Genetic mapping of Vibrio cholerae enterotoxin structural genes

Abstract

The structural genes which constitute the cholera toxin operon, ctxAB, were genetically mapped in the Vibrio cholerae El Tor strain RV79. This strain of V. cholerae contains two copies of the ctx operon located on a 7-kilobase-pair tandemly duplicated region. We began by isolating a vibriophage VcA1 insertion mutation in one of the two ctxA genes located in this region. The mutant carrying this ctxA::VcA1 insertion, DC24, was converted to a VcA1-facilitated donor by introduction of the conjugal plasmid pSJ15, which carries an inserted copy of a defective VcA1-like prophage. The donor characteristics of DC24(pSJ15) indicated that the ctxA::VcA1 insertion mutation was near the trp region of the V. cholerae chromosome. Subsequent RV79 three-factor crosses were performed between VcA1-facilitated donors and recipient strains carrying one of two structural gene mutations in ctx, either ActxA23P Kmr or Actx-7922. The former was constructed by an in vivo marker exchange procedure and could be scored either by its kanamycin resistance phenotype or by its lack of DNA sequences homologous to the ctxA region. The Actx-7922 mutation is a total deletion of both ctx copies of strain RV79. The three-factor cross data strongly suggest that the two ctx loci of RV79 map between the nal and his genes of V. cholerae in the trp nal his linkage group. Physical analysis and heterologous crosses between an RV79 El Tor donor and a 569B classical recipient indicates that one of the two 569B ctx operon copies maps in the same region as the RV79 ctx loci (i.e., linked to nal). Together with previously published observations, these data show that the ctx structural genes are not closely linked to other genes known to affect toxin production in V. cholerae.Toxinogenic strains of Vibrio cholerae produce an extracellular heat-labile protein that is primarily responsible for the diarrheal syndrome observed in Asiatic cholera (8). Cholera toxin is now recognized as the prototype for a growing family of protein enterotoxins that produce their toxic effects via the activation of adenylate cyclase in eucaryotic cells (4,5,9). The heat-labile enterotoxin of Escherichia coli also belongs to this family, and recent DNA sequence analysis has shown the heat-labile enterotoxin genes to be 78% homologous to the cholera toxin genes (5; deWilde, Nature [London], in press). However, the LT operon eltAB appears to be exclusively located on plasmids (7, 28), while the data suggest that the cholera toxin operon ctxAB is located on the bacterial chromosome. This conclusion has been based primarily on the absence of demonstrable plasmids in toxinogenic V. cholerae strains of either the classical or El Tor biotypes (15,22).Although a variety of laboratories have isolated mutants and genetically mapped mutations that affect toxin production in the highly toxinogenic classical strain 569B, all of these mutations have turned out to be regulatory mutations (2,10,12,17,19,20). The explanation for this failure to obtain structural gene mutations in ctx in this particular ...

Genetic analysis of the cholera toxin-positive regulatory gene toxR

“…This suggests that fmt lies between tox and his and is consistent with the results presented in Table 4. Therefore, it appears that the overall gene order is cys tox fmt his nal, with ctx lying between his and nal as previously determined (17). Thus, tox mutations are clearly in a locus distinct from the cholera toxin structural genes, ctxAB.…”

“…LB, brain heart infusion (BHI), Casamino Acids-yeast extract (CYE), and M63 minimal media were prepared as previously described (12,14). Mating experiments were performed as described by Sporecke et al (17). For selecting or ssoring resistance to 5-methyltryptophan, M63 plates containing 150 ,ug of 5methyltryptophan per ml were used.…”

“…In both cases the lowest-frequency class is that of the his' Fmts tox+ recombinants, suggesting that fmt lies between tox and his. Recent mapping data have shown that one copy of ctx in V. cholerae 569B strains is located between his and nal (17). To eliminate the possibility that ctx-related mutations are involved in the phenotype of tox mutants, nal and cys markers were included in a further cross.…”

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

“…Since arg and ilv are located close to each other on the circular V. cholerae genetic map (14,15,32), the data strongly suggest that the hly locus is positioned between the ilv and arg loci ( Table 2), with transfer occurring clockwise from the VcA-3 prophage integrated in or near the hly locus in SG54.…”

“…The presence of a VcA-3 prophage in or near the hly locus of strain HK-1 in SG23 provided a region of homology for VcA-1-mediated transfer (15) from the hly region of the V. cholerae SG54 chromosome into a number of auxotrophic El Tor recipient strains. The recipient strains used in these experiments carried auxotrophic mutations that have been mapped to five sites distributed around the V. cholerae chromosome (14,15,32). The recipient strains were resistant to spectinomycin, which permitted counterselection against the donor.…”