Sequence diversity and functional conservation of the origin of replication in lactococcal prolate phages

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P_E1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P_E1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P_E1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.