RepB protein of an Agrobacterium tumefaciens Ti plasmid binds to two adjacent sites between repA and repB for plasmid partitioning and autorepression

Plasmids of Agrobacterium tumefaciens replicate using the products of the repABC operon, which are highly conserved among plasmids and some chromosomes of the alpha-Proteobacteria. The products of repA and repB direct plasmid partitioning, while the repC gene encodes a replication initiator protein. The transcription of the repABC operon of tumour inducing (Ti) plasmids is both negatively autoregulated by the RepA and RepB proteins, and positively regulated by TraR. In the present study, we have identified a fourth gene (repD) in the repABC operon of an octopine-type Ti plasmid. repD is 78 codons in length, and maps between repA and repB genes. A repD-lacZ protein fusion demonstrated that repD is strongly expressed. Two identical binding sites for the RepB protein were found within the repD coding sequence, and these sites are required for plasmid stability and for maximal repression of repABC transcription. RepA protein enhances the binding of RepB at these binding sites, just as RepB increases the affinity of RepA for binding sites at the repABC P4 promoter. We propose that RepA and RepB form complexes that bind both sites, possibly causing a loop that is important for repression of the repABC operon. Binding at one or both sites may also be required for accurate plasmid partitioning.