Tripartite assembly of RND multidrug efflux pumps

NASA Astrophysics Data System (ADS)

Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

2016-02-01

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

2011-06-01

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

PubMed

Remis, Jonathan P; Wei, Dongguang; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H Ewa; Costerton, J William; Berleman, James E; Auer, Manfred

2014-02-01

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Resurrecting Inactive Antimicrobial Peptides from the Lipopolysaccharide Trap

PubMed Central

Mohanram, Harini

2014-01-01

Host defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, “trapped” by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action. PMID:24419338

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

PubMed

Murphy, C K; Klebba, P E

1989-11-01

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.

Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts

PubMed Central

Misra, Rajeev

2012-01-01

In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts. PMID:27335668

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

PubMed

Dong, Haohao; Tang, Xiaodi; Zhang, Zhengyu; Dong, Changjiang

2017-11-01

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 LptB 2 FG. 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. Copyright © 2017 Elsevier B.V. All rights reserved.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

PubMed Central

Pirbadian, Sahand; Barchinger, Sarah E.; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Shi, Liang; Gorby, Yuri A.; Golbeck, John H.; El-Naggar, Mohamed Y.

2014-01-01

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. PMID:25143589

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

PubMed

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894.

PubMed

Wang, L; Hu, X; Tao, G; Wang, X

2012-05-01

To investigate the role of lipopolysaccharide (LPS) structure in the stability of outer membrane and the ability of biofilm formation in Cronobacter sakazakii. A C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA_04107 encoding for heptosyltransferase I. LPS were purified from LWW02, and changes in their structure were confirmed by thin-layer chromatography and electrospray ionization mass spectrometry. Comparing with the wild-type strain BAA-894, slower growth, higher membrane permeability, higher surface hydrophobicity, stronger ability of autoaggregation and biofilm formation were observed for the mutant strain LWW02. The gene ESA_04107 encodes heptosyltransferase I in C. sakazakii ATCC BAA-894. The cleavage of LPS in C. sakazakii could cause its outer membrane defects and increase its ability to form biofilms. The study is important for understanding the pathogenic mechanism and efficient control of C. sakazakii. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

PubMed

Leuzzi, Rosanna; Nesta, Barbara; Monaci, Elisabetta; Cartocci, Elena; Serino, Laura; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-11-09

Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, David J.; Edwards, Marcus; White, Gaye F.

2012-06-01

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural featuresmore » of two of these outermembrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.« less

Outer Membrane Protein Folding and Topology from a Computational Transfer Free Energy Scale.

PubMed

Lin, Meishan; Gessmann, Dennis; Naveed, Hammad; Liang, Jie

2016-03-02

Knowledge of the transfer free energy of amino acids from aqueous solution to a lipid bilayer is essential for understanding membrane protein folding and for predicting membrane protein structure. Here we report a computational approach that can calculate the folding free energy of the transmembrane region of outer membrane β-barrel proteins (OMPs) by combining an empirical energy function with a reduced discrete state space model. We quantitatively analyzed the transfer free energies of 20 amino acid residues at the center of the lipid bilayer of OmpLA. Our results are in excellent agreement with the experimentally derived hydrophobicity scales. We further exhaustively calculated the transfer free energies of 20 amino acids at all positions in the TM region of OmpLA. We found that the asymmetry of the Gram-negative bacterial outer membrane as well as the TM residues of an OMP determine its functional fold in vivo. Our results suggest that the folding process of an OMP is driven by the lipid-facing residues in its hydrophobic core, and its NC-IN topology is determined by the differential stabilities of OMPs in the asymmetrical outer membrane. The folding free energy is further reduced by lipid A and assisted by general depth-dependent cooperativities that exist between polar and ionizable residues. Moreover, context-dependency of transfer free energies at specific positions in OmpLA predict regions important for protein function as well as structural anomalies. Our computational approach is fast, efficient and applicable to any OMP.

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

PubMed Central

Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

2015-01-01

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

PubMed Central

Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

PubMed

Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Consequences of Location-Dependent Organ of Corti Micro-Mechanics

PubMed Central

Liu, Yanju; Gracewski, Sheryl M.; Nam, Jong-Hoon

2015-01-01

The cochlea performs frequency analysis and amplification of sounds. The graded stiffness of the basilar membrane along the cochlear length underlies the frequency-location relationship of the mammalian cochlea. The somatic motility of outer hair cell is central for cochlear amplification. Despite two to three orders of magnitude change in the basilar membrane stiffness, the force capacity of the outer hair cell’s somatic motility, is nearly invariant over the cochlear length. It is puzzling how actuators with a constant force capacity can operate under such a wide stiffness range. We hypothesize that the organ of Corti sets the mechanical conditions so that the outer hair cell’s somatic motility effectively interacts with the media of traveling waves—the basilar membrane and the tectorial membrane. To test this hypothesis, a computational model of the gerbil cochlea was developed that incorporates organ of Corti structural mechanics, cochlear fluid dynamics, and hair cell electro-physiology. The model simulations showed that the micro-mechanical responses of the organ of Corti are different along the cochlear length. For example, the top surface of the organ of Corti vibrated more than the bottom surface at the basal (high frequency) location, but the amplitude ratio was reversed at the apical (low frequency) location. Unlike the basilar membrane stiffness varying by a factor of 1700 along the cochlear length, the stiffness of the organ of Corti complex felt by the outer hair cell remained between 1.5 and 0.4 times the outer hair cell stiffness. The Y-shaped structure in the organ of Corti formed by outer hair cell, Deiters cell and its phalange was the primary determinant of the elastic reactance imposed on the outer hair cells. The stiffness and geometry of the Deiters cell and its phalange affected cochlear amplification differently depending on the location. PMID:26317521

Detection of outer membrane vesicles in Synechocystis PCC 6803

PubMed Central

Pardo, Yehudah A.; Florez, Catalina; Baker, Kristopher M.; Schertzer, Jeffrey W.; Mahler, Gretchen J.

2015-01-01

It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well. PMID:26363014

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

PubMed Central

Goodwin, Edward C.; Motamedi, Nasim; Lipovsky, Alex; Fernández-Busnadiego, Rubén; DiMaio, Daniel

2014-01-01

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity. PMID:24732912

Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP.

PubMed

Iyer, Bharat Ramasubramanian; Mahalakshmi, Radhakrishnan

2016-09-06

The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gram-negative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. In addition, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix to folding and stability and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequence-dependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the W78F substitution in S. typhimurium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to that of E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins.

