Targeted mutations in the anaerobic methylotrophEubacterium limosumhave previously been obtained using CRISPR-based mutagenesis methods. In this study, a RelB-family toxin fromEubacterium callanderiwas 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 inEubacterium limosumB2. The genes targeted in this study were those encoding the histidine biosynthesis genehisI, the methanol methyltransferase and corrinoid proteinmtaAandmtaC, andmtcB, encoding an Mttb-family methyltransferase which has previously been shown to demethylate L-carnitine. A targeted deletion withinhisIbrought about the expected histidine auxotrophy, and deletions ofmtaAandmtaCboth abolished autotrophic growth on methanol. Deletion ofmtcBwas shown to abolish the growth ofE. limosumon 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 ofE. limosum.