Characterization of the Replicon from the Lactococcal Theta-Replicating Plasmid pJW563

The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kbEcoRI fragment. This DNA fragment was sequenced and a 1155-bp open reading frame,repB563, encoding a putative protein RepB563of 385 amino acids was found. An AT-rich noncoding region,repA563, was found upstream ofrepB563. This segment included several direct and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled torepB563. RepB563suppliedin transcould support replication of a plasmid containingrepA563and a truncatedrepB563. This observation suggests that RepB563is atrans-acting replication protein, andrepA563thecis-acting origin of replication.repA563,repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids. TherepADNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison revealed highly conserved areas among this family of plasmids. In addition, variable domains emerged, presumably having a plasmid specific function. pVS40 and pCI305 were plasmids with replication proteins showing high homology to RepB563. Despite this homology, replication fromrepA563could not be supported by the pVS40 or pCI305 replication protein suppliedin trans.Likewise, the pJW563 protein could not support replication from the pVS40 origin. pJW563 was found to be compatible with the pVS40 and pCI305 replicons. The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating plasmids. Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific, in many cases rendering the plasmids compatible.