scholarly journals Edge-strand of BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ryoji Miyazaki ◽  
Tetsuro Watanabe ◽  
Kohei Yoshitani ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Active Site ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Selective Permeability ◽  
Assembly Pathway ◽  
On And Off Pathways

The outer membrane (OM) of gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a β-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the β‐barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the β-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

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.

scholarly journals Edge strand of Escherichia coli BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways

2021 ◽  
Author(s):  
Ryoji Miyazaki ◽  
Tetsuro Watanabe ◽  
Kohei Yoshitani ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Site Directed Mutagenesis ◽  
Permeability Barrier ◽  
Bam Complex ◽  
Selective Permeability ◽  
Assembly Pathway ◽  
Novel Drugs ◽  
On And Off Pathways ◽  
Assembly Intermediate ◽  
And Function

The outer membrane (OM) of gram-negative bacteria is crucial for maintenance of cell viability as it functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of an essential OM protein, LptD, a β-barrel component of the lipopolysaccharide translocon. We have previously shown that BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions possibly on the β‐barrel assembly machinery (BAM) complex. However, the mechanistic details of how BepA recognizes and directs the LptD assembly intermediates to different pathways remains unclear. Here, we performed site-directed mutagenesis and crosslinking experiments to explore the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge strand located adjacent to the active site with LptD was crucial not only for proteolysis but also for assembly promotion of LptD. Site-directed crosslinking analysis indicated that the unstructured N-terminal half of the β-barrel-forming domain of an LptD assembly intermediate directly contacts with the BepA edge strand. Furthermore, the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the involvement of BepA in the maintenance of OM structure and function, which can be helpful in developing OM-targeted novel drugs.

scholarly journals The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

2020 ◽  
Author(s):  
Jacqueline Grimm ◽  
Handuo Shi ◽  
Wei Wang ◽  
Angela M. Mitchell ◽  
Ned S. Wingreen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
High Flux ◽  
Inner Membrane ◽  
Gram Negative ◽  
Selective Permeability ◽  
Delayed Cell Death ◽  
Inner Membrane Protein

AbstractThe outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and β-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ΔydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

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 Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli

2008 ◽  
Vol 190 (13) ◽  
pp. 4460-4469 ◽  
Author(s):  
Paola Sperandeo ◽  
Fion K. Lau ◽  
Andrea Carpentieri ◽  
Cristina De Castro ◽  
Antonio Molinaro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Gram Negative Bacteria ◽  
Membrane Structures ◽  
Cellular Compartment ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Assembly Pathway

ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

scholarly journals TheAcinetobacter baumanniiMla system and glycerophospholipid transport to the outer membrane

2018 ◽  
Author(s):  
Cassandra Kamischke ◽  
Junping Fan ◽  
Julien Bergeron ◽  
Hemantha D. Kulasekara ◽  
Zachary D. Dalebroux ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Atp Hydrolysis ◽  
Antibiotic Sensitivity ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Selective Permeability ◽  
Transposon Mutants

ABSTRACTThe outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screenedAcinetobacter baumanniitransposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system inE. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system toAcinetobacter baumanniiOM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

scholarly journals The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Cassandra Kamischke ◽  
Junping Fan ◽  
Julien Bergeron ◽  
Hemantha D Kulasekara ◽  
Zachary D Dalebroux ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Atp Hydrolysis ◽  
Antibiotic Sensitivity ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Selective Permeability ◽  
Transposon Mutants

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

scholarly journals Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins

2018 ◽  
Vol 115 (28) ◽  
pp. E6614-E6621 ◽  
Author(s):  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Carl J. Balibar ◽  
Juliana C. Malinverni ◽  
Ronald E. Painter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Nutrient Uptake ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Selective Inhibitor ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Underlying Basis

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a β-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

scholarly journals Spatiotemporal organization of BamA governs the pattern of outer membrane protein distribution in growing Escherichia coli cells

2021 ◽  
Author(s):  
Gideon Mamou ◽  
Patrick G. Inns ◽  
Dawei Sun ◽  
Renata Kaminska ◽  
Nicholas G. Housden ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Temporal Organization ◽  
Gram Negative Bacteria ◽  
Protein Distribution ◽  
Spatiotemporal Organization ◽  
Gram Negative ◽  
Bam Complex ◽  
K 12

AbstractThe outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on β-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the β-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/μm2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth.SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the β-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cell’s growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.

scholarly journals Peptidoglycan Remodeling EnablesEscherichiacoliTo Survive Severe Outer Membrane Assembly Defect

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Niccolò Morè ◽  
Alessandra M. Martorana ◽  
Jacob Biboy ◽  
Christian Otten ◽  
Matthias Winkle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Bacterial Cell ◽  
Cell Envelope ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Cross Links ◽  
Peptidoglycan Layer

ABSTRACTGram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show thatEscherichia colicells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCEIn Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show thatEscherichia colicells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.

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