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

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Restriction-modification (R-M) systems are crucial prokaryotic anti-phage defenses.
• R-M systems pose an autoimmunity risk due to potential self-DNA cleavage sites.
• High-temperature growth in Pseudomonas aeruginosa leads to temporary restriction endonuclease inactivation, mechanism unknown.

Purpose of the Study:

• Investigate the mechanism behind high-temperature inactivation of R-M systems in P. aeruginosa.
• Determine how bacteria prevent self-DNA targeting under these conditions.
• Understand the regulatory control and long-term effects of this phenomenon.

Main Methods:

• Utilized high-temperature growth conditions (>41°C) for P. aeruginosa.
• Employed proteomic analysis to identify degrading enzymes.
• Applied single-molecule, real-time sequencing and TadA-assisted N⁶-methyladenosine sequencing to assess DNA methylation.
• Monitored R-M system activity and methylation recovery upon return to normal temperatures (37°C).

Main Results:

• Two Lon-like proteases degrade type I restriction endonuclease and partially degrade methyltransferase at >41°C.
• This post-translational regulation prevents self-DNA targeting, linked to stable genomic hypomethylation.
• Following temperature shift to 37°C, full methylation recovery takes up to 60 bacterial generations, maintaining R-M system inactivation.

Conclusions:

• Type I R-M systems in P. aeruginosa are regulated post-translationally by Lon-like proteases at high temperatures.
• This regulation ensures genomic stability by preventing autotoxicity during periods of hypomethylation.
• A significant memory effect in methylation recovery contributes to sustained R-M system inactivation, safeguarding the genome.