Related Experiment Videos
Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction
1Department of Microbiology, University of Alberta, Edmonton, Canada.
Gene
|September 28, 1990
Summary
Researchers isolated a chloramphenicol-resistance determinant (CmR) from Campylobacter coli, identifying it as a chloramphenicol acetyltransferase (CAT) gene. This study also developed new Campylobacter cloning vectors, finding codon usage bias does not hinder gene expression in E. coli.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Characterization of antibiotic resistance mechanisms in Campylobacter is crucial for understanding bacterial adaptation.
- Campylobacter species are significant human pathogens, and their genetic manipulation is challenging.
Purpose of the Study:
- To isolate and sequence a chloramphenicol-resistance determinant (CmR) from Campylobacter coli.
- To investigate the expression and functional characteristics of the identified chloramphenicol acetyltransferase (CAT) gene in different bacterial hosts.
- To construct novel cloning vectors for Campylobacter species.
Main Methods:
- Cloning and nucleotide sequencing of the CmR determinant from Campylobacter coli plasmid pNR9589.
- Analysis of the gene product's putative amino acid sequence and comparison with known CAT proteins.
- Primer extension experiments to determine transcription initiation sites in Campylobacter coli and Escherichia coli.
- Construction and testing of new Campylobacter cloning vectors.
Main Results:
- Isolation and sequencing of a 621 bp open reading frame encoding a CAT protein with 43-57% identity to other CAT proteins.
- Constitutive expression of the cat gene in both C. coli and E. coli, with distinct transcription initiation sites.
- Identification of the aphA-3 gene (kanamycin resistance) downstream of the cat gene.
- High-level synthesis of the CAT polypeptide in E. coli maxicells despite different codon usage, indicating no significant impediment to Campylobacter gene expression in E. coli.
- Successful construction of new Campylobacter cloning vectors.
Conclusions:
- The identified chloramphenicol-resistance determinant confers resistance via chloramphenicol acetyltransferase activity.
- Codon usage bias does not prevent efficient expression of Campylobacter genes in E. coli.
- The developed Campylobacter cloning vectors facilitate genetic studies in these important pathogens.