A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

beta-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent beta-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent beta-barrel OMPs largely via its N-domain but with beta-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent beta-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving beta-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for beta-barrel OMP biogenesis.