Fig. 2
Effect of the major bacterial lipids on MelBSt protein expression and melibiose transport activities. a Melibiose transport with intact cells. Cells with varied lipid compositions without (open symbols) or with (filled symbols) MelBSt-expressing vector pK95 ΔAH/MelBSt/CHis10 were grown in LB as described in Methods. The melibiose transport assay in the absence or presence of 20 mM NaCl is described in the Methods. The intracellular melibiose was plotted against the incubation time. Inset, initial rate of transport within 30 s. Left column, strains AL95 (PE−) and AL95 with pDD72GM (PE+). Middle column, strains MG1655 (WT) and UE54 (PG− CL−). Right column, strains WK3110 (WT) and BKT12 (CL−). Black curves, the WT or strain AL95 with pDD72GM; red curves, the lipid-deficient strains. Error bar, SEM; and the number of tests = 4–6. b Membrane expression. An aliquot of cells prepared for the transport assay in panels a-c were used to prepare the crude membrane fraction as described in the Methods. Membrane proteins of 20 μg from each sample were analyzed with SDS-15%PAGE, and MelBSt was detected by western blot using Penta⋅His HRP antibody. c Melibiose fermentation. Cells with varied lipid compositions transformed with pK95 ΔAH/MelBSt/CHis10 were plated on the melibiose-containing MacConkey agar as described in Methods. Colonies on the MacConkey agar plates were grown at 37 °C, except for the strain AL95 with pDD72GM that was placed in a 30 °C incubator