Identification, sequencing, and enzymatic activity of the erythrose-4-phosphate dehydrogenase gene of Vibrio cholerae.

We have identified a gene in Vibrio cholerae (epd) which encodes an erythrose-4-phosphate dehydrogenase activity and is located immediately downstream of an iron-regulated virulence gene, irgA, and immediately upstream of a gene encoding phosphoglycerate kinase (pgk). Expression of epd in V. cholerae is not regulated by iron, nor is it required for virulence in an infant mouse model.

Use of the Vibrio cholerae irgA gene as a locus for insertion and expression of heterologous antigens in cholera vaccine strains.

Vibrio cholerae may be a particularly effective organism for use in delivering heterologous antigens to stimulate a common mucosal immune response. A live attenuated vaccine strain of V. cholerae was constructed from the ctxA deletion mutant 0395-N1, containing the B subunit of Shiga-like toxin I under the transcriptional control of the iron-regulated irgA promoter. The B subunit of Shiga-like toxin I is identical to the B subunit of Shiga toxin (StxB). irgA encodes the major iron-regulated outer membrane protein of V. cholerae, which is a known virulence factor for this organism. Clones of the structural gene irgA from the classical V. cholerae strain 0395, with the gene for the Shiga-like toxin I B subunit inserted under the control of the irgA promoter, were used to introduce an internal deletion of irgA into the chromosome of 0395-N1 by in vivo marker exchange, using the suicide vector plasmid pCVD442. This plasmid contains the sacB gene from Bacillus subtilis, which allowed positive selection for loss of plasmid sequences on exposure to sucrose. The construction of vaccine strains was confirmed by Southern hybridization studies and outer membrane protein analysis. The expression of StxB in the vaccine strain VAC2 following growth in high- or low-iron conditions was shown to be tightly iron-regulated by Western blot analysis and by quantification of StxB using a sandwich enzyme-linked immunosorbent assay. The production of StxB by VAC2 under low-iron conditions was greater than that of the reference strain Shigella dysenteriae 60R.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems.

Shiga-like toxin I (SLT-I) B-subunit expression was examined by using the trc promoter in two different constructs, pSBC32 and pSBC54, in which 710 bp of DNA downstream of the B subunit in pSBC32 was deleted. The trc promoter in pSBC54 was replaced also with the SLT-I iron-regulated promoter to create a third plasmid, pSBC61. SLT-I B-subunit expression was examined from all three plasmids following transfer into Escherichia coli JM105 and the cholera toxin A-subunit gene deletion mutant Vibrio cholerae 0395-N1. The SLT-I B subunit was expressed from all constructs. pSBC61 was regulated by elemental iron and produced equivalent amounts of SLT-I B subunit from both E. coli and V. cholerae. In contrast to the cholera toxin B subunit, virtually all released into the medium, the SLT-I B subunit was predominantly cell associated in the pSBC61 constructs. Both pSBC32 and pSBC54 were inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the E. coli background but not the V. cholerae background; however, when E. coli cultures were allowed to grow for 24 h, the yield of SLT-I B subunit was not increased by IPTG induction. Both pSBC32 and -54 expressed more SLT-I B subunit in the V. cholerae host than in the E. coli host. Scale-up to a 9.9-liter fermentor culture of V. cholerae 0395 N1 (pSBC32) resulted in the isolation of 220 mg of SLT-I B. The purified B subunit was identical, in terms of binding to Vero cells, stoichiometry after chemical cross-linking, and ability to inhibit cytotoxicity of intact Shiga toxin, to native SLT-I B subunit from E. coli O157:H7.

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.

Cloning, sequencing, and transcriptional regulation of the Vibrio cholerae fur gene.

