Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus

The gold standard to create gene deletions in Staphylococcus aureus is by homologous recombination using allelic exchange plasmids with a temperature sensitive origin of replication. A knockout vector that contains regions of homology is first integrated into the chromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by growth without antibiotic selection at low temperature, leading at a certain frequency to the excision of the plasmid and deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or more typically, a counter selection step is used. We here present the adaption of the counter selectable marker pheS*, coding for a mutated subunit of the phenylalanine-tRNA-synthetase for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analogue para-chlorophenylalanine (PCPA) into proteins and the addition of 20-40 mM PCPA to rich media leads to a drastic growth reduction of S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA to a complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we deleted the magnesium transporter gene mgtE in S. aureus USA300 LAC* (SAUSA300_0910/ SAUSA300_RS04895) and RN4220 (SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureus is mainly mediated by the presence of MgtE. This new plasmid will aid to efficiently and easily create gene knockouts in S. aureus.