Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P(E)1 abolished origin function. Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P(E)1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.