JOURNAL OF BACTERIOLOGY, Sept. 2008, p. 5953–5962 0021-9193/08/$08.00⫹0 doi:10.1128/JB.00569-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 17
Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters䌤 Alexandra R. Mey,* Elizabeth E. Wyckoff, Lindsey A. Hoover,† Carolyn R. Fisher, and Shelley M. Payne Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712-1095 Received 24 April 2008/Accepted 18 June 2008
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. 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 sys-
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 * Corresponding author. Mailing address: The University of Texas at Austin, Section of Molecular Genetics and Microbiology, Austin, TX 78712-1095. Phone: (512) 471-5204. Fax: (512) 471-7088. E-mail:
[email protected]. † Present address: Department of Biochemistry, The University of Wisconsin, Madison, WI 53706-1544. 䌤 Published ahead of print on 27 June 2008. 5953
5954
J. BACTERIOL.
MEY ET AL.
tems 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. MATERIALS AND METHODS Bacterial strains and plasmids and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 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 g 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 g of carbenicillin per ml, 50 g of kanamycin per ml, and 30 g of chloramphenicol per ml. For V. cholerae strains, the concentrations used were 125 g of carbenicillin per ml, 25 g of kanamycin per ml, 7.5 g of chloramphenicol per ml, and 75 g of streptomycin per ml. Electroporation of V. cholerae strains was carried out as described previously (41). 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. PCR. 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 pir-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
TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid
V. cholerae strains CA401 O395 ARM514 ARM591 LHV101 LHV103 N16961
Source or reference(s)
Description
Classical biotype Classical biotype O395 vciA::cam O395 ⌬vibB fbpA::cam O395 ⌬vibB fbpA::cam vciB::kan O395 ⌬fhuA El Tor biotype
16a 32 This study 67 This study This study J. Kaper
E. coli strains AB1515.24 AB1515 entB::Tn5 DH5␣(pir) Cloning strain, host strain for pGP704 derivatives, enterobactin producer H1771 MC4100 aroB feoB7 fhuF::plac Mu W3110 F– IN(rrnD-rrnE)1 W3110N W3110 Nalr ARM110 W3110N entF::cam ARM113 W3110N entF::cam tonB::kan ARM114 W3110N entF::cam feoB::kan
22 This This This This
S. flexneri strains SA101
26
SM193 SM193w SA167 SA167w
Nonvirulent, aerobactinproducing strain SM100 iucD::Tn5 sitA::cam feoB::tmp Nonvirulent derivative of SM193 SM100 iucD::Tn5 sitA::cam Nonvirulent derivative of SA167
Plasmids pACYC184 Medium-copy-number cloning vector; Cmr Tcr pHM5 Suicide vector pGP704 carrying sacB; Cbr Sucs pLAFR3 Cosmid cloning vector; Tcr pWKS30 Low-copy-number cloning vector; Cbr pAMH30 pACYC184 carrying vciA vciB pAMR42 pWKS30 carrying vciA pAMR43 pWKS30 carrying vciA vciB pAMR45 pWKS30 carrying vciB pAMS23 pHM5 carrying vciB::kan pAMS24 pHM5 carrying vciA::cam pCosA pLAFR3 carrying vciA vciB ⫹ flanking sequence pEG3 pWKS30 carrying S. flexneri sitABCD pFeo101 pWKS30 carrying V. cholerae feoABC pFbp1 pWKS30 carrying V. cholerae fbpABC pHit pWKS30 carrying H. influenzae hitABC pUH18E pT7-5 carrying E. coli feoABC pVciB pACYC184 carrying vciB
55 J. Kaper 18, 24 study study study study
49 67 49 This study
11 48 56 65 This study This This This This This This
study study study study study study
E. Gonzalez and L. Runyen-Janecky 67 67 This study 24 This study
VOL. 190, 2008
V. CHOLERAE VciB
5955
TABLE 2. Selected homologs of VciA and VciB Type of Vci homolog and species
VciA Aeromonas hydrophila Aeromonas salmonicida Pseudomonas fluorescens Burkholderia cenocepacia Neisseria meningitidis Vibrio parahaemolyticus
Name or gene identification of homolog
AHA_0461 ASA_3883 PFL_0310 Bcen2424_1655 NMC1887 VPA0150
V. vulnificus V. cholerae
DesA FhuA
VciB A. salmonicida A. hydrophila
ASA_3884 AHA_0460
B. cenocepacia
Bcen2424_3058
Azoarcus sp. strain EbN1 Xanthomonas campestris V. parahaemolyticus Photobacterium profundum Flavobacterium psychrophilum
ebA3935 XCV3088 VPA0157 P3TCK_07374 FP1923
Identity (%)
Similarity (%)
Ferrisiderophore receptor (p)c Ferrisiderophore receptor (p)c Ferrichrome receptor (p)d Ferrichrome receptor (p)d Ferrichrome receptor (p)d Ferric coprogen/rhodotorulic acid receptor (p)d Desferrioxamine B receptor (c) Ferrichrome receptor (c)
