Harris D. Bernstein

In 1987, ASM Press published the hefty two-volume book Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (1), which evolved after a second printed edition (2) into an online subscription-based publication called EcoSal and then into the online journal EcoSal Plus (3). The “Plus” refers to the inclusion of the rest of the Enterobacterales order, including Klebsiella, Yersinia, and Shigella. In the latest segment of this 40-year journey, EcoSal Plus has merged with Microbiology and Molecular Biology Reviews (MMBR), where it now continues as a collection within the journal, with a dedicated editorial board (4).

Virtually all integral outer membrane proteins (OMPs) produced by Gram-negative bacteria contain a unique ‘β barrel’ structure that serves as a membrane spanning domain. The universal b arrel a ssembly m achine (BAM) catalyzes OMP assembly (folding and membrane insertion) in vivo, and purified Escherichia coli BAM that is reconstituted into proteoliposomes catalyzes OMP assembly in vitro. Here we show that BAM also catalyzes the assembly of OMPs into outer membrane fractions (‘native OMs’) that are purified by optimized conventional methods. Interestingly, we found that OMP assembly was moderately impaired when native OMs were isolated from a mlaA ^- strain that is deficient in maintaining OM lipid homeostasis but was strongly reduced when native OMs were isolated from a pldA ^- strain that is deficient in a parallel pathway. We also found that the mlaA and pldA deletions altered the OM phospholipid profile to different degrees that correlated with the degree to which the mutations impaired OMP assembly. Taken together, our results provide direct evidence that the mla and pldA pathways play distinct roles in maintaining OM homeostasis and strongly suggest that OM phospholipids play a more significant role in OMP biogenesis than previously appreciated.

Outer membrane proteins (OMPs) produced by Gram-negative bacteria contain a cylindrical amphipathic β-sheet (“β-barrel”) that functions as a membrane spanning domain. The assembly (folding and membrane insertion) of OMPs is mediated by the heterooligomeric β-barrel assembly machine (BAM). The central BAM subunit (BamA) is an attractive antibacterial target because its structure and cell surface localization are conserved, it catalyzes an essential reaction, and potent bactericidal compounds that inhibit its activity have been described. Here we utilize mRNA display to discover cyclic peptides that bind to Escherichia coli BamA with high affinity. We describe three peptides that arrest the growth of BAM deficient E. coli strains, inhibit OMP assembly in live cells and in vitro, and bind to unique sites within the BamA β-barrel lumen. Remarkably, we find that if the peptides are added to cultures after a slowly assembling OMP mutant binds to BamA, they accelerate its biogenesis. The data strongly suggest that the peptides trap BamA in conformations that block the initiation of OMP assembly but favor a later assembly step. Molecular dynamics simulations provide further evidence that the peptides bind stably to BamA and function by a previously undescribed mechanism.

Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called the b arrel a ssembly m achine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of the Escherichia coli OmpC protein into the OM in vivo . We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction. IMPORTANCE: The assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called the b arrel a ssembly m achine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues in Escherichia coli OmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane.

The translocation and assembly module (TAM) has been proposed to play a crucial role in the assembly of a small subset of outer membrane proteins (OMPs) in Proteobacteria based on experiments conducted in vivo using tamA and tamB mutant strains and in vitro using biophysical methods. TAM consists of an OMP (TamA) and a periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). Here we examine the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. We find that TAM catalyzes the assembly of four model OMPs nearly as well as the β-barrel assembly machine (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our study provides direct evidence that TAM can function as an independent OMP insertase and describes a new method to gain insights into TAM function.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a β-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the β-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

The bacterial translocation a ssembly m odule (TAM) contains an outer membrane protein (OMP) (TamA) and an elongated periplasmic protein that is anchored to the inner membrane by a single α helix (TamB). TAM has been proposed to play a critical role in the assembly of a small subset of OMPs produced by Proteobacteria based on experiments conducted in vivo using tamA and/or tamB deletion or mutant strains and in vitro using biophysical methods. Recent genetic experiments, however, have strongly suggested that TAM promotes phospholipid homeostasis. To test the idea that TAM catalyzes OMP assembly directly, we examined the function of the purified E. coli complex in vitro after reconstituting it into proteoliposomes. Remarkably, we find that TAM catalyzes the assembly of four model OMPs nearly as well as the β- b arrel a ssembly m achinery (BAM), a universal heterooligomer that contains a TamA homolog (BamA) and that catalyzes the assembly of almost all E. coli OMPs. Consistent with previous results, both TamA and TamB are required for significant TAM activity. Our results provide strong evidence that although their peripheral subunits are unrelated, both BAM and TAM function as independent OMP insertases. Furthermore, our study describes a new method to gain insights into TAM function.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded β-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal β-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring β-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded β-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal β-barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring β-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

There is a clear need to develop novel broad-spectrum antibiotics to address the global problem of antimicrobial resistance. Multiple compounds that have strong antibacterial activity have recently been described that appear to inhibit the activity of the b arrel a ssembly m achinery (BAM), an essential complex that catalyzes the assembly (i.e., folding and membrane insertion) of outer membrane proteins (OMPs) in all Gram-negative bacteria.