB-dependent receptor, VciA. If this prediction is correct, VciA is the only remaining TonB-dependent receptor in V. cholerae for which the ligand has not been identified. The gene has a premature stop codon in the El Tor strain N16961 but appears to encode a full-length protein in many classical strains, including O395 and CA401. A BLAST search of the nonredundant NCBI and NMPDR databases showed that VciA is most similar to putative TonB-dependent ferrisiderophore receptors (Table (Table2).2). Among the receptors most closely related to VciA, several are annotated as ferrichrome receptors; however, the ligand assignments of those receptors have not been experimentally verified. Interestingly, the closest match to VciA in V. cholerae is the ferrichrome receptor FhuA (encoded by VC0200) (Table (Table2).2). To test whether vciA encodes a second ferrichrome receptor, an in-frame deletion was created in fhuA. This was done to avoid any possible polar effects on the downstream fhuBCD genes needed for transport of ferrichrome across the inner membrane. The fhuA mutant failed to use ferrichrome as an iron source, and the defect was suppressed when fhuA was supplied on a plasmid (data not shown). Thus, FhuA is likely the sole receptor for ferrichrome in V. cholerae. In Vibrio vulnificus, the gene product with the highest similarity to VciA is DesA, a receptor for the hydroxamate siderophore desferrioxamine B (Table (Table2);2); however, V. cholerae strain O395 did not use desferrioxamine B as a source of iron under the conditions tested (data not shown).

Selected homologs of VciA and VciB

The downstream open reading frame, vciB, encodes a predicted cytoplasmic membrane protein with three transmembrane helices and a large periplasmic loop. VciB has very few close homologs in the database and, strikingly, it is absent from all other Vibrio species sequenced to date. The closest sequence homolog of vciB is found in some species of Aeromonas, where it is also associated with a vciA homolog (Table (Table2);2); however, no function has been attributed to these genes in the aeromonads. In Vibrio parahaemolyticus, a predicted inner membrane protein with some similarity to VciB is encoded in the vicinity of, but not directly adjacent to, a gene encoding a putative hydroxamate receptor with homology to VciA (Table (Table22).

V. cholerae mutants in vciA (ARM514) and vciB (LHV101) were created and tested for growth using multiple iron sources. No defects in the use of vibriobactin, enterobactin, ferrichrome, heme, or inorganic iron were observed for either of these mutants (data not shown). Since V. cholerae encodes a large number of iron transport systems, many of which have partially redundant functions (35, 53, 68), it can be difficult to detect a phenotype for a mutant in any given system. To avoid these issues, the initial characterization of the Vci system was carried out in an E. coli iron transport mutant.

Characterization of the vci operon in E. coli.

To verify that the vciAB operon alone was responsible for growth stimulation of the E. coli siderophore mutant in low-iron medium, a smaller subclone containing only the vci genes, together with the upstream promoter region, was created. This clone, pAMR43, promoted growth of the E. coli entF strain ARM110 in iron-limited medium using the inorganic iron source FeSO4 (Fig. (Fig.1).1). Growth using the E. coli siderophore enterobactin was not affected by the vciAB genes, suggesting that the Vci system acts in a pathway distinct from enterobactin utilization. Further, the observed growth stimulation was not dependent on 2,3-dihydroxybenzoic acid (DHBA), a precursor to enterobactin produced by ARM110, since ARM110 carrying the vciAB genes did not use DHBA as a source of iron (data not shown). In addition, pAMR43 stimulated FeSO4 utilization in the DHBA-deficient E. coli strain AB1515.24, showing that the Vci system does not require DHBA production by the host strain (data not shown).

FIG. 1.
VciB is necessary and sufficient for enhanced growth of E. coli using FeSO4 as the iron source. The map shows the organization of the vci genes (thick arrows) and the locations of the proposed Fur-binding sequence (Fur box), the predicted transcriptional ...

Because vciA is predicted to encode a TonB-dependent receptor with an iron ligand, we anticipated that vciA would be required for the increase in iron utilization. Surprisingly, this was not the case. As shown in Fig. Fig.1,1, the plasmid pAMR42, containing only vciA, did not stimulate growth of the E. coli iron transport mutant ARM110, indicating that VciB is needed for the growth phenotype. In fact, vciA was found to be dispensable for iron acquisition, since an in-frame deletion within vciA in the context of the vciAB operon (encoded by pAMR45) did not diminish iron utilization (Fig. (Fig.1).1). Thus, VciB is necessary and sufficient for growth stimulation of E. coli ARM110 by inorganic iron.

