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), Sapitinib clinical trial hold at 220°C for 5 min. The identity of fatty acid methyl esters SC79 clinical trial were determined by comparing their retention times with identified fatty acid methyl ester standards (Sigma-Aldrich). The compositions

were expressed as weight percentages. Results Growth characteristics of S. aureus strain PDJ28 (ΔgpsA) The S. aureus gpsA gene (SA1306) was disrupted by the insertion of a Group II intron (see Methods). The insertion was confirmed by PCR genotyping showing the presence of the inactivating DNA insertion in the gpsA gene (Figure 1, inset). Strain PDJ28 was a click here glycerol or glycerol-PO4 auxotroph on agar plates (not shown). The growth of strain PDJ28 in RN media broth was followed after the removal of the glycerol supplement (Figure 1). The rate of cell growth immediately slowed, and then ceased after 90 min. These growth characteristics were similar to the growth phenotypes of the gpsA knockouts previously isolated in E. coli[30], B. subtilis[22] and S. aureus[20]. Figure 1 Growth phenotype of the gpsA knockout strain. S. aureus strain PDJ28 (ΔgpsA) was grown in RN medium to an OD600 of 0.5 and the cells were harvested and washed to remove the glycerol supplement. The culture was split and resuspended in media either with or without 0.1% glycerol, and growth was followed as a function of time. The growth curve is representative example of the

data obtained in duplicate experiments. The isothipendyl figure inset shows the multiplex PCR genotyping of the wild-type gpsA gene (528 bp) in strain RN4220 and the inactivated gpsA allele (394 bp) in strain PDJ28 as described under Methods. Alterations in membrane phospholipid homeostasis following glycerol removal The removal of the glycerol supplement from strain PDJ28 (ΔgpsA) had a significant impact on the membrane phospholipid composition. The metabolism of existing membrane phospholipids was determined by first labeling the cells with [14C]acetate in the presence of glycerol. The [14C]acetate and glycerol were then removed from the culture and the distribution of lipid classes examined after 30 min of glycerol deprivation by 2-dimensional thin-layer chromatography (Figure 2).