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

Abstract

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.Bacterial strains of the genus Lactococcus are widely used in the manufacture of cultured milk products. Bacteriophage attack of lactococcal starter strains may result in the lysis of susceptible strains and therefore in fermentation failure. Three genetically unrelated groups of lactococcal phages are responsible for most fermentation failures (23). These phages conform to one of two morphotypes: the prolate-headed c2 species and the isometric-headed 936 and P335 species (24). There are no known temperate phages in the c2 and 936 species groups, whereas the P335 species contains both temperate and lytic phages (24). Recently, the genomes of the small isometric 936 group phages sk1 (8) and bIL170 (11), the small isometric P335 group phages r1t (46), TP901-1 (4), Tuc2009 (GenBank accession no. AF109874), bIL285, bIL286, bIL309 (9), and BK5-T (3, 32), and the prolate-headed phages c2 (29) and bIL67 (43) have been sequenced and analyzed. Although there is a wealth of information at the nucleotide sequence level for these lactococcal phages, our current understanding of some aspects of phage biology, such as the replication of the phage genome, is relatively poor.The 22,163-bp double-stranded linear DNA genome of the prolate-headed phage c2 contains 39 open reading frames (ORFs), which are organized in two divergently oriented blocks consisting of the early genes and the late genes (29). These blocks are separated by a 611-bp noncoding region that contains the origin of replication (ori) in L...

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.…”

Lactococcus Ceduovirus Phages Isolated from Industrial Dairy Plants—From Physiological to Genomic Analyses

“…Based on studies of phage c2, the P E1 promoter was found to be involved in production of a non-coding transcript (P E1 -T) which may have a crucial role in initiating phage replication in a transcription-mediated fashion [23,24]. Both the length and the sequence and/or structure of P E1 -T were suggested to be crucial factors of each ori type-chimeric origins were non-functional [25].…”