Many genes involved in the transport of iron by bacteria as well as in pathogenesis are regulated by the environmental concentration of iron, with increased expression under low-iron conditions. In Escherichia coli, transcriptional regulation by iron depends on the fur gene. A virulence gene in Vibrio cholerae (irgA) is also transcriptionally regulated by iron, and the promoter of irgA contains a dyad repeat homologous to Fur binding sites in E. coli. Southern hybridization of V. cholerae chromosomal DNA, using an internal fragment of E. coli fur as a probe, showed a single hybridizing sequence under conditions of low stringency. We derived a restriction map in the vicinity of this hybridizing sequence; overlapping PstI and HindIII fragments were identified at the center of this map. The cloned PstI fragment failed to complement an E. coli fur mutant; sequence analysis revealed an open reading frame that began just downstream of the PstI site, suggesting that the clone was not functional because it lacked its promoter. The overlapping HindIII fragment contained the intact V. cholerae fur gene with its promoter and complemented an E. coli fur mutant. DNA sequencing of the HindIII fragment demonstrated a single open reading frame of 150 amino acids that was 76% homologous to E. coli Fur. Primer extension analysis localized two promoters for the V. cholerae fur gene; no significant homology to an E. coli Fur binding site was identified for either promoter. Northern blot analysis showed that the two fur transcripts were not strongly regulated by iron. These studies identify a gene in V. cholerae homologous in both function and sequence to the fur gene of E. coli, and we have designated this gene the fur gene of V. cholerae.

Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae.

We have previously described a virulence gene in Vibrio cholerae (irgA) that is more than 850-fold regulated in response to iron. Negative regulation of irgA by iron occurred at the transcriptional level, and there was a dyad symmetric nucleotide sequence in the vicinity of the irgA promoter homologous to the Fur binding site in Escherichia coli. When irgA was cloned into E. coli, we showed that transcription of irgA required 900 base pairs of DNA upstream of the irgA promoter that contained an open reading frame in inverse orientation to irgA. In the present study, we show that this upstream region of DNA encodes a gene in inverse orientation to irgA (named irgB) that is also negatively regulated by iron. Insertional inactivation of irgB on the V. cholerae chromosome leads to loss of expression of a chromosomal irgA'-'phoA fusion (in which the primes indicate truncated genes), which is restored to normal by provision of irgB on a plasmid in trans. DNA sequencing of irgB shows that the protein product (IrgB) is homologous to the LysR family of positive transcriptional activators, and secondary structure analysis of IrgB predicts a helix-turn-helix DNA binding motif. The promoters of irgB and irgA are divergent but overlap each other and the previously defined Fur-binding site. We propose a model for iron regulation of irgA expression in V. cholerae. In the presence of sufficient iron, transcription of both irgA and irgB is negatively regulated by a Fur-like protein. In low iron conditions, negative regulation of transcription is removed, and production of IrgB leads to positive transcriptional activation of irgA. It seems likely that the high induction ratio of irgA expression under low- and high-iron conditions (850-fold) relates to the fact that its cognate positive transcriptional activator (irgB) is itself negatively regulated by iron.

Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli fur system.

We have previously described an iron-regulated virulence determinant in Vibrio cholerae. Strain MBG40, which contains a TnphoA insertion mutation in the iron-regulated gene irgA, has reduced virulence in a newborn mouse model and has lost the major 77-kDa iron-regulated outer membrane protein. We report here the cloning of the irgA'-'phoA gene fusion, the sequencing of the 5'-proximal portion of irgA, and the definition of its promoter region by primer extension. The deduced amino acid sequence of the amino-terminal portion of IrgA is homologous to the ferrienterochelin receptor of Escherichia coli (FepA), suggesting that IrgA may be the iron-vibriobactin outer membrane receptor. Iron regulation of irgA in an E. coli background and that of the E. coli gene slt-IA in a V. cholerae background are reciprocal, suggesting a common mechanism of iron regulation. Regulation of irgA by iron in V. cholerae occurs at the transcriptional level, and there is an interrupted dyad symmetric sequence in the vicinity of the promoter that is homologous to Fur binding sites of E. coli. Unlike iron-regulated genes in E. coli, however, transcription of irgA requires an additional 900 bp of upstream DNA that contains an open reading frame in inverse orientation to irgA.

A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit.

We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter. The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography. The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit. B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells. This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.