PubMed

Heuck, Alexander; Schleiffer, Alexander; Clausen, Tim

2011-03-11

β-Barrel proteins are frequently found in the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. In Escherichia coli, these proteins are inserted in the outer membrane by the Bam (β-barrel assembly machinery) complex, a multiprotein machinery formed by the β-barrel protein BamA and the four peripheral membrane proteins BamB, BamC, BamD and BamE. The periplasmic part of BamA binds prefolded β-barrel proteins by a β-augmentation mechanism, thereby stabilizing the precursors prior to their membrane insertion. However, the role of the associated proteins within the Bam complex remains unknown. Here, we describe the crystal structure of BamB, a nonessential component of the Bam complex. The structure shows a typical eight-bladed β-propeller fold. Two sequence stretches of BamB were previously identified to be important for interaction with BamA. In our structure, both motifs are located in close proximity to each other and contribute to a conserved region forming a narrow groove on the top of the propeller. Moreover, crystal contacts reveal two interaction modes of how BamB might bind unfolded β-barrel proteins. In the crystal lattice, BamB binds to exposed β-strands by β-augmentation, whereas peptide stretches rich in aromatic residues can be accommodated in hydrophobic pockets located at the bottom of the propeller. Thus, BamB could simultaneously bind to BamA and prefolded β-barrel proteins, thereby enhancing the folding and membrane insertion capability of the Bam complex. Copyright © 2011 Elsevier Ltd. All rights reserved.

Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walian, P.J.

1989-11-01

One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing ofmore » these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)« less

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

Mitochondrial Protein Synthesis, Import, and Assembly

PubMed Central

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

Large-scale purification and biochemical characterization of crystallization-grade porin protein P from Pseudomonas aeruginosa.

PubMed

Worobec, E A; Martin, N L; McCubbin, W D; Kay, C M; Brayer, G D; Hancock, R E

1988-04-07

A large-scale purification scheme was developed for lipopolysaccharide-free protein P, the phosphate-starvation-inducible outer-membrane porin from Pseudomonas aeruginosa. This highly purified protein P was used to successfully form hexagonal crystals in the presence of n-octyl-beta-glucopyranoside. Amino-acid analysis indicated that protein P had a similar composition to other bacterial outer membrane proteins, containing a high percentage (50%) of hydrophilic residues. The amino-terminal sequence of this protein, although not homologous to either outer membrane protein, PhoE or OmpF, of Escherichia coli, was found to have an analogous protein-folding pattern. Protein P in the native trimer form was capable of maintaining a stable functional trimer after proteinase cleavage. This suggested the existence of a strongly associated tertiary and quaternary structure. Circular dichroism studies confirmed these results in that a large proportion of the protein structure was determined to be beta-sheet and resistant to acid pH and heating in 0.1% sodium dodecyl sulphate.

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State▿ †

PubMed Central

Zuber, Benoît; Chami, Mohamed; Houssin, Christine; Dubochet, Jacques; Griffiths, Gareth; Daffé, Mamadou

2008-01-01

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function. PMID:18567661

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

PubMed

Zuber, Benoît; Chami, Mohamed; Houssin, Christine; Dubochet, Jacques; Griffiths, Gareth; Daffé, Mamadou

2008-08-01

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

The Lipopolysaccharide of Brucella abortus BvrS/BvrR Mutants Contains Lipid A Modifications and Has Higher Affinity for Bactericidal Cationic Peptides

PubMed Central

Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S.; Koch, Michel H. J.; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

2005-01-01

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic β(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants. PMID:16077108

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

PubMed

Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D

2017-04-06

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles. Copyright © 2017 Elsevier Inc. All rights reserved.

Outer membrane proteins of pathogenic spirochetes

PubMed Central

Cullen, Paul A.; Haake, David A.; Adler, Ben

2009-01-01

Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg 2+ and Ca 2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-raymore » and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca 2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.« less

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

DOE PAGES

Clifton, Luke A.; Skoda, Maximilian W. A.; Le Brun, Anton P.; ...

2014-12-09

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg 2+ and Ca 2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-raymore » and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca 2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.« less

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

PubMed Central

2014-01-01

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration. PMID:25489959

Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module

PubMed Central

Selkrig, Joel; Belousoff, Matthew J.; Headey, Stephen J.; Heinz, Eva; Shiota, Takuya; Shen, Hsin-Hui; Beckham, Simone A.; Bamert, Rebecca S.; Phan, Minh-Duy; Schembri, Mark A.; Wilce, Matthew C.J.; Scanlon, Martin J.; Strugnell, Richard A.; Lithgow, Trevor

2015-01-01

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes. PMID:26243377

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

PubMed

Judd, Ellen M; Comolli, Luis R; Chen, Joseph C; Downing, Kenneth H; Moerner, W E; McAdams, Harley H

2005-10-01

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.

Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains.

PubMed

Bong, Ji-Hong; Yoo, Gu; Park, Min; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2014-11-01

The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane particle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The isolated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody-binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein-labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method. Copyright © 2014 Elsevier Inc. All rights reserved.

Improved purification of native meningococcal porin PorB and studies on its structure/function.

PubMed

Massari, Paola; King, Carol A; MacLeod, Heather; Wetzler, Lee M

2005-12-01

The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Modelling three-dimensional cochlear micromechanics within the guinea pig organ of Corti

NASA Astrophysics Data System (ADS)

Ni, Guangjian; Elliott, Stephen J.

2018-05-01

The active amplification process in the mammalian cochlea depends on a complex interaction between cells within the organ of Corti. A three-dimensional (3D) model was developed using the finite element method based on anatomy for the apical end in the guinea pig cochlea, which is comprised of 3D discrete hair cells, 3D continuous membranes and fluid. The basilar membrane, tectorial membrane and the reticular lamina are modelled with orthotropic materials. The Y-shape structures formed by the outer hair cell (OHC), the Deiters' cell and Deiters' cell phalangeal process are also included to account for the structural longitudinal coupling. The motion within the organ of Corti was first simulated in response to a pressure difference loading on the basilar membrane, in order to calculate the passive vibration pattern. Then, the outer hair cells somatic electromotility was implemented by applying a voltage across the OHC walls to investigate its contribution to membranes motion.

Subnanometer structure of an asymmetric model membrane: Interleaflet coupling influences domain properties

DOE PAGES

Heberle, Frederick A.; Marquardt, Drew; Doktorova, Milka; ...

2016-04-29

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order tomore » produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Lastly, our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.« less

BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly.

PubMed

Gu, Yinghong; Zeng, Yi; Wang, Zhongshan; Dong, Changjiang

2017-11-21

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli , the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C-β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C-β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.

PubMed

Rouse, Sarah L; Hawthorne, Wlliam J; Lambert, Sebastian; Morgan, Marc L; Hare, Stephen A; Matthews, Stephen

2016-12-01

Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF 106-430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Å resolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, β = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane β-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.

Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides

PubMed Central

Bhunia, Anirban; Mohanram, Harini; Domadia, Prerna N.; Torres, Jaume; Bhattacharjya, Surajit

2009-01-01

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane. PMID:19520860

Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability

PubMed Central

Kowata, Hikaru; Tochigi, Saeko; Takahashi, Hideyuki

2017-01-01

ABSTRACT The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli. The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients. IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less permeable to organic nutrients, with >20-fold lower permeability than the outer membrane of Escherichia coli. Such permeability appears to fit the cyanobacterial lifestyle, in which the diffusion pathway for inorganic solutes may suffice to sustain the autotrophic physiology, illustrating a link between outer membrane permeability and the cellular lifestyle. PMID:28696278

Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles.

PubMed

Kahnt, Jörg; Aguiluz, Kryssia; Koch, Jürgen; Treuner-Lange, Anke; Konovalova, Anna; Huntley, Stuart; Hoppert, Michael; Søgaard-Andersen, Lotte; Hedderich, Reiner

2010-10-01

Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.

Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins.

PubMed

Wanner, B L; Sarthy, A; Beckwith, J

1979-10-01

We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein IA (D. L. Diedrich, A. O. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus.

Detection of fibrils associated with Rickettsia rickettsii.

PubMed

Todd, W J; Burgdorfer, W; Wray, G P

1983-09-01

The ultrastructural appearance of the "halozone" formed at the interface between the spotted fever agent Rickettsia rickettsii and the cytoplasm of persistently infected cultured vole cells (Microtus pennsylvanicus) was studied by transmission electron microscopy. In sections of epoxy-embedded specimens stained with uranyl acetate and lead citrate, the halozone appeared clear and devoid of ultrastructural features. However, when unembedded preparations of whole infected cells were examined at 1,000 kV, fine structural features were observed within the halozone. These features, associated with the rickettsial outer membrane, were more clearly detectable when the infected cells were extracted with the detergent Triton X-100 before fixation. Under such conditions, long extensions of the rickettsial outer membrane, microfilament-like structures attached to that membrane, and extensive attachments between adjacent rickettsiae were seen. The fine structural features within the rickettsial halozone were also seen at 75 kV when unembedded sections were prepared from polyethylene glycol-embedded specimens. Thus, epoxy-embedding medium obscures the fine structural features within the halozone surrounding the rickettsiae in infected cells.

Detection of fibrils associated with Rickettsia rickettsii.

PubMed Central

Todd, W J; Burgdorfer, W; Wray, G P

1983-01-01

The ultrastructural appearance of the "halozone" formed at the interface between the spotted fever agent Rickettsia rickettsii and the cytoplasm of persistently infected cultured vole cells (Microtus pennsylvanicus) was studied by transmission electron microscopy. In sections of epoxy-embedded specimens stained with uranyl acetate and lead citrate, the halozone appeared clear and devoid of ultrastructural features. However, when unembedded preparations of whole infected cells were examined at 1,000 kV, fine structural features were observed within the halozone. These features, associated with the rickettsial outer membrane, were more clearly detectable when the infected cells were extracted with the detergent Triton X-100 before fixation. Under such conditions, long extensions of the rickettsial outer membrane, microfilament-like structures attached to that membrane, and extensive attachments between adjacent rickettsiae were seen. The fine structural features within the rickettsial halozone were also seen at 75 kV when unembedded sections were prepared from polyethylene glycol-embedded specimens. Thus, epoxy-embedding medium obscures the fine structural features within the halozone surrounding the rickettsiae in infected cells. Images PMID:6411620

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

The neuromechanics of hearing

NASA Astrophysics Data System (ADS)

Araya, Mussie K.; Brownell, William E.

2015-12-01

Hearing requires precise detection and coding of acoustic signals by the inner ear and equally precise communication of the information through the auditory brainstem. A membrane based motor in the outer hair cell lateral wall contributes to the transformation of sound into a precise neural code. Structural, molecular and energetic similarities between the outer hair cell and auditory brainstem neurons suggest that a similar membrane based motor may contribute to signal processing in the auditory CNS. Cooperative activation of voltage gated ion channels enhances neuronal temporal processing and increases the upper frequency limit for phase locking. We explore the possibility that membrane mechanics contribute to ion channel cooperativity as a consequence of the nearly instantaneous speed of electromechanical signaling and the fact that membrane composition and mechanics modulate ion channel function.

Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

PubMed

Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

2016-01-01

The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

Development of a novel drug release system, time-controlled explosion system (TES). I. Concept and design.

PubMed

Ueda, S; Hata, T; Asakura, S; Yamaguchi, H; Kotani, M; Ueda, Y

1994-01-01

A novel controlled drug release system. Time-Controlled Explosion System (TES) has been developed. TES has a four-layered spherical structure, which consists of core, drug, swelling agent and water insoluble polymer membrane. TES is characterized by a rapid drug release with a precisely programmed lag time; i.e. expansion of the swelling agent by water penetrating through the outer membrane, destruction of the membrane by stress due to swelling force and subsequent rapid drug release. For establishing the concept and development strategy, TES was designed using metoprolol and polystyrene balls (size: 3.2 mm in diameter) as a model drug and core particles. Among the polymers screened, low-substituted hydroxypropylcellulose (L-HPC) and ethylcellulose (EC) were selected for a swelling agent and an outer water insoluble membrane, respectively. The release profiles of metoprolol from the system were not affected by the pH of the dissolution media. Lag time was controlled by the thickness of the outer EC membrane; thus, a combination of TES particles possessing different lag times could offer any desired release profile of the model compound, metoprolol.

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

PubMed

Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

2015-11-13

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J.

2012-01-30

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin,more » and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.« less

Asymmetric distribution of charged lipids between the leaflets of a vesicle bilayer induced by melittin and alamethicin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qian, Shuo; Heller, William T

2011-01-01

Cellular membranes are complex mixtures of lipids, proteins, and other small molecules that provide functional, dynamic barriers between the cell and its environment, as well as between environments within the cell. The lipid composition of the membrane is highly specific and controlled in terms of both content and lipid localization. The membrane structure results from the complex interplay between the wide varieties of molecules present. Here, small-angle neutron scattering and selective deuterium labeling were used to probe the impact of the membrane-active peptides melittin and alamethicin on the structure of lipid bilayers composed of a mixture of the lipids dimyristoylmore » phosphatidylglycerol (DMPG) and chain-perdeuterated dimyristoyl phosphatidylcholine (DMPC). We found that both peptides enriched the outer leaflet of the bilayer with the negatively charged DMPG, creating an asymmetric distribution of lipids. The level of enrichment is peptide concentration-dependent and is stronger for melittin than it is for alamethicin. The enrichment between the inner and outer bilayer leaflets occurs at very low peptide concentrations and increases with peptide concentration, including when the peptide adopts a membrane-spanning, pore-forming state. The results suggest that these membrane-active peptides may have a secondary stressful effect on target cells at low concentrations that results from a disruption of the lipid distribution between the inner and outer leaflets of the bilayer that is independent of the formation of transmembrane pores.« less

Designed beta-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide.

