Mapping factors of phage specificity: the case of restriction-modification systems

After successful recognition and adsorption to bacterial receptors, phages need to overcome a myriad of intracellular defenses. Among these, restriction-modification (RM) systems are ubiquitous in bacteria, leading to the degradation of foreign DNA that contains specific sequence motifs. In recent years, PacBio sequencing has enabled the identification of RM motifs by analyzing methylation patterns, revealing a great diversity of RM specificities. However, this technology has limitations, including ambiguous motif identification and high costs. To overcome this, we propose a method to detect restricted DNA sequences upon DNA transfer using random sequences (RandSeq). This approach has been validated with Escherichia coli strains with known restricted motifs and has revealed unknown specificities. Further, we compared restriction efficiency based on DNA delivery by conjugation or transduction, revealing RM systems to be more effective against phages. By identifying targeted DNA motifs, this method holds the potential to assist in the rational design of phages for therapeutic and biotechnological applications.