Identification of Two Replicons in Phage-Plasmid P4

Abstract

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 alpha 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 alpha gene product, coexist in P4. The first replicon is made by the cnr and alpha genes and the ori1 and crr sites. The second is limited to the alpha 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 alpha-crr replicon was mapped within a 270-bp fragment in the first half of the alpha gene. The ori2 site was found to be dispensable in a replicon that contains ori1. A construct that besides crr and alpha carries also the cnr gene was unable to replicate, suggesting that Cnr not only controls replication from ori1, but also silences ori2.

Characterization of the oriI and oriII Origins of Replication in Phage-Plasmid P4

“…Bacterial strains, phages, and plasmids. The bacterial strains that were derivatives of E. coli C were C-1a (prototrophic [34]), C-2107 (polA12; temperature-sensitive mutation in DNA polymerase I [31]), C-2428 [(recA-srl)⌬5 (40)], C-1414 (rep1 [8]), C-2307 (dnaA46 [4]), and C-5582 [dnaG3(Ts) rpoD::Tn10 by P1 transduction from PC3 (44) in C-1a]. The bacterial strains that were derivatives of E. coli K-12 were CM748 [polyauxotrophic; dnaA203(Ts) (5)] and DH5␣ (polyauxotrophic; recA [18]).…”

“…In this same experiment, a second replication initiation site was detected within the ␣ coding region (close to the 6273-to-6906 P4 fragment). This might represent the initiation point of the alternative P4 replicon oriII (see Discussion) (40).…”

“…1]), in which the portions of the P4 genome constituting the oriI and oriII replicons, respectively, were ligated to the chloramphenicol resistance gene. In these plasmids, expression of the P4 cnr and/or ␣ gene is under the control of lacp (40). It was demonstrated (40) that both replicons depend on the ␣ protein for replication, whereas the role of the Cnr protein differs.…”

“…Replicon oriI requires the Cnr protein to avoid overreplication and cell killing. In fact, the construction of an oriI plasmid lacking the cnr gene failed, due to the absence of the negative regulation of replication (40). On the other hand, replicon oriII is inhibited by Cnr (40).…”

“…In fact, the construction of an oriI plasmid lacking the cnr gene failed, due to the absence of the negative regulation of replication (40). On the other hand, replicon oriII is inhibited by Cnr (40). This makes it rather unlikely that the oriII origin of replication is active in P4.…”