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

• DOI: 10.1099/mic.0.000791
• Publication Date: 2019-04-03T17:48:31Z
• Authors: Christopher F. Sc...; Sophie A. Howard; Angelika Gründling
• Keywords: Selectable marker; Gene knockout

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