General Transduction in Vibrio cholerae

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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“…Isolation of Kmr transductants. Stocks of vibriophage CP-Ti (23) were grown on SM201 by the method of Adams (1).…”

Section: Methodsmentioning

confidence: 99%

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Sporecke¹,

Castro²,

Mekalanos³

1984

J Bacteriol

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“…The fragments were inserted into pKAS32 (Skorupski and Taylor, 1996), generating pWEL89. To facilitate CPT1-mediated transduction of the mutation into a variety of strains after allelic exchange (Ogg et al, 1981), a kanamycin resistance gene was inserted between the two fragments in pWEL89 generating pWEL90. For DvpsR, primer VpsR1 (5′-GATCGGG-ATCCCAGAGCTGAACGATGCTGAAG) was used with VpsR2 (5′-GATCGGCGGCCGCAGTGCTCATGAACCTATA TTC) and VpsR3 (5′-GATCGGCGGCCGCATTCTAAG TTTTGCTGATTTAC) was used with VpsR4 (5′-GATCGGAA-TTCACTCAGCTGCCATTTTCCGC).…”

Section: Construction Of In-frame Deletionsmentioning

confidence: 99%

The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp‐ and VpsR‐mediated activation

Lin

Kovacikova

Skorupski³

2007

Molecular Microbiology

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SummaryHapR is a quorum sensing-regulated transcription factor that represses the virulence cascade in Vibrio cholerae by binding to a specific site centred at -71 in the aphA promoter, ultimately preventing activation of the tcpPH promoter on the Vibrio pathogenicity island. In an effort to elucidate the mechanism by which HapR represses aphA expression, we identified two transcriptional regulators, Lrp and VpsR, both of which activate the aphA promoter. Lrp, the leucineresponsive regulatory protein, binds to a region between -136 and -123 in the promoter to initiate aphA expression. VpsR, the response regulator that controls biofilm formation, binds to a region between -123 and -73 to activate aphA expression.

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“…Experiments were performed using a temperature-sensitive (ts) mutant of the V. cholerae transducing phage CP-T1ts [11,12], to determine whether VPI was transmissible by transduction to non-toxigenic V. cholerae O1 serogroup strains 468-83, GP6, V69 and 1528-79. In order to detect transduction of VPI, strain C6709A, an El Tor V. cholerae O1 serogroup strain, which contains a Kn marked copy of VPI was used.…”

Section: Cp-t1 Transductionmentioning

confidence: 99%

Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction

O'Shea

2002

FEMS Microbiology Letters

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Pathogenicity islands are large chromosomal regions encoding virulence genes that were acquired by horizontal gene transfer and are found in a wide range of pathogenic bacteria. In toxigenic Vibrio cholerae isolates the receptor for the cholera toxin encoding filamentous phage CTXP, the toxin-coregulated pilus, is part of the Vibrio pathogenicity island (VPI). In this paper, we show that the VPI can be transferred between O1 serogroup strains, the predominant cause of epidemic cholera, via a generalized transducing phage CP-T1. ß

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General Transduction in Vibrio cholerae

Cited by 41 publications

References 23 publications

Genetic mapping of Vibrio cholerae enterotoxin structural genes

Genetic mapping of Vibrio cholerae enterotoxin structural genes

The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp‐ and VpsR‐mediated activation

Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction

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