70 67 48 41 30 25
83 82 64 56 46 42
54 45 42 12 6 28
25 23
40 37
2 47; this study
Conserved hypothetical protein (p)c PepSY-associated TM helix family protein (p)c PepSY-associated TM helix domain protein (p)c,e Transmembrane protein (p)d Membrane protein (p)d Membrane protein (p)d Membrane protein (p)d Hypothetical protein (p)d
53 52
66 66
45 54
27
46
12
26 22 19 16 14
39 37 35 33 31
44 61 28 5 16
Annotationa
Reference(s)b
a
(p) indicates the annotation is putative; (c) indicates the annotation is confirmed. Unless the protein has been characterized, the reference is for the published genome sequence of the indicated organism. Annotation is from the NCBI database. d Annotation is from the NMPDR database. e Annotated as a putative membrane protein in the NMPDR database. b c
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 l 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)].
RESULTS 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 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 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 desfer-
5956
MEY ET AL.
J. BACTERIOL.
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 start site (right-angled arrow), and the putative transcriptional terminator sequence downstream of vciB. The brackets under the map delineate the DNA sequences (solid lines) included in each plasmid construct. The growth of E. coli strain ARM110 (W3110N entF) carrying the plasmids indicated was monitored as follows: strains were seeded at 106 cells per ml into molten LB agar containing 150 g EDDA per ml, and iron sources (5 l 10 mM FeSO4 and 5 l DH5␣ overnight culture [enterobactin]) were spotted onto the solidified agar. Following incubation for 24 h at 37°C, the diameter of each zone of growth was measured. The assays were repeated at least three times, and the average (with standard deviation) of three measurements is shown.
rioxamine B (Table 2); however, V. cholerae strain O395 did not use desferrioxamine B as a source of iron under the conditions tested (data not shown). 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 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 2). 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. 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). 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. 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. 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
VOL. 190, 2008
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 grown to late stationary phase in LB broth and then subcultured 1:200 into LB broth containing increasing concentrations of EDDA. Cultures were grown overnight at 37°C with aeration, and the optical densities at 650 nm (OD650) were measured. Shown here are the concentrations of EDDA required to completely inhibit growth of the parental strain ARM110 carrying the empty vector (5 g per ml) or pVciB (50 g per ml). The data represent the averages of three experiments. Standard deviations are indicated by the error bars.
control strain (Fig. 2A 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. 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 ironreplete medium (no added chelator) (Fig. 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
V. CHOLERAE VciB
5957
both the tonB mutant and the parental strain under ironreplete conditions, possibly due to overexpression of this presumed inner membrane protein. 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 3 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. 1 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 3), comparable to that of E. coli strain ARM110 expressing VciB (Fig. 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 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 2B 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
TABLE 3. VciB promotes iron utilization via Feo in S. flexneri Strain
SM193w (iucD sitA feoB) SA167w (iucD sitA)
Plasmid
pACYC184 pVciB pACYC184 pVciB
Zone of growth (mm) after 24 ha FeSO4
Aerobactin
11 (0.6) 12 (1.0) 10 (0.6) 29 (1.2)
20 (0.6) 20 (0.6) 19 (1.2) 19 (1.0)
a The assay conditions were as described in the legend to Fig. 1, except an SA101 overnight culture (5 l) was spotted as a source of aerobactin. The assays were repeated at least three times, and the average (and standard deviation) of three measurements is shown.
5958
MEY ET AL.
