Figure 1. R-IVET strategy employed in E. faecalis to identify promoters that are induced in vivo.

A: Modification of Lox2 strain occurring when a promoter is active. Chromosome of V583 strain was modified by integrating ermB gene and a promoterless copy of tetM gene, first of which is flanked by two copies of site-specific recombination sequences (loxP). A V583 genomic library was constructed by cloning DNA fragments in plasmid pCre2 upstream from cre gene. If an active promoter is thus cloned, the recombinase Cre would be expressed resulting in a homologous recombination event between both loxP sites. This triggers the irreversible excision of the ermB gene and tetM becomes functional. B: In vivo screening of E. faecalis V583 genomic library. After elimination of cells containing an active promoter under in vitro conditions, the resulting library is administrated to the animal host model. After infection, changes in the antibiotic resistance phenotype allow to select resolved bacteria containing an in vivo induced promoter.