Edinburgh Napier University

  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 035 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).