J. BACTERIOL.
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. 1. The following plasmids were used for expressing iron transport systems: pUH18E (E. coli feoABC); pFeo101 (V. cholerae feoABC); pEG3 (S. flexneri sitABCD); pFbp1 (V. cholerae fbpABC); pHit (H. influenzae hitABC). VciB was expressed from plasmid pVciB. The assays were repeated at least three times, and the averages of three measurements are shown. Standard deviations are indicated by the error bars.
stimulation was restored in the feoB mutant when the Feo system genes were supplied on a plasmid (Fig. 3). 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. 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. 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. 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 3). In the transport assays (Fig. 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. 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. 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
VOL. 190, 2008
V. CHOLERAE VciB
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, as described in Materials and Methods. Radiolabeled iron (55Fe) was added to the cells to begin the transport reaction, and cells were harvested, filtered, and washed after 5 min. The amount of 55Fe accumulated by the cells was determined by scintillation counting. The data represent the averages of three experiments. Standard deviations are indicated by the error bars.
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. DISCUSSION 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 2), Vibrio alginolyticus, and Vibrio splendidus (data not shown). A comparable organization is seen in many non-Vibrio genera as well, including species of
5959
Azoarcus (plant endophytes), Xanthomonas (plant pathogens), and Flavobacterium and Photobacterium (fish pathogens), where a putative inner membrane protein with homology to VciB (Table 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 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. 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)
5960
MEY ET AL.
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
J. BACTERIOL.
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. ACKNOWLEDGMENTS 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. REFERENCES 1. Adhikari, P., S. D. Kirby, A. J. Nowalk, K. L. Veraldi, A. B. Schryvers, and T. A. Mietzner. 1995. Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon. J. Biol. Chem. 270:25142–25149. 2. Aso, H., S. Miyoshi, H. Nakao, K. Okamoto, and S. Yamamoto. 2002. Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions. FEMS Microbiol. Lett. 212:65–70. 3. Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M. Tomita, B. L. Wanner, and H. Mori. 21 February 2006, posting date. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2:2006.0008.. 4. Barchini, E., and R. E. Cowart. 1996. Extracellular iron reductase activity produced by Listeria monocytogenes. Arch. Microbiol. 166:51–57. 5. Bartlett, D., S. Ferriera, J. Johnson, S. Kravitz, A. Halpern, K. Remington, K. Beeson, B. Tran, Y.-H. Rogers, R. Friedman, and J. C. Venter. 2006, posting date. Photobacterium profundum 3TCK whole genome shotgun sequence. Direct submission to NCBI. NCBI, Bethesda, MD. 6. Bentley, S. D., G. S. Vernikos, L. A. Snyder, C. Churcher, C. Arrowsmith, T. Chillingworth, A. Cronin, P. H. Davis, N. E. Holroyd, K. Jagels, M. Maddison, S. Moule, E. Rabbinowitsch, S. Sharp, L. Unwin, S. Whitehead, M. A. Quail, M. Achtman, B. Barrell, N. J. Saunders, and J. Parkhill. 2007. Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18. PLoS Genet. 3:e23. 7. Boulette, M. L., and S. M. Payne. 2007. Anaerobic regulation of Shigella flexneri virulence: ArcA regulates Fur and iron acquisition genes. J. Bacteriol. 189:6957–6967. 8. Boyer, E., I. Bergevin, D. Malo, P. Gros, and M. F. Cellier. 2002. Acquisition of Mn(II) in addition to Fe(II) is required for full virulence of Salmonella enterica serovar Typhimurium. Infect. Immun. 70:6032–6042. 9. Butterton, J. R., J. A. Stoebner, S. M. Payne, and S. B. Calderwood. 1992. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J. Bacteriol. 174:3729– 3738. 10. Cartron, M. L., S. Maddocks, P. Gillingham, C. J. Craven, and S. C. Andrews. 2006. Feo—transport of ferrous iron into bacteria. Biometals 19: 143–157. 11. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134:1141–1156.
