FIG. 2.
Essential nature of the ltaS gene for LTA biosynthesis. (A) Loss of LTA in ΔltaS mutant cells. Cells were metabolically labeled with 32Pi, and 32P-labeled LTA was extracted, separated by 15% SDS-PAGE, and detected by autoradiography. Lanes: 1, RN4220/pKE515; 2, M0674 (ΔltaS)/pM101/pKE515; 3, M0674/pM101/pSltaS. (B) Quantification of LTA by ELISA. Levels of LTA in S. aureus cell culture were quantified by ELISA using an anti-polyglycerolphosphate antibody. Means from three independent experiments are presented along with standard deviations. Bars: 1, RN4220/pKE515; 2, M0674/pM101/pKE515; 3, M0674/pM101/pSltaS. (C) The ltaS gene is dispensable for WTA biosynthesis. WTA was extracted from stationary-phase S. aureus cells, separated by PAGE (18%), and visualized by 0.5% alcian blue staining. Lanes: 1, RN4220; 2, M0674/pM101; 3, M0702 (ΔtagO). (D) Decreased turnover rate of membrane PG in an S. aureus ΔltaS mutant. Cells were metabolically pulse-labeled with 32Pi for 1 h and incubated further in fresh medium for the indicated periods. Total lipids were extracted and separated by silica gel thin-layer chromatography (chloroform-methanol-acetic acid, 65:25:10). 32P-labeled PG was quantified by autoradiography. Filled circle, RN4220/pKE515; open circle, M0674/pM101/pKE515; filled triangle, M0674/pM101/pSltaS. Data are means and standard deviations from three independent experiments.