Disruption of the tethering between the outer membrane and the peptidoglycan impairs peptidoglycan maintenance and osmotolerance in Escherichia coli

Details

Supervisors

Collet, Jean-François ; Dupont-Gillain, Christine C.

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

The cell envelope of Escherichia coli, a Gram-negative bacterium, is a three-layered structure composed of an inner membrane, a peptidoglycan (PG) layer, and an outer membrane (OM). The OM is tethered to the PG through both covalent and non-covalent interactions. While the PG layer has traditionally been considered the sole load-bearing element constraining internal turgor pressure, recent studies have demonstrated that the OM, and more recently, its physical connection to the PG, also plays a mechanical role in maintaining envelope integrity, especially under osmotic stress. Building on evidence that OM-PG tethering is essential to withstand hypoosmotic shocks, this master thesis investigates whether these structural connections are also required during long-term adaptation to hyperosmotic environments. Using E. coli strains with disrupted OM-PG interactions, we observed impaired growth and survival under high salt concentrations, both on NaCl gradient plates and in liquid cultures. These phenotypes were associated with compromised PG synthesis, potentially due to impaired activation of the bifunctional synthases PBP1A and PBP1B. We propose that altered membrane organization and increased outer membrane vesiculation due to OM-PG detachment may disrupt the spatial interaction between these synthases and their OM-anchored lipoprotein activators. Whole-genome analysis of suppressor mutants recovering growth under high salt conditions revealed compensatory mutations in genes associated with PG biosynthesis, notably mrcB (coding PBP1B) and mraY, highlighting the central role of PG integrity in osmotolerance. Furthermore, we observed consistent activation of the regulator capsule synthesis (Rcs) phosphorelay, a stress-responsive system, in OM-PG disconnected mutants under hyperosmotic conditions. Although this response conferred only modest improvements in survival, our preliminary results suggest it may sense and partially mitigate structural defects by activating envelope stress adaptation pathways. Overall, our results highlight the physiological importance of OM-PG tethering for E. coli tolerance to hyperosmotic stresses and provide new insights into envelope-centered survival strategies.