Summary
Replication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small ori RK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus ‘incompatible” with RK2 replicons. However, the 393 bp ori RK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on ori RK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII oriRK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from ori RK2is required or may simply reflect the strong selective pressure for establishment of the incoming ori RK2 plasmid and poor ability of the HpaII ori RK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII ori RK2 may be designated inc 1. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc 2 region, to distinguish it from inc 1, above, that overlaps but does not coincide with the 393 bp HpaII ori RK2. This inc 2 region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete ori RK2. Also, only part of the inc 2 region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for ori RK2.
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Thomas, C.M., Stalker, D.M. & Helinski, D.R. Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol Gen Genet 181, 1–7 (1981). https://doi.org/10.1007/BF00338996
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DOI: https://doi.org/10.1007/BF00338996