A gene containment strategy based on a restriction–modification system

Engineering barriers to the spread of specific genes are of great interest both to increase the predictability of recombinant microorganisms used for environmental applications and to study the role of gene transfer in the adaptation of microbial communities to changing environments. We report here a new gene containment circuit based on a toxin–antidote pair that targets the cell DNA, i.e. the type II Eco RI restriction–modification system. The set‐up involved linkage of the ecoRIR lethal gene encoding the Eco RI endonuclease (toxin) to the contained character in a plasmid and chromosomal insertion of the ecoRIM gene encoding the cognate Eco RI methylase (antidote) that protects the target DNA from restriction. Transfer of the contained character to a recipient cell lacking the antidote caused Eco RI‐mediated chromosomal breaks, leading to cell death, thereby preventing gene spread. Using transformation and conjugation as mechanisms of DNA transfer and different environmentally relevant bacteria as recipients, we have shown that the potentially universal Eco RI‐based containment system decreases gene transfer frequencies by more than four orders of magnitude. Analyses of the survivors escaping killing revealed a number of possible inactivation mechanisms.