fig4

Figure 4. The Bam complex in OMVs is properly assembled, with BamA surface exposed. (A) ProtK treatment of Bam-containing OMVs. The cartoon depicts the rationale of the experiment: OMVs with the BamA subunit of the BAM complex inserted into the OMV membrane, with its extracellular loops exposed. ProtK (orange shapes) is externally added, making these extracellular loops accessible to proteolytic cleavage. The panel on the right shows the relevant part of a CBB-stained gel of OMVs from BL21(DE3) omp8 cells containing BAM, either treated with ProtK or left untreated as a control. The asterisk () indicates a BamA band resulting from ProtK degradation. Lane 3 contains OMVs treated with Triton X-100 and ProtK, demonstrating the sensitivity of proteins, including BAM subunits, when the OMVs are solubilized. The expected positions of the BAM subunits are indicated on the right; (B) Site-specific labeling of BamA with an engineered cysteine. A cysteine was engineered between Gln753-Tyr754 of BamA, localized in an extracellular loop, allowing labeling on intact OMVs. The cartoon depicts the experiment design: OMVs carrying BAM with the engineered BamA^cys are first incubated with a MBP linker that binds the exposed cysteine residue. As a control, OMVs containing BAM with non-mutated BamA are used. The samples are analyzed by SDS-PAGE and Western blotting, with detection using a Strep-HRP. The panel shows the Ponceau stain of the blot (left) and a merged luminescence and bright-light image (right). Lane 2 contains the Precision Plus Protein^TM prestained marker (Bio-Rad), which is visible on the right panel due to the merged images. The expected position of BamA is indicated on the right; (C) Pull-down of BAM using His-tagged BamE. The BAM complex encoded by pJH114 includes a His-tagged BamE [BamE(His)], enabling pull-down using TALON resin. The blot shows samples from this pull-down experiment using OMVs solubilized with DDM. The lanes contain the FT, W, and two elution fractions (E1 and E2). The expected positions of the BAM subunits are depicted on the right. OMV: Outer membrane vesicle; BAM: β-barrel assembly machinery; ProtK: proteinase K; CBB: Coomassie brilliant blue; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Triton X-100: a non-ionic surfactant; MBP: Maleimide-PEG-Biotin; Strep-HRP: Streptavidin-HRP conjugate; DDM: n-dodecyl-β-D-maltoside; FT: flow-through; W: wash; E1/E2: elution fractions 1 and 2.