Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex.

UNLABELLED: Feo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. The feo operon in Vibrio cholerae is made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linked in vivo in V. cholerae. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport. IMPORTANCE: The Feo system is the major route for ferrous iron transport in bacteria. In this work, the Vibrio cholerae Feo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complex in vivo. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly.

Identification and Characterization of a Putative Manganese Export Protein in Vibrio cholerae.

UNLABELLED: Manganese plays an important role in the cellular physiology and metabolism of bacterial species, including the human pathogen Vibrio cholerae The intracellular level of manganese ions is controlled through coordinated regulation of the import and export of this element. We have identified a putative manganese exporter (VC0022), named mneA (manganese exporter A), which is highly conserved among Vibrio spp. An mneA mutant exhibited sensitivity to manganese but not to other cations. Under high-manganese conditions, the mneA mutant showed an almost 50-fold increase in intracellular manganese levels and reduced intracellular iron relative to those of its wild-type parent, suggesting that the mutant's manganese sensitivity is due to the accumulation of toxic levels of manganese and reduced iron. Expression of mneA suppressed the manganese-sensitive phenotype of an Escherichia coli strain carrying a mutation in the nonhomologous manganese export gene, mntP, further supporting a manganese export function for V. cholerae MneA. The level of mneA mRNA was induced approximately 2.5-fold after addition of manganese to the medium, indicating regulation of this gene by manganese. This study offers the first insights into understanding manganese homeostasis in this important pathogen. IMPORTANCE: Bacterial cells control intracellular metal concentrations by coordinating acquisition in metal-limited environments with export in metal-excess environments. We identified a putative manganese export protein, MneA, in Vibrio cholerae An mneA mutant was sensitive to manganese, and this effect was specific to manganese. The mneA mutant accumulated high levels of intracellular manganese with a concomitant decrease in intracellular iron levels when grown in manganese-supplemented medium. Expression of mneA in trans suppressed the manganese sensitivity of an E. coli mntP mutant. This study is the first to investigate manganese export in V. cholerae.

Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of V. cholerae iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in V. cholerae. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source in vitro, we demonstrate that heme is not available to V. cholerae in the infant mouse intestine.

Catechol Siderophore Transport by Vibrio cholerae.

UNLABELLED: Siderophores, small iron-binding molecules secreted by many microbial species, capture environmental iron for transport back into the cell. Vibrio cholerae synthesizes and uses the catechol siderophore vibriobactin and also uses siderophores secreted by other species, including enterobactin produced by Escherichia coli. E. coli secretes both canonical cyclic enterobactin and linear enterobactin derivatives likely derived from its cleavage by the enterobactin esterase Fes. We show here that V. cholerae does not use cyclic enterobactin but instead uses its linear derivatives. V. cholerae lacked both a receptor for efficient transport of cyclic enterobactin and enterobactin esterase to promote removal of iron from the ferrisiderophore complex. To further characterize the transport of catechol siderophores, we show that the linear enterobactin derivatives were transported into V. cholerae by either of the catechol siderophore receptors IrgA and VctA, which also transported the synthetic siderophore MECAM [1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene]. Vibriobactin is transported via the additional catechol siderophore receptor ViuA, while the Vibrio fluvialis siderophore fluvibactin was transported by all three catechol receptors. ViuB, a putative V. cholerae siderophore-interacting protein (SIP), functionally substituted for the E. coli ferric reductase YqjH, which promotes the release of iron from the siderophore in the bacterial cytoplasm. In V. cholerae, ViuB was required for the use of vibriobactin but was not required for the use of MECAM, fluvibactin, ferrichrome, or the linear derivatives of enterobactin. This suggests the presence of another protein in V. cholerae capable of promoting the release of iron from these siderophores. IMPORTANCE: Vibrio cholerae is a major human pathogen and also serves as a model for the Vibrionaceae, which include other serious human and fish pathogens. The ability of these species to persist and acquire essential nutrients, including iron, in the environment is epidemiologically important but not well understood. In this work, we characterize the ability of V. cholerae to acquire iron by using siderophores produced by other organisms. We resolve confusion in the literature regarding its ability to use the Escherichia coli siderophore enterobactin and identify the receptor and TonB system used for the transport of several siderophores. The use of some siderophores did not require the ferric reductase ViuB, suggesting that an uncharacterized ferric reductase is present in V. cholerae.

FeoA and FeoC are essential components of the Vibrio cholerae ferrous iron uptake system, and FeoC interacts with FeoB.

