A ΦKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases

Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which ΦKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

Bacterial resistance to antimicrobial medication is escalating, and yet new antibiotics are not readily available. Without novel antibiotics, phage therapy has emerged as a viable response to the antibiotic resistance. Ideally, phage will achieve broad host range through layered anti-defense strategies that ensure their replicative success. Here we describe a broad-acting mechanism that allows Phikmvvirus phages to evade nuclease targeting. Through a phage encoded DNA ligase, gp17, ΦKMV phage seems to repair at predicted cut sites, often with high fidelity but occasionally leaving scars reminiscent of NHEJ repair. Active phage DNA ligases also support nuclease evasion by a distinct phage, DMS3. These findings describe phage escape through faithful repair and identify a potentially interesting gene for phage therapy.