Reconstruction of the Gram-Negative Bacterial Outer-Membrane Bilayer

Area of Science:

• Microbiology
• Biophysics
• Biochemistry

Background:

• Gram-negative bacteria possess a unique outer membrane (OM) with an asymmetric lipid composition, featuring lipopolysaccharides (LPS) in the outer leaflet.
• Understanding the OM's structure and function is crucial for developing new antimicrobial strategies.

Purpose of the Study:

• To reconstruct a model of the Escherichia coli outer membrane lipid bilayer (OMLB) using a droplet interface bilayer (DIB) system.
• To investigate the translocation of bacteriocin colicin E9 (colE9) through the porin OmpF embedded in the model OMLB.

Main Methods:

• Reconstruction of the OMLB using the DIB technique, creating a bilayer separating two aqueous droplets in oil.
• Insertion of the trimeric porin OmpF into the model OMLB.
• Monitoring the translocation of colicin E9 (colE9) through OmpF in the OMLB.

Main Results:

• Colicin E9 exhibited multiple failed attempts to engage with OmpF in the OMLB before successful translocation, unlike in LPS-free bilayers.
• The rate of the second step of colE9 translocation was three times slower in the OMLB compared to LPS-free bilayers.
• Colicin E9 dissociated from the OMLB upon reversal of the membrane potential.

Conclusions:

• The DIB approach is effective for creating model OMLBs with physiologically relevant lipids.
• The model OMLB exhibits distinct properties compared to simple lipid bilayers, influenced by LPS.
• The model OMLB serves as a valuable platform for screening antibiotic properties.