Regulation of the Beta-barrel Assembly Machine complex in Escherichia coli

Abstract

The outer membrane of Escherichia coli functions as a selectively permeable barrier between the bacterium and its external environment, allowing for the influx of ions and nutrients and efflux of waste while preventing the entry of harmful small molecules and toxins. The outer membrane is composed of an asymmetric lipid bilayer, with phospholipids in the inner leaflet and lipopolysaccharide in the outer leaflet, as well as membrane associated lipoproteins and integral -barrel proteins that serve as channels and pores through the membrane. The assembly of the outer membrane is a complex process that must be tightly regulated to maintain the barrier function during cell growth.

This work focuses on the assembly of -barrel outer membrane proteins (OMPs) by the -barrel assembly machine (Bam) complex. The Bam complex assembles diverse OMP substrates in the absence of an obvious energy source and without interrupting the integrity of outer membrane lipid bilayer, but the mechanism is still unknown. By using mutants of essential proteins BamA and BamD, we sought to understand the role that interaction and communication between these two proteins plays in OMP assembly. We identified mutants that bypass the need for this interaction as well as mutants that do not interact productively, and we found that the interaction between these two proteins is important for coordinating the conformational changes in both proteins necessary for OMP assembly, thus allowing for activation of both proteins during the assembly process.

Additionally, we sought to characterize these conformational changes in BamA. We used genetic and biochemical analysis to characterize mutations that lock BamA in a variety of functionally distinct conformations and tested their ability to assemble both wildtype and assembly-defective OMP substrates. Mutations that lock BamA in an active conformation can assemble defective substrates but also cause dominant membrane permeability defects, suggesting that regulation of BamA conformation is necessary for maintaining the integrity of the permeability barrier during outer membrane biogenesis.

Taken together, this work provides additional insight into the mechanistic details of the OMP assembly process and the need for regulation of the assembly process to maintain membrane integrity.