fig3

Figure 3. OMVs contain fully folded β-barrel structures of BamA and trimeric PhoE. (A) Relevant parts of CBB-stained semi-native SDS-PAGE gels showing native (N) and denatured (D) samples of OMVs and CEs from BL21(DE3) WT and BL21(DE3) omp8 cells containing either EV or BAM, induced with 100 µM IPTG (+) or not induced (-). The OD₆₀₀ equivalents of the cultures loaded are indicated. Closed and open arrowheads mark the positions of folded () and unfolded () BamA, respectively. The positions of BamB, BamC, and BamD are indicated on the right. The open asterisk denotes the position of unfolded OmpF () present in the WT samples; (B) Coomassie-stained semi-native gels showing native (N) and denatured (D) samples of OMVs from BL21(DE3) omp8 cells expressing PhoE induced with 100 µM IPTG. The positions of monomeric denatured PhoE (mon-PhoE) and native trimeric PhoE (tri-PhoE) are indicated; the trimer runs as a smear due to its association with LPS; (C) Native and denatured samples from CEs and OMVs obtained from BL21(DE3) omp8 cells expressing BAM induced with 100 or 500 µM IPTG. Shown are relevant parts of two gels run in parallel (one for CEs and one for OMVs). The positions of folded () and unfolded () BamA are indicated. The position of unfolded BamA in native samples is indicated by a closed asterisk (). OMV: Outer membrane vesicle; BAM: β-barrel assembly machinery; PhoE: outer membrane porin PhoE; CBB: Coomassie brilliant blue; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WT: wild type; EV: empty vector; IPTG: isopropyl β-D-1-thiogalactopyranoside; OD600: optical density at 600 nm; OmpF: outer membrane porin F; LPS: lipopolysaccharide; CEs: cell envelopes.