Figure 7. An assembly-defective LptD mutant is rescued by a compensatory mutation in the BamA interior wall.

(A) Expression of LptD^ΔD330 confers outer membrane permeability defects, while changes in BamA can suppress LptD^ΔD330 associated-defects. MC4100 or bamA^E470G 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) LptD^ΔD330, like LptD4213, stalls on BamA during assembly. BamA^E470G alleviates stalling of LptD^ΔD330, as judged by a reduction in crosslinking efficiency, but does not alleviate stalling of LptD4213. (C) LptD^ΔD330 can adopt the mature disulfide bonded state. MC4100 or bamA^E470G 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) LptD^ΔD330 is slow to mature into the functional disulfide bond configuration, while BamA^E470G alleviates LptD^ΔD330 assembly defects. MC4100 or bamA^E470G 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 LptD^ΔD330-associated defects are shown in Figure 7—figure supplement 1 and Figure 7—figure supplement 2.

Figure 7—figure supplement 1. LptD^ΔD330 partially phenocopies LptD4213-associated assembly defects.

(A) Residue D330 in LptD resides in β-strand seven, and its side chain is oriented into the β-barrel interior. The first and last β-strands are colored in tan, the region deleted in LptD4213 is colored in pink, and LptE is colored in green. (B) LptD^ΔD330 confers outer membrane permeability to vancomycin even in the presence of a functional copy of LptD (+0.2% arabinose). A compensatory intragenic mutation, N274I, restores permeability defects associated with both LptD4213 and LptD^ΔD330.

Figure 7—figure supplement 2. Compensatory mutations in BamA can rescue outer membrane permeability defects associated with stalled LptD substrates.

Changes in BamA can suppress LptD^ΔD330-associated defects. A BamA depletion strain was transformed with a plasmid encoding a BamA variant (including bamA^E470G) and another plasmid encoding either WT or mutant lptD alleles. Plating assays were performed in the presence of arabinose (i.e., with expression of both the wild-type chromosomal copy of BamA and the plasmid-borne copy of BamA) or in the absence of glucose (i.e., without expression of the wild-type chromosomal copy of BamA, but with expression of the plasmid-borne copy of BamA). Strains were plated on LB supplemented with 20 μg/mL vancomycin.