Figure 5.

Removal of PlsX from the membrane blocks phospholipid synthesis. A, rescue of cells expressing PlsX^L254E by supplementation with B. subtilis fatty acids. Strains DS28 (Pxyl-plsX), DS69 (Pxyl-plsX amyE::Physpank-gfp-plsX), and DS160 (Pxyl-plsX amyE::Physpank-gfp-plsX^L254), were grown in LB medium containing 0.5 mm IPTG and 1 mg/ml of BSA, and in the presence or absence of 0.4% xylose and/or fatty acid methyl esters (FAMEs) (2 mg/ml). −FAMEs, black lines/squares; + FAMEs, red lines/circles. Data are representative of at least two independent experiments. B, lipid synthesis in cells expressing PlsX mutants. Strains were grown in LB medium in the presence of 1 mm IPTG to induce the expression of the different PlsX variants. The cultures were supplemented with xylose (0.4%) to induce the expression of WT PlsX (PlsXwt) or PlsY (in strain VD211 Pxyl-plsY). Strains DS28, DS160, DS171, and VD211 were labeled with 2 μCi/ml of [¹⁴C]acetate for 30 min during the transition from log to stationary phase. Strains DS69 and DS170 grown to midexponential phase were labeled with 2 μCi/ml of [¹⁴C]acetate for 30 min. Cells were harvested and total lipids were quantified in a scintillation counter. Bars represent the mean ± S.D. from three independent experiments. C, fatty acids accumulate in strains expressing PlsX that does not associate with the membrane. Total lipids of cells labeled with[¹⁴C]acetate as described in panel B were analyzed by TLC on preadsorbent Silica Gel G layers (Merck) developed with hexane-ethyl ether-acetic acid (60/40/1, v/v/v). Each lane was loaded with about 1,000 cpm of labeled lipids. The radioactive lipid species were identified by their co-migration with standards. FFA, free fatty acid; DAGs, diacylglycerols; PL, phospholipid; Unk, unknown. The example shown is typical of three independent experiments.