New insights into the β-barrel Assembly Machinery mechanism and its interaction with the stress sensor lipoprotein RcsF in Escherichia coli
(2021)
Files
ThesisRodriguezAlonsoJune2021.pdf
Restricted Access - Adobe PDF
- 35.37 MB
Details
- Authors
- Supervisors
- Collet, Jean-François
- Abstract
- The cell envelope of Gram-negative bacteria is essential for their survival. It represents the first line of defense and a physical barrier between the cell and its surroundings, providing protection against hostile conditions, and allowing the adaptation to changes in the environment. This elaborate structure is composed of an outer membrane (OM) and an inner membrane (IM), separated by a viscous compartment, the periplasmic space. In the periplasm lies the cell wall, a sacculus with a mesh-like structure made of peptidoglycan (PG). The Rcs (Regulator of capsule synthesis) responds to OM and PG damage in enterobacteria. 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 mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF–OMP complexes. Here I report the crystal structure of the key BAM component, BamA, in complex with RcsF. In this complex, BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep inside the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export during conformational cycling of BamA and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity. In addition, we provide insights into the mechanism of BAM: we found that flexibility and proper coordination between the POTRA and β-barrel domains of BamA is important for BAM’s function. Finally, we identified suppressor mutations in the β-barrel domain of BamA that alleviate the toxicity caused by decreasing the flexibility of the hinge region between POTRA and the β-barrel. Altogether, these discoveries contribute to our understanding of the BAM complex at the molecular level, shedding new light on its essential function in the assembly of the bacterial cell envelope.
- Affiliations
Citations
Rodriguez Alonso, R. (2021). New insights into the β-barrel Assembly Machinery mechanism and its interaction with the stress sensor lipoprotein RcsF in Escherichia coli. https://hdl.handle.net/2078.5/108152