Figure 4.

HPLC analysis of membrane extracts (Bligh and Dyer procedure) prepared from E. coli FB8 cells carrying either the empty pASK vector (A) or pASK plasmids expressing the wild-type PcaM1 (B), its isolated cytotoxic domain (C) or the D222A mutant (D), respectively. Cells were grown exponentially in 2YT medium, and PcaM1 variants’ expression was induced at OD₆₀₀ = 0.2 by the addition of anhydrotetracycline. Cells were harvested just before the onset of lysis and treated by sonication. Membrane extracts were prepared and isoprenoids extracted and analyzed by HPLC as described in the text.