VciB-mediated iron utilization is TonB independent.

E. coli ARM110 encodes several TonB-dependent receptors, leaving open the possibility that an endogenous receptor with the same ligand as VciA was compensating for the loss of VciA in ARM110 expressing only VciB. If this is the case, stimulation of iron acquisition by VciB should be TonB dependent. To test this hypothesis, an E. coli entF tonB mutant strain, ARM113, was tested for its ability to resist iron stress in the presence and absence of VciB. In this assay, strains were grown in liquid medium containing increasing concentrations of the iron chelator EDDA. The concentration of EDDA at which growth of the parental strain (ARM110) expressing VciB was completely inhibited was approximately 10-fold higher than for the vector control strain (Fig. (Fig.2A2A and data not shown), showing that VciB greatly increases resistance to the iron chelator. In the entF tonB double mutant (ARM113), VciB stimulated growth to an extent similar to that in the parental strain, demonstrating that TonB is not required for VciB-mediated iron utilization (Fig. (Fig.2A).2A). These data are consistent with the observation that VciA is not needed for the iron utilization phenotype and show that the iron source(s) used by VciB crosses the outer membrane in a TonB-independent manner. All strains grew well in iron-replete medium (no added chelator) (Fig. (Fig.2A),2A), showing that the positive effect of VciB is limited to iron stress conditions and is not an effect on growth in general. In fact, expression of vciB from a plasmid caused a minor decrease in the growth of both the tonB mutant and the parental strain under iron-replete conditions, possibly due to overexpression of this presumed inner membrane protein.

FIG. 2.
VciB-mediated resistance to iron stress in E. coli requires Feo but not TonB. The E. coli entF strain ARM110 and its tonB derivative ARM113 (A) or ARM110 and its feoB derivative ARM114 (B), carrying either pACYC184 (vector) or pVciB (encoding VciB), were ...

VciB does not encode a complete transport system.

In previous work, we used the Shigella flexneri strain SM193w to study new V. cholerae iron transport systems (67). This strain lacks the iron acquisition systems (Iuc, Feo, and Sit) needed for utilization of iron sources present in LB medium and grows poorly unless supplied with a functional iron transport system or with an iron source it can use, such as the siderophore aerobactin (49). To test whether VciB is a complete transporter, pAMR45 was introduced into SM193w. The presence of VciB did not stimulate growth of SM193w under any conditions tested, indicating that VciB by itself is unlikely to function as an iron permease (Table (Table33 and data not shown). Supplying vciA in addition to vciB did not change these results (data not shown). VciB also failed to promote iron uptake in the E. coli iron transport mutant H1771 (18, 24) at a level sufficient to turn off the iron-repressible fhuE-lacZ fusion present in that strain (data not shown). It was noted that both SM193w and H1771 carry mutations in the genes encoding the ferrous iron transport system Feo, while none of the strains that supported VciB-mediated iron utilization (Fig. (Fig.11 and data not shown) were defective for Feo, suggesting that VciB may require Feo activity. This was tested using SM193w and its isogenic Feo+ strain, SA167w. Whereas no VciB-dependent growth stimulation using FeSO4 was observed for SM193w, a very robust increase in growth was exhibited by SA167w expressing VciB under those conditions (Table (Table3),3), comparable to that of E. coli strain ARM110 expressing VciB (Fig. (Fig.1).1). From these data we conclude that VciB function requires an auxiliary factor, which in S. flexneri can be supplied by the Feo system.

VciB promotes iron utilization via Feo in S. flexneri

VciB stimulates iron utilization via ferrous iron transport systems.

To test whether VciB function in E. coli is also dependent upon Feo, a feoB mutation was introduced into strain ARM110. The resulting entF feoB mutant was assessed for its ability to grow at a low concentration of iron in the presence and absence of VciB. Figure Figure2B2B shows that, while VciB greatly increased growth of the parental strain in the presence of the iron chelator EDDA, it had no effect on the ability of the feo mutant to grow under conditions of iron stress. No differences in growth were observed between the parental strain and the feoB mutant in the absence of VciB under any of the conditions tested, showing that the feoB mutant is not more sensitive to iron chelators than its parental strain under these conditions. VciB-mediated growth stimulation was restored in the feoB mutant when the Feo system genes were supplied on a plasmid (Fig. (Fig.33).

