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.     

Haemolysin genes of Vibrio cholerae: presence of homologous DNA in non-haemolytic O1 and haemolytic non-O1 strains

Abstract The structural gene for the haemolysin and two accessory genes from a Vibrio cholerae O1 El Tor strain have previously been cloned in Escherichia coli K-12 to give the plasmid pPM431. This plasmid has been used as a probe with a variety of O1 and non-O1 Vibrio cholerae strains to examine by Southern DNA hybridisations for the presence of homologous DNA. Such experiments show that the DNA homologous to that present in pPM431 is present in all of the 20 strains examined, whether they were haemolytic or non-haemolytic, implying that the genes were present but not expressed in non-haemolytic strains. Using a variety of restriction enzymes to cut the chromosomal DNA of different V. cholerae strains and probing with pPM431, it was possible to distinguish O1 and non-O1 strains, as well as haemolytic or non-haemolytic strains. This variability between hly⁺ and hly⁻ may be indicative of a change in the regulatory region of the haemolysin genes. The results also imply a high degree of homology of the haemolysin of O1 and non-O1 strains.     

Identification and nucleotide sequence determination of the gene responsible for Ogawa serotype specificity of V. cholerae 01.

The gene encoding a protein of 27 kDa, which is specifically expressed in Vibrio cholerae of serotype Ogawa, was identified and its nucleotide sequence determined. The plasmid carrying this gene was found to convert serotype specificity from Inaba to Ogawa when introduced into the Escherichia coli DH5(pVCI112) cell which harbors a cloned 20-kilobase genomic DNA fragment of V. cholerae NIH35A3 and expresses the 01 antigen of Inaba serotype.     

Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

Hemolysin from Vibrio cholerae eltor: cloning and expression of genes in Escherichia coli]

A 6.56-kb V. cholerae eltor DNA fragment encoding hemolysin synthesis was cloned in pUC18. The resultant recombinant plasmid pES4H (9.25 kb) was mapped by restriction analysis and shown to express in different E. coli strains as well as in nonhemolytic V. cholerae strains. Application of the cloned fragment as a molecular probe revealed homologous sequences in all V. cholerae strains tested independently on their biotypes, hemolytic activity and presence of vct-genes in their genomes while none of other Vibrio species and related microorganisms contained such sequences. A recombinant E. coli strain, a V. cholerae eltor hemolysin producer, was constructed. The simultaneous expression of hemolytic and toxinogenic properties by the same V. cholerae strains is discussed.     

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.     

Molecular cloning and characterization of mannitol-1-phosphate dehydrogenase from Vibrio cholerae

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Comparative analysis of the hemolysin genes of Vibrio cholerae non-01, V. mimicus, and V. hollisae that are similar to the tdh gene of V. parahaemolyticus

The genes encoding the hemolysins similar to the thermostable direct hemolysin (tdh gene) of Vibrio parahaemolyticus were cloned from chromosomes of V. mimicus and V. hollisae. These cloned hemolysin genes and previously cloned tdh genes of V. parahaemolyticus and V. cholerae non-01 were compared by physical mapping and by hybridization with oligodeoxyribonucleotide probes. The nucleotide sequences in the coding regions of all the cloned hemolysin genes were very homologous and had only minor variations but the sequences flanking the homolysin genes were dissimilar, indicating that the hemolysin genes have a common ancestor and suggesting that they may have been transferred between Vibrio species as a descrete genetic unit.     

A 14-kilodalton inner membrane protein of Vibrio cholerae biotype e1 tor confers resistance to group IV choleraphage infection to classical vibrios.

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.     

Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

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.     

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Vibrio cholerae El Tor RV79 is phenotypically nonhemolytic; however, strongly hemolytic convertants are occasionally observed on blood agar plates. We have cloned DNA sequences corresponding to the hemolysin determinant from RV79 (Hly+) in the lambda L47.1 and pBR322 vectors. A 2.3-kilobase fragment of V. cholerae DNA was found to be necessary for hemolytic activity. This cloned DNA sequence was used as a probe in Southern blot hybridization analysis of chromosomal restriction digests of a variety of El Tor and classical biotype V. cholerae strains. In all cases, DNA fragments with the same electrophoretic mobilities hybridized to the Hly probe. The results presented demonstrate that the cloned hemolysin determinant is the hly locus. By using mutator vibriophage VcA-3 insertion to promote high-frequency transfer, the hly locus was mapped between arg and ilv on the V. cholerae RV79 chromosome.     

Isolation and characterization of the Vibrio cholerae recA gene

A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments.     

Serogroup conversion of Vibrio cholerae in aquatic reservoirs

The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.