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

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
DOI: 10.1099/mic.0.000791 Publication Date: 2019-04-03T17:48:31Z
ABSTRACT
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 .
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