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.     

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

The human immune system can directly lyse invading micro‐organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single‐particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram‐negative bacteria. Recent functional studies on Gram‐negative bacteria elucidate that local assembly of MAC pores by surface‐bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase‐generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gram‐negative bacteria. Furthermore, this review elaborates on how MAC‐dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram‐negative bacteria.     

Biogenesis of bacterial membrane vesicles

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo . Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein–peptidoglycan (OM–PG) and OM protein–inner membrane protein (OM–PG–IM) interactions within the envelope structure. Modulation of OM–PG and OM–PG–IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM–PG and OM–PG–IM associations within the envelope structure, thus releasing OM as MVs.     

Polymyxins induce lipid scrambling and disrupt the homeostasis of Gram-negative bacteria membrane

Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.     

How complement kills E. coli. I. Location of the lethal lesion

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.     

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.     

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.     

Turgor pressure, membrane tension and the control of exocytosis in higher plants

Both turgor pressure and differences in membrane tension are capable of providing an energy input into exocytosis, the process of fusion of Golgi vesicles with the cell membrane in plants. It is shown that the contribution of turgor pressure is much larger than that of membrane tension, so that the exocytotic process is not likely on thermodynamic grounds to be reversible unless another source of energy is made available. However, recycling of membrane material as flattened, empty vesicles is energetically possible and is likely to be favoured when the magnitude of membrane tension in the cell membrane is low. Thus the outward flows of membrane and cell wall material are in principle linked to turgor, whereas membrane tension influences the inward flow of membrane material.     

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10-20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10-20% sucrose was observed to have no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis "bays," visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as "spot welds" between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.     

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.     

Turgor Pressure Regulation in Valonia utricularis: Effect of Cell Wall Elasticity and Auxin

The electrical membrane resistance rho(0) of the marine alga Valonia utricularis shows a marked maximum in dependence on the turgor pressure. The critical pressure, P(c), at which the maximum occurs, as well as its absolute value, rho(0) (max), are strongly volume-dependent. Both P(c) and rho(0) (max), increase with decreasing cell volume. It seems likely, that these relationships reflect the elastic properties of the cell wall, because the volumetric elastic modulus, epsilon, is also volume-dependent, increasing hyperbolically with cell volume. Both P(c) and rho(0) (max) can be affected by external application of indole-3-acetic acid at concentrations of 2.10(-7)m to 2 .10(-5)m. The critical pressure is shifted by 1.2 to 6 bars toward higher pressures and the maximum membrane resistance increased up to 5.6-fold. During the course of the experiments (up to 4 hours), however, IAA had no effect on the volumetric elastic modulus, epsilon.The maximum in membrane resistance is discussed in terms of a pressure-dependent change of potassium fluxes. The volume dependence of P(c) and rho(0) (max) suggests that not only turgor pressure but also epsilon must be considered as a regulating parameter during turgor pressure regulation. On this basis a hypothesis is presented for the transformation of both, a pressure signal and of changes in the elastic properties of the cell wall into alterations of ion fluxes. It is assumed that the combined effects of tension and compression of the membranes as well as the interaction between membrane and cell wall opposingly change the number of transport sites for K(+) providing a turgor-sensing mechanism that regulates ion fluxes. The IAA effects demonstrated are consistent with this view, suggesting that the basic mechanisms for turgor pressure regulation and growth regulation are similar.Any relation connecting growth rate with turgor pressure should be governed by two parameters, i.e. by a yielding pressure, at which cell growth starts, and by the critical pressure, at which it ceases again.     

Theoretical and experimental exclusion of errors in the determination of the elasticity and water transport parameters of plant cells by the pressure probe technique

The volumetric elastic modulus of the cell wall and the hydraulic conductivity of the cell membranes were measured on ligatured compartments of different sizes of Chara corallina internodes using the pressure probe technique. The ratio between intact cell surface area and the area of puncture in the cell wall and membrane introduced by the microcapillary of the pressure probe was varied over a large range by inserting microcapillaries of widely varying diameters in different sized compartments. The relationship of the elastic modulus and the hydraulic conductivity to turgor pressure was independent of the ratio of intact cell surface area to the area of injury. The increase in the hydraulic conductivity below 2 bar turgor pressure and the volume dependence of the elastic modulus were shown to be the same as those observed in intact nonligatured cells. Theoretical considerations of the possible influence of injury of the cell wall and cell membrane around the inserted microcapillary on the measurement of the water transport and cell wall parameters do not explain the experimental findings. Thus, mechanical artifacts, if at all present, are too small to account for the observed dependence of the hydraulic conductivity and the elastic modulus on turgor pressure. The pressure probe technique thus represents an accurate method for measuring water transport parameters in both giant algal cells and in tissue cells of higher plants.     