FIG. 3.
VciB activity depends upon a ferrous, but not ferric, iron transport system. The assay conditions were as described in the legend to Fig. Fig.1.1. The following plasmids were used for expressing iron transport systems: pUH18E (E. coli feoABC); ...

VciB, a V. cholerae gene product, is unlikely to have a specific requirement for E. coli or S. flexneri Feo. Rather, VciB may function with Feo transporters in general or with multiple inorganic iron transport systems. To verify that VciB is active with its native V. cholerae Feo system, the E. coli entF feoB mutant was supplied with the V. cholerae feoABC genes on a plasmid, and the resulting strain was tested for growth using FeSO4 as the iron source in the presence or absence of VciB. The results, shown in Fig. Fig.3,3, clearly demonstrate that V. cholerae Feo is as effective as E. coli Feo in supporting VciB function. To determine whether VciB specifically requires a Feo-type transporter, S. flexneri Sit, a ferrous iron ABC transport system unrelated to Feo (25, 49; E. E. Wyckoff, unpublished data), was tested for its ability to function with VciB. The Sit system facilitated a level of VciB-dependent growth similar to that by E. coli and V. cholerae Feo, showing that VciB is active with a non-Feo ferrous transporter as well (Fig. (Fig.3).3). In contrast, neither of the ferric iron transport systems tested, V. cholerae Fbp (67) or Haemophilus influenzae Hit (1), restored the VciB-dependent growth stimulation around FeSO4 (Fig. (Fig.3).3). Both the fbpABC and the hitABC clones used in these studies encode complete functional iron transporters, as demonstrated by their ability to support growth of an S. flexneri iron transport mutant (67) (data not shown). All strains grew well using enterobactin as the iron source, regardless of which iron transport system was expressed (data not shown). Taken together, these data show that VciB activity depends upon the presence of a ferrous, but not ferric, iron transport system.

VciB promotes iron transport via Feo.

The data presented thus far are consistent with a model in which VciB stimulates iron transport through its cognate ferrous iron transporter. It is also conceivable, however, that VciB increases the efficiency with which iron brought in via a ferrous iron uptake system is used by the cell. To test whether VciB increases net transport of iron into the cell, iron transport assays were carried out with S. flexneri SA167w in the presence and absence of VciB. This strain was chosen because it lacks all of its major iron transport systems except Feo, thus minimizing background iron uptake that could potentially confound the assay. In addition, the presence of VciB in SA167w greatly enhanced utilization of FeSO4 in growth assays (Table (Table3).3). In the transport assays (Fig. (Fig.4),4), the amount of radiolabeled iron accumulated by the cells was consistently two- to threefold higher in the presence of VciB. To rule out the possibility that the observed iron accumulation was due to periplasmic iron binding activity by VciB rather than to active transport of iron into the cell, the assays were repeated with the isogenic feoB mutant, SM193w. As shown in Fig. Fig.4,4, no net increase in VciB-mediated iron accumulation was observed with SM193w, indicating that active transport via a ferrous transporter is required for VciB-dependent iron accumulation by the cell.

FIG. 4.
VciB stimulates iron transport through Feo. S. flexneri strains SA167w (iucD sitA) and SM193w (iucD sitA feoB) carrying either pACYC184 (vector control) or pVciB (encoding VciB) were grown to late exponential phase and resuspended in transport buffer, ...

VciB is not required for growth of V. cholerae in the host environment.

The environment of the host small intestine is expected to be largely anaerobic or microaerophilic. In this environment, the ferrous form of iron is likely to predominate, and the V. cholerae Vci system could thus be relevant for growth in vivo. In addition, a screen for V. cholerae genes induced in the human host showed that the vciAB promoter is activated in the host environment (27), suggesting that these genes may be involved in survival in the human intestine. To test whether vciB plays a role in in vivo growth, a competition assay using infant mice was performed. In this assay, eight infant mice were inoculated intragastrically with equal numbers of the V. cholerae vciB mutant LHV101 and its parental strain, ARM591. Both of these strains contain mutations in siderophore production and in the ferric uptake system Fbp; this strain background was chosen in order to maximize the effect of ferrous iron uptake via VciB on growth in the host environment. The competitive index (the output ratio normalized to the input ratio) of the vciB mutant to the parental strain following growth for 24 h in infant mice was calculated to be 0.8 ± 0.2, showing that the mutant had no significant defect in growth within the infant mouse intestine. While expression of vciB in vivo may contribute to iron acquisition in this host, it is likely that other systems are involved in this process as well.


