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.     

Structural insight into lipopolysaccharide transport from the Gram-negative bacterial inner membrane to the outer membrane

Lipopolysaccharide (LPS) is an important component of the outer membrane (OM) of Gram-negative bacteria, playing essential roles in protecting bacteria from harsh environments, in drug resistance and in pathogenesis. LPS is synthesized in the cytoplasm and translocated to the periplasmic side of the inner membrane (IM), where it matures. Seven lipopolysaccharide transport proteins, LptA-G, form a trans‑envelope complex that is responsible for LPS extraction from the IM and transporting it across the periplasm to the OM. The LptD/E of the complex transports LPS across the OM and inserts it into the outer leaflet of the OM. In this review we focus upon structural and mechanistic studies of LPS transport proteins, with a particular focus upon the LPS ABC transporter LptB₂FG. This ATP binding cassette transporter complex consists of twelve transmembrane segments and has a unique mechanism whereby it extracts LPS from the periplasmic face of the IM through a pair of lateral gates and then powers trans‑periplasmic transport to the OM through a slide formed by either of the periplasmic domains of LptF or LptG, LptC, LptA and the N-terminal domain of LptD. The structural and functional studies of the seven lipopolysaccharide transport proteins provide a platform to explore the unusual mechanisms of LPS extraction, transport and insertion from the inner membrane to the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli

Biogenesis of the outer membrane (OM) in Gram‐negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well‐studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans‐envelope Tol‐Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol‐Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol‐Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol‐Pal complex may promote OM invagination during cell division.     

Characterization of lipopolysaccharide transport protein complex

Lipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.     

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.     

Concentration-dependent oligomerization and oligomeric arrangement of LptA

Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-by-side dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.     

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.     

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.     

Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Disruption of LptA oligomerization and affinity of the LptA–LptC interaction

The lipopolysaccharide (LPS)‐rich outer membrane (OM) is a unique feature of Gram‐negative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB₂. Structures of the periplasmic protein LptA and the soluble portion of the membrane‐associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end‐to‐end. LptA and LptC have been shown to associate in vivo and are expected to form a similar protein–protein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C‐terminal β‐strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (K_d = 15 µM) and isothermal titration calorimetry (K_d = 14 µM). K_d values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 µM) and for WT LptA oligomerization (29 µM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization.     

YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli

Recent advances in the study of bacterial membranes have led to the identification of a multicomponent YaeT complex in the outer membrane (OM) of Gram-negative bacteria that is involved in the targeting and folding of beta-barrel outer membrane proteins (OMPs). In Escherichia coli, this complex consists of an essential OMP, YaeT, and three OM lipoproteins, YfgL, NlpB and YfiO. YfiO is the only essential lipoprotein component of the complex. We show that this lipoprotein is required for the proper assembly and/or targeting of OMPs to the OM but not the assembly of lipopolysaccharides (LPS). Depletion of YfiO causes similar phenotypes as does the depletion of YaeT, and we conclude that YfiO plays a critical role in YaeT-mediated OMP folding. We demonstrate that YfiO and YfgL directly interact with YaeT in vitro, while NlpB interacts directly with YfiO. Genetic analysis verifies the importance of YfiO and its interactions with NlpB in maintaining the functional integrity of the YaeT complex.     

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.     

Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

The presence of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria creates a permeability barrier that prevents the entry of most currently available antibiotics. The seven lipopolysaccharide transport (Lpt) proteins involved in transporting and assembling this glycolipid are essential for growth and division in Escherichia coli; therefore, inhibiting their functions leads to cell death. LptB, the ATPase that provides energy for LPS transport and assembly, forms a complex with three other inner membrane (IM) components, LptC, F, and G. We demonstrate that inhibitors of pure LptB can also inhibit the full IM complex, LptBFGC, purified in detergent. We also compare inhibition of LptB and the LptBFGC complex with the antibiotic activity of these compounds. Our long-term goal is to develop tools to study inhibitors of LPS biogenesis that could serve as potentiators by disrupting the OM permeability barrier, facilitating entry of clinically used antibiotics not normally used to treat Gram-negative infections, or that can serve as antibiotics themselves.     

Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae

Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens.     

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.     

Direct Measurements of the Outer Membrane Stage of Ferric Enterobactin Transport: POSTUPTAKE BINDING*

When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (∼35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of ∼5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33–35 kcal/mol. The accumulation of FeEnt in the periplasm required the binding protein and inner membrane permease components of its overall transport system; postuptake binding assays on strains devoid of FepB, FepD, or FepG did not show uptake of FeEnt through the OM. However, fluorescence labeling data implied that FepA was active in the ΔfepB strain, suggesting that FeEnt entered the periplasm but then leaked out. Further experiments confirmed this futile cycle; cells without FepB transported FeEnt across the OM, but it immediately escaped through TolC.     

Variation of amino acid properties in all-beta globular and outer membrane protein structures

Discriminating outer membrane (OM) proteins from globular proteins is an important task. The structural analysis of β-strands dominating globular (all-β) proteins and OM proteins provides useful insight to distinguish between them. In this work, we analyze the characteristic features of the 20 amino acid residues in all-β and OM proteins. We set up numerical indices for several properties of amino acid residues, such as, conformational parameters, surrounding hydrophobicity, accessible surface area and reduction in accessibility, and inter-residue contacts. We found that all the aromatic residues prefer to be in β-strands of both globular and OM proteins. The surrounding hydrophobicity of aromatic and non-polar amino acid residues in globular proteins is significantly higher than that of OM proteins. The residues Trp, Arg, Phe and Gln show a remarkable difference of reduction in accessibility between all-β globular (βG) and OM proteins. The positively charged residues, Lys and Arg in the membrane part of OM proteins have more number of contacts than globular proteins. Further, the behavior of the 20 amino acid residues in β-strand segments of globular and OM proteins have been discussed. The parameters developed in this work can be used for identifying transmembrane β-strands in OM proteins and for discriminating βG proteins from OM proteins.     

Treponema pallidum and the quest for outer membrane proteins

Treponema pallidum, the syphilis spirochaete, has a remarkable ability to evade the humoral and cellular responses it elicits in infected hosts. Although formerly attributed to the presence of an outer coat comprised of serum proteins and/or mucopolysaccharides, current evidence indicates that the immuno-evasiveness of this bacterium is largely the result of its unusual molecular architecture. Based upon a combination of molecular, biochemical, and ultrastructural data, it is now believed that the T. pallidum outer membrane (OM) contains a paucity of poorly immunogenic transmembrane proteins (‘rare outer membrane proteins’) and that its highly immunogentc proteins are lipoproteins anchored predominantly to the periplasmic leaflet of the cytoplasmic membrane. The presence in the T. pallidum OM of a limited number of transmembrane proteins has profound implications for understanding syphilis pathogenesis as well as treponemal physiology. Two major strategies for molecular characterization of rare outer membrane proteins have evolved. The first involves the identification of candidate OM proteins as fusions with Escherichia coli alkaline phosphatase. The second involves the characterization of candidate OM proteins identified in outer membranes isolated from virulent T. pallidum. Criteria to define candidate OM proteins and for definitive identification of rare OM proteins are proposed as a guide for future studies.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.