Bacterial strains and media
S. aureus strain RN4220 was obtained from Richard Novick . Strain PDJ28 (ΔgpsA) was constructed as described previously . A group II intron was inserted at bp 42 of the gpsA gene using the primer design software and plasmid system provided by Sigma-Aldrich (Targetron system) . The presence of the insertions was verified by multiplex PCR using opposing primers located in the gpsA gene outside the intron insertion site and one primer inside the intron. The wild-type allele yields a product of 528 bp and the disrupting gene gives a product of 394 bp. RN minimal medium was used for broth cultures and consisted of M9 salts, 1 mM MgSO4, 10 mM CaCl2, 15 μM vitamin B1, 32 μM vitamin B3, 0.1% casein hydrolysate, 0.4% glucose, 0.1 mg/l biotin, 2 mg/l pantothenic acid, 10 μM FeCl2, 6 mg/l citrate, 10 mg/l MnCl2, 4 μg/l L-tryptophan, and 0.1 mg/l lipoic acid.
Phospholipids and fatty acids were labeled by the addition of 50 μCi [1-14C]acetate (50 Ci/mol) per 10 ml culture. For labeling of lipids before glycerol starvation, RN media supplemented with 0.1% glycerol and 50 μCi [1-14C]acetate (1 Ci/mol) per 10 ml culture was inoculated with strain PDJ28 to OD600 = 0.05 and grown to OD600 = 0.6. The cells were pelleted and washed with 50 ml RN media and used to inoculate cultures in RN media with and without 0.1% glycerol supplement for indicated time. For labeling of lipids after glycerol starvation, cells were washed as previously described, grown for 30 minutes before addition of 50 μCi [1-14C]acetate (50 Ci/mol) per 10 ml culture for indicated time. 10 ml samples were centrifuged, washed with 10 ml RN media and 10 ml H2O. Pellets were resuspended in 100 μl H2O and lipids extracted through the addition of 360 μl of chloroform:methanol:HCl (1/2/0.02) and incubated at room temperature for 20 minutes. 120 μl chloroform and 120 μl 2 M KCl were added to separate phases and after centrifugation, the organic phase was removed and radioactivity quantified by scintillation counting.
Radiolabelled lipids were analyzed by 1-dimensional and 2-dimensional thin-layer chromatography. The 1-dimensional system used to separate phospholipids from diacylglycerol and fatty acid was Silica Gel G layers developed with chloroform:methanol:acetic acid (98/2/1) and visualized using Bioscan imaging detector. The 2-dimensional system also employed Silica Gel G layers and was developed first with chloroform:methanol:water (65/25/4) and secondly tetrahydrofuran/dimethoxyethane/methanol/4 M ammonium hydroxide (10/6/4/1). The resulting thin-layer plate exposed to a PhosphoImager screen and visualized using a Typhoon 9200.
Lipid mass spectrometry
Mass spectrometry of phospholipids was performed using a Finnigan TSQ Quantum (Thermo Electron, San Jose, CA) triple quadrupole mass spectrometer. Samples were prepared in 50:50 (v/v) chloroform:methanol. The instrument was operated in the negative ion mode. Ion source parameters were as follows: spray voltage of 3,000 V, capillary temperature of 270°C, capillary offset of 35 V, and the tube lens offset was set by infusion of polytyrosine tuning and calibration solution (Thermo Electron, San Jose, CA) in the electrospray mode. Acquisition parameters were as follows: full scan, scan range 600 – 100 m/z, scan time 0.5 s, peak width Q1 0.7 FWHM. Instrument control and data acquisition was performed with the Finnigan™ Xcalibur™ software (Thermo Electron, San Jose, CA).
