Multigenerational proteolytic inactivation of restriction upon subtle genomic hypomethylation in Pseudomonas aeruginosa

Restriction-modification (R-M) systems protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage defences, R-M systems pose a major risk of autoimmunity, exacerbated by the presence of hundreds to thousands of potential cleavage sites in the bacterial genome. Pseudomonas aeruginosa strains experience the temporary inactivation of restriction endonucleases following growth at high temperatures, but the reason and mechanisms for this phenomenon are unknown. Here we report that P. aeruginosa type I restriction endonuclease is degraded and the methyltransferase is partially degraded, by two Lon-like proteases when replicating at >41 °C. This post-translational regulation prevents self-DNA targeting, which is a risk due to stable genomic hypomethylation, as demonstrated by single-molecule, real-time sequencing and TadA-assisted N⁶-methyladenosine sequencing. When cells grown at >41 °C are returned to 37 °C, full genomic methylation does not fully recover for up to 60 bacterial generations, and thus restriction activity remains off for the duration. Our findings demonstrate that type I R-M is tightly regulated post-translationally with a long memory effect that ensures genomic stability and mitigates autotoxicity.