Figure 2: The autotransporter passenger secretion channel is located within the BamA lumen.

(A) E. coli BL21(DE3) expressing ^HisBamA_N427CBCDE and ^MBP−76EspP with cysteine substitutions at the indicated residues were mock treated (−) or treated with 4-DPS (+). Intermolecular disulfide-bonds (•) between cysteine pairs were detected by double-immunoblotting with antibodies against the N-terminus of ^MBP−76EspP (αStrepII, top) or the C-terminus of BamA (αBamA_C, bottom). Right, example of detected ^HisBamA_N427C•^MBP−76EspP_T984C magnified from boxed region of immunoblot (and αStrepII (cyan) and αBamA_C (red) signals overlaid). Asterisk: non-specific side-reactions. (B) Disulfide-bond levels (mean ± SEM) between ^MBP−76EspP and ^HisBamA for all cysteine substitutions defined in Figure 1. Experiments (N=3) conducted as in A except that immunoblots were probed with αStrepII only. Statistical tests are in Table S1. (C) Model of the autotransporter secretion channel based on disulfide-mapping. ‘1’ and ‘2’ portray the dynamicity of the passenger (orange) as it traverses the OM through the luminal space within the BamA-EspP hybrid-barrel. Some passenger residue positions used in A and B are depicted (orange/blue circles). EspP α-helix residue N1026 (purple circle) is also shown. BamA β1 (red), β2 (black), and β3 (green) are shown with luminal residues N427 (red circle), G443 (gray circle), and G457 (green circle), respectively. MBP prevents ^MBP−76EspP residues close to S953 from entry into the channel. (D) Surface-view cross-section of C. BamA β16 residues N805 (tan circle), I806 (yellow circle), and T809 (white circle), and EspP β1 (cyan square) are shown.