A gene containment strategy based on a restriction–modification system

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Abstract
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 EcoRI restriction–modification system. The set‐up involved linkage of the ecoRIR lethal gene encoding the EcoRI endonuclease (toxin) to the contained character in a plasmid and chromosomal insertion of the ecoRIM gene encoding the cognate EcoRI methylase (antidote) that protects the target DNA from restriction. Transfer of the contained character to a recipient cell lacking the antidote caused EcoRI‐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 EcoRI‐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.