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: EXPRESSION; 0301 basic medicine; 570; Staphylococcus aureus; Genetic Vectors; 610; homologous recombination; Microbiology; Gene Knockout Techniques; 03 medical and health sciences; Bacterial Proteins; C-DI-AMP; Humans; counter selection; PCPA; Cloning, Molecular; 0303 health sciences; Science & Technology; DELETION; REPRESSOR; Staphylococcal Infections; S. aureus; GENE; 3. Good health; mgtE; ESCHERICHIA-COLI; MAGNESIUM TRANSPORT; pIMAY*; Phenylalanine-tRNA Ligase; allelic exchange plasmid; Genetic Engineering; Life Sciences & Biomedicine; SYSTEM; RESISTANCE; Genome, Bacterial; ALLELIC REPLACEMENT; Plasmids

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 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 the deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or, more typically, a counterselection step is used. We present here the adaptation 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 drastic growth reduction for

S. aureus

and supplementing chemically defined medium with 2.5–5 mM PCPA leads to complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we delete 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 the efficient and easy creation of gene knockouts in

S. aureus

.