Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

Abstract

The biogenesis of the outer membrane (OM) of Escherichia coli is a conserved and vital process. The assembly of integral ␤-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric ␤-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, lowabundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations in bamD that lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wildtype BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM. IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral ␤-barrel proteins (OMPs) are assembled by the ␤-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins. The assembly of integral ␤-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria is essential for cell growth and viability. OMP targeting and OM integration depend on an assembly pathway comprising semiredundant periplasmic chaperones and the heteropentameric OM-associated ␤-barrel assembly machine (Bam). The primary constituents of the periplasmic chaperone network in Escherichia coli are the proline isomerases SurA and FkpA, the bifunctional protease/chaperone DegP, and the prefoldin-like chaperone Skp. The Bam complex is composed of BamA (itself a ␤-barrel) and four associated lipoproteins, BamB, BamC, BamD, and BamE (1-3).Although OMPs require the Bam complex for folding into the OM, the individ...

Mitochondrial-bacterial hybrids of BamA/Tob55 suggest variable requirements for the membrane integration of β-barrel proteins

“…This assumption is supported by a recent report describing a ternary complex formed by BamA (mainly via POTRA domain 2), SurA, and β-barrel nascent protein35. Our observations are also in line with previous works suggesting that the individual or combinatorial deletion of periplasmic chaperones or non-essential Bam components differentially affect the assembly of various groups of β-barrel proteins, proposing that there are classes of substrates with variable dependencies for efficient assembly363738.…”

“…In sharp contrast, OmpA was detected in these cells in slightly elevated amounts. These findings complement very recent observations where lower levels of Bam components resulted in differential effect on the levels of various OM proteins38. Of note, Mahoney et al .…”

“…These observations are in line with recent findings of Mahoney et al . in E. coli cells where in contrast to other bacterial β-barrel proteins, OmpA was not directly affected by strong depletion of BamA and BamD38. Altogether, these observations, suggest that OmpA can be efficiently integrated into the membrane even by low levels of a functional insertion complex and without major help from chaperones and POTRA domains.…”

Outer Membrane Biogenesis

“…Several lines of evidence suggest that although non-essential, these lipoproteins may be required for assembly of specific substrates. For example, assembly of large, difficult substrates (such as LptD, TolC, autotransporters) is not affected by single mutations in non-essential components, (10; 58; 78). In contrast, deletion of bamB strongly affects high volume trimeric substrates, such as porins and the maltodextrin transporter LamB (10).…”

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

“…Molecular dynamics simulations (20, 21) and other studies (22) that suggest that the BamA β-barrel causes thinning of the adjacent membrane have led to an alternative model in which the Bam complex catalyzes OMP assembly by creating a local deformation of the lipid bilayer. To further complicate matters, the finding that the absence of non-essential Bam complex subunits or reductions in the level of BamA or BamD differentially affects the assembly of individual OMPs (23,24) raises the possibility that there are multiple parallel folding pathways that rely on the Bam complex components and periplasmic chaperones in different ways.…”