Unraveling the outer membrane protein quality control network in Escherichia coli

Abstract

The outer membrane (OM) of Gram-negative bacteria is an essential permeability barrier that prevents the entry of harmful molecules such as antibiotics while permitting the passage of nutrients. This membrane contains lipids and proteins, many of which are ß-barrel outer membrane proteins (OMPs). Prior to their assembly by the ß-barrel assembly machine (Bam), ß-barrels must be maintained in a folding-competent state as they traverse the aqueous periplasmic compartment, where conditions often mirror the cell’s hostile environment.

Many of the factors, such as the chaperone SurA and protease DegP, which contribute to the maintenance of OMP assembly, have been identified. However, due to their essential role in OM integrity, these and other factors comprise a redundant network of quality control that is difficult to unravel. The work in this thesis describes a series of experiments using informed double mutant construction and complementary biochemistry to reveal the functional roles of periplasmic factors.

We present molecular-level results revealing a role for two heretofore-unrecognized domains of the SurA chaperone in the regulation of chaperone function and OMP assembly. We show that the role of SurA in the biogenesis of essential OMPs is complemented by other chaperones, including the holdase Skp. Through the use of well-characterized mutant OMP substrates, we demonstrate that DegP, but also the proteases BepA and YcaL degrade an essential OMP at distinct points of Bam-mediated assembly. Our work presented here provides a more comprehensive understanding of the multifaceted process through which assembly and quality control factors function together with the Bam complex to preserve the integrity of OMP assembly.