A new simple method to introduce marker-free deletions in the Bacillus subtilis genome.

A genetic tool to introduce marker-free deletions is essential for multiple manipulations of genomes. We report a simple and efficient method to create marker-free deletion mutants of Bacillus subtilis through transformation with recombinant PCR products, using the Escherichia coli mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counter-selection tool. Our method will be applicable to any bacterium in which introduction of the mazF cassette into the genome by double crossover homologous recombination is possible.