scholarly journals Breaking the permeability barrier ofEscherichia coliby controlled hyperporination of the outer membrane.

2016 ◽  
pp. AAC.01882-16 ◽  
Author(s):  
Ganesh Krishnamoorthy ◽  
David Wolloscheck ◽  
Jon W. Weeks ◽  
Cameron Croft ◽  
Valentin V. Rybenkov ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Antibacterial Agents ◽  
Efflux Transporters ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Active Efflux ◽  
Efflux Inhibition ◽  
Membrane Barrier ◽  
Inhibitory Activities ◽  
Synergistic Relationship

In Gram-negative bacteria, a synergistic relationship between slow passive uptake of antibiotics across the outer membrane and active efflux transporters creates a permeability barrier, which efficiently reduces effective concentrations of antibiotics in cells and hence, their activities. To analyze the relative contributions of the active efflux and the passive barrier in activities of antibiotics, we constructedEscherichia colistrains with controllable permeability of the outer membrane. The strains express a large pore that does not discriminate compounds based on their hydrophilicity and sensitizes cells to a variety of antibacterial agents. We found that efficacies of antibiotics in these strains are specifically affected by either active efflux, slow uptake, or both, and reflect differences in the properties of the outer membrane barrier, repertoire of efflux pumps and inhibitory activities of antibiotics. Our results identify antibiotics which are the best candidates for potentiation of activities through efflux inhibition and permeabilization of the outer membrane.

scholarly journals Synergy between Active Efflux and Outer Membrane Diffusion Defines Rules of Antibiotic Permeation into Gram-Negative Bacteria

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Ganesh Krishnamoorthy ◽  
Inga V. Leus ◽  
Jon W. Weeks ◽  
David Wolloscheck ◽  
Valentin V. Rybenkov ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Efflux Pumps ◽  
Antibacterial Activities ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux ◽  
Membrane Barrier ◽  
Biological Determinants ◽  
Resistant Strains

ABSTRACT Gram-negative bacteria are notoriously resistant to antibiotics, but the extent of the resistance varies broadly between species. We report that in significant human pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Burkholderia spp., the differences in antibiotic resistance are largely defined by their penetration into the cell. For all tested antibiotics, the intracellular penetration was determined by a synergistic relationship between active efflux and the permeability barrier. We found that the outer membrane (OM) and efflux pumps select compounds on the basis of distinct properties and together universally protect bacteria from structurally diverse antibiotics. On the basis of their interactions with the permeability barriers, antibiotics can be divided into four clusters that occupy defined physicochemical spaces. Our results suggest that rules of intracellular penetration are intrinsic to these clusters. The identified specificities in the permeability barriers should help in the designing of successful therapeutic strategies against antibiotic-resistant pathogens. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery. IMPORTANCE Multidrug-resistant strains of Gram-negative pathogens rapidly spread in clinics. Significant efforts worldwide are currently directed to finding the rules of permeation of antibiotics across two membrane envelopes of these bacteria. This study created the tools for analysis of and identified the major differences in antibacterial activities that distinguish the permeability barriers of P. aeruginosa, A. baumannii, Burkholderia thailandensis, and B. cepacia. We conclude that synergy between active efflux and the outer membrane barrier universally protects Gram-negative bacteria from antibiotics. We also found that the diversity of antibiotics affected by active efflux and outer membrane barriers is broader than previously thought and that antibiotics cluster according to specific biological determinants such as the requirement of specific porins in the OM, targeting of the OM, or specific recognition by efflux pumps. No universal rules of antibiotic permeation into Gram-negative bacteria apparently exist. Our results suggest that antibiotic clusters are defined by specific rules of permeation and that further studies could lead to their discovery.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals Trans-envelope multidrug efflux pumps of Gram-negative bacteria and their synergism with the outer membrane barrier

2018 ◽  
Vol 169 (7-8) ◽  
pp. 351-356 ◽  
Author(s):  
Helen I. Zgurskaya ◽  
Valentin V. Rybenkov ◽  
Ganesh Krishnamoorthy ◽  
Inga V. Leus
Keyword(s):  
Outer Membrane ◽  
Efflux Pumps ◽  
Gram Negative Bacteria ◽  
Multidrug Efflux ◽  
Gram Negative ◽  
Multidrug Efflux Pumps ◽  
Membrane Barrier

scholarly journals Outer Membrane Lipid Secretion and the Innate Immune Response to Gram-Negative Bacteria

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Nicole P. Giordano ◽  
Melina B. Cian ◽  
Zachary D. Dalebroux
Keyword(s):  
Outer Membrane ◽  
Mammalian Cells ◽  
Lipid A ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Host Immunity ◽  
Host Recognition ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative

