Genetic techniques
Oligonucleotides used in this study were synthesized by Integrated DNA
Technologies (IDT; Coralville, IA) and are listed in Table S2.Genomic DNAs (gDNAs) were sequencing using Illumina whole genome
sequencing (SeqCenter; Pittsburgh, PA) and results were analyzed using
BRESEQ software (Barrick et al., 2009). Plasmids used in this study are
listed in Table S3 and were constructed using Gibson assembly
as described previously (Gibson et al., 2009) and verified by Sanger
sequencing (Azenta Life Sciences) or whole plasmid sequencing
(Plasmidsaurus; Eugene, OR). E. coli NEB 5α (NEB) was used as a
host for plasmid constructions, which were then transferred intoS. aureus strain RN4220 by electroporation as previously
described (Schenk & Laddaga, 1992). Plasmids and marked mutations
were moved between S. aureus strains via Φ85-mediated
transduction (Novick, 1991).
Construction of the lpdA::kan+ strain:To construct the lpdA::kan+ strain (SRB3000),
we moved the transposon insertion containing the KmRmarker from the S. aureus SH1000 strain VKS102 (Singh et al.,
2008) into the S. aureus USA300 LAC SRB2421 (lpdA ::φNΣ)
via Φ85-mediated transduction (Novick, 1991). Recombinants were selected
on TSA Km medium and scored for Em susceptibility. We then confirmed the
allele by PCR.