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Cloning, sequencing, and expression of the Escherichia coli cytolethal distending toxin genes
C L Pickett1, D L Cottle, E C Pesci
1Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington 40536-0084.
Abstract:
A limited number of Escherichia coli isolates which produce an apparently novel toxin, termed cytolethal distending toxin (CDT), have been reported. The toxic activity produced by these strains causes certain cultured cell lines to become slowly distended and then disintegrate. DNA was isolated from the CDT-producing E. coli strain, 9142-88, and cloned into a cosmid vector. Plasmid DNA from a toxin-positive transductant was further subcloned until a plasmid with a 4-kb insert which still encoded the toxin activity was obtained. Nucleotide sequencing of a portion of this insert revealed the presence of three adjacent open reading frames. Further subcloning and deletion analysis suggested that the products of all three open reading frames may be required for toxin activity. Minicell experiments identified the products of all three open reading frames. The three proteins had predicted sizes of 27,753,29,531, and 19,938 Da, and all three appeared to have strong consensus leader sequences. None of the three predicted proteins had significant homology to known proteins.
Insights
Researchers identified a novel toxin, cytolethal distending toxin (CDT), produced by Escherichia coli. The toxin requires three genes to function, producing proteins with no known homology.
Area of Science:
- Microbiology
- Molecular Biology
- Toxicology
Background:
- Escherichia coli can produce a novel toxin, cytolethal distending toxin (CDT).
- CDT causes characteristic cell distension and disintegration in susceptible cultured cell lines.
Purpose of the Study:
- To genetically characterize the cytolethal distending toxin (CDT) from Escherichia coli.
- To identify the genetic elements responsible for CDT production and understand the mechanism of its toxicity.
Main Methods:
- Cloning of DNA from a CDT-producing E. coli strain into a cosmid vector.
- Subcloning and deletion analysis to narrow down the DNA region encoding toxin activity.
- Nucleotide sequencing of the relevant DNA insert.
- Minicell experiments to identify protein products.
Main Results:
- A 4-kb DNA fragment containing three adjacent open reading frames was identified as responsible for CDT activity.
- All three open reading frames appear to be necessary for toxin production.
- The three genes encode proteins of predicted sizes 27,753, 29,531, and 19,938 Da.
- None of the predicted proteins showed homology to known proteins, indicating a novel toxin mechanism.
Conclusions:
- The cytolethal distending toxin (CDT) from Escherichia coli is encoded by a three-gene operon.
- The novel proteins produced by these genes are essential for the observed cytotoxic effects.
- This study elucidates the genetic basis of a newly identified bacterial toxin.