Melittin-Induced Permeabilization,Re-sealing,and Re-permeabilization of E. coli Membranes

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ～4 s of OM permeabilization, the CM invaginates to form inward facing “periplasmic bubbles.” Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ～20 s, first the OM and then the CM re-seals to GFP. Some 2–20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.     

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.     

Dynamical Organization of Compositionally Distinct Inner and Outer Membrane Lipids of Mycobacteria

Mycobacterium species, including Mycobacterium tuberculosis, employs atypical long (C_60–90) and branched lipids to produce a complex cell wall and localizes these toward distinct spatial locations, inner membrane (IM) and outer membrane (OM), thus forming a robust permeability barrier. The properties and functional roles of these spatially orchestrated membrane platforms remain unknown. Herein, we report the distinctive lateral organization, fluidity, and lipid domain architecture of protein-free membranes reconstituted from IM and OM lipids in vitro from M. smegmatis (Msm) underscored by their lipid packing and lipid dynamics. We show that Msm OM, against common notion, is more dynamic and fluid compared with IM and reveal the role of cell wall-associated peptidoglycans and lipoarabinomannan on the Msm OM organization. Overall, these studies indicate that mycobacterial species may regulate their overall membrane functionality by regulating the synthesis of these complex arrays of lipids. Based on the structure-function relationship drawn here, documented alteration in the mycobacterial lipidome during cellular infection and/or drug treatment could reflect a mechanism to fine-tune M. tuberculosis membrane properties to its advantage. These findings are expected to inspire development of lipid-centric therapeutic approaches targeted toward its membrane.     

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.     

Identification and characterization of Cor413im proteins as novel components of the chloroplast inner envelope

Plastids are surrounded by two membrane layers, the outer and inner envelope membranes, which have various transport and metabolic activities. A number of envelope membrane proteins have been identified by biochemical approaches and have been assigned to specific functions. Despite those efforts, the chloroplast envelope membrane is expected to contain a number of as yet unidentified proteins that may affect specific aspects of plant growth and development. In this report, we identify and characterize a novel class of inner envelope membrane proteins, designated as Cor413 chloroplast inner envelope membrane group (Cor413im). Both in vivo and in vitro studies indicate that Cor413im proteins are targeted to the chloroplast envelope. Biochemical analyses of Cor413im1 demonstrate that it is an integral membrane protein in the inner envelope of chloroplasts. Quantitative real-time PCR analysis reveals that COR413IM1 is more abundant than COR413IM2 in cold-acclimated Arabidopsis leaves. The analyses of T-DNA insertion mutants indicate that a single copy of COR413IM genes is sufficient to provide normal freezing tolerance to Arabidopsis. Based on these data, we propose that Cor413im proteins are novel components that are targeted to the chloroplast inner envelope in response to low temperature.     

Outer membrane lipoprotein biogenesis: Lol is not the end

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.     

Timing of TolA and TolQ Recruitment at the Septum Depends on the Functionality of the Tol-Pal System

Efficient cell division of Gram-negative bacteria requires the presence of the Tol-Pal system to coordinate outer membrane (OM) invagination with inner membrane invagination (IM) and peptidoglycan (PG) remodeling. The Tol-Pal system is a trans-envelope complex that connects the three layers of the cell envelope through an energy-dependent process. It is composed of the three IM proteins, TolA, TolQ and TolR, the periplasmic protein TolB and the OM lipoprotein Pal. The proteins of the Tol-Pal system are dynamically recruited to the cell septum during cell division. TolA, the central hub of the Tol-Pal system, has three domains: a transmembrane helix (TolA₁), a long second helical periplasmic domain (TolA₂) and a C-terminal globular domain (TolA₃). The TolQR complex uses the PMF to energize TolA, allowing its cyclic interaction via TolA₃ with the OM TolB-Pal complex. Here, we confirm that TolA₂ is sufficient to address TolA to the site of constriction, whereas TolA₁ is recruited by TolQ. Analysis of the protein localization as function of the bacterial cell age revealed that TolA and TolQ localize earlier at midcell in the absence of the other Tol-Pal proteins. These data suggest that TolA and TolQ are delayed from their septal recruitment by the multiple interactions of TolA with TolB-Pal in the cell envelope providing a new example of temporal regulation of proteins recruitment at the septum.     

Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites.

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.