FIG 2.

S. Typhimurium requires clsA for CL biosynthesis during logarithmic growth, while clsB and clsC contribute to CL production during stationary phase. (A) The wild-type (WT) and cls-mutant S. Typhimurium isolates were cultured to either the logarithmic (LOG) or stationary (STAT) phase of growth. Glycerophospholipids (GPL) were extracted from the total membrane fractions and separated by one-dimensional thin-layer chromatography (1D-TLC). PA, phosphatidic acid. (B) The GPLs derived from total membrane of fractions of log-phase WT, ΔclsA, and ΔclsA bacteria carrying either an empty vector or a vector encoding the clsA operon were separated and visualized by 1D-TLC. (C and D) GPLs were extracted from the total membranes of the WT and cls-mutant S. Typhimurium at the stationary phase of growth and quantified by normal-phase liquid-chromatography tandem mass spectrometry (LC-MS/MS). The quantities from four independent biological replicates are presented as the average amount of lipid per extract ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by Bonferroni posttest was used. Asterisks indicate a significant difference for the ΔclsABC triple mutant relative to the wild type (*, P < 0.05; **, P < 0.01). (E) Two-dimensional (2D)-TLC analysis of the GPLs that were extracted from the total membranes of the wild-type and ΔclsABC-mutant S. Typhimurium. The empty circle denotes the predicted migration position of CL in the ΔclsABC-mutant extract; however, this genotype is devoid of detectable CL. acyl-PGl, acylphosphatidylgycerol.