Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is th e only phage protein present. However, 100% excision is observed when the p hage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the T P901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the f amily of extended resolvases. Orf7 is a basic protein of 64 amino acids, an d the corresponding gene (orf7) is the third gene in the early lytic operon . This location of an excisionase gene of a temperate bacteriophage has nev er been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes . In order to detect very low frequencies of excision, a method for positiv e selection of loss of genetic material based upon the upp gene (encoding u racil phosphoribosyltransferase) was designed, since upp mutants are resist ant to fluorouracil. By using this system, frequencies of excision on the o rder of 10(-5) per cell could easily be measured. The described selection p rinciple may be of general use for many organisms and also for types of del etion events other than excision.