Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages

Abstract

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 E 1-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 E 1-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 E 1-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.Lactococcus lactis is widely used in the production of fermented products by the dairy and food industries. Large numbers of bacteriophages infect this bacterium, causing the lysis of the host and fermentation failure. The most frequently encountered are two species of small phages with isometric heads and B1 morphology, named 936 and P335, and one species of phages with prolate heads and a B2 morphology, named c2 (32). Each of the species comprises numerous homologous phages.

Isolation of Lactococcal Prolate Phage-Phage Recombinants by an Enrichment Strategy Reveals Two Novel Host Range Determinants

“…The two prolate phages (c2 and bIL67) whose genome sequences have been determined to date have an overall identity of 80% (36,54). Both 20-to 22-kbp double-stranded DNA (dsDNA) genomes code for two blocks of divergently oriented open reading frames (ORFs), transcribed early and late, separated by a noncoding region that serves as an origin of replication (51,53,61). The early ORFs are detectable 2 min postinfection.…”

Essentiality of the Early Transcript in the Replication Origin of the Lactococcal Prolate Phage c2

“…It has been shown that transcription of a divergent region downstream of P E 1 occurs in the three groups of lactococcal prolate phages into which our collection was subdivided (40). Here we demonstrate that production of the P E 1 transcript is required for phage c2 origin function in a plasmid model system.…”

“…To investigate this, scanning mutagenesis was performed with the DNA sequence coding for the transcripts made from P E 1. The 10-, 13-, and 48-bp deletions used were designed to disrupt the predicted secondary structures (40) in the transcripts made from P E 1 (pLP217 to pLP223). Of the seven deletion plasmids constructed, only pLP217 replicated in L. lactis (Fig.…”

“…The product was cloned into the EcoRI site of pVA891 to generate pLP214. Plasmid pLP216 was created by cloning a PCR product (template pLP207; primers ori15 and ori16) into the NcoI and PstI site of plasmid pVA⍀, which contained a transcriptional terminator (⍀ terminator) (16,40). Plasmids pLP215 and pLP217 through pLP223 were made by inverse PCR by using pUC-203 as the template and primers ori30 and ori31 (pLP215), ori44 and ori45 (pLP217), ori42 and ori43 (pLP218), ori40 and ori41 (pLP219), ori38 and ori39 (pLP220), ori36 and ori37 (pLP221), ori34 and ori35 (pLP222), and ori32 and ori33 (pLP223).…”

“…It is also highly conserved in the closely related prolate phages bIL67 and 197 (47), in contrast to the region downstream of P E 1, which is not conserved. It has recently been shown that a large panel of lactococcal prolate phage could be divided into three distinct groups based on the sequence of the P E 1 transcript (40). All three ori types supported plasmid replication.…”