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Cloning and analysis of the Clostridium perfringens tetracycline resistance plasmid, pCW3
Abstract:
Clostridium perfringens strain CW92 carries pCW3, a conjugative 47-kb plasmid that confers inducible resistance to tetracycline. The plasmid was examined by restriction endonuclease analysis and by cloning each of the five ClaI fragments of pCW3 in Escherichia coli, using pBR322. Analysis of the recombinant plasmids allowed the deduction of a detailed restriction map of pCW3. The tetracycline resistance determinant of pCW3 was mapped by examining the phenotype of recombinant E. coli clones derived from the cloning, into pUC vector plasmids, of EcoRI fragments from pCW3. The C. perfringens tetracycline resistance determinant was expressed in E. coli and was shown to be located on two juxtaposed EcoRI fragments which together encompass a 4-kb region of pCW3. Deletion experiments showed that the tetracycline resistance gene, and/or its control regions, contained internal EcoRI and SphI sites. E. coli strains that carried recombinant plasmids with only the 4-kb region were found to express tetracycline resistance constitutively. In contrast, recombinant plasmids harboring a 10.5-kb ClaI fragment of pCW3, that included the 4-kb region, coded for an inducible tetracycline resistance phenotype. The existence of a negatively regulated resistance gene, similar to that proposed for several other bacteria is postulated.
Insights
Clostridium perfringens plasmid pCW3 confers tetracycline resistance. Researchers mapped this resistance to a 4-kb region, finding it inducible in E. coli, suggesting a regulated gene.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Clostridium perfringens strain CW92 harbors the conjugative plasmid pCW3.
- This plasmid (pCW3) confers inducible resistance to tetracycline.
- Understanding tetracycline resistance mechanisms is crucial in combating bacterial infections.
Purpose of the Study:
- To detail the restriction map of the pCW3 plasmid.
- To identify and characterize the tetracycline resistance determinant of pCW3.
- To investigate the regulation of tetracycline resistance conferred by pCW3.
Main Methods:
- Restriction endonuclease analysis of the pCW3 plasmid.
- Cloning of pCW3 fragments into Escherichia coli vectors (pBR322 and pUC).
- Phenotypic analysis of recombinant E. coli clones for tetracycline resistance.
- Deletion experiments to delineate the resistance gene and its regulatory elements.
Main Results:
- A detailed restriction map of the 47-kb pCW3 plasmid was constructed.
- The tetracycline resistance determinant was localized to a 4-kb region comprising two EcoRI fragments.
- Constitutive tetracycline resistance was observed with the 4-kb region alone, while inducible resistance was seen with a larger 10.5-kb ClaI fragment.
Conclusions:
- The tetracycline resistance gene and its control regions within pCW3 possess internal restriction sites.
- A negatively regulated gene system, similar to those in other bacteria, is proposed for the inducible tetracycline resistance.
- This study elucidates the genetic basis of tetracycline resistance in Clostridium perfringens.