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Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing
1Institute for Virus Research, Kyoto University, Japan.
Abstract:
Mini-F plasmids cannot replicate in Escherichia coli strains (delta rpoH) lacking sigma 32, presumably because transcription of the repE gene encoding the replication initiator protein (RepE protein) depends mostly on RNA polymerase containing sigma 32. We have isolated and characterized mini-F mutants able to replicate in delta rpoH cells. Contrary to the initial expectation, five mutants with mutations in the repE coding region that produce altered RepE proteins were obtained. The mutations caused replacement of a single amino acid: the 92nd glutamic acid was replaced by lysine (repE10, repE16, and repE25) or glycine (repE22) or the 109th glutamic acid was replaced by lysine (repE26). These plasmids overproduced RepE protein and exhibited very high copy numbers. Two major activities of mutated RepE proteins have been determined in vivo; the autogenous repressor activity was significantly reduced, whereas the initiator activity was much enhanced in all mutants. These results indicate the importance of a small central region of RepE protein for both initiator and repressor activities. Thus the decreased repE transcription in delta rpoH cells can be compensated for by an increased initiator activity and a decreased repressor activity of RepE, resulting in the increased synthesis of hyperactive RepE protein.
Insights
Mini-F plasmids require sigma 32 for replication initiation protein (RepE) gene transcription. Mutated RepE proteins with enhanced initiator activity compensate for reduced transcription, enabling replication in sigma 32 deficient Escherichia coli.
Area of Science:
- Molecular Biology
- Bacteriology
Background:
- Mini-F plasmids depend on the repE gene for replication.
- The repE gene's transcription in Escherichia coli primarily relies on RNA polymerase containing sigma 32 (σ32).
- E. coli strains lacking σ32 (ΔrpoH) cannot replicate mini-F plasmids due to insufficient RepE protein synthesis.
Purpose of the Study:
- To isolate and characterize mini-F plasmids capable of replicating in ΔrpoH E. coli strains.
- To investigate the functional alterations in the replication initiator protein (RepE) that permit replication in the absence of σ32.
Main Methods:
- Isolation and characterization of mini-F mutants.
- Site-directed mutagenesis of the repE gene.
- In vivo analysis of RepE protein activities (initiator and repressor).
Main Results:
- Five mini-F mutants with single amino acid substitutions in the repE gene were identified.
- Mutations at positions 92 (Glu to Lys/Gly) and 109 (Glu to Lys) resulted in altered RepE proteins.
- These altered RepE proteins exhibited significantly reduced autogenous repressor activity and greatly enhanced initiator activity.
- Mutant plasmids overproduced RepE protein, leading to very high copy numbers.
Conclusions:
- A central region of the RepE protein is crucial for both its initiator and repressor functions.
- Enhanced RepE initiator activity and reduced repressor activity can compensate for decreased repE transcription in ΔrpoH cells.
- This compensation mechanism allows for mini-F plasmid replication even in the absence of σ32.