PubMed

Bhunia, Anirban; Mohanram, Harini; Domadia, Prerna N; Torres, Jaume; Bhattacharjya, Surajit

2009-08-14

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like beta-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nM concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the beta-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate beta-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.

Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane.

PubMed

Wang, Yan; Andole Pannuri, Archana; Ni, Dongchun; Zhou, Haizhen; Cao, Xiou; Lu, Xiaomei; Romeo, Tony; Huang, Yihua

2016-05-06

The partially de-N-acetylated poly-β-1,6-N-acetyl-d-glucosamine (dPNAG) polymer serves as an intercellular biofilm adhesin that plays an essential role for the development and maintenance of integrity of biofilms of diverse bacterial species. Translocation of dPNAG across the bacterial outer membrane is mediated by a tetratricopeptide repeat-containing outer membrane protein, PgaA. To understand the molecular basis of dPNAG translocation, we determined the crystal structure of the C-terminal transmembrane domain of PgaA (residues 513-807). The structure reveals that PgaA forms a 16-strand transmembrane β-barrel, closed by four loops on the extracellular surface. Half of the interior surface of the barrel that lies parallel to the translocation pathway is electronegative, suggesting that the corresponding negatively charged residues may assist the secretion of the positively charged dPNAG polymer. In vivo complementation assays in a pgaA deletion bacterial strain showed that a cluster of negatively charged residues proximal to the periplasm is necessary for biofilm formation. Biochemical analyses further revealed that the tetratricopeptide repeat domain of PgaA binds directly to the N-deacetylase PgaB and is critical for biofilm formation. Our studies support a model in which the positively charged PgaB-bound dPNAG polymer is delivered to PgaA through the PgaA-PgaB interaction and is further targeted to the β-barrel lumen of PgaA potentially via a charge complementarity mechanism, thus priming the translocation of dPNAG across the bacterial outer membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

PubMed

Villa, Riccardo; Martorana, Alessandra M; Okuda, Suguru; Gourlay, Louise J; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel; Polissi, Alessandra

2013-03-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

PubMed Central

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, Louise J.; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel

2013-01-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components. PMID:23292770

The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.

PubMed

Wenz, Lena-Sophie; Opaliński, Lukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

2014-06-01

The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. © 2014 The Authors.

The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

NASA Astrophysics Data System (ADS)

Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

2015-08-01

Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

Structure and Function of the Haemophilus influenzae Autotransporters

PubMed Central

Spahich, Nicole A.; St. Geme, Joseph W.

2011-01-01

Autotransporters are a large class of proteins that are found in the outer membrane of Gram-negative bacteria and are almost universally implicated in virulence. These proteins consist of a C-terminal β-domain that is embedded in the outer membrane and an N-terminal domain that is exposed on the bacterial surface and is endowed with effector function. In this article, we review and compare the structural and functional characteristics of the Haemophilus influenzae IgA1 protease and Hap monomeric autotransporters and the H. influenzae Hia and Hsf trimeric autotransporters. All of these proteins play a role in colonization of the upper respiratory tract and in the pathogenesis of H. influenzae disease. PMID:22919571

Adaptations in rod outer segment disc membranes in response to environmental lighting conditions.

PubMed

Rakshit, Tatini; Senapati, Subhadip; Parmar, Vipul M; Sahu, Bhubanananda; Maeda, Akiko; Park, Paul S-H

2017-10-01

The light-sensing rod photoreceptor cell exhibits several adaptations in response to the lighting environment. While adaptations to short-term changes in lighting conditions have been examined in depth, adaptations to long-term changes in lighting conditions are less understood. Atomic force microscopy was used to characterize the structure of rod outer segment disc membranes, the site of photon absorption by the pigment rhodopsin, to better understand how photoreceptor cells respond to long-term lighting changes. Structural properties of the disc membrane changed in response to housing mice in constant dark or light conditions and these adaptive changes required output from the phototransduction cascade initiated by rhodopsin. Among these were changes in the packing density of rhodopsin in the membrane, which was independent of rhodopsin synthesis and specifically affected scotopic visual function as assessed by electroretinography. Studies here support the concept of photostasis, which maintains optimal photoreceptor cell function with implications in retinal degenerations. Copyright © 2017 Elsevier B.V. All rights reserved.

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

PubMed

Harner, Max; Neupert, Walter; Deponte, Marcel

2011-07-15

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

Deuterium Labeling Together with Contrast Variation Small-angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

PubMed Central

Zaccai, Nathan R.; Sandlin, Clifford W.; Hoopes, James T.; Curtis, Joseph E.; Fleming, Patrick J.; Fleming, Karen G.; Krueger, Susan

2016-01-01

In gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archeal prefoldins and the mitochondrial Tim chaperones, that is α-helical ‘tentacles’ extend from a β-strand ‘body’ to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis and structure modeling. PMID:26791979

Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins.

PubMed

Zaccai, Nathan R; Sandlin, Clifford W; Hoopes, James T; Curtis, Joseph E; Fleming, Patrick J; Fleming, Karen G; Krueger, Susan

2016-01-01

In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling. © 2016 Elsevier Inc. All rights reserved.

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell.

PubMed

Drulis-Kawa, Zuzanna; Dorotkiewicz-Jach, Agata; Gubernator, Jerzy; Gula, Grzegorz; Bocer, Tomasz; Doroszkiewicz, Wlodzimierz

2009-02-09

The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy.

PubMed

Oh, Yoo Jin; Plochberger, Birgit; Rechberger, Markus; Hinterdorfer, Peter

2017-06-01

Lipopolysaccharide (LPS) on gram-negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 μg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano-resolution approaches in microbiology. Copyright © 2017 John Wiley & Sons, Ltd.

TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Z.; Xu C.; Benning, C.

The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminalmore » {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.« less

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes

DOE PAGES

Jacobson, Kurt H.; Gunsolus, Ian L.; Kuech, Thomas R.; ...

