Figure 5.

A triple charge-inversion mutation in the hydrophilic channel within MlaA results in gain-of-function phenotypes. A, a representative structure of MlaA from all-atomistic MD simulations (as in Fig. 3, top view) with position of the channel depicted by an asterisk. Negatively charged residues mutated to arginine are highlighted (magenta, in sticks). The figure was generated using the program VMD (55). B, analysis of SDS/EDTA sensitivity of WT and ΔmlaA strains producing indicated MlaA variants at low levels from the pET23/42 vector (p) (12). Serial dilutions of respective cultures were spotted on LB agar plates containing ampicillin (Amp), supplemented with or without 0.50% SDS and 0.5/0.8 mm EDTA, as indicated, and incubated overnight at 37 °C. C, representative thin-layer chromatography (TLC)/autoradiographic analysis of ³²P-labeled lipid A extracted from exponential phase cultures of strains described in B. As a positive control for lipid A palmitoylation, WT cells were treated with 25 mm EDTA for 10 min prior to extraction. Equal amounts of radioactive material were spotted for each sample. Average percentages of palmitoylation of lipid A, and the standard deviations were quantified from triplicate experiments and plotted below. For Student's t tests: *, p < 0.005 compared with WT with empty vector; **, p < 0.001 compared with WT p-mlaA–His.