ABSTRACT The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that consists of inner leaflet phospholipids and outer leaflet lipopolysaccharides (LPS). The asymmetric character and unique biochemistry of LPS molecules contribute to the OM’s ability to function as a molecular permeability barrier that protects the bacterium against hazards in the environment. Assembly and regulation of the OM have been extensively studied for understanding mechanisms of antibiotic resistance and bacterial defense against host immunity; however, there is little knowledge on how Gram-negative bacteria release their OMs into their environment to manipulate their hosts. Discoveries in bacterial lipid trafficking, OM lipid homeostasis, and host recognition of microbial patterns have shed new light on how microbes secrete OM vesicles (OMVs) to influence inflammation, cell death, and disease pathogenesis. Pathogens release OMVs that contain phospholipids, like cardiolipins, and components of LPS molecules, like lipid A endotoxins. These multiacylated lipid amphiphiles are molecular patterns that are differentially detected by host receptors like the Toll-like receptor 4/myeloid differentiation factor 2 complex (TLR4/MD-2), mouse caspase-11, and human caspases 4 and 5. We discuss how lipid ligands on OMVs engage these pattern recognition receptors on the membranes and in the cytosol of mammalian cells. We then detail how bacteria regulate OM lipid asymmetry, negative membrane curvature, and the phospholipid-to-LPS ratio to control OMV formation. The goal is to highlight intersections between OM lipid regulation and host immunity and to provide working models for how bacterial lipids influence vesicle formation.

scholarly journals A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

scholarly journals Efflux Pumps of Burkholderia thailandensis Control the Permeability Barrier of the Outer Membrane

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Ganesh Krishnamoorthy ◽  
Jon W. Weeks ◽  
Zhen Zhang ◽  
Courtney E. Chandler ◽  
Haotian Xue ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Efflux Pumps ◽  
Underlying Mechanism ◽  
Antibacterial Activities ◽  
Permeability Barrier ◽  
Contributing Factors ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Active Efflux

ABSTRACT Burkholderia comprises species that are significant biothreat agents and common contaminants of pharmaceutical production facilities. Their extreme antibiotic resistance affects all classes of antibiotics, including polycationic polymyxins and aminoglycosides. The major underlying mechanism is the presence of two permeability barriers, the outer membrane with modified lipid A moieties and active drug efflux pumps. The two barriers are thought to be mechanistically independent and act synergistically to reduce the intracellular concentrations of antibiotics. In this study, we analyzed the interplay between active efflux pumps and the permeability barrier of the outer membrane in Burkholderia thailandensis. We found that three efflux pumps, AmrAB-OprA, BpeEF-OprC, and BpeAB-OprB, of B. thailandensis are expressed under standard laboratory conditions and provide protection against multiple antibiotics, including polycationic polymyxins. Our results further suggest that the inactivation of AmrAB-OprA or BpeAB-OprB potentiates the antibacterial activities of antibiotics not only by reducing their efflux, but also by increasing their uptake into cells. Mass spectrometry analyses showed that in efflux-deficient B. thailandensis cells, lipid A species modified with 4-amino-4-deoxy-l-aminoarabinose are significantly less abundant than in the parent strain. Taken together, our results suggest that changes in the outer membrane permeability due to alterations in lipid A structure could be contributing factors in antibiotic hypersusceptibilities of B. thailandensis cells lacking AmrAB-OprA and BpeAB-OprB efflux pumps.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals DpaA Detaches Braun’s Lipoprotein from Peptidoglycan

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Impermeable Barrier

ABSTRACT Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Federica A. Falchi ◽  
Elisa A. Maccagni ◽  
Simone Puccio ◽  
Clelia Peano ◽  
Cristina De Castro ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Antibiotic Sensitivity ◽  
Major Constituent ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Increased Sensitivity

ABSTRACTIn Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction oflptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting thatlptA41affects LPS transport. Indeed,lptA41is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors oflptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion inmlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation inopgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCELipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective inlptAthat exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

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

2016 ◽  
Vol 198 (14) ◽  
pp. 1984-1992 ◽  
Author(s):  
Tara F. Mahoney ◽  
Dante P. Ricci ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mechanistic Model ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Bam Complex ◽  
Periplasmic Chaperones

ABSTRACTThe biogenesis of the outer membrane (OM) ofEscherichia coliis a conserved and vital process. The assembly of integral β-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-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, low-abundance, 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 inbamDthat 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 wild-type 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 asEscherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-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.

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