2015-07-24

We report that design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations, and assessment of the potential implications of nanoparticle release into the environment require understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the lipid-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) andmore » second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. Association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Lastly, our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.« less

Structure-Function Aspects of Membrane Associated Prokaryotic DNA replication

DTIC Science & Technology

1994-09-01

Membrane associated DNA replication in prokaryotes has been studied intensively using two model systems, Bacillus subtilis and plasmid RK2 cultured...in its Escherichia coli host. In the former a new membrane protein that had previously been found to act as an inhibitor of DNA replication was...prior to a round of DNA replication . In the latter, plasmid DNA replication has been found to be associated with the inner but not outer membrane of

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

PubMed Central

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human factor-H and is predicted to form a membrane-spanning β-barrel

PubMed Central

Dyer, Adam; Brown, Gemma; Stejskal, Lenka; Laity, Peter R.; Bingham, Richard J.

2015-01-01

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane, is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The outer membrane protein A (OmpA) domain is an eight-stranded membrane-spanning β-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work, we describe the identification of four novel OmpA-like β-barrels from Borrelia afzelii, the most common cause of erythema migrans (EM) rash in Europe. Structural characterization of one these proteins (BAPKO_0422) by SAXS and CD indicate a compact globular structure rich in β-strand consistent with a monomeric β-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25×45 Å (1 Å=0.1 nm). Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal phenylalanine residue commonly found in Proteobacterial OM proteins is replaced by isoleucine/leucine or asparagine. BAPKO_0422 is demonstrated to bind human factor H (fH) and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value. PMID:26181365

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts 1

PubMed Central

Moeller, Curt H.; Mudd, J. Brian

1982-01-01

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:16662716

The novel 2Fe–2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain

PubMed Central

Conlan, Andrea R.; Paddock, Mark L.; Axelrod, Herbert L.; Cohen, Aina E.; Abresch, Edward C.; Wiley, Sandra; Roy, Melinda; Nechushtai, Rachel; Jennings, Patricia A.

2009-01-01

A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe–2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004 ▶), Am. J. Physiol. Endocrinol. Metab. 286, E252–E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4 Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane. PMID:19574633

Iodination of Escherichia coli with chloramine T: selective labeling of the outer membrane lipoprotein.

PubMed Central

Munford, R S; Gotschlich, E C

1977-01-01

Iodination of Escherichia coli cells with chloramine T preferentially labels the free and murein-bound forms of the outer membrane lipoprotein. Iodination for 15 s at 15 degrees C labels the two forms of the lipoprotein almost exclusively, whereas iodination for 60 s at 25 degrees C also labels the other major outer membrane proteins. Chloramine T iodination is a rapid, simple technique for labeling the outer membrane lipoprotein. PMID:400793

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Structural and functional insights into the lipopolysaccharide ABC transporter LptB2FG.

PubMed

Dong, Haohao; Zhang, Zhengyu; Tang, Xiaodi; Paterson, Neil G; Dong, Changjiang

2017-08-09

The cell surface of most Gram-negative bacteria contains lipopolysaccharide that is essential for their viability and drug resistance. A 134-kDa protein complex LptB 2 FG is unique among ATP-binding cassette transporters because it extracts lipopolysaccharide from the external leaflet of the inner membrane and propels it along a filament that extends across the periplasm to directly deliver lipopolysaccharide into the external leaflet of the outer membrane. Here we report the crystal structure of the lipopolysaccharide transporter LptB 2 FG from Klebsiella pneumoniae, in which both LptF and LptG are composed of a β-jellyroll-like periplasmic domain and six α-helical segments in the transmembrane domain. LptF and LptG form a central cavity containing highly conserved hydrophobic residues. Structural and functional studies suggest that LptB 2 FG uses an alternating lateral access mechanism to extract lipopolysaccharide and traffic it along the hydrophobic cavity toward the transporter's periplasmic domains.Lipopolysaccharides (LPS) are synthesized at the periplasmic side of the inner membrane of Gram-negative bacteria and are then extracted by the LptB 2 FG complex during the first step of LPS transport to the outer membrane. Here the authors present the LptB 2 FG structure, which supports an alternating lateral access mechanism for LPS extraction.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

PubMed

Yang, Chih-Wei; Wu, Mai-Szu; Pan, Ming-Jeng; Hsieh, Wang-Ju; Vandewalle, Alain; Huang, Chiu-Ching

2002-08-01

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

PubMed Central

Hönlinger, A; Bömer, U; Alconada, A; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Pfanner, N

1996-01-01

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase. Images PMID:8641278

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

PubMed

May, Janine M; Owens, Tristan W; Mandler, Michael D; Simpson, Brent W; Lazarus, Michael B; Sherman, David J; Davis, Rebecca M; Okuda, Suguru; Massefski, Walter; Ruiz, Natividad; Kahne, Daniel

2017-12-06

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Infectious polymorphic toxins delivered by outer membrane exchange discriminate kin in myxobacteria.

PubMed

Vassallo, Christopher N; Cao, Pengbo; Conklin, Austin; Finkelstein, Hayley; Hayes, Christopher S; Wall, Daniel

2017-08-18

Myxobacteria are known for complex social behaviors including outer membrane exchange (OME), in which cells exchange large amounts of outer membrane lipids and proteins upon contact. The TraA cell surface receptor selects OME partners based on a variable domain. However, traA polymorphism alone is not sufficient to precisely discriminate kin. Here, we report a novel family of OME-delivered toxins that promote kin discrimination of OME partners. These SitA lipoprotein toxins are polymorphic and widespread in myxobacteria. Each sitA is associated with a cognate sitI immunity gene, and in some cases a sitB accessory gene. Remarkably, we show that SitA is transferred serially between target cells, allowing the toxins to move cell-to-cell like an infectious agent. Consequently, SitA toxins define strong identity barriers between strains and likely contribute to population structure, maintenance of cooperation, and strain diversification. Moreover, these results highlight the diversity of systems evolved to deliver toxins between bacteria.

Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells

PubMed Central

Hadis, Mohammed; Alderwick, Luke

2017-01-01

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections. PMID:29186191

[Biochemical characteristics and antigenic structures of Chlamydia].

PubMed

Puy, H; Fuentes, V; Eb, F; Orfila, J

1989-01-01

New biotechnology in immunology and molecular biology has enabled the identification and definition of the structure of glycolipids and especially membrane proteins of Chlamydia. Chlamydia antigen lipopolysaccharide, major outer membrane protein, protein 74 kDa, eukaryotic cell binding protein and cysteine rich proteins are all carriers of antigenic determinants, genus, species or type specific. They are very usefull for diagnosis of Chlamydial infections and epidemiological studies. These membranous antigens have an important role in the pathogenesis of these bacteries. Finally these studies have contributed to the isolation of a new species: C. pneumoniae (TWAR strains).

Effects of beta-cyclodextrin on the structure of sphingomyelin/cholesterol model membranes.

