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Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers
R H Durland1, A Toukdarian, F Fang
1Center for Molecular Genetics, University of California, San Diego, La Jolla 92093.
Abstract:
Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.
Insights
Researchers isolated mutated trfA genes from plasmid RK2, increasing its copy number in E. coli. These mutations in the replication protein TrfA suggest a regulatory role in controlling plasmid replication initiation.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Plasmid RK2 replication is tightly regulated.
- The trfA gene encodes the essential replication protein TrfA.
- Understanding replication control is crucial for plasmid stability and applications.
Purpose of the Study:
- To isolate and characterize mutations in trfA that increase plasmid RK2 copy number.
- To investigate the mechanism by which these mutations affect replication control.
- To identify the regulatory domain of the TrfA protein.
Main Methods:
- Isolation and sequencing of high-copy-number (copy-up) mutants of trfA.
- Mapping of mutations within the trfA gene.
- Amino acid substitution analysis.
- Trans-complementation and dominance tests.
Main Results:
- Six single-base transition mutations in trfA were identified, each causing an amino acid substitution.
- Mutations were located in a small region of the TrfA protein, suggesting a critical functional domain.
- Mutant trfA alleles exhibited dominant effects, increasing copy number even in the presence of wild-type trfA.
- Excess mutant TrfA protein trans-complemented to increase copy number of RK2 derivatives.
Conclusions:
- Mutations in trfA affect a regulatory activity of the TrfA protein.
- This regulatory activity is a key factor in controlling the initiation of RK2 replication.
- The identified mutations provide insights into the mechanism of plasmid copy number control.