Genetic modifications to temperate Enterococcus faecalis phage φEf1 1 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection

-Ef11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcius faecalis strain 'recovered from an infected root canal, and the phi Ef11 prophage is widely disseminated among strains of E. faecalis Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by phi Ef11 as a first ste,pin the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of phi Ef11 in E., faecalis JH2-2 yielded a variant that produced largo, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between phi Ef11 and a defective OFL1C-like prophage located in the E. faecalis :JH2-2 chromosome. The recombinant possessed five ORFs of the defective OFL1C-Ilike prophage place of six ORFs of the OEf11 genome. Deletiori of the putative lysogeny gene,module;(ORFs 31-36) and {enlacement of the putative cro promoter from the recombinant phage genorne with :a nisin-indubible promoter resulted in no loss of virus infectivity. The :genetic construct incorporating all the aforementioned phi Ef11 genomic modifications resulted in the generation of a variant that was incapable of lySOgeny and insensitive to repressor, rendering it virulent and :highly lytic, with a notably extended host range.