PubMed

Jablin, Michael S; Flasiński, Michał; Dubey, Manish; Ratnaweera, Dilru R; Broniatowski, Marcin; Dynarowicz-Łatka, Patrycja; Majewski, Jarosław

2010-09-08

The interaction of beta-cyclodextrin (beta-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after beta-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by beta-CD, indicating that beta-CD is unable to remove sphingomyelin or complexed Chol. However, beta-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by beta-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of beta-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by beta-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Comparative study on histological structures of the vitelline membrane of hen and duck egg observed by cryo-scanning electron microscopy.

PubMed

Chung, Wen-Hsin; Lai, Kung-Ming; Hsu, Kuo-chiang

2010-02-10

The histological structures of the vitelline membranes (VM) of hen and duck eggs were observed by cryo-scanning electron microscopy (cryo-SEM), and the chemical characteristics were also compared. The outer layer surface (OLS) of duck egg VM showed networks constructed by fibrils and sheets (0.1-5.2 microm in width), and that of hen egg presented networks formed only by sheets (2-6 microm in width). Thicker fibrils (0.5-1.5 microm in width) with different arrangement were observed on the inner layer surface (ILS) of duck egg VM as compared to those (0.3-0.7 microm in width) of hen egg VM. Upon separation, the outer surface of the outer layer (OSOL) and the inner surface of the inner layer (ISIL) of hen and duck egg VMs were quite similar to fresh VM except that the OSOL of duck egg VM showed networks constructed only by sheets. Thin fibrils interlaced above a bumpy or flat structure were observed at the exposed surface of the outer layer (ESOL) of hen and duck egg VMs. The exposed surfaces of inner layers (ESIL) of hen and duck egg VMs showed similar structures of fibrils, which joined, branched, and ran in straight lines for long distances up to 30 microm; however, the widths of the fibrils shown in ESOL and ESIL of duck egg VM were 0.1 and 0.7-1.4 microm, respectively, and were greater than those (<0.1 and 0.5-0.8 microm) of hen egg VM. The continuous membranes of both hen and duck egg VMs were still attached to the outer layers when separated. The content of protein, the major component of VM, was higher in duck egg VM (88.6%) than in hen egg VM (81.6%). Four and six major SDS-soluble protein patterns with distinct localization were observed in hen and duck egg VMs, respectively. Overall, the different histological structures of hen and duck egg VMs were suggested to be majorly attributable to the diverse protein components.

Electrophoretic characterisation of the outer membrane proteins of Yersinia pestis isolated in north-east Brazil.

PubMed Central

Abath, F. G.; Almeida, A. M.; Ferreira, L. C.

1989-01-01

The outer membrane proteins of 38 Yersinia pestis isolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts among Y. pestis strains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed. Images Fig. 2 PMID:2606164

Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of Porphyromonas gingivalis

PubMed Central

Sawada, Koichi; Kokeguchi, Susumu; Hongyo, Hiroshi; Sawada, Satoko; Miyamoto, Manabu; Maeda, Hiroshi; Nishimura, Fusanori; Takashiba, Shogo; Murayama, Yoji

1999-01-01

Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected in P. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalis strains. PMID:10531208

Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jingzhi; Qian, Xinguo; Hu, Junbin

2010-11-03

The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away,more » and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.« less

White Feces Syndrome of Shrimp Arises from Transformation, Sloughing and Aggregation of Hepatopancreatic Microvilli into Vermiform Bodies Superficially Resembling Gregarines

PubMed Central

Sriurairatana, Siriporn; Boonyawiwat, Visanu; Gangnonngiw, Warachin; Laosutthipong, Chaowanee; Hiranchan, Jindanan; Flegel, Timothy W.

2014-01-01

Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes. Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium. When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined. PMID:24911022

White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines.

PubMed

Sriurairatana, Siriporn; Boonyawiwat, Visanu; Gangnonngiw, Warachin; Laosutthipong, Chaowanee; Hiranchan, Jindanan; Flegel, Timothy W

2014-01-01

Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes. Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium. When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined.

Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

PubMed

Barnard, Travis J; Dautin, Nathalie; Lukacik, Petra; Bernstein, Harris D; Buchanan, Susan K

2007-12-01

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.

A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability▿

PubMed Central

Bou Raad, Roland; Méniche, Xavier; de Sousa-d'Auria, Celia; Chami, Mohamed; Salmeron, Christophe; Tropis, Marielle; Labarre, Cecile; Daffé, Mamadou; Houssin, Christine; Bayan, Nicolas

2010-01-01

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum. PMID:20363942

[Ultrastructural organization of cytoplasmatic membrane of Anaerobacter polyendosporus studied by electron microscopic cryofractography].

PubMed

Duda, V I; Suzina, N E; Dmitriev, V V

2001-01-01

Anaerobacter polyendosporus cells do not have typical mesosomes. However, the analysis of this anaerobic multispore bacterium by electron microscopic cryofractography showed that its cytoplasmic membrane contains specific intramembrane structures in the form of flat lamellar inverted lipid membranes tenths of nanometers to several microns in size. It was found that these structures are located in the hydrophobic interior between the outer and inner leaflets of the cytoplasmic membrane and do not contain intramembrane particles that are commonly present on freeze-fracture replicas. The flat inverted lipid membranes were revealed in bacterial cells cultivated under normal growth conditions, indicating the existence of a complex-type compartmentalization in biological membranes, which manifests itself in the formation of intramembrane compartments having the appearance of vesicles and inverted lipid membranes.

Process for making ceramic hot gas filter

DOEpatents

Connolly, Elizabeth Sokolinski; Forsythe, George Daniel; Domanski, Daniel Matthew; Chambers, Jeffrey Allen; Rajendran, Govindasamy Paramasivam

2001-01-01

A ceramic hot-gas candle filter having a porous support of filament-wound oxide ceramic yarn at least partially surrounded by a porous refractory oxide ceramic matrix, and a membrane layer on at least one surface thereof. The membrane layer may be on the outer surface, the inner surface, or both the outer and inner surface of the porous support. The membrane layer may be formed of an ordered arrangement of circularly wound, continuous filament oxide ceramic yarn, a ceramic filler material which is less permeable than the filament-wound support structure, or some combination of continuous filament and filler material. A particularly effective membrane layer features circularly wound filament with gaps intentionally placed between adjacent windings, and a filler material of ceramic particulates uniformly distributed throughout the gap region. The filter can withstand thermal cycling during backpulse cleaning and is resistant to chemical degradation at high temperatures.

