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Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation in Pseudomonas aeruginosa

ABSTRACT: Restriction-modification (R-M) systems protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage defenses, R-M systems pose a significant risk of auto-immunity, 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 (tiREN) upon growth at high temperatures, but the mechanisms and implications of this are unknown. Here, we report that P. aeruginosa Type I restriction endonuclease (HsdR) is degraded, and the methyltransferase (HsdMS) is partially degraded, by two Lon-like proteases when replicating above 41 °C. This post-translational regulation prevents self-DNA targeting and leads to partial genomic hypomethylation, as demonstrated by SMRT sequencing and eTAM-seq. Interestingly, upon return to 37 ºC, restriction activity and full genomic methylation do not fully recover for up to 60 bacterial generations. Our findings demonstrate that Type I R-M is tightly regulated post-translationally with a long memory effect that ensures genomic stability and mitigates auto-toxicity.

ORGANISM(S): Pseudomonas aeruginosa PAO1

PROVIDER: GSE301453 | GEO | 2025/07/09

REPOSITORIES: GEO

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Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation

Project description:In Pseudomonas aeruginosa, restriction inactivation upon growth at high temperatures was previously described, however, which system is being inactivated, the underlying mechanism, as well as the timing of recovery, remain unknown. Here, we report that P. aeruginosa Type I methyltransferase (HsdMS) and restriction endonuclease (HsdR) components are degraded by two Lon-like proteases when replicating above 41 °C, which induces partial genome hypomethylation and simultaneously prevents self-targeting, respectively. Interestingly, upon return to 37 °C, methyltransferase activity returns gradually, with restriction activity not fully recovering for over 60 bacterial generations, representing the longest bacterial memory to our knowledge. Forced expression of HsdR over the first 45 generations is toxic, demonstrating the fitness benefit of HsdR inactivation.

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Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation in Pseudomonas aeruginosa

Project description:Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation in Pseudomonas aeruginosa

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Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation

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