Fig. 4.

The LptE interaction site at residues 529–538 of LptD is required for proper folding of LptD and assembly of the LptD/E complex. (A) Ten-fold serial dilutions of overnight cultures (normalized by optical density) on LB containing 25 μg/mL kanamycin without (Top) or with 0.5% SDS and 0.75 mM EDTA. The WT and Δ529–538 strains harbored a chromosomal ΔlptD2∷kan deletion as well as pET23/42lptD or its derivative pET23/42lptDΔ529–538, respectively. (B) Western blot analysis, using the indicated antibodies, of total membrane extracts or eluates from Ni-NTA chromatography of these extracts from strains expressing low levels of LptE–His and either wild-type LptD or LptDΔ529–538 in a chromosomal ΔlptD background. β-mercaptoethanol (βME) reduces the disulfide bonds found in LptD, decreasing its apparent molecular weight. Membrane extracts were normalized by total protein content; equal volumes of Ni-NTA eluates were loaded. The asterisk denotes a protein that cross-reacts with the anti-LptD antibody and serves as a loading control.