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.