It's a trap : new findings into the role of outer membrane proteins in the Rcs stress response system of Escherichia coli

The cell envelope of Gram-negative bacteria is essential for their survival. It serves as their initial line of defense as well as a physical barrier between the cell and its surrounding environment. Furthermore, the cell envelope protects bacteria by allowing them to respond to changes in their environment. This complex structure comprises an outer membrane (OM) and an inner membrane (IM) separated by a viscous compartment, the periplasmic space. The cell wall, a sacculus with a mesh-like structure comprised of peptidoglycan, is found in the periplasm (PG). The Rcs (Regulator of capsule synthesis) system responds to OM and PG damage in Enterobacteriaceae. The OM lipoprotein RcsF senses stress and activates the signaling cascade. The β-Barrel Assembly Machinery (BAM) inserts OM β-barrel proteins (OMPs) in the OM of Gram-negative bacteria. BAM also most likely exports RcsF to the cell surface, probably by forming RcsF-OMP complexes. The main objective of my thesis project was to investigate the relationship between RcsF and its OMP partners. Hereby by dissecting the protein-protein interaction interface (PPI) between OmpA, an abundant E. coli OMP, and RcsF, I identified an unexpected role of OmpA in the Rcs stress response system. In this complex, OmpA, folded as a two-domain protein, interacts via its periplasmic domain with the globular domain of RcsF. This mode of interaction between OmpA and RcsF does not allow OmpA to be the vehicle for the surface exposure of RcsF. Instead, my results suggest that OmpA plays the role of a periplasmic buffer for the Rcs system: The periplasmic domain of OmpA, when interacting with RcsF, prevents the interaction of RcsF with IgaA, the Rcs repressor. To identify the PPI of the OmpA-RcsF complex, I used a site-specific photo-crosslinking method based on non-natural photoactivable amino acids. By doing so, my work identified a flaw in this considered foolproof method, which I investigated to raise the attention of scientists working on protein-protein interaction or protein complexes on the probable existence of artifacts.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202