Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2

Replication of the broad-host-range, IncP alpha plasmid RK2 requires two pl asmid loci: trfA, the replication initiator gene, and oriV, the origin of r eplication. While these determinants are sufficient for replication in a wi de variety of bacteria, they do not confer the stable maintenance of parent al RK2 observed in its hosts. The product of the incC gene has been propose d to function in the stable maintenance of RK2 because of its relatedness t o the ParA family of ATPases, some of which are known to be involved in the active partition of plasmid and chromosomal DNA. Here we show that IncC ha s the properties expected of a component of an active partition system. The smaller polypeptide product of incC (IncC2) exhibits a strong, replicon-in dependent incompatibility phenotype with RK2. This incompatibility phenotyp e requires the global transcriptional repressor, KorB, and the target for i ncC-mediated incompatibility is a KorB-binding site (O-B). We found that Ko rB and IncC interact in vivo by using the yeast two-hybrid system and in vi tro by using partially purified proteins. Elevated expression of the incC a nd korB genes individually has no obvious effect on Escherichia coli cell g rowth, but their simultaneous overexpression is toxic, indicating a possibl e interaction of IncC-KorB complexes with a vital host target. A region of RK2 bearing incC, korB, and multiple KorB-binding sites is able to stabiliz e an unstable, heterologous plasmid in an incC-dependent manner. Finally, e levated levels of IncC2 cause RK2 to aggregate, indicating a possible role for IncC in plasmid pairing. These findings demonstrate that IncC, KorB, an d at least one KorB-binding site are components of an active partition syst em for the promiscuous plasmid RK2.