CRISPR-Cas in Pseudomonas aeruginosa provides transient population-level immunity against high phage exposures

Abstract

The prokaryotic adaptive immune system, CRISPR-Cas, requires the acquisition of spacer sequences that target invading mobile genetic elements such as phages. Previous work has identified ecological variables that drive the evolution of CRISPR-based immunity of the model organism Pseudomonas aeruginosa PA14 against its phage DMS3vir, resulting in rapid phage extinction. However, it is unclear if and how stable such acquired immunity is within bacterial populations, and how this depends on the environment. Here we examine the dynamics of CRISPR spacer acquisition and loss over a 30-day evolution experiment, and identify conditions that tip the balance between long-term maintenance of immunity versus invasion of alternative resistance strategies that support phage persistence. Specifically, we find that both the initial phage dose and reinfection frequencies determine whether or not acquired CRISPR immunity is maintained in the long term, and whether or not phage can co-exist with the bacteria. At the population genetics level, emergence and loss of CRISPR immunity is associated with high levels of spacer diversity that subsequently decline due to invasion of bacteria carrying pilus-associated mutations. Together these results provide high resolution of the dynamics of CRISPR immunity acquisition and loss and demonstrate that the cumulative phage burden determines the effectiveness of CRISPR over ecologically relevant timeframes.