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.     

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.     

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

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 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.     

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.     

Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Molecular characterization of nosA, a Pseudomonas stutzeri gene encoding an outer membrane protein required to make copper-containing N2O reductase.

A Pseudomonas stutzeri gene (nosA) encoding an outer membrane protein was cloned into the broad-host-range vector pRK290 and expressed in a mutant lacking the protein. Deletion analysis identified the approximate extent of the nosA region which was sequenced, and it was found to contain an open reading frame encoding 683 amino acids including a presumed signal sequence of 44 amino acids. The putative processed form had a molecular weight of 70,218, characteristics typical of outer membrane proteins, and considerable amino acid sequence homology with Escherichia coli BtuB. A short stretch of amino acids was homologous with the E. coli TonB-dependent outer membrane proteins, BtuB, IutA, FepA, and FhuA, suggesting a homologous function: interaction with a periplasmic protein or uptake of a specific substrate.     

Characterization of the Plesiomonas shigelloides genes encoding the heme iron utilization system

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.     

Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV

The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.     

The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake

A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.     

Cloning and initial characterization of the Bordetella pertussis fur gene

In several Gram-negative pathogens the fur (ferric uptake regulator) gene product controls the expression of many genes involved in iron uptake and virulence. To facilitate the study of iron-regulated gene expression in Bordetella pertussis, we cloned the fur gene from this organism. The B. pertussis fur gene product was 54% identical to the Escherichia coli Fur and complemented two E. coli fur mutants. As with the E. coli fur gene, sequences upstream of the B. pertussis fur were homologous to the consensus Fur-binding site and to the consensus catabolite activator protein binding site.     

Identification of an operon required for ferrichrome iron utilization in Vibrio cholerae

Mutagenesis of Vibrio cholerae with TnphoA, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to Escherichia coli FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the E. coli fhu operon, the V. cholerae ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.