(A) Expression of LptDΔD330 confers outer membrane permeability defects, while changes in BamA can suppress LptDΔD330 associated-defects. MC4100 or bamAE470G cells were transformed with plasmids that express WT or mutant lptD alleles. Plating assays were performed on LB supplemented with 50 μg/mL vancomycin. (B) LptDΔD330, like LptD4213, stalls on BamA during assembly. BamAE470G alleviates stalling of LptDΔD330, as judged by a reduction in crosslinking efficiency, but does not alleviate stalling of LptD4213. (C) LptDΔD330 can adopt the mature disulfide bonded state. MC4100 or bamAE470G cells expressing WT or mutant lptD alleles were harvested and analyzed via immunoblotting of cell lysates. When analyzed in the absence of reducing agent, LptD migrates at a molecular weight that reflects its state of assembly. Assembly of LptD involves the conversion of a reduced form to a form containing a disulfide bond between consecutive cysteines (designated [1,2]-LptD) that is then converted to the mature form, which contains disulfide bonds between nonconsecutive cysteines (designated [1,3][2,4]-LptD for the order in which the cysteines appear in the sequence). The [1,3][2,4] disulfide configuration reflects properly folded, functional LptD. (D) LptDΔD330 is slow to mature into the functional disulfide bond configuration, while BamAE470G alleviates LptDΔD330 assembly defects. MC4100 or bamAE470G cells expressing FLAG-tagged LptD(WT/ΔD330) were pulsed with [35S]methionine and chased with cold methionine. Samples were subsequently immunoprecipitated using α-FLAG beads and analyzed by autoradiography. The asterisk below each autoradiograph represents the time point at which approximately 50% of the substrate has converted to the mature form (containing the [1,3][2,4] disulfide bond configuration). Other compensatory mutations that restore LptDΔD330-associated defects are shown in Figure 7—figure supplement 1 and Figure 7—figure supplement 2.