Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage phi LC3

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococcus lacti s subsp. cremoris phage phi LC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate la ctococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phi LC3. Gel mobility s hift assays showed that the partially purified Orf286 protein bound specifi cally to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Or f286 protects two distinct sites within this region. Sequence analysis of t he intergenic region revealed two putative, divergently oriented promoters, P-1 and P-2; orf286 and orf63 are probably transcribed from P1 and P-2 res pectively. In vivo analyses of P-1 and P-2 using beta -galactosidase as a r eporter enzyme in L. lactis showed that transcription from P-1 was represse d while transcription from P-2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in re gulating the genetic switch between lytic and lysogenic growth of phi LC3.