FIG 6.

Expressing the clsB operon from a plasmid is sufficient to restore CL biosynthesis to the Δ-clsABC mutant. (A) Whole-cell lysates were collected from stationary-phase cultures of the wild type and the ΔclsABC mutants expressing an empty vector (pEmpty) and the ΔclsABC mutant expressing a plasmid-borne copy of the clsB operon (pclsB). GPLs were extracted from equivalent amounts of total membrane and were separated and visualized by LC-MS. (B) GPLs from stationary-phase cultures were extracted from total membranes of bacteria and separated by TLC. (C) GPLs were extracted from whole-cell lysates, and the abundance of individual CL and PGl molecules was quantified by LC-MS/MS as the ng of GPL per μL of extract ± SEM. Four biological replicates were analyzed. A two-way ANOVA followed by a Tukey’s multiple-comparison test was used to test significance. Asterisks indicate a significant difference relative to the wild type, ****, P < 0.0001; **, P < 0.01. ns, not significant.