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Published on: January 27, 2021
Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump
1Department of Biotechnology, Institute of Medical Biology, University of Tromsø, Norway.
Abstract:
A novel gene designated cmr, which mapped to 18.8 min of the Escherichia coli K-12 genome, was shown to mediate resistance to chloramphenicol when it was expressed from a multicopy vector. The accumulation of chloramphenicol was significantly less in cells overexpressing cmr than in control cells harboring the vector without insert. After the addition of a proton motive force blocker, the level of accumulation of chloramphenicol in the resistant cells rapidly approached the levels found in sensitive cells carrying only the chromosomal cmr. Northern (RNA) blot analyses revealed that the cmr gene is expressed as a 1.3-kb transcript. This size corresponds very well with a predicted size of 1,293 nucleotides (nt) based on the mapping of the transcription initiation site to a G residue 24 nt upstream of the start codon and the presence of a putative rho-independent terminator sequence ending 36 nt downstream of the 1,233-nt open reading frame encoding the putative Cmr protein. The 411-residue-long derived amino acid sequence contains 12 putative transmembrane segments and displays significant sequence similarities to several known drug resistance protein sequences of the major facilitator family. We provide evidence strongly suggesting that the resistance mediated by Cmr involves active exclusion of chloramphenicol.
Insights
A newly discovered gene, cmr, in Escherichia coli K-12 confers resistance to chloramphenicol. This gene actively excludes the antibiotic, significantly reducing its accumulation in bacterial cells.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Chloramphenicol is a broad-spectrum antibiotic.
- Antibiotic resistance is a growing global health concern.
- Understanding resistance mechanisms is crucial for developing new treatments.
Purpose of the Study:
- To identify and characterize a novel gene conferring chloramphenicol resistance in Escherichia coli.
- To elucidate the mechanism of chloramphenicol resistance mediated by the identified gene.
Main Methods:
- Gene mapping and cloning in Escherichia coli K-12.
- Gene expression analysis using Northern (RNA) blot.
- Chloramphenicol accumulation assays with and without proton motive force blockers.
- Sequence analysis of the novel gene and its protein product.
Main Results:
- A novel gene, cmr, was identified and mapped to the Escherichia coli K-12 genome.
- Overexpression of cmr significantly reduced chloramphenicol accumulation in bacterial cells.
- Northern blot analysis confirmed cmr expression as a 1.3-kb transcript.
- Sequence analysis revealed Cmr protein shares similarities with major facilitator family drug resistance proteins.
- Evidence suggests active exclusion of chloramphenicol as the resistance mechanism.
Conclusions:
- The novel cmr gene confers chloramphenicol resistance in Escherichia coli.
- The Cmr protein likely functions as an efflux pump, actively excluding chloramphenicol.
- This finding contributes to understanding antibiotic resistance mechanisms in bacteria.
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