Transcriptional analysis of the genetic elements involved in the lysogeny/lysis switch in the temperate lactococcal bacteriophage ϕLC3, and identification of the Cro-like protein ORF76

A transcriptional analysis of the lysogeny-related genes of the temperate bacteriophage Lactococcus lactis ϕLC3 was performed using Northern blot hybridization during lysogeny and lytic infection by the phage. The lysogeny-related gene cluster was found to contain four promoters (P1, P2, Pint and P173), while the P87 promoter directed transcription of orf80 and the putative gene orf87, which are located between the integrase gene and the cell lysis genes. The start sites of the transcripts were determined by primer extension. The divergently oriented lysogenic P1 and lytic P2 promoters located in the genetic switch region are responsible for transcription of orf286 which encodes the phage repressor, and the genes orf63 - orf76 - orf236 - orf110 - orf82 - orf57, respectively, while orf173 is transcribed from P173. orf76 was identified as the gene encoding the Cro-like protein of ϕLC3, and it was shown that ORF76 is able to bind specifically to the genetic switch region, albeit with lower affinity than does the phage repressor ORF286. ORF76 also competed with ORF286 for binding to this region. The functionality of P1 and P2, and their regulation by ORF286 and ORF76, was investigated using a reporter gene. In general, P2 was a stronger promoter than P1, but expression from both promoters, especially P2, was regulated and modulated by flanking sequences and the presence of orf286 and orf76. ORF286 and ORF76 were both able to repress transcription from P1 and P2, while ORF286 was able to stimulate its own synthesis by tenfold. This work reveals the complex interplay between the regulatory elements that control the genetic switch between lysis and lysogeny in ϕLC3 and other temperate phages of Lactococcus.