Identification of two replicons in phage-plasmid P4

DNA replication of phage-plasmid P4 proceeds bidirectionally from the ori1 site (previously named ori), but requires a second cis-acting region, crr. Replication depends on the product of the P4 α gene, a protein with primase and helicase activity, that binds both ori1 and crr. A negative regulator of P4 DNA replication, the Cnr protein, is required for copy number control of plasmid P4. Using a plasmid complementation test for replication, we found that two replicons, both dependent on the α gene product, coexist in P4. The first replicon is made by the cnr and α genes and the ori1 and crr sites. The second is limited to the α and crr region. Thus, in the absence of the ori1 region, replication can initiate at a different site. By deletion mapping, a cis-acting region, ori2, essential for replication of the α-crr replicon was mapped within a 270-bp fragment in the first half of the α gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and α carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.