Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Antimicrobial activity of lysozyme isoforms: Key molecular features

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry‐heating increasing lysozyme activity. Dry‐heated lysozyme (DH‐L) is a mixture of isoforms (isoaspartyl, native‐like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH‐L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH‐L against E. coli , indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.     

Structural properties of the outer membrane and the regular surface protein of Comamonas acidovorans

The outer membrane of Comamonas acidovorans, formerly Pseudomonas acidovorans, contains a regularly arrayed surface protein. The tetragonal lattice (p4 symmetry, unit cell dimensions a = B = 10.5 nm is composed of a single type of polypeptide. It forms dimeric morphological complexes as revealed by means of electron microscopy in conjunction with image processing, STEM mass determination, and IR analysis. The surface protein has tightly associated carbohydrates and behaves like a glycoprotein in electrophoresis and IR spectroscopy. The outer membrane proteins Omp21 and Omp32 are not regularly arrayed. Omp32 has the characteristic attributes of an intrinsic outer membrane protein, such as moderate hydrophobicity, a high β-structure content, and a typical solubilization behavior. It forms channels in black lipid membranes and it, therefore, represents the major porin of C. acidovorans.     

Neuronal membrane lipid asymmetry.

The outer surface of intact synaptosomes was covalently labelled with trinitrobenzenesulfonic acid prior to isolation of the synaptic plasma membrane. Analysis of the membrane lipid demonstrated an asymmetric distribution of phospholipids across the synaptosomal plasma membrane. In addition, the fatty acyl composition of phosphatidylethanolamine from this neuronal membrane fraction was also distributed asymmetrically. The data are consistent with a $\text{de novo}$ generation of phospholipid asymmetry independent of serum lipid exchange processes. This structural asymmetry may have important consequences for neurotransmission.     

A two‐component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram‐negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2‐keto‐3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. F. novicida KdtA is bi‐functional, but F. novicida contains a membrane‐bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co‐transcribed genes. KdoH1 (related to sialidases) has a single predicted N‐terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non‐ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose‐deficient E. coli mutant that makes Kdo₂‐lipid A as its sole LPS, accumulates Kdo‐lipid A when expressing the both hydrolase components, and 1‐dephospho‐Kdo‐lipid A when expressing both the hydrolase and the Francisella lipid A 1‐phosphatase (LpxE).     

Sheath and outer membrane components from the cyanobacterium Fischerella sp. PCC 7414

A purified sheath fraction and an outer membrane fraction were obtained from the cyanobacterium Fischerella sp. PCC 7414. The sheath had a fine structure with osmiophilic fibers running in parallel to the cell surface in two distinct layers. The sheath fraction contained mainly neutral sugars (Glc, Man, Gal, Xyl, Fuc, 2-O-methylhexose), GlcN, uronic acids, and minor components such as amino acids, sulfate, phosphate, and fatty acids. The protein moiety was removable from the sheath fraction by treatment with boiling sodium dodecyl sulfate. The presence of three different 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to GlcN indicated the presence of lipopolysaccharide in the outer membrane. One major (M_r 50,000) and two minor (M_r 54,000 and 65,000) proteins were detected as constituents of the outer membrane.Abbreviations A₂pm diaminopimelic acid - GLC gas-liquid chromatography - GlcN glucosamine - Ino inositol - MurN muramic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Interaction of peptides and proteins with bacterial surface glycolipids: a comparison of glycosphingolipids and lipopolysaccharides

The bacterial cell wall of Gram-negative bacteria consists, in addition to the cytoplasmic membrane, of another permeability barrier, the outer membrane. The lipid distribution between both sides of this membrane is strictly asymmetric. The outer leaflet is made up of glycolipids, usually lipopolysaccharides. In Sphingomonas spp glycosphingolipids were found to substitute for lipopolysaccharides. In this review, it is shown by an electrophysiological approach that glycosphingolipid can replace lipopolysaccharide with respect to its function as antigenic surface structure as well as to its contribution to the diffusion barrier properties of the outer membrane. This review is focused on: (i) the function of porins, as examples of transmembrane proteins, in the different glycolipid environments; (ii) the interaction of polymyxin B with the outer membrane, as an example of polycationic antibacterial peptides; and (iii) the activation of the human complement system by lipopolysaccharides and glycosphingolipids. Received 14 April 1999/ Accepted in revised form 19 June 1999     

Occurrence,localization, and possible significance of an ornithine-containing lipid in Paracoccus denitrificans

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.Non standard abbreviations TLC thin-layer chromatography - Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride - TMS trimethylsilyl - TFA triluoroacetyl - NPPN ninhydrin-positive, phosphorus-negative - ECL equivalent chain length     

Selective removal of lipids from the outer membrane layer of human erythrocytes without hemolysis. Consequences for bilayer stability and cell shape

