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Proteins encoded by the trans-acting replication and maintenance regions of broad host range plasmid RK2
Abstract:
The broad host range plasmid RK2 has previously been found to contain three separate regions of the genome involved in replication and maintenance in Escherichia coli (C. M. Thomas, R. Meyer, D. R. Helinski, 1980, J. Bacteriol. 141, 213-222). They include the origin of replication (oriRK2) and the trfA region which encodes a trans-acting function required for replication. The third region (trfB), although not essential for replication, supplies a function involved in the maintenance of plasmid RK2. Using the maxicell system of labeling plasmid-specific proteins, we have identified all of the proteins encoded by two miniplasmid derivatives of RK2 which contain only the regions oriRK2, trfA, and trfB. To determine which region specifies each protein, RK2/mini-ColE1 hybrid plasmids were used which contain various restriction fragments of the mini-RK2 replicon. The trfA region appears to encode three proteins designated A1 (39,000 MW), A2 (31,000 MW), and A3 (14,000 MW). Analysis of proteins synthesized by plasmids containing deleted forms of the trfA region indicates that the A2 protein is the essential trfA-encoded replication protein of plasmid RK2. The proteins A1 and A3 may be the products specified by the genes tra3 (involved in transmissibility) and kilB1 (involved in host-cell viability) which also map in the trfA region. The trfB region specifies two proteins designated B1 (36,000 MW) and B2 (30,000 MW). These may be the products of the two kil-override (kor) genes located in the trfB region which have been implicated in plasmid maintenance.
Insights
Researchers identified key proteins essential for plasmid RK2 replication and maintenance in Escherichia coli. The trfA region encodes the essential replication protein A2, while trfB specifies proteins involved in plasmid stability.
Area of Science:
- Molecular Biology
- Bacteriology
- Genetics
Background:
- Plasmid RK2, a broad host range replicon, possesses distinct genomic regions (oriRK2, trfA, trfB) crucial for its replication and maintenance in Escherichia coli.
- The trfA region encodes a trans-acting replication function, while trfB provides a non-essential but important maintenance function.
Purpose of the Study:
- To identify and characterize plasmid-specific proteins encoded by the oriRK2, trfA, and trfB regions of plasmid RK2.
- To determine the specific roles of these proteins in plasmid replication and maintenance.
Main Methods:
- Utilized the maxicell system for labeling plasmid-specific proteins.
- Constructed and analyzed RK2/mini-ColE1 hybrid plasmids with various restriction fragments of the mini-RK2 replicon.
- Examined proteins synthesized by plasmids with deleted forms of the trfA region.
Main Results:
- Identified three proteins (A1, A2, A3) encoded by the trfA region, with molecular weights of 39,000, 31,000, and 14,000 MW, respectively.
- Determined that the A2 protein (31,000 MW) is the essential replication protein encoded by trfA.
- Identified two proteins (B1, B2) encoded by the trfB region (36,000 and 30,000 MW), potentially involved in plasmid maintenance via kil-override (kor) genes.
Conclusions:
- The A2 protein is essential for plasmid RK2 replication.
- Proteins A1 and A3 may be involved in transmissibility (tra3) and host-cell viability (kilB1).
- The trfB-encoded proteins B1 and B2 are likely products of kor genes, contributing to plasmid maintenance.