VOL. 190, 2008 12. Copeland, A., S. Lucas, A. Lapidus, K. Barry, J. C. Detter, T. del Rio Glavina, N. Hammon, S. Israni, S. Pitluck, P. Chain, S. Malfatti, M. Shin, L. Vergez, J. Schmutz, F. Larimer, M. Land, L. Hauser, N. Kyrpides, E. Kim, J. J. LiPuma, C. F. Gonzalez, K. Konstantinidis, J. M. Tiedje, and P. Richardson. 2006, posting date. Complete sequence of chromosome 1 of Burkholderia cenocepacia HI2424. Direct submission to NCBI. NCBI, Bethesda, MD. 13. Cox, C. D. 1980. Iron reductases from Pseudomonas aeruginosa. J. Bacteriol. 141:199–204. 14. Crosa, J. H., A. R. Mey, and S. M. Payne (ed.). 2004. Iron transport in bacteria. ASM Press, Washington, DC. 15. Deneer, H. G., V. Healey, and I. Boychuk. 1995. Reduction of exogenous ferric iron by a surface-associated ferric reductase of Listeria spp. Microbiology 141:1985–1992. 16. Duchaud, E., M. Boussaha, V. Loux, J. F. Bernardet, C. Michel, B. Kerouault, S. Mondot, P. Nicolas, R. Bossy, C. Caron, P. Bessieres, J. F. Gibrat, S. Claverol, F. Dumetz, M. Le Henaff, and A. Benmansour. 2007. Complete genome sequence of the fish pathogen Flavobacterium psychrophilum. Nat. Biotechnol. 25:763–769. 16a.Gardner, E. W., S. T. Lyles, and C. E. Lankford. 1964. A comparison of virulence of Vibrio cholerae strains for the embryonated egg. J. Infect. Dis. 114:412–416. 17. Griffiths, G. L., S. P. Sigel, S. M. Payne, and J. B. Neilands. 1984. Vibriobactin, a siderophore from Vibrio cholerae. J. Biol. Chem. 259:383–385. 18. Hantke, K. 1987. Ferrous iron transport mutants in Escherichia coli K12. FEMS Microbiol. Lett. 44:53–57. 19. Hantke, K. 1981. Regulation of ferric iron transport in Escherichia coli K-12: isolation of a constitutive mutant. Mol. Gen. Genet. 182:288–292. 20. Henderson, D. P., and S. M. Payne. 1994. 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. 176:3269–3277. 21. Henderson, D. P., and S. M. Payne. 1993. Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin. Mol. Microbiol. 7:461–469. 22. Hill, C., and B. Harnish. 1981. Inversions between ribosomal RNA genes of Escherichia coli. Proc. Natl. Acad. Sci. USA 78:7069–7072. 23. Homuth, M., P. Valentin-Weigand, M. Rohde, and G. F. Gerlach. 1998. Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis. Infect. Immun. 66:710–716. 24. Kammler, M., C. Schon, and K. Hantke. 1993. Characterization of the ferrous iron uptake system of Escherichia coli. J. Bacteriol. 175:6212–6219. 25. Kehres, D. G., A. Janakiraman, J. M. Slauch, and M. E. Maguire. 2002. SitABCD is the alkaline Mn2⫹ transporter of Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:3159–3166. 26. Lawlor, K. M., P. A. Daskaleros, R. E. Robinson, and S. M. Payne. 1987. Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds. Infect. Immun. 55:594–599. 27. Lombardo, M. J., J. Michalski, H. Martinez-Wilson, C. Morin, T. Hilton, C. G. Osorio, J. P. Nataro, C. O. Tacket, A. Camilli, and J. B. Kaper. 2007. An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers. Proc. Natl. Acad. Sci. USA 104:18229–18234. 28. Makino, K., K. Oshima, K. Kurokawa, K. Yokoyama, T. Uda, K. Tagomori, Y. Iijima, M. Najima, M. Nakano, A. Yamashita, Y. Kubota, S. Kimura, T. Yasunaga, T. Honda, H. Shinagawa, M. Hattori, and T. Iida. 2003. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of Vibrio cholerae. Lancet 361:743–749. 29. Marlovits, T. C., W. Haase, C. Herrmann, S. G. Aller, and V. M. Unger. 2002. The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria. Proc. Natl. Acad. Sci. USA 99:16243–16248. 30. McHugh, J. P., F. Rodriguez-Quinones, H. Abdul-Tehrani, D. A. Svistunenko, R. K. Poole, C. E. Cooper, and S. C. Andrews. 2003. Global irondependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J. Biol. Chem. 278:29478–29486. 31. McNeil, L. K., C. Reich, R. K. Aziz, D. Bartels, M. Cohoon, T. Disz, R. A. Edwards, S. Gerdes, K. Hwang, M. Kubal, G. R. Margaryan, F. Meyer, W. Mihalo, G. J. Olsen, R. Olson, A. Osterman, D. Paarmann, T. Paczian, B. Parrello, G. D. Pusch, D. A. Rodionov, X. Shi, O. Vassieva, V. Vonstein, O. Zagnitko, F. Xia, J. Zinner, R. Overbeek, and R. Stevens. 2007. The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation. Nucleic Acids Res. 35:D347–D353. 32. Mekalanos, J. J., D. J. Swartz, G. D. Pearson, N. Harford, F. Groyne, and M. de Wilde. 1983. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature 306:551–557. 33. Mey, A. R., S. A. Craig, and S. M. Payne. 2005. Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect. Immun. 73:5706–5719. 34. Mey, A. R., and S. M. Payne. 2003. Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors. J. Bacteriol. 185:1195–1207. 35. Mey, A. R., and S. M. Payne. 2001. Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptors. Mol. Microbiol. 42:835– 849.
