Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

Abstract

It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, ori RK2, located at 12 kb on the genome, is also required. A segment of DNA containing ori RK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from ori RK2. Deletion of the trfB region in trans to an ori RK2 plasmid does not correlate with alteration in copy number or stability of the ori RK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori RK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the ori RK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from ori RK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori RK2, trfA, and trfB.