In this section
@article{Schuster:2019:10.1099/mic.0.000791,
author = {Schuster, C and Howard, S and Grundling, A},
doi = {10.1099/mic.0.000791},
journal = {Microbiology},
pages = {572--584},
title = {Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus},
url = {http://dx.doi.org/10.1099/mic.0.000791},
volume = {165},
year = {2019}
}
TY - JOUR
AB - The gold standard tocreate gene deletions in Staphylococcus aureusis by homologous 16recombination using allelic exchange plasmids with a temperature sensitive origin of replication. A knockout vectorthat containsregions of homologyis first integrated into thechromosome 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 20growth without antibiotic selection at lowtemperature, leading at a certain frequencyto the excision of the plasmid and deletionof the gene of interest.To detector 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 markerpheS, 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 analoguepara-chlorophenylalanine(PCPA)into proteins and the addition of 20-40 m M PCPAto rich medialeads to a drasticgrowth reductionof 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, wedeletedthe magnesium transporter gene mgtEin S. aureusUSA300 LAC(SAUSA300_0910/ SAUSA300_RS04895) and RN4220(SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureusis mainly mediated by the presence of MgtE.This new plasmid will aidto efficiently and easily create gene knockouts in S. aureus.
AU - Schuster,C
AU - Howard,S
AU - Grundling,A
DO - 10.1099/mic.0.000791
EP - 584
PY - 2019///
SN - 1350-0872
SP - 572
TI - Use of the counter selectable marker PheS for genome engineering in Staphylococcus aureus
T2 - Microbiology
UR - http://dx.doi.org/10.1099/mic.0.000791
UR - http://hdl.handle.net/10044/1/68669
VL - 165
ER -
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