Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Toxin-antitoxin (TA) systems are genetic modules crucial for plasmid stability.
• Post-segregational killing (PSK) is the proposed mechanism, but direct evidence was lacking.

Purpose of the Study:

• To directly visualize and confirm post-segregational killing (PSK) at the single-cell level.
• To investigate the molecular mechanisms and consequences of PSK in plasmid stabilization.

Main Methods:

• Developed a novel meganuclease-driven plasmid curing system for single-cell visualization.
• Utilized the ccd toxin-antitoxin system to induce and observe PSK.
• Analyzed DNA damage, SOS response, and cell death in plasmid-free segregants.

Main Results:

• Directly visualized PSK, showing DNA damage, filamentation, and SOS response induction in plasmid-free cells.
• Demonstrated that while most cells died, some repaired PSK-induced damage via SOS-dependent mechanisms.
• Observed that ccd-induced damage activated lambdoid prophages, enhancing cell killing.

Conclusions:

• PSK is a general and effective mechanism for TA system-mediated plasmid stabilization.
• The study provides the first direct evidence and visualization of PSK at the single-cell level.
• PSK involves DNA damage and can be influenced by other cellular responses like prophage activation.