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

Nathan Fraikin, Laurence Van Melderen

Nucleic acids research 2024 Feb 28

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Toxin-antitoxin (TA) systems are small selfish genetic modules that increase vertical stability of their replicons. They have long been thought to stabilize plasmids by killing cells that fail to inherit a plasmid copy through a phenomenon called post-segregational killing (PSK) or addiction. While this model has been widely accepted, no direct observation of PSK was reported in the literature. Here, we devised a system that enables visualization of plasmid loss and PSK at the single-cell level using meganuclease-driven plasmid curing. Using the ccd system, we show that cells deprived of a ccd-encoding plasmid show hallmarks of DNA damage, i.e. filamentation and induction of the SOS response. Activation of ccd triggered cell death in most plasmid-free segregants, although some intoxicated cells were able to resume growth, showing that PSK-induced damage can be repaired in a SOS-dependent manner. Damage induced by ccd activates resident lambdoid prophages, which potentiate the killing effect of ccd. The loss of a model plasmid containing TA systems encoding toxins presenting various molecular mechanisms induced different morphological changes, growth arrest and loss of viability. Our experimental setup enables further studies of TA-induced phenotypes and suggests that PSK is a general mechanism for plasmid stabilization by TA systems. © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Nathan Fraikin, Laurence Van Melderen. Single-cell evidence for plasmid addiction mediated by toxin-antitoxin systems. Nucleic acids research. 2024 Feb 28;52(4):1847-1859