Exploitation of a Type 1 Toxin-Antitoxin System as an Inducible Counter-Selective Marker for Genome Editing in the Acetogen .

Targeted mutations in the anaerobic methylotroph have previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin from was placed under the control of an anhydrotetracycline-sensitive promoter, forming an inducible counter-selective system. This inducible system was coupled with a non-replicative integrating mutagenesis vector to create precise gene deletions in B2. The genes targeted in this study were those encoding the histidine biosynthesis gene , the methanol methyltransferase and corrinoid protein and , and , encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion within brought about the expected histidine auxotrophy, and deletions of and both abolished autotrophic growth on methanol. Deletion of was shown to abolish the growth of on L-carnitine. After an initial selection step to isolate transformant colonies, only a single induction step was required to obtain mutant colonies for the desired targets. The combination of an inducible counter-selective marker and a non-replicating integrative plasmid allows for quick gene editing of .