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
AUTHORS (3)
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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