Osmoporin OmpC forms a complex with MlaA to maintain outer membrane lipid asymmetry in Escherichia coli

Gram‐negative bacteria can survive in harsh environments in part because the asymmetric outer membrane (OM) hinders the entry of toxic compounds. Lipid asymmetry is established by having phospholipids (PLs) confined to the inner leaflet of the membrane and lipopolysaccharides (LPS) to the outer leaflet. Perturbation of OM lipid asymmetry, characterized by PL accumulation in the outer leaflet, disrupts proper LPS packing and increases membrane permeability. The multi‐component Mla system prevents PL accumulation in the outer leaflet of the OM via an unknown mechanism. Here, we demonstrate that in Escherichia coli, the Mla system maintains OM lipid asymmetry with the help of osmoporin OmpC. We show that the OM lipoprotein MlaA interacts specifically with OmpC and OmpF. This interaction is sufficient to localize MlaA lacking its lipid anchor to the OM. Removing OmpC, but not OmpF, causes accumulation of PLs in the outer leaflet of the OM in stationary phase, as was previously observed for MlaA. We establish that OmpC is an additional component of the Mla system; the OmpC‐MlaA complex may function to remove PLs directly from the outer leaflet to maintain OM lipid asymmetry. Our work reveals a novel function for the general diffusion channel OmpC in lipid transport.     

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.     

Allosteric β‐propeller signalling in TolB and its manipulation by translocating colicins

The Tol system is a five‐protein assembly parasitized by colicins and bacteriophages that helps stabilize the Gram‐negative outer membrane (OM). We show that allosteric signalling through the six‐bladed β‐propeller protein TolB is central to Tol function in Escherichia coli and that this is subverted by colicins such as ColE9 to initiate their OM translocation. Protein–protein interactions with the TolB β‐propeller govern two conformational states that are adopted by the distal N‐terminal 12 residues of TolB that bind TolA in the inner membrane. ColE9 promotes disorder of this ‘TolA box’ and recruitment of TolA. In contrast to ColE9, binding of the OM lipoprotein Pal to the same site induces conformational changes that sequester the TolA box to the TolB surface in which it exhibits little or no TolA binding. Our data suggest that Pal is an OFF switch for the Tol assembly, whereas colicins promote an ON state even though mimicking Pal. Comparison of the TolB mechanism to that of vertebrate guanine nucleotide exchange factor RCC1 suggests that allosteric signalling may be more prevalent in β‐propeller proteins than currently realized.     

Structural Insight into Phospholipid Transport by the MlaFEBD Complex from P. aeruginosa

The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4–3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaF₂E₂B₂D₆, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.     

Kinetic basis for the competitive recruitment of TolB by the intrinsically disordered translocation domain of colicin E9

TolB and Pal are members of the Tol-Pal system that spans the cell envelope of Gram-negative bacteria and contributes to the stability and integrity of the bacterial outer membrane (OM). Lipoylated Pal is tethered to the OM and binds the β-propeller domain of periplasmic TolB, which, as recent evidence suggests, disengages TolB from its interaction with other components of the Tol system in the inner membrane. Antibacterial nuclease colicins such as colicin E9 (ColE9) also bind the β-propeller domain of TolB in order to catalyze their translocation across the bacterial OM. In contrast to Pal, however, colicin binding to TolB promotes its interaction with other components of the Tol system. Here, through a series of pre-steady-state kinetic experiments utilizing fluorescence resonance energy transfer pairs within the individual protein-protein complexes, we establish the kinetic basis for such 'competitive recruitment' by the TolB-binding epitope (TBE) of ColE9. Surprisingly, the 16-residue disordered ColE9 TBE associates more rapidly with TolB than Pal, a folded 13-kDa protein. Moreover, we demonstrate that calcium ions, which bind within the confines of the TolB β-propeller domain tunnel and are known to increase the affinity of the TolB-ColE9 complex, do not exert their influence through long-range electrostatic effects, as had been predicted, but through short-range effects that slow the dissociation rate of ColE9 TBE from its complex with TolB. Our study demonstrates that an intrinsically disordered protein undergoing binding-induced folding can compete effectively with a globular protein for a common target by associating more rapidly than the globular protein.     

Programmed cell death and the pathogenesis of tissue injury induced by type A Francisella tularensis

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol–Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.     

Tol Energy-Driven Localization of Pal and Anchoring to the Peptidoglycan Promote Outer-Membrane Constriction

During cell division, gram-negative bacteria must coordinate inner-membrane invagination, peptidoglycan synthesis and cleavage and outer-membrane (OM) constriction. The OM constriction remains largely enigmatic, and the nature of this process, passive or active, is under debate. The proton-motive force-dependent Tol–Pal system performs a network of interactions within these three compartments. Here we confirm that the trans-envelope Tol–Pal complex accumulates at constriction site in Escherichia coli. We show that the inner-membrane complex composed of TolA, TolQ and TolR recruits the OM complex TolB–Pal to the septum, in an energy-dependent process. Pal recruitment then allows its binding to peptidoglycan and subsequently OM constriction. Our results provide evidence that the constriction of the OM is an energized process.     

Characterization of lptA and lptB, two essential genes implicated in lipopolysaccharide transport to the outer membrane of Escherichia coli

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic sigma factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.     

The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins

The Tol proteins are involved in outer membrane stability of Gram-negative bacteria. The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane. These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA. The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope. Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.     

