Auxotrophic mutations induced by Vibrio cholerae mutator phage VcA1

Johnson, S

doi:10.1016/0378-1097(81)90047-1

Cited by 6 publications

(14 citation statements)

References 5 publications

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“…Isolation and characterization of a VcAl insertion mutation in ctxA. VcAl is a temperate vibriophage with biological properties resembling those of phage Mu in E. coli (13). Johnson et al (13) have recently described a gene transfer system that uses inserted VcA1 prophages as portable regions of homology between the V. cholerae conjugal sex factor P and the V. cholerae chromosome.…”

Section: Resultsmentioning

confidence: 99%

“…VcAl is a temperate vibriophage with biological properties resembling those of phage Mu in E. coli (13). Johnson et al (13) have recently described a gene transfer system that uses inserted VcA1 prophages as portable regions of homology between the V. cholerae conjugal sex factor P and the V. cholerae chromosome. In this system, the chromosomal location of a VcA1 prophage can be inferred from the donor characteristics of the corresponding lysogenic strain when it carries the plasmid pSJ15.…”

Section: Resultsmentioning

confidence: 99%

“…Since pSJ15 contains an inserted copy of a defective VcA1-like prophage, transient integration of pSJ15 into the V. cholerae chromosome presumably occurs, directed by homologous crossover events between the chromosomal and plasmid copies of VcA1. Thus, the location and orientation of the VcA1 prophage on the chromosome determines the origin and polarity of transfer of genetic markers by one of these donors (13). Such donor strains containing a chromosomal VcA1 prophage and plasmid pSJl5 are referred to as VcA1facilitated donors.…”

Section: Resultsmentioning

confidence: 99%

“…Three different ctx structural gene alterations were used as genetic markers in this analysis. Initially, an insertion mutation of the mutagenic vibriophage VcA1 (13) into the ctxA gene was used to construct a VcA1-facilitated donor by the method of Johnson et al (13). The ctxA::VcA1facilitated donor strain, DC24(pSJ15), was shown to transfer trp+ at a high frequency to RV79 recipients, indicating that the chromosomal ctxA::VcA1 insertion in this strain was near the trp region on the chromosome.…”

Section: Discussionmentioning

confidence: 99%

“…After growth in LB broth at 37°C for 2 h, the cells were pelleted by centrifugation at 4,500 rpm for 5 min in a Sorvall SS34 rotor. The cells were suspended in an equal volume of M63 salts and plated onto M63 plates supplemented with 200 ,ug of nutrient broth (Difco) to give isolated colonies (13,33). After incubation at 37°C for 2 days, tiny colonies were transferred with toothpicks onto TYCC plates.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Sporecke¹,

Castro²,

Mekalanos³

1984

J Bacteriol

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Molecular epidemiological studies of United States Gulf Coast Vibrio cholerae strains: integration site of mutator vibriophage VcA-3

Goldberg¹,

Murphy²

1983

Infect Immun

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Environmental and clinical Vibrio cholerae 0-1 strains isolated from the U.S. Gulf Coast region were found to be lysogenic for a vibriophage which we have designated VcA-3. Comparison of VcA-3 with the previously described vibriophages VcA-1 and VcA-2 has shown that VcA-1 and VcA-3 are homoimmune, have extensive sequence homology, but have markedly different restriction endonuclease digestion patterns. VcA-3 was found to randomly integrate into the V. cholerae RV79 chromosome and to introduce stable auxotrophic mutations. We show that all U.S. Gulf Coast environmental and clinical isolates that are lysogenic for VcA-3, including both tox+ and tox-isolates, contain the prophage integrated at an identical chromosomal site. Given the known stability of temperate mutator bacteriophages, these results suggest that there is a clonal relationship among the V. cholerae 0-1 strains examined in this study, including the tox+ and tox-isolates.

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High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype

Mostow

Richardson

1990

Infect Immun

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The species Vibrio cholerae contains within it two biotypes, classical and El Tor, both of which are motile. Phenotypic expression of motility was unaffected by type of growth medium, salt concentration, pH, or temperature of incubation. However, seven strains of classical V. cholerae produced spontaneous nonmotile mutants at an unusually high frequency (ca. 10-4), while no mutants were detected for all three El Tor strains examined. No revertants of these nonmotile mutants were detected. Four independent mutants of classical strain 395 were isolated to characterize this phenomenon. By transmission electron microscopy, one of the nonmotile mutants was found to be flagellated, while the other three were found to be aflagellate. Chromosomal DNA from the mutants and parental wild-type strain 395 was exatnined by Southern blot analysis with, as probes, V. cholerae mutagenic prophages VcA-1 and VcA-2 and six cloned motility gene regions isolated from transposon insertion motility mutants of strains 395 and N16961 (El Tor, Inaba). The parental wild-type strain and all of the mutants exhibited the same pattern of bands when probed with VcA-1 and VcA-2 DNAs. Four of the cloned motility gene regions hybridized to the same fragments of DNA in both the wild-type and mutant isolates. However, two other probes detected a new fragment for a single aflagellate mutant. The observations that spontaneous nonmotile mutants occurred at a high frequency and that these mutants did not revert at a detectable frequency suggested that a genetic event is involved. The phenomenon appears to be limited to classical V. cholerae and may explain why classical V. cholerae is only sporadically associated with disease in the current pandemic.

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Auxotrophic mutations induced by Vibrio cholerae mutator phage VcA1

Cited by 6 publications

References 5 publications

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Molecular epidemiological studies of United States Gulf Coast Vibrio cholerae strains: integration site of mutator vibriophage VcA-3

High-frequency spontaneous mutation of classical Vibrio cholerae to a nonmotile phenotype

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