Ceramic hot-gas filter

DOEpatents

Connolly, Elizabeth Sokolinski; Forsythe, George Daniel; Domanski, Daniel Matthew; Chambers, Jeffrey Allen; Rajendran, Govindasamy Paramasivam

1999-01-01

A ceramic hot-gas candle filter having a porous support of filament-wound oxide ceramic yarn at least partially surrounded by a porous refractory oxide ceramic matrix, and a membrane layer on at least one surface thereof. The membrane layer may be on the outer surface, the inner surface, or both the outer and inner surface of the porous support. The membrane layer may be formed of an ordered arrangement of circularly wound, continuous filament oxide ceramic yarn, a ceramic filler material which is less permeable than the filament-wound support structure, or some combination of continuous filament and filler material. A particularly effective membrane layer features circularly wound filament with gaps intentionally placed between adjacent windings, and a filler material of ceramic particulates uniformly distributed throughout the gap region. The filter can withstand thermal cycling during backpulse cleaning and is resistant to chemical degradation at high temperatures.

Ceramic hot-gas filter

DOEpatents

Connolly, E.S.; Forsythe, G.D.; Domanski, D.M.; Chambers, J.A.; Rajendran, G.P.

1999-05-11

A ceramic hot-gas candle filter is described having a porous support of filament-wound oxide ceramic yarn at least partially surrounded by a porous refractory oxide ceramic matrix, and a membrane layer on at least one surface thereof. The membrane layer may be on the outer surface, the inner surface, or both the outer and inner surface of the porous support. The membrane layer may be formed of an ordered arrangement of circularly wound, continuous filament oxide ceramic yarn, a ceramic filler material which is less permeable than the filament-wound support structure, or some combination of continuous filament and filler material. A particularly effective membrane layer features circularly wound filament with gaps intentionally placed between adjacent windings, and a filler material of ceramic particulates uniformly distributed throughout the gap region. The filter can withstand thermal cycling during back pulse cleaning and is resistant to chemical degradation at high temperatures.

The antiepileptic drug diphenylhydantoin affects the structure of the human erythrocyte membrane.

PubMed

Suwalsky, Mario; Mennickent, Sigrid; Norris, Beryl; Villena, Fernando; Cuevas, Francisco; Sotomayor, Carlos P

2004-01-01

Phenytoin (diphenylhydantoin) is an antiepileptic agent effective against all types of partial and tonic-clonic seizures. Phenytoin limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord neurons maintained in vitro. This effect is mediated by a slowing of the rate of recovery of voltage activated Na+ channels from inactivation. For this reasons it was thought of interest to study the binding affinities of phenytoin with cell membranes and their perturbing effects upon membrane structures. The effects of phenytoin on the human erythrocyte membrane and molecular models have been investigated in the present work. This report presents the following evidence that phenytoin interacts with cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that phenytoin perturbed a class of lipids found in the outer moiety of cell membranes; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the polar head groups and acyl chains of the erythrocyte membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes the formation of echinocytes was observed, due to the insertion of phenytoin in the outer monolayer of the red cell membrane. This is the first time that an effect of phenytoin on the red cell shape is described. However, the effects of the drug were observed at concentrations higher than those currently found in plasma when phenytoin is therapeutically administered.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Structural basis for maintenance of bacterial outer membrane lipid asymmetry.

PubMed

Abellón-Ruiz, Javier; Kaptan, Shreyas S; Baslé, Arnaud; Claudi, Beatrice; Bumann, Dirk; Kleinekathöfer, Ulrich; van den Berg, Bert

2017-12-01

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds 1 , 2 . The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet 1-3 . This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane 4 . The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C 5,6 , but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

Outer-membrane Transport of Aromatic Hydrocarbons as a First Step in Biodegradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hearn,E.; Patel, D.; van den Berg, B.

Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-strandedmore » {beta}-barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.« less

Structural and mechanistic insights into phospholipid transfer by Ups1-Mdm35 in mitochondria

NASA Astrophysics Data System (ADS)

Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

2015-08-01

Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1-Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1-Mdm35 with and without PA. The Ups1-Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes.

Sub-nm 3D observation of human hair melanin by high-voltage STEM.

PubMed

Imai, Takehito; Higuchi, Kimitaka; Yamamoto, Yuta; Arai, Shigeo; Nakano, Takashi; Tanaka, Nobuo

2016-04-01

The ultrastructure of melanin granules in human hair was studied using 1,000 kV high-voltage scanning transmission electron microscopy to successfully reconstruct three-dimensional images of the whole melanin granule. It was revealed that the melanin granule was composed of a membrane-like outer structure that included many spherical vesicles, and an inner matrix containing a sheet-like structure in the elongated direction of the melanin granule and a sheet-like arrays structure in the cross direction. The outer structure of the melanin granule was maintained even after exposure to hair-bleaching agents to decompose the melanin granule, suggesting that the outer structure was a highly robust structure and composition compared with the inner matrix . © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

PubMed Central

Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G.; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D.

2015-01-01

We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprCN and TprCC) orthologous to regions in the major outer sheath protein (MOSPN and MOSPC) of Treponema denticola and that TprCC is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSPC-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSPN-like domains are tethered within the periplasm. TprF, which does not contain a MOSPC-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSPN and MOSPC-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSPN-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

Three-Dimensional Imaging of the Mouse Organ of Corti Cytoarchitecture for Mechanical Modeling

NASA Astrophysics Data System (ADS)

Puria, Sunil; Hartman, Byron; Kim, Jichul; Oghalai, John S.; Ricci, Anthony J.; Liberman, M. Charles

2011-11-01

Cochlear models typically use continuous anatomical descriptions and homogenized parameters based on two-dimensional images for describing the organ of Corti. To produce refined models based more closely on the actual cochlear cytoarchitecture, three-dimensional morphometric parameters of key mechanical structures are required. Towards this goal, we developed and compared three different imaging methods: (1) A fixed cochlear whole-mount preparation using the fluorescent dye Cellmask®, which is a molecule taken up by cell membranes and clearly delineates Deiters' cells, outer hair cells, and the phalangeal process, imaged using confocal microscopy; (2) An in situ fixed preparation with hair cells labeled using anti-prestin and supporting structures labeled using phalloidin, imaged using two-photon microscopy; and (3) A membrane-tomato (mT) mouse with fluorescent proteins expressed in all cell membranes, which enables two-photon imaging of an in situ live preparation with excellent visualization of the organ of Corti. Morphometric parameters including lengths, diameters, and angles, were extracted from 3D cellular surface reconstructions of the resulting images. Preliminary results indicate that the length of the phalangeal processes decreases from the first (inner most) to third (outer most) row of outer hair cells, and that their length also likely varies from base to apex and across species.

An evaluation of bacterial contamination of barriers used in periapical tissue regeneration: Part 2--Bacterial penetration.

