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-kb EcoRI fragment. This DNA fragment was sequenced and a 115 5-bp open reading frame, repB(563), encoding a putative protein RepB(5 63) of 385 amino acids was found. An AT-rich noncoding region, repA(56 3), was found upstream of repB(563). This segment included several dir ect and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translation ally coupled to repB(563). RepB(563) supplied in trans could support r eplication of a plasmid containing repA(563) and a truncated repB(563) . This observation suggests that RepB(563) is a trans-acting replicati on protein, and repA(563) the cis-acting origin of replication. repA(5 63), repB(563), and the beginning of ORF X showed high homology to sim ilar regions in a family of lactococcal theta-replicating plasmids. Th e repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison re vealed highly conserved areas among this family of plasmids. In additi on, variable domains emerged, presumably having a plasmid specific fun ction. pVS40 and pCI305 were plasmids with replication proteins showin g high homology to RepB(563). Despite this homology, replication from repA(563) could not be supported by the pVS40 or pCI305 replication pr otein supplied in trans. Likewise, the pJW563 protein could not suppor t 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 pla smids. Despite the high homology between their replicons, the interact ion between the replication origin and the protein is highly specific, in many cases rendering the plasmids compatible. (C) 1995 Academic Pr ess, Inc.