Genetic analysis of the lytic replicon of bacteriophage P1. II. Organization of replicon elements.

The region of bacteriophage P1 DNA containing a lytic (vegetative) replicon has been identified by cloning P1 fragments into a phage lambda vector. We present the sequence of that replicon. Using a novel fusion vector containing two P1 loxP recombination sites, we have developed a transformation assay for replicon function and have used that assay to identify some of the components of the P1 lytic replicon. Among those components is a transcription promoter, P53, whose activity is essential for replicon function. When that promoter is inactivated by the binding of P1 repressor to an operator site, Op53, whose sequence overlaps the promoter, replicon function is blocked. The P53 promoter can be replaced for replicon function by other promoters and, when the lacZ promoter was used, the extent of replication was shown to be proportional to promoter activity. Two open reading frames are located downstream from P53. The promoter-proximal reading frame is 266 amino acid residues long and is not essential for replicon function. In fact, expression of that open reading frame either interferes with plasmid establishment after transformation or is lethal to cells. The promoter-distal reading frame, designated the repL open reading frame, is either 269 or 281 amino acid residues long and is essential for replicon function. Insertion of a Tn5 transposon into the 266 amino acid residue open reading frame inactivates the cloned lytic replicon probably by interfering with the transcription of the repL open reading frame from P53. In P1, this Tn5 insertion mutation completely blocks lytic replication, indicating that the replicon identified here is either the only P1 lytic replicon or, if not, is at least necessary for the function of any other lytic replicon. A four base insertion in the repL open reading frame has largely the same inhibitory effect on phage lytic replication as the Tn5 insertion.