Quantification of Staphylococcus aureus Phospholipids

Phospholipids were isolated and quantified as described recently (20 (link), 21 (link)). Bacterial overnight cultures grown in MHB to an optical density at 600 nm (OD₆₀₀) of 0.05 were incubated in 100 ml fresh MHB until the exponential-growth phase (OD₆₀₀ of 0.5 to 1) was reached. After adjusting S. aureus strains to equal optical densities, the Bligh-Dyer method (37 (link)) was used to extract lipids with a chloroform-methanol-sodium acetate buffer (20 mM, pH 4.6) mixture (1:1:1 [vol/vol/vol]). Isolated lipids were vacuum dried, resuspended in chloroform-methanol (2:1 [vol/vol), and spotted onto silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany) with a Linomat 5 sample application unit (Camag, Berlin, Germany). Polar lipids were separated in an ADC 2 developing chamber (Camag, Berlin, Germany) with a chloroform-methanol-water (65:25:4 [vol/vol/vol]) running solvent. Phospholipids were detected by staining of phosphate groups with molybdenum blue, and the LysPG content was determined in relation to the total phospholipid content by densitometry analysis performed with ImageJ (http://rsbweb.nih.gov/ij/docs/guide/index.html) as described recently (20 (link), 21 (link)).