A different approach was used by Paoletti ARS-1620 cost et al. [23] who inactivated plsY in B. subtilis with an intact plsY gene under control of a regulated promoter. In this model, the inactivation of PlsY activity is not immediate or complete, but rather the strain must be grown for hours to deplete pre-existing PlsY protein. Nonetheless, fatty acid accumulation was detected

in PlsY-depleted cells [23]. These earlier EX 527 cost experiments did not investigate the effect of glycerol deprivation on either the membrane lipid composition or the level or composition of the lipid precursor pools. Because knowledge of these metabolic intermediates will provide insight into the role of PlsY in pathway

regulation, we constructed a gpsA knockout in S. aureus to more precisely investigate the regulation of FASII and phospholipid metabolism in the absence of PlsY activity. The cessation of phospholipid synthesis does not blunt the continued metabolism of the principle membrane phospholipid, phosphatidylglycerol (PtdGro), resulting in a marked disruption of membrane phospholipid homeostasis. Long-chain acyl-acyl carrier protein (ACP) and malonyl-CoA accumulate following the block at PlsY, but fatty acid synthesis continues at a reduced rate reflected by the accumulation of intracellular fatty acids. Methods Bacterial strains and media S. aureus strain RN4220 was obtained from Richard Novick [24]. Strain PDJ28 (ΔgpsA) was constructed as described previously [25]. A group II intron was inserted at bp 42 of the gpsA gene using the primer design software and JNK-IN-8 plasmid system provided by Sigma-Aldrich (Targetron system) [26]. 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 SPTLC1 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. Metabolic labeling 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.