The ferrous iron transport system Feo is widely distributed among bacterial species, yet its physical structure and mechanism of iron transport are poorly understood. In Vibrio cholerae, the feo operon consists of three genes, feoABC. feoB encodes an 83-kDa protein with an amino-terminal GTPase domain and a carboxy-terminal domain predicted to be embedded in the inner membrane. While FeoB is believed to form the pore for iron transport, the roles of FeoA and FeoC are unknown. In this work, we show that FeoA and FeoC, as well as the more highly conserved FeoB, are all required for iron acquisition by V. cholerae Feo. An in-frame deletion of feoA, feoB, or feoC eliminated iron acquisition. The loss of transport activity in the feoA and feoC mutants was not due to reduced transcription of the feo operon, suggesting that these two small proteins are required for activity of the transporter. feoC was found to encode a protein that interacts with the cytoplasmic domain of FeoB, as determined using the BACTH bacterial two-hybrid system. Two conserved amino acids in FeoC were found to be necessary for the interaction with FeoB in the two-hybrid assay, and when either of these amino acids was mutated in the context of the entire feo operon, iron acquisition via Feo was reduced. No interaction of FeoA with FeoB or FeoC was detected in the BACTH two-hybrid assay.

The Vibrio cholerae VctPDGC system transports catechol siderophores and a siderophore-free iron ligand.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron. It transports the catechol siderophores vibriobactin, which it synthesizes and secretes, and enterobactin. These siderophores are transported across the inner membrane by one of two periplasmic binding protein-dependent ABC transporters, VctPDGC or ViuPDGC. We show here that one of these inner membrane transport systems, VctPDGC, also promotes iron acquisition in the absence of siderophores. Plasmids carrying the vctPDGC genes stimulated growth in both rich and minimal media of a Shigella flexneri mutant that produces no siderophores. vctPDGC also stimulated the growth of an Escherichia coli enterobactin biosynthetic mutant in low iron medium, and this effect did not require feoB, tonB or aroB. A tyrosine to phenylalanine substitution in the periplasmic binding protein VctP did not alter enterobactin transport, but eliminated growth stimulation in the absence of a siderophore. These data suggest that the VctPDGC system has the capacity to transport both catechol siderophores and a siderophore-free iron ligand. We also show that VctPDGC is the previously unidentified siderophore-independent iron transporter in V. cholerae, and this appears to complete the list of iron transport systems in V. cholerae.

Genetics and virulence association of the Shigella flexneri sit iron transport system.

The sit-encoded iron transport system is present within pathogenicity islands in all Shigella spp. and some pathogenic Escherichia coli strains. The islands contain numerous insertion elements and sequences with homology to bacteriophage genes. The Shigella flexneri sit genes can be lost as a result of deletion within the island. The formation of deletions was dependent upon RecA and occurred at relatively high frequency. This suggests that the sit region is inherently unstable, yet sit genes are maintained in all of the clinical isolates tested. Characterization of the sitABCD genes in S. flexneri indicates that they encode a ferrous iron transport system, although the genes are induced aerobically. The sit genes provide a competitive advantage to S. flexneri growing within epithelial cells, and a sitA mutant is outcompeted by the wild type in cultured epithelial cells. The Sit system is also required for virulence in a mouse lung model. The sitA mutant was able to infect the mice and induce a protective immune response but was avirulent compared to its wild-type parent strain.

Genetics and environmental regulation of Shigella iron transport systems.

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.

Vibrio cholerae VciB promotes iron uptake via ferrous iron transporters.

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.

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments.

Iron is an essential element for Vibrio spp., but the acquisition of iron is complicated by its tendency to form insoluble ferric complexes in nature and its association with high-affinity iron-binding proteins in the host. Vibrios occupy a variety of different niches, and each of these niches presents particular challenges for acquiring sufficient iron. Vibrio species have evolved a wide array of iron transport systems that allow the bacteria to compete for this essential element in each of its habitats. These systems include the secretion and uptake of high-affinity iron-binding compounds (siderophores) as well as transport systems for iron bound to host complexes. Transporters for ferric and ferrous iron not complexed to siderophores are also common to Vibrio species. Some of the genes encoding these systems show evidence of horizontal transmission, and the ability to acquire and incorporate additional iron transport systems may have allowed Vibrio species to more rapidly adapt to new environmental niches. While too little iron prevents growth of the bacteria, too much can be lethal. The appropriate balance is maintained in vibrios through complex regulatory networks involving transcriptional repressors and activators and small RNAs (sRNAs) that act posttranscriptionally. Examination of the number and variety of iron transport systems found in Vibrio spp. offers insights into how this group of bacteria has adapted to such a wide range of habitats.

Iron acquisition in Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.

Characterization of ferric and ferrous iron transport systems in Vibrio cholerae.

