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.     

Palaeontological evidence for membrane fusion between a unit membrane and a half-unit membrane

Membrane fusion is of fundamental importance for many biological processes and has been a topic of intensive research in past decades with several models being proposed for it. Fossils had previously not been considered relevant to studies on membrane fusion. But here two different membrane fusion patterns are reported in the same well-preserved fossil plant from the Miocene (15–20 million years old) at Clarkia, Idaho, US. Scanning electron microscope, transmission electron microscope, and traditional studies reveal the vesicles in various states (even transient semi-fusion) of membrane fusion, and thus shed new light on their membrane structure and fusion during exocytoses. The new evidence suggests that vesicles in plant cells may have not only a unit membrane but also a half-unit membrane, and that a previously overlooked membrane fusion pattern exists in plant cells. This unexpected result from an unexpected material not only marks the first evidence of on-going physiological activities in fossil plants, but also raises questions on membrane fusion in recent plants.     

Paleontological evidence for membrane fusion between a unit membrane and a half-unit membrane

Membrane fusion is of fundamental importance for many biological processes and has been a topic of intensive research in past decades with several models being proposed for it. Fossils had previously not been considered relevant to studies on membrane fusion. But here two different membrane fusion patterns are reported in the same well-preserved fossil plant from the Miocene (15-20 million years old) at Clarkia, Idaho, US. Scanning electron microscope, transmission electron microscope, and traditional studies reveal the vesicles in various states (even transient semi-fusion) of membrane fusion, and thus shed new light on their membrane structure and fusion during exocytoses. The new evidence suggests that vesicles in plant cells may have not only a unit membrane but also a half-unit membrane, and that a previously overlooked membrane fusion pattern exists in plant cells. This unexpected result from an unexpected material not only marks the first evidence of on-going physiological activities in fossil plants, but also raises questions on membrane fusion in recent plants.     

Three-dimensional structure of a membrane-microtubule complex

The unicellular algae Distigma proteus contain a group of aligned microtubules associated with their cell membrane. The association is maintained in isolated membrane fragments. The membrane-microtubule complex also includes a crystalline array of membrane particles. The major peptide component of this array was identified by labeling whole cells with radioiodine. The entire complex of membrane, particles, and microtubules is sufficiently well ordered to permit reconstruction from electron micrographs by Fourier techniques. A three-dimensional model of the membrane array at a nominal resolution of 2.5 nm has been calculated. Some similarities were apparent between lattice spacings in the membrane array and in microtubules. Analysis of these lattice correlations suggests a way in which the array of membrane particles may serve as scaffolding for microtubule attachment.     

Altered accumulation of a membrane protein unique to a membrane-deoxyribonucleic acid complex in a dna initiation mutant of Bacillus subtilis.

Membrane-deoxyribonucleic acid complexes (M-bands) have been isolated from Bacillus subtilis by their affinity for crystals of Mg2+-Sarkosyl. The membrane proteins of these complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the membrane protein composition of M-band and unfractionated membrane revealed three protein components of 125,000 (mac-1), 57,000 (mac-2), and 42,000 (mac-3) daltons unique to M-band membrane. Growth of a temperature-sensitive dna initiation mutant at the restrictive temperature resulted in an accumulation in the membrane of mac-2. This accumulation did not begin, however, until cell growth had nearly ceased, some 3 to 4 h after the cessation of deoxyribonucleic acid synthesis. Upon return of the mutant to the permissive temperature, mac-2 did not begin to return to normal levels until after the first round of deoxyribonucleic acid synthesis. A protein of 30,000 daltons, common to both M-band and whole membrane, was found to disappear from the membrane when the mutant was grown at the restrictive temperature. This disappearance is the result of increased degradation or removal from the membrane followed by a decreased rate of synthesis or insertion.     

