Supplementary Figure 8: TamA/BamA structural homology and TamB production of mature/functional Ag43 on the cell surface.

(a) Crystal structures of the insertase domains of BamA (pdb entry 4C4V, cyan) and TamA (pdb entry 4C00, green) solved from E. coli. The extracellular loop 6 is drawn in magenta, and the conserved VRGY motif required for the function of the translocases from Omp85 family is shown in stick representation. BamA and TamA belong to the Omp85 family and share a similar fold mechanism. Their β-barrel domains reveal an unusual cracked-barrel architecture created by the lack of H-bonds between strands 1 and 16 that are loosely associated to create access from the barrel lumen to the hydrophobic lipid phase. As well, their cracked-barrel architecture is suggested to promote a lateral opening of the β-barrel to insert OMP within the membrane (Noinaj et al., Nature, 501, 385-390, 2013). (b) (i) Transmission-electron micrograph showing immunogold-labelling of His-tagged Ag43 autotransporters on the surface (dots are demarcated in arrows; zoom-in of the dot shown in top right panel) of wild-type (WT), ΔtamA and ΔtamB E. coli cells previously transformed with an empty or Ag43 expressing plasmids; scale bar, 100 nm. (ii) Full electron micrographs of the indicated strains observed at a magnification of 40,000 x 1.5; scale bar, 300 nm. (c, d) Release of α-Ag43 fragment from the bacterial cell surface under thermal denaturation. The supernatant preparations from heat-treated bacterium (incubated at 25 or 55 °C for 5 min) were loaded onto SDS-PAGE (c) and analyzed using MALDI-TOF mass spectrometry (observed masses listed in d; n.d. for not detected). (e) Mass determination of the heat-released Ag43 passenger domain from the bacterial surface. Supernatant fractions following a 5 min incubation at 55 °C of WT (top and middle) and ΔtamB (bottom) E. coli cells analyzed using MALDI-TOF to detect Ag43 α-domain release from the bacterial cell surface under thermal denaturation.