Vibrio cholerae has multiple iron acquisition systems, including TonB-dependent transport of heme and of the catechol siderophore vibriobactin. Strains defective in both of these systems grow well in laboratory media and in the infant mouse intestine, indicating the presence of additional iron acquisition systems. Previously uncharacterized potential iron transport systems, including a homologue of the ferrous transporter Feo and a periplasmic binding protein-dependent ATP binding cassette (ABC) transport system, termed Fbp, were identified in the V. cholerae genome sequence. Clones encoding either the Feo or the Fbp system exhibited characteristics of iron transporters: both repressed the expression of lacZ cloned under the control of a Fur-regulated promoter in Escherichia coli and also conferred growth on a Shigella flexneri mutant that has a severe defect in iron transport. Two other ABC transporters were also evaluated but were negative by these assays. Transport of radioactive iron by the Feo system into the S. flexneri iron transport mutant was stimulated by the reducing agent ascorbate, consistent with Feo functioning as a ferrous transporter. Conversely, ascorbate inhibited transport by the Fbp system, suggesting that it transports ferric iron. The growth of V. cholerae strains carrying mutations in one or more of the potential iron transport genes indicated that both Feo and Fbp contribute to iron acquisition. However, a mutant defective in the vibriobactin, Fbp, and Feo systems was not attenuated in a suckling mouse model, suggesting that at least one other iron transport system can be used in vivo.

Iron and pathogenesis of Shigella: iron acquisition in the intracellular environment.

Shigella species are able to grow in a variety of environments, including intracellularly in host epithelial cells. Shigella have a number of different iron transport systems that contribute to their ability to grow in these diverse environments. Siderophore iron uptake systems, heme transporters, and ferric and ferrous iron transport systems are present in these bacteria, and the genes encoding some of these systems appear to have spread among the Shigella species by horizontal transmission. Iron is not only essential for growth of Shigella but also plays an important role in regulation of metabolic processes and virulence determinants in Shigella. This regulation is mediated by the repressor protein Fur and the small RNA RyhB.

Iron and fur regulation in Vibrio cholerae and the role of fur in virulence.

Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.

Shigella dysenteriae ShuS promotes utilization of heme as an iron source and protects against heme toxicity.

Shigella dysenteriae serotype 1, a major cause of bacillary dysentery in humans, can use heme as a source of iron. Genes for the transport of heme into the bacterial cell have been identified, but little is known about proteins that control the fate of the heme molecule after it has entered the cell. The shuS gene is located within the heme transport locus, downstream of the heme receptor gene shuA. ShuS is a heme binding protein, but its role in heme utilization is poorly understood. In this work, we report the construction of a chromosomal shuS mutant. The shuS mutant was defective in utilizing heme as an iron source. At low heme concentrations, the shuS mutant grew slowly and its growth was stimulated by either increasing the heme concentration or by providing extra copies of the heme receptor shuA on a plasmid. At intermediate heme concentrations, the growth of the shuS mutant was moderately impaired, and at high heme concentrations, shuS was required for growth on heme. The shuS mutant did not show increased sensitivity to hydrogen peroxide, even at high heme concentrations. ShuS was also required for optimal utilization of heme under microaerobic and anaerobic conditions. These data are consistent with the model in which ShuS binds heme in a soluble, nontoxic form and potentially transfers the heme from the transport proteins in the membrane to either heme-containing or heme-degrading proteins. ShuS did not appear to store heme for future use.

HutZ is required for efficient heme utilization in Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, requires iron for growth. One mechanism by which it acquires iron is the uptake of heme, and several heme utilization genes have been identified in V. cholerae. These include three distinct outer membrane receptors, two TonB systems, and an apparent ABC transporter to transfer heme across the inner membrane. However, little is known about the fate of the heme after it enters the cell. In this report we show that a novel heme utilization protein, HutZ, is required for optimal heme utilization. hutZ (open reading frame [ORF] VCA0907) is encoded with two other genes, hutW (ORF VCA0909) and hutX (ORF VCA0908), in an operon divergently transcribed from the tonB1 operon. A hutZ mutant grew poorly when heme was provided as the sole source of iron, and the poor growth was likely due to the failure to use heme efficiently as a source of iron, rather than to heme toxicity. Heme oxygenase mutants of both Corynebacterium diphtheriae and C. ulcerans fail to use heme as an iron source. When the hutWXZ genes were expressed in the heme oxygenase mutants, growth on heme was restored, and hutZ was required for this effect. Biochemical characterization indicated that HutZ binds heme with high efficiency; however, no heme oxygenase activity was detected for this protein. HutZ may act as a heme storage protein, and it may also function as a shuttle protein that increases the efficiency of heme trafficking from the membrane to heme-containing proteins.

Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence.

The gram-negative enteric pathogen Vibrio cholerae requires iron for growth. V. cholerae has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make. One siderophore that V. cholerae transports, but does not make, is enterobactin. Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA. In this study, two candidate enterobactin receptor genes, irgA (VC0475) and vctA (VCA0232), were identified by analysis of the V. cholerae genomic sequence. A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin. When either irgA or vctA was supplied on a plasmid, the ability of the irgA vctA double mutant to use enterobactin was restored. This indicates that both VctA and IrgA transport enterobactin. We also identify the genes vctPDGC, which are linked to vctA and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230). An irgA::TnphoA mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of V. cholerae infection (M. B. Goldberg, V. J. DiRita, and S. B. Calderwood, Infect. Immun. 58:55-60, 1990). In this work, a new irgA mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose. These data indicate that the virulence defect in MBG40 is not due to the loss of irgA function and that irgA is unlikely to be an important virulence factor.