The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site OB3 but other KorB-binding sites form destabilizing complexes in the absence of Ob3

Summary: The sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54·0 to 60·0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids. This Par region encodes the central control operon ( korA, incC, korB, korF and korG ) and the associated genes kfrA, upf54.8 and upf54.4 . Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype. incC encodes two polypeptides from alternative translational starts. A deletion of the start of the operon showed that only IncC2, the shorter product is essential for partitioning. Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (O b s) which were candidate cis -acting sequences needed for stability. Only inactivation of O B 3, which lies between upf54.4 and upf54.8 , resulted in an increased rate of segregational loss. However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region. Either inactivation of korB or deletion of O b 1 from this O b 3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone. Thus KorB can act on O b 1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules. This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes.