Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated pheS for Counterselection

Gene alteration/deletion by allelic exchange is the preferred strategy for gene manipulation in bacteria. Here we present the fundamentals for an efficient allelic exchange gene deletion method in the bacterial pathogen Listeria monocytogenes. Combining vector generation by Gibson assembly with a counterselection system based on the mutated phenylalanine synthetase (pheS*) makes the generation of gene deletion mutants straightforward and time efficient.