V. CHOLERAE VciB
5961
36. Mey, A. R., E. E. Wyckoff, V. Kanukurthy, C. R. Fisher, and S. M. Payne. 2005. Iron and fur regulation in Vibrio cholerae and the role of fur in virulence. Infect. Immun. 73:8167–8178. 37. Mey, A. R., E. E. Wyckoff, A. G. Oglesby, E. Rab, R. K. Taylor, and S. M. Payne. 2002. Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence. Infect. Immun. 70:3419– 3426. 38. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 39. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575–2583. 40. Naikare, H., K. Palyada, R. Panciera, D. Marlow, and A. Stintzi. 2006. Major role for FeoB in Campylobacter jejuni ferrous iron acquisition, gut colonization, and intracellular survival. Infect. Immun. 74:5433–5444. 41. Occhino, D. A., E. E. Wyckoff, D. P. Henderson, T. J. Wrona, and S. M. Payne. 1998. Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes. Mol. Microbiol. 29:1493–1507. 42. Paulsen, I. T., C. M. Press, J. Ravel, D. Y. Kobayashi, G. S. Myers, D. V. Mavrodi, R. T. DeBoy, R. Seshadri, Q. Ren, R. Madupu, R. J. Dodson, A. S. Durkin, L. M. Brinkac, S. C. Daugherty, S. A. Sullivan, M. J. Rosovitz, M. L. Gwinn, L. Zhou, D. J. Schneider, S. W. Cartinhour, W. C. Nelson, J. Weidman, K. Watkins, K. Tran, H. Khouri, E. A. Pierson, L. S. Pierson III, L. S. Thomashow, and J. E. Loper. 2005. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23:873–878. 43. Poch, M. T., and W. Johnson. 1993. Ferric reductases of Legionella pneumophila. Biometals 6:107–114. 44. Rabus, R., M. Kube, J. Heider, A. Beck, K. Heitmann, F. Widdel, and R. Reinhardt. 2005. The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch. Microbiol. 183:27–36. 45. Reith, M. E., R. K. Singh, B. Curtis, J. Boyd, A. Bouevitch, J. Kimball, J. Munholland, C. Murphy, D. Sarty, J. Williams, J. Nash, S. Johnson, and L. Brown. 2007, posting date. The genome sequence of Aeromonas salmonicida subsp. salmonicida A449. Direct submission to NCBI. NCBI, Bethesda, MD. 46. Rogers, H. J. 1973. Iron-binding catechols and virulence in Escherichia coli. Infect. Immun. 7:445–456. 47. Rogers, M. B., J. A. Sexton, G. J. DeCastro, and S. B. Calderwood. 2000. Identification of an operon required for ferrichrome iron utilization in Vibrio cholerae. J. Bacteriol. 182:2350–2353. 48. Runyen-Janecky, L. J., M. Hong, and S. M. Payne. 1999. Virulence plasmidencoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation. Infect. Immun. 67:1415– 1423. 49. Runyen-Janecky, L. J., S. A. Reeves, E. G. Gonzales, and S. M. Payne. 2003. Contribution of the Shigella flexneri Sit, Iuc, and Feo iron acquisition systems to iron acquisition in vitro and in cultured cells. Infect. Immun. 71:1919– 1928. 50. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 51. Schroder, I., E. Johnson, and S. de Vries. 2003. Microbial ferric iron reductases. FEMS Microbiol. Rev. 27:427–447. 52. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47–56. 53. Seliger, S., A. Mey, A. Valle, and S. Payne. 2001. The two TonB systems of Vibrio cholerae: redundant and specific functions. Mol. Microbiol. 39:801– 812. 