(1) Treatment of erythrocytes with phospholipase A₂ from bee venom cleaves about 55% of the phosphatidylcholine in the outer membrane lipid layer without changing the discoid shape of the cells. All of the fatty acids and 80% of the lysophosphatidylcholine produced under this conditions can be sequentially extracted by bovine serum albumin without hemolysis of the cells. (2) The cells remain discoid up to extraction of all of the fatty acids and 15% of the lysophosphatidylcholine. Removal of a higher fraction of lysophosphatidylcholine induces formation of stomatocytes and sphero-stomatocytes, probably going along with an internalization of membrane vesicles. Stomatocytosis can be explained on the basis of the ‘bilayer couple hypothesis’ (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. 71, 4457–4461). The shape change will compensate for the differences in surface pressure between the two leaflets induced by selective removal of material from the outer leaf of the bilayer. (3) Increasing the shear modulus of the membrane by diamide prevents this compensatory shape change even after extraction of up to 80% of the lysophosphatidylcholine, which amounts to a loss of 34% of the phospholipids of the outer membrane layer or 22% of its area. This leads to the interesting situation of a membrane possibly having a strikingly diminished ratio of the numbers of phospholipid molecules in the outer to that in the inner lipid layer. A marked difference in surface pressures should arise in this situation, unless other compensatory mechanisms become operative. Evidence for a compensation for outer lipid loss by a constriction of the inner layer has been obtained. A compensation by transbilayer reorientation of phospholipids could not be demonstrated. This latter observation supports the concept of a stabilisation of the asymmetric phospholipid arrangement by proteins such as spectrin.     

Carbohydrates in the liver microsome fraction of rats with hypovitaminosis A

Accessibility for trypsin and sodium deoxycholate was determined in carbohydrates of glycoproteins in the liver microsome fraction of rats, which were kept for 80 days on retinol-deficient diet and received optimal amounts of vitamin A. It was found that the prevailing amount of hexose- and glucosamine-containing glycoproteins is located on the outer surface of membrane vesicles and only a smaller part of these proteins is submerged into the lipid layer of the membrane or is located on its inner surface. Above a half of protein bound with fucose and neuraminic acid is located in the lipid layer. Retinol deficiency leads to translocation of a portion of fucose- and hexose-containing proteins on the outer surface of vesicles and to a decrease of the share of these proteins in the hydrophobic membrane zone.     

Leptospira: a spirochaete with a hybrid outer membrane

Leptospira is a genus of spirochaetes that includes organisms with a variety of lifestyles ranging from aquatic saprophytes to invasive pathogens. Adaptation to a wide variety of environmental conditions has required leptospires to acquire a large genome and a complex outer membrane with features that are unique among bacteria. The most abundant surface‐exposed outer membrane proteins are lipoproteins that are integrated into the lipid bilayer by amino‐terminal fatty acids. In contrast to many spirochaetes, the leptospiral outer membrane also includes lipopolysaccharide and many homologues of well‐known beta‐barrel transmembrane outer membrane proteins. Research on leptospiral transmembrane outer membrane proteins has lagged behind studies of lipoproteins because of their aberrant behaviour by Triton X‐114 detergent fractionation. For this reason, transmembrane outer membrane proteins are best characterized by assessing membrane integration and surface exposure. Not surprisingly, some outer membrane proteins that mediate host–pathogen interactions are strongly regulated by conditions found in mammalian host tissues. For example, the leptospiral immunoglobulin‐like (Lig) repeat proteins are dramatically induced by osmolarity and mediate interactions with host extracellular matrix proteins. Development of molecular genetic tools are making it possible to finally understand the roles of these and other outer membrane proteins in mechanisms of leptospiral pathogenesis.     

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.     

Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae.

Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction. Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis. In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components. Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface. Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide. This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content.     

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Fine structure of the cuticle of the black widow spider with reference to surface lipids

The surface and transverse sections of the cephalothorax, abdomen, and walking leg cuticle of the black widow spider, Latrodectus hesperus, were examined by scanning and transmission electron microscopy. Cuticle that was untreated prior to normal EM preparative procedures was compared with cuticle subjected to lipid solvents and/or concentrated alkali. The surface of untreated dorsal cephalothorax cuticle contained droplets and a lipid film that obscured fine surface detail. Immersing the cuticle in chloroform: methanol removed the droplets and lipid film, exposing previously covered openings to dermal gland ducts. An epicuticle, exocuticle, and endocuticle were present in all transverse sections of cuticle as was a complex system of pore and wax canals that connected the epidermis with the cuticle surface. The epicuticle of the walking leg was composed of three sublayers: outer membrane, outer epicuticle, and the dense homogeneous layer. A cuticulin layer was not observed. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax/pore canals.     

Uptake of a fluorescent-labeled fatty acid by spiroplasma floricola cells

12-(1-pyrene)dodecanoic fatty acid (P12) uptake by Spiroplasma floricola BNR-1 cells was characterized with regard to its kinetics, specificity, metabolism and susceptibility to protein and lipid inhibitors. The uptake process depended on temperature and pH, and exhibited biphasic saturation kinetics with a very low (2.7 M) and a high (37 M) apparent K _m value. Lauric, myristic, palmitic, stearic and oleic fatty acids did not compete with P12 for transport. The fluorescence of P12 was exclusively recovered in the neutral lipid fraction, suggesting that this fatty acid is not further utilized for phospholipid biosynthesis. Valinomycin, carbonylcyanide m-chlorophenyldrazone (CCCP), dicyclohexylcarbodiimide (DCCD), and pronase strongly reduced P12 uptake by cells, but not by membrane vesicles, affecting the high affinity (low K _m) component of the uptake system. Uptake of P12 by cells, as well as by membrane vesicles, was very sensitive to glutaraldehyde, chlorpromazine, phospholipase A₂₁ and ascorbate with FeCl₃, which affected the low affinity (high K _m) component of a transport system. Digitonin stimulated P12 uptake. We suggest that the incorporation of P12 into spiroplasma cell membrane is a two-step process: a high specificity energy-dependent and protease-sensitive binding to the outer surface of membrane, and a low specificity and energy-independent diffusion and partition into the membrane lipid environment.     

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.