Mass spectrometry malonyl-CoA measurement
Cultures of strain PDJ28 were grown in RN medium supplemented with 0.1% glycerol to OD600 = 0.6. Cells were pelleted and washed with 50 ml RN medium to remove glycerol and used to inoculate RN medium with and without 0.1% glycerol. Cultures were grown for 120 minutes and harvested at room temperature. Cells were extracted using the Bligh and Dyer method , and 50 pmol of [13C3]malonyl-CoA (Stable Isotope Products; Isotec) was added. The aqueous phase was applied to a 100-mg 2-(2-pyridyl)ethyl functionalized silica gel column (Supelco) equilibrated with 2% acetic acid in methanol/water (1:1) . The column was washed two times with 1 ml of equilibration buffer and 1 ml water. CoAs were eluted with 1 ml of 50% acetonitrile containing 15 mM ammonium hydroxide. Mass spectrometry of acyl-CoA was performed using a Finnigan TSQ Quantum (Thermo Electron) triple-quadrupole mass spectrometer . The instrument was operated in positive mode using single ion monitoring (SIM) neutral loss scanning corresponding to the loss of the phosphoadenosine diphosphate from CoA species. The ion source parameters were as follows: spray voltage, 4,000 V; capillary temperature, 250°C; capillary offset, -35 V; sheath gas pressure, 10; auxiliary gas pressure, 5; and tube lens offset was set by infusion of the polytyrosine tuning and calibration in electrospray mode. Acquisition parameters were as follows: scan time, 0.5 s; collision energy, 30 V; peak width Q1 and Q3, 0.7 FWHM; Q2 CID gas, 0.5 mTorr; source CID, 10 V; neutral loss, 507.0 m/z; SIM mass of 855 m/z with a scan width of 10 m/z to capture the signals from both light and heavy malonyl-CoA, and SIM mass of 810 m/z with a scan width of 6 m/z to capture the signal of acetyl-CoA.
Cultures of strain PDJ28 (ΔgpsA) and parent S. aureus strain RN4220 cells were grown to OD600 = 0.5 in RN minimum media with 1% glycerol supplementation at 37°C with rigorous shaking (225 rpm), and then split into 50 ml aliquots. Cells were washed twice with RN media. For PDJ28 without glycerol supplement and strain RN4220, cells were suspended in 50 ml of RN media. Strain PDJ28 was grown in RN media with 1% glycerol supplementation. Cells were grown for the indicated amount of time, pelleted, and resuspended in 125 μl of 25% sucrose and 50 mM Tris pH 7.0 on ice. Lysostaphin (25 μl of a 5 mg/ml) was added to the mixture, and incubated on ice for 15 minutes. Finally, the cells were lysed by adding 200 μl of 10% Triton X-100, 62.5 mM EDTA, and 50 mM Tris–HCl pH 7.5. The lysed cells were centrifuged at 40,000 g for 30 minutes. The supernatant, in native loading buffer, was loaded onto a 2.5 M urea, 15% acrylamide gel. The amount of supernatant loaded in each sample is adjusted to OD600 such that total protein is similar for each lane.
Cultures of strain PDJ28 cells were grown in RN media with 1% glycerol supplement at 37°C with rigorous shaking (225 rpm). Cells were grown to OD600 of 0.5, aliquoted to 50 ml cultures, and washed twice with RN media. Then, one cell aliquot was grown in RN minimum media and another aliquot was grown in RN minimum media supplemented with 1% glycerol for an additional 2 hours. Cells were washed with phosphate-buffered slaine three times and harvested for lipids using the method of Bligh and Dyer . The free fatty acids were separated from the other lipid species by thin-layer chromatography. Briefly, the lipid extract was loaded onto Silica Gel G plates (Analtech) and chromatographed in chloroform:methanol:acetic acid (98/2/1) solvent mixture. The silica gel at Rf of 0.7 or higher was scraped off the plate to collect the free fatty acid fractions. The scraped silica was added to 1 ml water, and extracted 3 times with 1 ml hexane. The hexane fractions were collected and evaporated to obtain the free fatty acid samples. Fatty acid methyl esters were generated from the collected fatty acids by bringing up the fatty acid samples in 5 ml of 2% anhydrous HCl in anhydrous methanol and incubating overnight. The reaction was evaporated under nitrogen and brought up in 1 ml of distilled water. The water phase was extracted 3 times with hexanes. The hexane fractions were pooled and evaporated over nitrogen. The fatty acid methyl esters were analyzed by a Hewlett-Packard model 5890 gas chromatograph equipped with a flame ionization detector, and separated on 30 m × 0.536 mm × 0.50 μm DB-225 capillary column. The injector was set at 250°C, and the detector was at 300°C. The temperature program was as followed: initial temp 70°C for 2 min, rate of 20°C/min for 5 min (final 170°C), rate of 2°C/min for 10 min (final 190°C), hold at 190°C for 5 min, rate of 2°C/min for 15 min (final 220°C), hold at 220°C for 5 min. The identity of fatty acid methyl esters were determined by comparing their retention times with identified fatty acid methyl ester standards (Sigma-Aldrich). The compositions were expressed as weight percentages.