54. Seshadri, R., S. W. Joseph, A. K. Chopra, J. Sha, J. Shaw, J. Graf, D. Haft, M. Wu, Q. Ren, M. J. Rosovitz, R. Madupu, L. Tallon, M. Kim, S. Jin, H. Vuong, O. C. Stine, A. Ali, A. J. Horneman, and J. F. Heidelberg. 2006. Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades. J. Bacteriol. 188:8272–8282. 55. Staab, J. F., and C. F. Earhart. 1990. EntG activity of Escherichia coli enterobactin synthase. J. Bacteriol. 172:6403–6410. 56. Stasakawicz, B., D. Dahlbeck, N. Keen, and C. Napoli. 1987. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 169:5789–5794. 57. Stoebner, J. A., J. R. Butterton, S. B. Calderwood, and S. M. Payne. 1992. Identification of the vibriobactin receptor of Vibrio cholerae. J. Bacteriol. 174:3270–3274. 58. Stoebner, J. A., and S. M. Payne. 1988. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect. Immun. 56:2891–2895. 59. Stojiljkovic, I., M. Cobeljic, and K. Hantke. 1993. Escherichia coli K-12 ferrous iron uptake mutants are impaired in their ability to colonize the mouse intestine. FEMS Microbiol. Lett. 108:111–115. 60. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833–2837. 61. Thieme, F., R. Koebnik, T. Bekel, C. Berger, J. Boch, D. Buttner, C. Caldana, L. Gaigalat, A. Goesmann, S. Kay, O. Kirchner, C. Lanz, B. Linke, A. C. McHardy, F. Meyer, G. Mittenhuber, D. H. Nies, U. Niesbach-Klosgen, T.
5962
MEY ET AL.
Patschkowski, C. Ruckert, O. Rupp, S. Schneiker, S. C. Schuster, F. J. Vorholter, E. Weber, A. Puhler, U. Bonas, D. Bartels, and O. Kaiser. 2005. Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J. Bacteriol. 187:7254–7266. 62. Tsolis, R. M., A. J. Baumler, F. Heffron, and I. Stojiljkovic. 1996. Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella typhimurium in the mouse. Infect. Immun. 64:4549–4556. 63. Vartivarian, S. E., and R. E. Cowart. 1999. Extracellular iron reductases: identification of a new class of enzymes by siderophore-producing microorganisms. Arch. Biochem. Biophys. 364:75–82. 64. Velayudhan, J., N. J. Hughes, A. A. McColm, J. Bagshaw, C. L. Clayton, S. C. Andrews, and D. J. Kelly. 2000. Iron acquisition and virulence in Helicobacter pylori: a major role for FeoB, a high-affinity ferrous iron transporter. Mol. Microbiol. 37:274–286.
J. BACTERIOL. 65. Wang, R. F., and S. R. Kushner. 1991. Construction of versatile low-copynumber vectors for cloning, sequencing and gene expression in Escherichia coli. Gene 100:195–199. 66. Wyckoff, E. E., D. Duncan, A. G. Torres, M. Mills, K. Maase, and S. M. Payne. 1998. Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria. Mol. Microbiol. 28:1139–1152. 67. Wyckoff, E. E., A. R. Mey, A. Leimbach, C. F. Fisher, and S. M. Payne. 2006. Characterization of ferric and ferrous iron transport systems in Vibrio cholerae. J. Bacteriol. 188:6515–6523. 68. Wyckoff, E. E., A. R. Mey, and S. M. Payne. 2007. Iron acquisition in Vibrio cholerae. Biometals 20:405–416. 69. Wyckoff, E. E., M. Schmitt, A. Wilks, and S. M. Payne. 2004. HutZ is required for efficient heme utilization in Vibrio cholerae. J. Bacteriol. 186: 4142–4151.
