Genetic mapping of toxin regulatory mutations in Vibrio cholerae

Mekalanos, John J.; Sublett, R D; Romig, W. R.

doi:10.1128/jb.139.3.859-865.1979

Cited by 37 publications

(33 citation statements)

References 24 publications

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“…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 V. cholerae strain is presumably related to the fact that the ctx operon is duplicated in 569B (22, 25).…”

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Genetic mapping of Vibrio cholerae enterotoxin structural genes

Sporecke¹,

Castro²,

Mekalanos³

1984

J Bacteriol

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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 ...

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“…The plasmid content of a strain is given in parentheses after its name in the text.Media. LB and CYE media, BHI broth, and M63 minimal medium have been described previously (20,21). Adenine and thiamine were always added to M63 minimal medium to a final concentration of 20 and 2.5 ,ug/ml, respectively.…”

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confidence: 99%

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Sporecke¹,

Castro²,

Mekalanos³

1984

J Bacteriol

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“…Hypertoxinogenic mutants, htx, of strain 569B that result in a threeto sevenfold increase in toxin production have also been isolated (12). Unlinked mutations that suppress the a The indicated recipient strains were each mated to V. cholerae JM1(pSJ13), and His+ recombinants were selected and scored for toxin production (tox+) by the ganglioside filter assay (11).…”

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confidence: 99%

“…Media and mating procedures. 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).…”

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Genetic analysis of the cholera toxin-positive regulatory gene toxR

Miller¹,

Mekalanos²

1985

J Bacteriol

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Southern blot analysis with a toxR-specific gene probe indicates that Vibrio chokrae 569B has a 1.2-kilobase deletion near the tox-R gene. Heterologous conjugative crosses were carried out between the El Tor strain RV79 and 569B tox mutants. Tox+ recombinants showed the same linkage properties to the his locus as to the previously mapped tox locus of 569B. Southern blot analysis with the toxR probe of the Tox+ recombinants obtained in these heterologous crosses showed that these recombinants had replaced' the V. cholerae-569B (recipient) toxR DNA with the V. cholerae RV79 (donor) toxR DNA, indicating that tox and toxR are the same locus. However, the Tox+ recombinants synthesized an amount of toxin intermediate between the level observed for wild-type RV79 and 569B strains, suggesting there is a difference in the ability of toxR genes from different strains to activate ctx. About half of the mutations which suppress the phenotype of hypertoxinogenic locus htx are unlinked to htx and in addition have a hypotoxinogenic phenotype relative to that of the wild type. Most of these hypotoxinogenic, second-site suppressors show a linkage to his similar to the linkage of toxR to his and are therefore probably mutations in toxR. These results indicate that the toxR gene product is required for ctx expression and that a functional toxR gene is required for the effect of an htx mutation to be seen.

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