Practical Method for Isolation of Phage Deletion Mutants

The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus).This research study was supported by grants AGL2015-65673-R (Ministry of Science and Innovation, Spain), EU ANIWHA ERA-NET (BLAAT ID: 67), GRUPIN14-139 (Program of Science, Technology and Innovation 2013-2017 and FEDER EU funds, Principado de Asturias, Spain). PG and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14) and the bacteriophage network FAGOMA II