Identification and characterization of an active plasmid partition mechanism for the novel Lactococcus lactis plasmid pCI2000

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed, The nucleotide sequence of one 5.6-kb EcoRI fragment which was c apable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmi ds considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated repA, is indeed required for replication. The results also suggest that the origin of replication is located outside repA. Upstre am and divergently transcribed from repA, an ORF that showed significant (4 8 to 64%) homology to a number of proteins that are required for faithful s egregation of chromosomal or plasmid DNA of gram-negative bacteria was iden tified. Gene interruption and transcomplementation experiments showed that this ORF, designated parA, is required for stable inheritance of pCI2000 an d is active in trans. This is the first example of such a partitioning mech anism for plasmids in gram-positive bacteria.