The lipopolysaccharide transport system of Gram-negative bacteria

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex lipid with important biological activities. In the host it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM. The chemical structure of LPS and its biosynthetic pathways have been fully elucidated. By contrast only recently details of the transport and assembly of LPS into the OM have emerged. LPS is synthesized in the cytoplasm and at the inner leaflet of the IM and needs to cross two different compartments, the IM and the periplasm, to reach its final destination at the OM. This review focuses on recent studies that led to our present understanding of the protein machine implicated in LPS transport and in assembly at the cell surface.     

Biosynthesis and dynamics of lipids in Plasmodium-infected mature mammalian erythrocytes

The asexual development of Plasmodium within the mature mammalian erythrocyte is associated with intense membrane biogenesis, notably to ensure the increase in the size of the parasite and of the parasitophorous vacuolar membranes PVM. A considerable increase in the content of most lipids except cholesterol [namely, phospholipids PL, neutral lipids, and fatty acids FA] occurs. The PL composition and the constitutive FAs of the parasite differ markedly from the original host cell membrane. Particularly notable is the absence of cholesterol and sphingomyelin SM from the parasite membranes. How can the parasite obtain such a quantity of new lipid molecules in a host cell totally devoid of any lipid biosynthetic activity? Like the normal erythrocyte, the infected cell is unable to synthesize cholesterol or FAs. In contrast, it exhibits an intense biosynthesis of neutral lipids and a bewildering variety of PL biosyntheses. Phosphatidylcholine PC is synthesized by a de novo pathway, and also by methylation of phosphatidylethanolamine PE, which itself originates from de novo biosynthesis or from decarboxylation of phosphatidylserine PS. Hence, interference with this intense and specific PL metabolism could provide the basis for a new malaria chemotherapy. Indeed, compounds that interfere with the entry of the plasmatic precursors (FAs or polar heads) or with their metabolism are lethal to the parasite. Lastly, we focus on the structural modifications of the host cell membrane with respect to lipids, including increased fluidity and enhanced transbilayer mobility of PLs. Possible modifications in the asymmetric distribution of PLs in the host cell membrane are discussed in light of the various methods used and their limits. The capacity of infected cells to take up and metabolize large quantities of exogenous vesicles of PLs accounts for the intense dynamics of lipids in the infected erythrocytes.     

Polar localization of Escherichia coli chemoreceptors requires an intact Tol–Pal complex

Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans‐envelope Tol–Pal complex, a widely conserved component of the cell envelope of Gram‐negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in Escherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol–Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol–Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E. coli Tol–Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co‐ordinate cell division and segregation of the chemosensory machinery.     

Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli

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.     

Absence of YhdP,TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria provides the cell with a formidable barrier that excludes external threats. The two major constituents of this asymmetric barrier are lipopolysaccharide (LPS) found in the outer leaflet, and glycerophospholipids (GPLs) in the inner leaflet. Maintaining the asymmetric nature and balance of LPS to GPLs in the OM is critical for bacterial viability. The biosynthetic pathways of LPS and GPLs are well characterized, but unlike LPS transport, how GPLs are translocated to the OM remains enigmatic. Understanding this aspect of cell envelope biology could provide a foundation for new antibacterial therapies. Here, we report that YhdP and its homologues, TamB and YdbH, members of the “AsmA-like” family, are critical for OM integrity and necessary for proper GPL transport to the OM. The absence of the two largest AsmA-like proteins (YhdP and TamB) leads to cell lysis and antibiotic sensitivity, phenotypes that are rescued by reducing LPS synthesis. We also find that yhdP, tamB double mutants shed excess LPS through outer membrane vesicles, presumably to maintain OM homeostasis when normal anterograde GPL transport is disrupted. Moreover, a yhdP, tamB, ydbH triple mutant is synthetically lethal, but if GPL transport is partially restored by overexpression of YhdP, the cell shape adjusts to accommodate increased membrane content as the cell accumulates GPLs in the IM. Our results therefore suggest a model in which “AsmA-like” proteins transport GPLs to the OM, and when hindered, changes in cell shape and shedding of excess LPS aids in maintaining OM asymmetry.     

Regulated assembly of the transenvelope protein complex required for lipopolysaccharide export

Gram-negative bacteria are impervious to many drugs and environmental stresses because they possess an outer membrane (OM) containing lipopolysaccharide (LPS). LPS is biosynthesized at the cytoplasmic (inner) membrane and is transported to the OM by an unknown mechanism involving the LPS transport proteins, LptA-G. These proteins have been proposed to form a bridge between the two membranes; however, it is not known how this bridge is assembled to prevent mistargeting of LPS. We use in vivo photo-cross-linking to reveal the specific protein-protein interaction sites that give rise to the Lpt bridge. We also show that the formation of this transenvelope bridge cannot proceed before the correct assembly of the LPS translocon in the OM. This ordered sequence of events may ensure that LPS is never transported to the OM if it cannot be translocated across it to the cell surface.     

New insights into the Lpt machinery for lipopolysaccharide transport to the cell surface: LptA-LptC interaction and LptA stability as sensors of a properly assembled transenvelope complex

Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport.     

DipM links peptidoglycan remodelling to outer membrane organization in Caulobacter

Cell division in Gram‐negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Here, we identify DipM, a putative LytM endopeptidase in Caulobacter crescentus, and show that it plays a critical role in maintaining cell envelope architecture during growth and division. DipM localized to the division site in an FtsZ‐dependent manner via its PG‐binding LysM domains. Although not essential for viability, ΔdipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. Cryo electron tomography of sacculi isolated from cells depleted of DipM revealed marked thickening of the PG as compared to wild type, which we hypothesize leads to loss of trans‐envelope contacts between components of the Tol–Pal complex. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope.     

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

E.?coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.     

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.