Molecular analysis of the replication origin of the Lactococcus lactis plasmid pCJ305

The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (RepB) essential for plasmid replication. Gel retardation assays, using lactococcal crude cell extracts in which RepB was overproduced, were used to demonstrate that the replication protein interacts with DNA sequences within the origin region. IR1 was identified as a RepB binding site. The -35 region of the repB promoter is contained within the loop of the potential stem-loop structure of IR1, suggesting autoregulation of repB. The pCI305 RepB failed to interact with DNA sequences within the minimal replicons of nine other members of the pCI305 family of plasmids and it was concluded that this DNA-protein interaction was replicon specific. In vivo studies were performed to determine the role of the three-and-one-half copies of the 22-bp sequences. When this sequence was provided in trans on a compatible vector, it resulted in the loss of pCI305 from the cell population (incompatibility).