Schematic of an allelic-exchange procedure that uses plasmid pIMAY* and, by way of example, the generation of a tagO gene deletion. In A, the allelic-exchange plasmid with the gene deletion (ΔtagO) and 1 kb upstream and downstream gene fragments is shown along with the corresponding Staphylococcus aureus chromosomal tagO gene region. The plasmid has a temperature-sensitive origin (ts ori) of replication in S. aureus. (B) Growth at 37°C in the presence of antibiotic selection (chloramphenicol [cam] in this example) selects for strains that have undergone the first homologous-recombination event and now carry the plasmid integrated into the chromosome. (C) Following growth at 28°C and a second homologous-recombination event, strains either contain the wild-type gene copy or the desired gene deletion. The second homologous-recombination and plasmid-excision event is often selected for using a counterselectable marker. When using the allelic-exchange plasmid pIMAY*, the phenylalanine analog p-chlorophenylalanine (PCPA) can be used for counterselection, which is toxic for strains containing the plasmid.