# Dependence of post-segregational killing mediated by Type II restriction–modification systems on the lifetime of restriction endonuclease effective activity

## TL;DR

This study shows that post-segregational killing by RM systems depends on how long restriction enzymes stay active after plasmid loss.

## Contribution

The study introduces a CRISPR-based method to study PSK and reveals that enzyme lifetime determines PSK occurrence.

## Key findings

- Plasmids with EcoRV, Eco29kI, and EcoRI RM systems cause PSK and SOS response upon loss.
- Esp1396I RM system does not cause PSK due to short enzyme activity lifetime.
- Mathematical modeling explains PSK dynamics based on enzyme lifetimes and replication cycles.

## Abstract

Plasmid-borne Type II restriction–modification (RM) systems mediate
post-segregational killing (PSK). PSK is thought to be caused by the
dilution of restriction and modification enzymes during cell division,
resulting in accumulation of unmethylated DNA recognition sites and their
cleavage by restriction endonucleases. PSK is the likely reason for
stabilization of plasmids carrying RM systems in the absence of selection
for plasmid maintenance. In this study, we developed a CRISPR
interference-based method to eliminate RM-carrying plasmids and study
PSK-related phenomena with minimal perturbation to the Escherichia
coli host. Plasmids carrying the EcoRV, Eco29kI, and EcoRI RM
systems were highly stable, and their loss resulted in SOS response and PSK.
In contrast, plasmids carrying the Esp1396I system were poorly stabilized;
their loss led to a temporary cessation of growth, followed by full
recovery. We demonstrate that this unusual behavior is due to a limited
lifetime of the Esp1396I restriction endonuclease activity, which, upon
Esp1396I plasmid loss, disappears approximately after two cycles of cell
division, i.e., before unmethylated sites appear in significant numbers. Our
results indicate that whenever PSK induced by a loss of RM systems, and,
possibly, other toxin–antitoxin systems, is considered, the lifetimes
of individual system components and the growth rate of host cells shall be
taken in account. Mathematical modeling shows, that unlike the situation
with classical toxin–antitoxin systems, RM system-mediated PSK is
possible when the lifetimes of restriction endonuclease and
methyltransferase activities are similar, as long as the toxic restriction
endonuclease activity persists for more than two chromosome replication
cycles.

It is widely accepted that many Type II restriction–modification
(RM) systems mediate post-segregational killing (PSK) if plasmids that
encode them are lost. In this study, we harnessed an inducible
CRISPR-Cas system to remove RM plasmids from Escherichia
coli cells to study PSK while minimally perturbing cell
physiology. We demonstrate that PSK depends on restriction endonuclease
activity lifetime and is not observed when it is less than two
replication cycles. We present a mathematical model that explains
experimental data and shows that unlike the case of
toxin–antitoxin-mediated PSK, the loss of an RM system induced
PSK even when the RM enzymes have identical lifetimes.