Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.     

A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [(3)H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores.     

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

Vibrio cholerae was found to have two sets of genes encoding TonB, ExbB and ExbD proteins. The first set ( tonB1, exbB1, exbD1 ) was obtained by complementation of a V. cholerae tonB mutant. In the mutant, a plasmid containing these genes permitted transport via the known V. cholerae high-affinity iron transport systems, including uptake of haem, vibriobactin and ferrichrome. When chromosomal mutations in exbB1 or exbD1 were introduced into a wild-type V. cholerae background, no defect in iron transport was noted, indicating the existence of additional genes that can complement the defect in the wild-type background. Another region of the V. cholerae chromosome was cloned that encoded a second functional TonB/Exb system ( tonB2, exbB2, exbD2 ). A chromosomal mutation in exbB2 also failed to exhibit a defect in iron transport, but a V. cholerae strain that had chromosomal mutations in both the exbB1 and exbB2 genes displayed a mutant phenotype similar to that of an Escherichia coli tonB mutant. The genes encoding TonB1, ExbB1, ExbD1 were part of an operon that included three haem transport genes ( hutBCD ), and all six genes appeared to be expressed from a single Fur-regulated promoter upstream of tonB1 . A plasmid containing all six genes permitted utilization of haem by an E. coli strain expressing the V. cholerae haem receptor, HutA. Analysis of the hut genes indicated that hutBCD, which are predicted to encode a periplasmic binding protein (HutB) and cytoplasmic membrane permease (HutC and HutD), were required to reconstitute the V. cholerae haem transport system in E. coli. In V. cholerae , the presence of hutBCD stimulated growth when haemin was the iron source, but these genes were not essential for haemin utilization in V. cholerae .     

Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols.

The chromosomally encoded Vibrio anguillarum fur gene was characterized. The amino acid sequence of the Fur protein showed a very high degree of homology with those of V. cholerae and V. vulnificus. The degree of homology was lower, although still high, with the Escherichia coli and Yersinia pestis Fur amino acid sequences, while the lowest degree of homology was found with the Pseudomonas aeruginosa Fur protein. The C-terminal portion of Fur is the least conserved region among these Fur proteins. Within this portion, two regions spanning amino acids 105 to 121 and 132 to the end are the least conserved. A certain degree of variation is also present in the N termini spanning amino acids 28 to 46. Regulation of expression of the V. anguillarum fur gene by iron was not detected by immunoblot analysis. Mutations in the cloned fur gene were generated either by site-directed mutagenesis (the Lys-77 was changed to a Gly to generate the derivative FurG77) or by insertion of a DNA fragment harboring the aph gene in the same position. FurG77 was impaired in its ability to regulate a reporter gene with the Fur box in its promoter, while the insertion mutant was completely inactive. V. anguillarum fur mutants were obtained by isolating manganese-resistant derivatives. In one of these mutants, which encoded a Fur protein with an apparent lower molecular weight, the regulation of the production of catechols and synthesis of the outer membrane protein FatA were partially lost. In the case of another mutant, no protein was detected by anti-Fur serum. This derivative showed a total lack of regulation of biosynthesis of catechols and FatA protein by iron.     

A dominant-negative fur mutation in Bradyrhizobium japonicum

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis.

Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was constructed by marker exchange. This strain was unable to produce vibriobactin or DHBA, confirming that in V. cholerae VibA catalyzes an early step in vibriobactin biosynthesis.     

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.     

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.