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 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. Overall design: Samples were collected from cultures grown overnight at either 43°C or 37°C, as well as after recovery at 37°C for 18 and 54 hours following incubation at 43 °C

限制性修饰（Restriction-modification, R-M）系统通过识别并降解入侵的外源DNA，抵御噬菌体感染。然而，与众多原核生物抗噬菌体防御系统类似，R-M系统存在显著的自身免疫风险，而细菌基因组中存在成百上千个潜在酶切位点，进一步加剧了这一风险。铜绿假单胞菌菌株在高温培养时会发生限制性核酸内切酶（restriction endonucleases, RE）的暂时性失活（tiREN），但该现象的具体机制与潜在影响仍未可知。本研究发现，铜绿假单胞菌的I型限制性核酸内切酶（HsdR）与甲基转移酶（HsdMS），在41℃以上增殖时会被两种Lon样蛋白酶降解，其中甲基转移酶发生部分降解。该翻译后调控机制可阻止R-M系统靶向自身DNA，并引发基因组部分低甲基化，这一结论通过单分子实时测序（Single-Molecule Real-Time sequencing, SMRT）与eTAM-seq得到验证。有趣的是，当培养温度重回37℃后，限制性核酸内切酶活性与基因组完全甲基化状态在长达60个细菌世代内均无法完全恢复。本研究结果表明，I型R-M系统受到严格的翻译后调控，并通过长效记忆效应保障基因组稳定性，同时减轻自身毒性。实验整体设计：样本分别采集自于43℃或37℃过夜培养的菌液，以及经43℃孵育后在37℃分别恢复培养18小时与54小时的菌液。