Escherichia coli exhibits a membrane-related response to a small arginine- and tryptophan-rich antimicrobial peptide

Since multiresistant bacterial strains are more widespread and the victim numbers steadily increase, it is very important to possess a broad bandwidth of antimicrobial substances. Antibiotics often feature membrane-associated effect mechanisms. So, we present a membrane proteomic approach to shed light on the cellular response of Escherichia coli as model organism to the hexapeptide MP196, which is arginine and tryptophan rich. Analyzing integral membrane proteins are still challenging, although various detection strategies have been developed in the past. In particular, membrane proteomics in bacteria have been conducted very little due to the special physical properties of these membrane proteins. To obtain more information on the cellular response of the new compound group of small peptides, the tryptophan- and arginine-rich hexapeptide MP196 was subject to a comprehensive quantitative membrane proteomic study on E. coli by means of metabolic labeling in combination with membrane lipid analyses. This study provides in total 767 protein identifications including 185 integral membrane proteins, from which 624 could be quantified. Among these proteins, 134 were differentially expressed. Thereby, functional groups such as amino acid and membrane biosynthesis were affected, stress response could be observed, and the lipid composition of the membrane was significantly altered. Especially, the strong upregulation of the envelope stress induced protein. Spy indicates membrane damage, as well as the downregulation of the mechano-sensitive channel MscL beside others. Finally, the exceptional downregulation of transport systems strengthens these findings.     

Light-induced potential and current across a large bacteriorhodopsin-asolectin planar membrane stabilized on a polyacrylamide gel surface

A phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface. Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membrane-forming solution.     

Comparison between a moving bed membrane bioreactor and a conventional membrane bioreactor on membrane fouling

A membrane bioreactor filled with carriers instead of activated sludge named a moving bed membrane bioreactor (MBMBR) was investigated to minimize the effect of suspended solids on membrane fouling. The MBMBR and a conventional membrane bioreactor (CMBR) were operated in parallel for about two months. Unexpectedly, the rate of membrane fouling in MBMBR was about three times of that in CMBR. MBMBR showed a higher cake layer resistance than CMBR due to plenty of filamentous bacteria inhabited in suspended solids in MBMBR. Protein and polysaccharide contents of soluble EPS in MBMBR were obviously larger than those in CMBR. It could be speculated that the overgrowth of filamentous bacteria in MBMBR resulted in severe cake layer and induced a large quantity of EPS, which deteriorated the membrane fouling.     

From coiled tubules to a secretory canaliculus: a new model for membrane transformation and acid secretion by gastric parietal cells

The acid-secreting gastric parietal cell has a unique secretory membrane system. This membrane system exists in an inactive (non-secreting) and an active (secreting) form. The current accepted model to explain the transformation events associated with the conversion of the non-secreting membrane to the secreting membrane, and vice versa, invokes membrane recycling of elongated vesicle structures. However, recent studies employing cryopreparation have shown that the non-secreting membrane in these cells is actually a complex network of helically coiled tubules. Here, we present an alternative model to explain how the membrane in parietal cells is activated to secrete HCl.     

Shear-Induced Migration of a Transmembrane Protein within a Vesicle

Biomembranes feature phospholipid bilayers and serve as the interface between cells or organelles and the extracellular and/or cellular environment. Lipids can move freely throughout the membrane; the lipid bilayer behaves like a fluid. Such fluidity is important in terms of the actions of membrane transport proteins, which often mediate biological functions; membrane protein motion has attracted a great deal of attention. Because the proteins are small, diffusion phenomena are often in play, but flow-induced transport has rarely been addressed. Here, we used a dissipative particle dynamics approach to investigate flow-induced membrane protein transport. We analyzed the drift of a membrane protein located within a vesicle. Under the influence of shear flow, the protein gradually migrated toward the vorticity axis via a random walk, and the probability of retention around the axis was high. To understand the mechanism of protein migration, we varied both shear strength and protein size. Protein migration was induced by the balance between the drag and thermodynamic diffusion forces and could be represented by the Péclet number. These results improve our understanding of flow-induced membrane protein transport.     

On the role of anisotropy of membrane constituents in formation of a membrane neck during budding of a multicomponent membrane

The expression for the isotropic membrane bending energy was generalized for the case of a multicomponent membrane where the membrane constituents (single molecules or small complexes of molecules-membrane inclusions) were assumed to be anisotropic. Using this generalized expression for the membrane energy it was shown that the change of intrinsic shape of membrane components may induce first-order-like shape transitions leading to the formation of a membrane neck. The predicted discontinuous membrane shape transition and the concomitant lateral segregation of membrane components were applied to study membrane budding. Based on the results presented we conclude that the budding process might be driven by accumulation of anisotropic membrane components in the necks connecting the bud and the parent membrane, and by accumulation of isotropic (conical) membrane components on the bud. Both processes may strongly depend on the intrinsic shape of membrane components and on the direct interactions between them.     

Characterization of a targeting motif for a flagellar membrane protein in Leishmania enriettii.

