A method for mapping phage-inducible promoters for use in bacteriophage-triggered defense systems

Abstract

We have recently developed a novel bacteriophage-protection system for Lactococcus lactis, based on a two-component genetic ‘trap’. An inducible promoter from a lytic bacteriophage is used to activate a lethal gene after infection, killing the host cell and halting phage proliferation. To expand the potential use of this novel defense strategy, promoters specific to any particular phage of interest must be available for fusion to a universal death gene. A method to localize regulated promoters within the context of the total phage genome was evaluated. The ‘capping’ activity of the vaccinia virus guanylyltransferase was exploited to label newly synthesized mRNA extracted from infected cells at sequential time points over the course of a phage infection. The labeled mRNAs were then used as probes in Southern hybridization reactions to identify restriction fragments in the phage genome where new transcripts were initiated during progression of the phage lytic cycle. This method has been used successfully in our laboratory to map the general location of a number of inducible promoters on the genomes of bacteriophages attacking lactic acid bacteria. Once identified and cloned, small fragments encoding inducible promoters can be partially sequenced and primer extension reactions carried out on phage RNA, isolated over the course of an infection, to pinpoint the precise location of the promoter. In this study, we illustrate the use of the capping method to map the phage-inducible promoter on the genome of the lactococcal bacteriophage skl. This approach provides a rapid and efficient means to identify promoter regions on the genomes of relatively uncharacterized phages. These promoters can then be used in a variety of applications, including phage-triggered defenses and inducible gene expression systems.