A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters pR and pL. ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter pL by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either pL is open and pR is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed pL fusion containing orf4 and orf5 was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages
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