The vci locus is not widely distributed among bacterial species; the vciAB operon is not found in any sequenced genomes apart from those of V. cholerae and a few species of Aeromonas. Interestingly, however, genes encoding proteins with some similarity to VciA and VciB are present adjacent to the proposed tonB2 locus (41) of several Vibrio species, including V. parahaemolyticus (Table (Table2),2), Vibrio alginolyticus, and Vibrio splendidus (data not shown). A comparable organization is seen in many non-Vibrio genera as well, including species of Azoarcus (plant endophytes), Xanthomonas (plant pathogens), and Flavobacterium and Photobacterium (fish pathogens), where a putative inner membrane protein with homology to VciB (Table (Table2)2) is encoded near genes for TonB, TonB-dependent receptors, PiuC (iron uptake factor), and ABC transporters with putative metal ion ligands (data not shown). This organization may imply that VciB-like proteins play a role in iron acquisition in other organisms as well, and it will be interesting to determine whether any of these putative VciB homologs has a function in iron utilization similar to that of V. cholerae VciB.

In BLAST analyses, particularly those carried out against the genomes of other Vibrio species, VciA is most similar to receptors either shown to be or predicted to be involved in the transport of ferrichrome or other hydroxamate siderophores (Table (Table2).2). For example, the closest match to VciA in V. cholerae is the documented ferrichrome receptor FhuA(47), and in V. vulnificus, it is DesA, a receptor known to be required for the utilization of desferrioxamine B (2). At least one database has already annotated the gene encoding VciA in strain O395 as a ferrichrome receptor gene (fig|345073.6.peg.3158 at http://www.nmpdr.org/). Our data show that VciA is unlikely to function in ferrichrome utilization, but it is nevertheless suggestive that VciA is most similar to these hydroxamate receptors. Although the V. cholerae strain used in this study did not use the hydroxamate siderophores ferrioxamine B, aerobactin, and schizokinen as a source of iron under the conditions tested (A. R. Mey, unpublished results), we cannot rule out the possibility that VciA has another hydroxamate ligand. The ligand for VciA may also be a nonhydroxamate siderophore, or a nonsiderophore iron compound. It does not appear, however, that VciA is involved in the transport of heme, vibriobactin, or enterobactin, since the receptors for those ligands have all been identified and receptor mutations have been created that abolish the use of those iron sources (9, 35, 37, 57).

Our data show that VciB enhances iron uptake via a ferrous iron transport system, and there are several models that could account for this. VciB may directly stimulate the activity of its cognate ferrous iron transporter, or it may be an iron-binding protein that delivers ferrous iron directly to ferrous iron transport systems. Both of these models imply a direct interaction between VciB and the transporter, which is difficult to reconcile with the observed lack of specificity of VciB for its ferrous iron transport partner. A more likely hypothesis is that VciB acts to increase the amount of ferrous iron available for transport by ferrous iron transporters. VciB could accomplish this by trapping ferrous iron in the vicinity of a ferrous iron transporter; however, VciB does not appear to be capable of trapping large quantities of periplasmic iron, since there was no net VciB-dependent iron accumulation in the absence of transport through Feo (Fig. (Fig.4).4). A more attractive model is that VciB acts as a ferric iron reductase, effectively increasing the local concentration of ferrous iron available for transport by a ferrous iron uptake system. Because much of the iron present in the diverse environments occupied by V. cholerae is in the ferric form, either in insoluble ferric chelates or complexed with siderophores or host iron-binding proteins, ferric iron reductase activity may give this organism access to a much greater pool of iron than would otherwise be available. Many bacterial pathogens produce extracellular (secreted or surface-bound) or periplasmic ferric iron reductases in order to take advantage of one or several ferric iron complexes present in their distinct environments (4, 13, 15, 23, 43, 51, 63). Not only is this a powerful strategy for exploiting a large variety of iron sources, but it is also less metabolically expensive than synthesizing a specific transport machinery for each compound used. VciB, a putative inner membrane protein with a large periplasmic domain (a prediction that is supported by the observation that VciB cooperates with an inner membrane transport complex), could be ideally situated to reduce ferric iron complexes in the periplasm. Alternatively, VciB may represent a completely novel mechanism of iron uptake via a ferrous iron transport system.

