Flexibility in repression and cooperativity by KorB of broad host range IncP-1 plasmid RK2.

KorB, encoded by plasmid RK2, belongs to the ParB family of active partitioning proteins. It binds to 12 operators on the RK2 genome and was previously known to repress promoters immediately adjacent to operators O(B)1, O(B)10 and O(B)12 (proximal) or up to 154 bp away (distal) from O(B)2, O(B)9 and O(B)11. To achieve strong repression, KorB requires a cooperative interaction with one of two other plasmid-encoded repressors, KorA or TrbA. Reporter gene assays were used in this study to test whether the additional KorB operators may influence transcription and to test how KorB acts at a distance. The distance between O(B)9 and trbBp could be increased to 1.6kb with little reduction in repression or cooperativity with TrbA. KorB was also able to repress the promoter and cooperate with TrbA when the O(B) site was placed downstream of trbBp. This suggested a potential regulatory role for O(B) sites located a long way from any known promoter on RK2. O(B)4, 1.9kb upstream of traGp, was shown to mediate TrbA-potentiated KorB repression of this promoter, but no effect on traJp upstream of O(B)4 was observed, which may be due to the roadblocking or topological influence of the nucleoprotein complex formed at the adjacent transfer origin, oriT. Repression and cooperativity were alleviated significantly when a lac operator was inserted between O(B)9 and trbBp in the context of a LacI+ host, a standard test for spreading of a DNA-binding protein. On the other hand, a standard test for DNA looping, movement of the operator to the opposite face of the DNA helix from the natural binding site, did not significantly affect KorB repression or cooperativity with TrbA and KorA over relatively short distances. While these results are more consistent with spreading as the mechanism by which KorB reaches its target, previous estimates of KorB molecules per cell are not consistent with there being enough to spread up to 1kb from each O(B). A plausible model is therefore that KorB can do both, spreading over relatively short distances and looping over longer distances.

The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site O(B)3 but other KorB-binding sites form destabilizing complexes in the absence of O(B)3.

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.

Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751.

The central control region (Ctl) of IncP plasmids is associated with two phenotypes: the coordinate expression of replication and transfer genes; and the ability to increase the segregational stability of a low-copy number test plasmid. This region of the IncP beta plasmid R751 shows significant sequence divergence from the IncP alpha plasmid RK2 sequence, and two genes, korF and korG, present in the IncP alpha region are missing in the IncP beta Ctl. In other respects the organization of the Ctl is basically the same. Although the two key global regulatory genes korA and korB are highly conserved, studies on their ability to repress transcription from a variety of IncP alpha and IncP beta plasmid promoters suggest differences in operator recognition by KorA and synergy with other repressors. The products of kfrA, upf54.8 and upf54.4 genes are conserved; KfrA shows least conservation and, while retaining the ability to act as a transcriptional repressor, appears to have completely different DNA-binding specificity. The genes required for the plasmid segregational stabilization (partitioning) phenotype--incC, korB and the korB operator OB3--are conserved and contribute to a more efficient plasmid stabilization than the IncP alpha equivalents. This may indicate that the Ctl plays an especially important role in partitioning of IncP beta plasmids, since they lack the second stability region (parlmrs) found in IncP alpha plasmids.