Dynamic Association of BAM Complex Modules Includes Surface Exposure of the Lipoprotein BamC

Webb, Chaille T; Selkrig, Joel; Perry, Andrew; Noinaj, Nicholas; Buchanan, Susan K.; Lithgow, Trevor

doi:10.1016/j.jmb.2012.05.035

Cited by 81 publications

(67 citation statements)

References 43 publications

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“…In addition, at each time-point, an aliquot was treated with proteinase K to digest the population of molecules that were surface exposed: preliminary experiments were done to titrate the amount of proteinase K in use, so as to be sufficient to cleave the periplasmic protein SurA and the lipoprotein BamD that are exposed to the periplasm, but only when polymyxin B was also present (Fig. 3c); polymyxin B is an antibiotic that selectively permeabilizes the outer membrane 36,37 .…”

Section: Resultsmentioning

confidence: 99%

“…The first aliquot was added to a tube containing a 10% (w/v) final concentration of trichloroacetic acid (TCA) and placed on ice. The second aliquot was treated with 200 mg ml À 1 proteinase K for 20 min on ice to digest Pet exposed on the cell surface and the third aliquot was treated with 200 mg ml À 1 polymyxin B for 10 min on ice as previously described 36,37 before proteinase K treatment. Phenylmethanesulfonyl fluoride (PMSF) at 2 mM final concentration was used to stop the protease reaction before TCA precipitation.…”

Section: Methodsmentioning

confidence: 99%

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A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

et al. 2014

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Bacterial autotransporters comprise a 12-stranded membrane-embedded b-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring b-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

et al. 2014

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“…It should also be noted that when we treated proteoliposomes with trypsin after performing an assembly reaction we did not observe a protected fragment of BamC that might result from the transfer of the lipoprotein across the lipid bilayer during activation of the Bam complex (Fig. S2C, "post-assay") (52).…”

Section: The Bam Complex Catalyzes Efficient Folding Of Espp Into a Wmentioning

confidence: 89%

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

Hussain

Bernstein

2018

Journal of Biological Chemistry

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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

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“…Four lipoproteins (BamB-E) are part of the Bam complex, of which BamD is essential for the assembly and/or insertion of proteins in the OM (Malinverni et al, 2006). BamC is surface exposed (Webb et al, 2012). How the lipid moiety is orientated in the OM is still an open question.…”

Section: Lipoproteins Involved In Key Processes Of Envelope Biosynthesismentioning

confidence: 99%

The molecular mechanism of bacterial lipoprotein modification—How, when and why?

Buddelmeijer¹

2015

FEMS Microbiology Reviews

110

117

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One-sentence summary: This review highlights recent findings on the mechanism by which proteins are lipidated and discusses the enzymes involved, substrate specificities among bacterial species and the role of lipoprotein modification in the biogenesis of the cell envelope. Editor: Mecky Pohlschroder ABSTRACTPosttranslational modification of proteins by lipidation is a common process in biological systems. Lipids provide protein stability, interaction with other membrane components, and in some cases, due to reversibility of the process, a mechanism for regulating protein localization and function. Bacterial lipoproteins possess fatty acids at their amino-termini that are derived from phospholipids, and this lipid moiety anchors the proteins into the membrane. These lipids, as is the case for lipopolysaccharides and lipoteichoic acids, play an important role in signaling of the innate immune system through the interaction with Toll-like receptors. Over the past three years, tremendous progress has been made in understanding the mechanism by which lipoproteins become lipidated. Advanced methodology in mass spectrometry, proteomics and genome-wide analyses allowed precise characterization of lipoprotein modifications and the identification of the enzymes catalyzing the reactions in diverse bacterial species. This review will highlight new findings on bacterial lipoprotein modification with focus on the reaction mechanisms and the role of lipoproteins in cell envelope homeostasis.

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Dynamic Association of BAM Complex Modules Includes Surface Exposure of the Lipoprotein BamC

Cited by 81 publications

References 43 publications

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

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

The molecular mechanism of bacterial lipoprotein modification—How, when and why?

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