How ferrous iron accumulates in the periplasm in sufficient quantities to be transported via inner membrane ferrous iron transport systems, particularly under aerobic conditions at neutral pH, is not well understood. Our study of V. cholerae VciB is the first report of a protein in bacteria which appears to directly cooperate with Feo and other ferrous transporters, possibly by increasing the availability of their substrate in the periplasm. Understanding the mechanism underlying this cooperation may shed some light on how transporters, such as Feo, operate. In E. coli, as in other species, the Feo system genes are subject to Fur regulation; however, anaerobically, these genes are upregulated by FNR, even in the presence of iron (24). Presumably, this ensures expression of the Feo system under conditions favoring the ferrous form of iron (anaerobiosis) while maintaining iron- and Fur-dependent repression of the other, predominantly ferric, iron transport systems. Aerobically, the feo genes are induced in response to low iron (24, 30), indicating that the Feo system is needed under conditions favoring the ferric form of iron as well. This is supported by the observation that the Feo system works well aerobically in E. coli (24) and in other species (8, 49, 67). Extracellular or periplasmic ferric iron reductase activity, as perhaps supplied by VciB in V. cholerae, would greatly increase the efficiency of iron uptake via Feo aerobically. In support of this model, extracytoplasmic ferric iron reductase activity in H. pylori has been proposed as the basis for high-affinity ferric iron uptake via Feo in this organism (64).

The promoter controlling the vci genes was shown to be active in the human host (27), suggesting a role for the Vci system in vivo; however, we did not find that vciB was required for virulence in the infant mouse model of V. cholerae infection. This could be due to V. cholerae expressing multiple iron uptake systems in vivo, the loss of only one of which may not impair growth. We have shown previously that disrupting a single system, such as siderophore-mediated iron uptake or heme acquisition, is not detrimental for survival in the infant mouse, and even the loss of multiple systems does not significantly affect the ability of V. cholerae to colonize infant mice (68). It is also possible that the ligand used by the Vci system is not found in the infant mouse intestine or that the vci genes are not expressed well in that environment. Our preliminary data indicate that the expression of the vci genes, although regulated by iron and Fur, may be subject to additional regulation; compared with other iron transport genes, the vci genes are poorly expressed in the laboratory, even under iron-limiting conditions (Mey, unpublished). It is conceivable that the vci genes respond to a signal that is present in the human host but not in the mouse. One reason to expect that a system such as the Vci system, which increases iron uptake via a ferrous iron transporter, is important for in vivo iron acquisition is that the lumen of the human intestine is likely anaerobic or microaerophilic, conditions known to promote the ferrous form of iron and the expression of ferrous iron transport systems such as Feo (7, 24) while repressing ferric iron uptake systems (7). In several pathogens of the digestive tract, ferrous iron uptake via Feo is critical for establishing gastric (64) or intestinal infection in the host (40, 59, 62).

V. cholerae maintains itself in the natural environment as well as in the human host and must therefore be able to adapt to a wide range of changing conditions and iron sources. The importance of iron for V. cholerae is reflected in the sheer number of genes involved in iron uptake in this organism, with more than 1% of its total genome content being dedicated to iron acquisition (68). This allows V. cholerae to take advantage of the full range of iron sources that it may encounter in its life cycle and to compete efficiently for iron when this element is scarce. Further characterization of the Vci system in V. cholerae may bring new information about how this organism transitions so successfully between diverse environments and could uncover a novel pathway for iron acquisition in microbial pathogens in general.


We are greatly indebted to Tim Mietzner for sharing plasmids and to Svetlana Gerdes for invaluable training in using the National Microbial Pathogen Data Resource database.

This work was supported by National Institutes of Health grant AI50669.


[down-pointing small open triangle]Published ahead of print on 27 June 2008.


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