The surface membranes of eukaryotic flagella and cilia are contiguous with the plasma membrane. Despite the absence of obvious physical structures that could form a barrier between the two membrane domains, the lipid and protein compositions of flagella and cilia are distinct from the rest of the cell surface membrane. We have exploited a flagellar glucose transporter from the parasitic protozoan Leishmania enriettii as a model system to characterize the first targeting motif for a flagellar membrane protein in any eukaryotic organism. In this study, we demonstrate that the flagellar membrane-targeting motif is recognized by several species of Leishmania. Previously, we demonstrated that the 130 amino acid NH(2)-terminal cytoplasmic domain of isoform 1 glucose transporter was sufficient to target a nonflagellar integral membrane protein into the flagellar membrane. We have now determined that an essential flagellar targeting signal is located between amino acids 20 and 35 of the NH(2)-terminal domain. We have further analyzed the role of specific amino acids in this region by alanine replacement mutagenesis and determined that single amino acid substitutions did not abrogate targeting to the flagellar membrane. However, individual mutations located within a cluster of five contiguous amino acids, RTGTT, conferred differences in the degree of targeting to the flagellar membrane and the flagellar pocket, implying a role for these residues in the mechanism of flagellar trafficking.     

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

Membrane lipids: it's only a phase

All biological membranes contain lipids that prefer to adopt a non-bilayer phase. Recent results suggest that, in the thylakoid membrane, membrane proteins force all the lipids to adopt a bilayer structure, and that the non-bilayer-forming lipids in the thylakoid membrane serve to drive the formation of membrane stacks.     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

MemO: a consensus approach to the annotation of a protein's membrane organization

Membrane organization describes the relationship of proteins to the membrane, that is, whether the protein crosses the membrane or is integral to the membrane and its orientation with respect to the membrane. Membrane organization is determined primarily by the presence of two features which target proteins to the secretory pathway: the endoplasmic reticulum signal peptide and the ?-helical transmembrane domain. In order to generate membrane organization annotation of high quality, confidence and throughput, the Membrane Organization (MemO) pipeline was developed, incorporating consensus feature prediction modules with integration and annotation rules derived from biological observations. The pipeline classifies proteins into six categories based on the presence or absence of predicted features: Soluble, intracellular proteins; Soluble, secreted proteins; Type I membrane proteins; Type II membrane proteins; Multi-span membrane proteins and Glycosylphosphatidylinositol anchored membrane proteins. The MemO pipeline represents an integrated strategy for the application of state-of-the-art bioinformatics tools to the annotation of protein membrane organization, a property which adds biological context to the large quantities of protein sequence information available.     

Nystatin as a probe for investigating the electrical properties of a tight epithelium

We show how the antibiotic nystatin may be used in conjunction with microelectrodes to resolve transepithelial conductance Gt into its components: Ga, apical membrane conductance; Gbl, basolateral membrane conductance; and Gj, junctional conductance. Mucosal addition of nystatin to rabbit urinary bladder in Na+-containing solutions caused Gt to increase severalfold to ca. 460 micrometerho/muF, and caused the transepithelial voltage Vt to approach +50 mV regardless of its initial value. From measurements of Gt and the voltage-divider ratio as a function of time after addition or removal of nystatin, values for Ga, Gbl, and Gj of untreated bladder could be obtained. Nystatin proved to have no direct effect on Gbl or Gj but to increase Ga by about two orders of magnitude, so that the basolateral membrane then provided almost all of the electrical resistance in the transcellular pathway. The nystatin channel in the apical membrane was more permeable to cations than to anions. The dose-response curve for nystatin had a slope of 4.6. Use of nystatin permitted assessment of whether microelectrode impalement introduced a significant shunt conductance into the untreated apical membrane, with the conclusion that such a shunt was negligible in the present experiments. Nystatin caused a hyperpolarization of the basolateral membrane potential in Na+- containing solutions. This may indicate that the Na+ pump in this membrane is electrogenic.     

Some considerations on a carrier mechanism as a model for ion accumulation

A mechanism is described which accounts for the active transport of Na⁺ ions through a membrane. It is assumed that at one side of the membrane the ion combines with a carrier ion, the resulting carrier compound then diffuses through the membrane and decomposes at the other side of the membrane. The free diffusion of the ions is also taken into account. The time rate of accumulation of the ion in question at the latter side of the membrane is calculated in terms of the concentrations of the ion at both sides of the membrane.     

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589- 4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.     

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.