Bacterial strains, growth media, and culture conditions
Strains used in the present study are listed in Table S1 . To
routinely cultivate S. aureus strains, tryptic soy broth (TSB)
containing 0.25% (wt/vol) dextrose (BD Biosciences) or complete
chemically defined medium (CDM, pH 6.5) were used (Sheldon et al.,
2014). Briefly, CDM medium was formulated with alanine (672 μM),
arginine (287 μM), aspartic acid (684 μM), cysteine (166 μM), glutamic
acid (680 μM), glycine (670 μM), histidine (129 μM), isoleucine (228
μM), leucine (684 μM), lysine (342 μM), methionine (20 μM),
phenylalanine (240 μM), proline (690 μM), serine (285 μM), threonine
(260 μM), tryptophan (50 μM), tyrosine (275 μM), valine (684 μM),
thiamine (56 μM), nicotinic acid (10 μM), biotin (0.04 μM), pantothenic
acid (2.3 μM), MgCl2 (1,000 μM), CaCl2(100 μM), monopotassium phosphate (40,000 μM), dipotassium phosphate
(14,700 μM), sodium citrate dehydrate (1,400 μM), magnesium sulfate (400
μM), ammonium sulfate (7,600 μM), and glucose (27,753 μM). Blood agar
plates were used to propagate the lpdA mbcS double mutant and its
derivatives to mitigate the severe growth defect on TSB.Escherichia coli strains were grown in lysogeny broth (LB) medium
without glucose (10 g L-1 tryptone, 5 g
L-1 yeast extract, and 10 g L-1sodium chloride) (Bertani, 1951). When necessary, media were
solidified with agar at 1.5% [w/v] and supplemented with
antibiotics at the following concentrations to maintain selection:
ampicillin (Ap) 100 μg ml-1, chloramphenicol (Cm) 10
μg ml-1, erythromycin (Em) 10 μg
ml-1, kanamycin (Km) 100 μg ml-1 or
tetracycline (Tc) 1.5 μg ml-1. When required, media
were supplemented with the short branched-chain carboxylic acidsi C4, i C5, anda C5 (Sigma-Aldrich), with the branched-chain
fatty acids a 15:0 (Avanti Polar Lipids) and a 17:0
(Sigma-Aldrich), or with the branched-chain aldehyde
2-methylbutyraldehyde (Sigma-Aldrich) to a final concentration of 0.5
mM. For genetic complementation, TSB was supplemented with 50 ng
ml-1 anhydrotetracycline (aTc). Unless otherwise
noted, all strains were grown at 37oC. A double-back
dilution scheme was used to grow cells for growth curves and GC-FAME
analysis to ensure steady-state growth. Briefly, for growth curve
experiments, overnight cultures were used to inoculate precultures in
disposable 16x125 mm borosilicate glass tubes (Fisher Scientific) to an
optical density at 600 nm (OD600) of 0.05 and incubated
with rotation. Cell growth was monitored by measuring
OD600 using an Amersham Ultraspec 2100 Pro UV-visible
spectrophotometer. After streaking from frozen stocks, overnight
cultures were inoculated from single colonies and were used to initiate
pre-cultures that were grown to exponential phase to an
OD600 of 1.0 and diluted into fresh TSB to an
OD600 of 0.05 and continued to grow. Exponential-phase
samples were collected at an OD600 of 0.5±0.2. Samples
were inoculated into a fresh medium in a 96-well plate to an
OD600 of 0.05 and cell density was monitored over time
in a computer-controlled Synergy H1 plate reader (BioTek/Agilent)
running Gen5 software ver3.14. Cells for GC-FAME analysis were grown
following the same scheme as above but in 125-mL DeLong shake flasks
with 12.5 mL TSB (10:1 flask/medium ratio) and incubated in a gyratory
water bath shaking. Exponential-phase samples were collected at an
OD600 of 0.5 ± 0.1. Cells were grown to stationary phase
in disposable borosilicate glass tubes for reporter and chemical
complementation assays.