The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

Abstract

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "b-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires nonphysiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the b-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an a-helix embedded inside the EspP b-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. __________________________ Gram-negative bacteria, a class that includes many pathogenic and emerging antibiotic-resistant organisms, are bound by a double cell membrane. Most of the proteins that reside in the outer membrane (OM) 2 , which serves as a robust barrier, have a distinctive "b-barrel" architecture. A bbarrel is essentially a b-sheet rolled into a closed cylinder that has a hydrophobic exterior and a hydrophilic interior and that is held together by hydrogen bonds between the first and last bstrands. The b-barrel scaffold is found exclusively in the OM of bacteria and organelles of bacterial origin. OM proteins (OMPs) mediate many different functions vital for bacterial survival and range considerably in size from 8-26 b-strands (1, 2). Some OMPs form oligomers (3), while others contain a segment that is embedded inside the bbarrel or a separately folded domain that is displayed on either the periplasmic or extracellular side of the OM (1).OMPs must first traverse the inner membrane (IM) and the periplasmic space, which is devoid of ATP (4), before they attain their final structure in the OM. After OMPs are transported across the IM in an unfolded conformation through the Sec translocon, a variety of periplasmic chaperones including SurA, Skp, and DegP prevent their aggregation in the periplasm (5-7). Integration into the OM is then catalyzed by the heterooligomeric β-barrel assembly machinery (Bam) complex (8,9). In E. coli, the Bam complex is composed of BamA, a b-barrel protein that contains five http://www.jbc.org/cgi

Cracking Open Bacterial Membrane Vesicles

“…The authors suggested that DegP can control the protein composition of OMVs by acting as a chaperone for certain proteins. Although the determinants of protein composition in bacterial MVs are still largely unknown, it is possible that the Bam complex, which catalyzes the assembly of outer membrane proteins, may, in part, determine protein cargo selection (Bonnington and Kuehn, 2014;Hussain and Bernstein, 2018). Haurat et al (2011) reported that anionic LPS (A-LPS) plays a critical role in OMV protein cargo selection in Porphyromonas gingivalis.…”

Proteins in the periplasmic space and outer membrane vesicles ofRhizobium etliCE3 grown in minimal medium are largely distinct and change with growth phase

“…BamD, the outer membrane protein assembly factor that forms part of the Bam complex, was also present in OMVs. Although the reconstituted E. coli Bam complex is able to insert proteins into artificial membrane vesicles (63), it is not known whether Bam complex components can do the same in natural OMVs. Porin RHE_RS04090 also has a sequence indicative of an OM beta-barrel protein.…”

Isolate Specific Cold Response of Yersinia enterocolitica in Transcriptional, Proteomic, and Membrane Physiological Changes

“…To investigate the regulatory factors of membrane fluidity in Y. enterocolitica, groups of genes regarding outer membrane proteins and lipid A biosynthesis were selected according to previous studies (Dekker, 2000;Nikaido, 2003;Barria et al, 2013;Hussain and Bernstein, 2018;Robinson, 2019). Transcriptional changes in these genes were investigated under cold stress for 2 h in three isolates with the primers listed in Supplementary Table S3.…”