Fig. 3. LptM promotes LptD oxidative maturation.

a Schematic representation of mature LptD primary sequence indicating the position and oxidation state of Cys residues in LptD^Red (top, left), LptD^C1-C2 (middle, left) or fully oxidized LptD^Ox (bottom, left). A typical migration pattern corresponding to different oxidative states of LptD by non-reducing SDS-PAGE is represented on the right. b The total protein contents of wild-type (lptM⁺) and ΔlptM strains transformed with an empty vector pCtrl, and the complementation strain ΔlptM transformed with pLptM^His were separated by non-reducing SDS-PAGE and analyzed by Western blotting using the indicated antisera. **Indicates a non-identified protein band. Data are representative of three independent experiments. c The total protein contents of wild-type and the indicated mutant strains were separated by reducing or non-reducing SDS-PAGE and analyzed by Western blotting as in (b). Red, empty arrowheads indicate reduced LptD (LptD^Red), whereas filled, gray arrowheads indicate LptD^C1-C2. **Indicates a non-identified protein band that migrates slightly faster than LptD^Red. Data are representative of three independent experiments. d Purification of LptE^His from ΔlptM cells that overproduced LptDE^His (lane 1) or from wild-type cells that overproduced LptDME^His (lane 2) or ΔlptM cells that overproduced the indicated LptD mutant variant of LptDE^His (3-5). *Refers to the LptD Cys-to-Ser variant: wild-type LptD^CCCC, LptD^CCSS, LptD^CSCS or LptD^SCSC, as specified on the top of each gel lane. Data are representative of three independent experiments. The signals of any LptD forms in lanes 1 and 2 were quantified. The amount of intermediate forms (LptD^Red + LptD^C1-C2 + LptD^C2-C4) was normalized to the overall amount of all LptD forms (LptD^Red + LptD^C1-C2 + LptD^C2-C4 + LptD^ox). Data are represented as means ± s.e.m. (n = 3 independent experiments). Source data are provided as Source Data file. e LptDE^His containing either wild-type (LptD^CCCC) or LptD^CCSS co-overproduced together with LptM or alone in wild-type or ΔlptM cells were Ni-affinity purified and resolved by BN-PAGE, prior to Coomassie Brilliant Blue staining. *Indicates the plasmid-encoded LptD variant, wild-type LptD^CCCC, LptD^CCSS, as specified on the top of each gel lane. Data are representative of three independent experiments.