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1.
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.  相似文献   

2.
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 .  相似文献   

3.
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.  相似文献   

4.
Iron is essential for the survival of almost all bacteria. Vibrio cholerae acquires iron through the secretion of a catecholate siderophore called vibriobactin. At present, how vibriobactin chelates ferric ion remains controversial. In addition, the mechanisms underlying the recognition of ferric vibriobactin by the siderophore transport system and its delivery into the cytoplasm specifically have not been clarified. In this study, we report the high-resolution structures of the ferric vibriobactin periplasmic binding protein ViuP and its complex with ferric vibriobactin. The holo-ViuP structure reveals that ferric vibriobactin does not adopt the same iron coordination as that of other catecholate siderophores such as enterobactin. The three catechol moieties donate five, rather than six, oxygen atoms as iron ligands. The sixth iron ligand is provided by a nitrogen atom from the second oxazoline ring. This kind of iron coordination results in the protrusion of the second catechol moiety and renders the electrostatic surface potential of ferric vibriobactin less negatively polarized compared with ferric enterobactin. To accommodate ferric vibriobactin, ViuP has a deeper subpocket to hold the protrusion of the second catechol group. This structural characteristic has not been observed in other catecholate siderophore-binding proteins. Biochemical data show that siderocalin, which is part of the mammalian innate immune system, cannot efficiently sequester ferric vibriobactin in vitro, although it can capture many catecholate siderophores with high efficiency. Our findings suggest that the unique iron coordination found in ferric vibriobactin may be utilized by some pathogenic bacteria to evade the siderocalin-mediated innate immune response of mammals.  相似文献   

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7.
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.  相似文献   

8.
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.  相似文献   

9.
The two TonB systems in Vibrio cholerae were found to have unique as well as common functions. Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome. However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V. cholerae. Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization. This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source. Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size. An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2. A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low-osmolarity medium, but could not use haemin in high osmolarity. This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport. Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.  相似文献   

10.
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.  相似文献   

11.
Vibriobactin, a siderophore from Vibrio cholerae   总被引:14,自引:0,他引:14  
A novel siderophore (microbial iron transport compound) has been isolated from low iron cultures of Vibrio cholerae. Belonging to the catecholamide family of chelators, it has been shown to contain three residues of 2,3-dihydroxybenzoic acid and two residues of threonine. Both threonine moieties are present in the form of oxazoline rings. Furthermore, the polyamine backbone of the molecule was proved to be not spermidine, but the rare N-(3-aminopropyl)-1,3-diaminopropane, norspermidine. The structure of the new siderophore has been determined to be N-[3-(2,3-dihydroxybenzamido)propyl]-1, 3-bis[2,3-dihydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]prop ane. The compound has been given the trivial name vibriobactin. Mutants defective in the synthesis and utilization of vibriobactin were isolated. In an iron-limited environment V. cholerae was found to respond more strongly to vibriobactin, agrobactin, and ferrichrome than to enterobactin.  相似文献   

12.
Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium.  相似文献   

13.
14.
The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.  相似文献   

15.
Vibrio cholerae synthesizes the catechol siderophore vibriobactin. In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes. vibD encodes a phosphopantetheinyl transferase, and vibH encodes a novel nonribosomal peptide synthase. Both VibD and VibH are required for vibriobactin biosynthesis.  相似文献   

16.
17.
A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.  相似文献   

18.
Liu X  Du Q  Wang Z  Liu S  Li N  Chen Y  Zhu C  Zhu D  Wei T  Huang Y  Xu S  Gu L 《FEBS letters》2012,586(8):1240-1244
VctP, one of the two essential siderophore-binding PBPs from the pathogen Vibrio cholerae, plays an important role in the transport of enterobactin and vibriobactin, which have quite different configurations of iron coordination, from the periplasm to the inner membrane. The current study reports the crystal structure of VctP from V. cholerae N16961 at 1.7 Å resolution. A structural comparison of VctP with its homologues and the results of molecular docking indicate that enterobactin and vibriobactin share the same binding pocket. Significantly, a basic triad consisting of Arg137, Arg226 and Arg270 is used to balance the three negative charges of ferric-enterobactin, while a basic dyad consisting of Arg137 and Arg270 is used to balance the two negative charges of ferric-vibriobactin.  相似文献   

19.
C Amaro  R Aznar  E Alcaide    M L Lemos 《Applied microbiology》1990,56(8):2410-2416
A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore) as well as some strains of Vibrio anguillarum deficient in the anguibactin-mediated system. The chromatographic analyses and absorption spectra of siderophores extracted from culture supernatants suggest that vibriobactin may be produced by the strains examined. Interestingly, some strains also produced hydroxamate-type compounds, as determined by chemical tests, and were able to promote the growth of an aerobactin-deficient strain of Escherichia coli. These results were confirmed by the absorption spectra and chromatographic analyses of the culture extracts. The synthesis of iron-regulated outer membrane proteins in representative strains was also examined. The molecular sizes of the main induced proteins ranged from 70 to 78 kilodaltons. These results indicate that several iron uptake mechanisms which could be involved in environmental survival and pathogenicity are present in environmental V. cholerae non-O1 strains.  相似文献   

20.
This work reports the utilization of an in vivo expression technology system to identify in vivo-induced (ivi) genes in Yersinia ruckeri after determination of the conditions needed for its selection in fish. Fourteen clones were selected, and the cloned DNA fragments were analyzed after partial sequencing. In addition to sequences with no significant similarity, homology with genes encoding proteins putatively involved in two-component and type IV secretion systems, adherence, specific metabolic functions, and others were found. Among these sequences, four were involved in iron acquisition through a catechol siderophore (ruckerbactin). Thus, unlike other pathogenic yersiniae producing yersiniabactin, Y. ruckeri might be able to produce and utilize only this phenolate. The genetic organization of the ruckerbactin biosynthetic and uptake loci was similar to that of the Escherichia coli enterobactin gene cluster. Genes rucC and rupG, putative counterparts of E. coli entC and fepG, respectively, involved in the biosynthesis and transport of the iron siderophore complex, respectively, were analyzed further. Thus, regulation of expression by iron and temperature and their presence in other Y. ruckeri siderophore-producing strains were confirmed for these two loci. Moreover, 50% lethal dose values 100-fold higher than those of the wild-type strain were obtained with the rucC isogenic mutant, showing the importance of ruckerbactin in the pathogenesis caused by this microorganism.  相似文献   

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