Assembly of multimeric outer membrane proteins in Escherichia coli
thesisposted on 03.03.2017, 01:24 by Dunstan, Rhys Alexander
Gram-negative bacterial outer membranes incorporate proteins of at least three well characterized architectures: β-barrel proteins, lipoproteins and multimeric secretion pores. Assembly of β-barrel proteins into the outer membrane is mediated by the β-barrel assembly machinery (BAM), consisting of an essential core β-barrel BamA and four accessory lipoproteins (BamBCDE). Lipoproteins are targeted and subsequently anchored to the outer membrane by covalently attached lipid modifications by the localization of lipoproteins (Lol) machinery. Secretins are amongst the most characterized multimeric outer membrane proteins, and often rely on a small lipoprotein, termed pilotin, to catalyze the efficient translocation of secretin monomers to the outer membrane for assembly into a functional secretion pore. Through the use of cryo-EM and X-ray crystallography it has become clear that oligomeric outer membrane proteins can adopt either α-helical or β-stranded transmembrane conformations, yet their mechanism of assembly remains unclear. Chapter two describes the identification and characterisation of a novel pilotin AspS for the secretin GspD from the type 2 secretion system of enteropathogenic Escherichia coli. Sucrose density fractionation and novel time course assays show that AspS is required for the targeting of GspD to the outer membrane and for the efficient assembly of GspD multimers. The structure of AspS was solved, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to pilotins from other secretion syetems. Chapter three describes the optimization of a bamA depletion strain of E. coli to monitor outer membrane protein assembly. Assays were performed to analyse bacterial viability, morphology and functionality of the BAM after bamA depletion. Chapter four focusses on analysing the role of BamA in assembling secretion channels GspD, Wza and CsgG, using the depletion regime described in Chapter three. Assembly assays demonstrate that GspD, Wza and CsgG can assemble into the outer membrane in the absence of BamA. These assays also showed that periplasmic proteins CsgE and CsgF could enhance the assembly efficiency of both the mature CsgG multimer and a possible CsgG assembly intermediate.