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Replication control genes of plasmid pE194
R Villafane1, D H Bechhofer, C S Narayanan
1Department of Microbiology, Public Health Research Institute, City of New York, Inc., New York 10016.
Abstract:
pE194, a 3.7-kilobase plasmid, confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. The previously identified cop and repF genes of pE194 have been further localized by molecular cloning and mutational analysis together with DNA sequencing. The CfoIB fragment of pE194 is capable of autonomous replication and contains both genes. Most of this region has been resequenced, and two errors reported in a previous study have been corrected. The corrected sequence indicates that the replication region contains a single large open reading frame, which we propose encodes the repF product. Northern blot (RNA blot) analysis of this region detected six transcripts, all transcribed in the same direction as one another and opposite to repF. A 350-base transcript is synthesized from the region containing cop. No in vivo transcript for the repF gene was detected, but a protein was observed in an in vitro transcription-translation system which appears to be its product. An ochre mutation was inserted in the putative repF open reading frame, and a nonsense fragment was detected in the in vitro system. When carried passively on a pUB110 replicon, this mutant product appears capable of inhibiting pE194 replicons in trans. The pE194 origin of replication has been localized to within 200 bases.
Insights
The pE194 plasmid
Area of Science:
- Molecular Biology
- Genetics
- Antimicrobial Resistance
Background:
- The pE194 plasmid confers resistance to macrolide, lincosamide, and streptogramin B antibiotics.
- Understanding plasmid replication mechanisms is crucial for combating antibiotic resistance.
Purpose of the Study:
- To precisely localize and characterize the replication genes (cop and repF) and origin of replication of the pE194 plasmid.
- To correct previous sequence errors and elucidate the functional roles of identified genes.
Main Methods:
- Molecular cloning and mutational analysis
- DNA sequencing
- Northern blot (RNA blot) analysis
- In vitro transcription-translation assays
Main Results:
- The CfoIB fragment of pE194 was confirmed to contain both cop and repF genes and support autonomous replication.
- A single large open reading frame, proposed to encode the repF product, was identified in the corrected sequence.
- Six transcripts were detected, all transcribed opposite to repF, with a 350-base transcript from the cop region.
- A mutant repF product, when expressed from a different replicon, inhibited pE194 replication in trans.
- The pE194 origin of replication was localized to a 200-base region.
Conclusions:
- The study provides a corrected and detailed map of the pE194 replication region.
- The repF gene product plays a role in plasmid replication and can exert a trans-acting inhibitory effect.
- The findings contribute to understanding plasmid replication control mechanisms relevant to antibiotic resistance.