Toxin/antitoxin systems induce persistence and work in concert with restriction/modification systems to inhibit phage

Myriad bacterial anti-phage systems have been described and often the mechanism of programmed cell death is invoked for phage inhibition. However, there is little evidence of “suicide” under physiological conditions for these systems. Instead of death to stop phage propagation, we show here that persister cells, i.e., transiently-tolerant, dormant, antibiotic-insensitive cells, are formed and survive using the Escherichia coli C496_10 tripartite toxin/antitoxin system MqsR/MqsA/MqsC to inhibit T2 phage. Specifically, MqsR/MqsA/MqsC inhibited T2 phage by 10⁵-fold and reduced T2 titers by 3,000-fold. During T2 phage attack, in the presence of MqsR/MqsA/MqsC, evidence of persistence includes the single-cell physiological change of reduced metabolism (via flow cytometry), increased spherical morphology (via transmission electron microscopy), and heterogeneous resuscitation. Critically, we found restriction-modification systems (primarily EcoK McrBC) work in concert with the toxin/antitoxin system to inactivate phage, likely while the cells are in the persister state. Hence, a phage attack invokes a stress response similar to antibiotics, starvation, and oxidation, which leads to persistence, and this dormant state likely allows restriction/modification systems to clear phage DNA.