PubMed

Sharma, Priya; Mickel, André K; Chogle, Sami; Sharma, Prem Nath; Han, Yiping W; Jones, Jefferson J

2008-03-01

To compare the relative penetration of Prevotella melaninogenica and Enterococcus faecalis through 3 guided tissue regeneration membranes: Atrisorb, Lambone, and OsseoQuest. It was hypothesized that OsseoQuest would show increased bacterial penetration when compared to Lambone and Atrisorb. Centrifuge tubes containing trypticase soy broth were sealed with circular sections of membranes and placed in test tubes containing culture media. The bacterial penetration was assessed by passage of bacteria from the outer tube culture media to the inner centrifuge tube media through the membrane. After incubation for 4 and 48 hours, the media from the outer and inner tubes were compared for bacterial count. P melaninogenica exhibited 91% penetration for Lambone in 2 days, while OsseoQuest displayed 87% penetration with E faecalis in the same time. Atrisorb displayed a minimal penetration with both bacteria (2%). Atrisorb displayed the least bacterial penetration, which may be attributed to membrane structure, chemical configuration, hydrophobicity, and porosity of tested membranes.

Acute epidural-like appearance of an encapsulated solid non-organized chronic subdural hematoma.

PubMed

Prieto, Ruth; Pascual, José M; Subhi-Issa, Issa; Yus, Miguel

2010-01-01

We report the exceptional case of an encapsulated solid non-organized chronic subdural hematoma (SDH) in a 67-year-old woman that was admitted with acute hemiplegia followed by rapid deterioration in consciousness 5 months after a minor head trauma. Computed tomography (CT) showed an extracerebral biconvex shaped hyperdense mass that led to the misdiagnosis of an acute epidural hematoma. Urgent craniotomy revealed an encapsulated mass filled with solid fresh clot in the subdural space. Complete evacuation of this SDH, including both its inner and outer membranes, was achieved, and the patient recovered successfully. Histological analysis confirmed that the content of the hematoma corresponded to a newly formed clot that was enclosed between an inner membrane, composed of two collagen layers, and an outer membrane with a three layered structure. Chronic SDH may seldom present as an encapsulated solid non-organized lesion that consists of a fibrous capsule enclosing a fresh clot and lacking the thick fibrous septations that typically connect the inner and outer membranes of organized chronic SDH. This entity mimics the clinical course and radiological appearance of acute epidural hematomas and should be considered in the differential diagnosis of extracerebral hyperdense biconvex shaped lesions.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

PubMed

Valguarnera, Ezequiel; Spera, Juan M; Czibener, Cecilia; Fulgenzi, Fabiana R; Casabuono, Adriana C; Altabe, Silvia G; Pasquevich, Karina A; Guaimas, Francisco; Cassataro, Juliana; Couto, Alicia S; Ugalde, Juan E

2018-03-28

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

PubMed Central

Kim, JiHyun; Huang, Zhen; St. Clair, Johnna R.; Brown, Deborah A.; London, Erwin

2016-01-01

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

PubMed

Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

2016-12-06

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

PubMed Central

Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

2017-01-01

Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

Tomographic Structural Changes of Retinal Layers after Internal Limiting Membrane Peeling for Macular Hole Surgery.

PubMed

Faria, Mun Yueh; Ferreira, Nuno P; Cristóvao, Diana M; Mano, Sofia; Sousa, David Cordeiro; Monteiro-Grillo, Manuel

2018-01-01

To highlight tomographic structural changes of retinal layers after internal limiting membrane (ILM) peeling in macular hole surgery. Nonrandomized prospective, interventional study in 38 eyes (34 patients) subjected to pars plana vitrectomy and ILM peeling for idiopathic macular hole. Retinal layers were assessed in nasal and temporal regions before and 6 months after surgery using spectral domain optical coherence tomography. Total retinal thickness increased in the nasal region and decreased in the temporal region. The retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL) showed thinning on both nasal and temporal sides of the fovea. The thickness of the outer plexiform layer (OPL) increased. The outer nuclear layer (ONL) and outer retinal layers (ORL) increased in thickness after surgery in both nasal and temporal regions. ILM peeling is associated with important alterations in the inner retinal layer architecture, with thinning of the RNFL-GCL-IPL complex and thickening of OPL, ONL, and ORL. These structural alterations can help explain functional outcome and could give indications regarding the extent of ILM peeling, even though peeling seems important for higher rate of hole closure. © 2017 S. Karger AG, Basel.

Cardiolipin effects on membrane structure and dynamics.

PubMed

Unsay, Joseph D; Cosentino, Katia; Subburaj, Yamunadevi; García-Sáez, Ana J

2013-12-23

Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson's disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

A nonlinear cochlear model with the outer hair cell piezoelectric activity

NASA Astrophysics Data System (ADS)

Jiang, Xiaoai; Grosh, Karl

2003-10-01

In this paper we present a simple cochlear model which captures the most important aspect of nonlinearity in the cochlea-the nonlinearity caused by the piezoelectric-like activity of outer hair cells and the variable conductance of the outer hair cell stereocilia. A one-dimensional long-wave model is built to simulate the dynamic response of the fluid-loaded basilar membrane. The basilar membrane is simulated as isolated linear oscillators along the cochlear length, and its motion is coupled with the fluid pressure and the nonlinear force produced by the outer hair cells. As the basilar membrane moves, the fluid shears stereocilia, and the resulting ion flow changes the transmembrane potential of the outer hair cells and subsequently their length, leading to further movement of the basilar membrane. The piezoelectric-like activity of the outer hair cell is simulated by a current source, and stereocilia motion is modeled as a varying conductance that changes as the basilar membrane moves. A solution in the time domain will be presented. [Work supported by NIH.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Shuyan; Sun, Cancan; Tan, Kemin

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystalmore » structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.« less

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

NASA Astrophysics Data System (ADS)

Casuso, Ignacio; Khao, Jonathan; Chami, Mohamed; Paul-Gilloteaux, Perrine; Husain, Mohamed; Duneau, Jean-Pierre; Stahlberg, Henning; Sturgis, James N.; Scheuring, Simon

2012-08-01

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

The structure of the β-barrel assembly machinery complex

DOE PAGES

Bakelar, Jeremy; Buchanan, Susan K.; Noinaj, Nicholas

2016-01-08

β-Barrel outer membrane proteins (OMPs) are found in the outer membranes of Gram-negative bacteria and are essential for nutrient import, signaling, and adhesion. A 200-kilodalton five-component complex called the β-barrel assembly machinery (BAM) complex has been implicated in the biogenesis of OMPs. In this paper, we report the structure of the BAM complex from Escherichia coli, revealing that binding of BamCDE modulates the conformation of BamA, the central component, which may serve to regulate the BAM complex. The periplasmic domain of BamA was in a closed state that prevents access to the barrel lumen, which indicates substrate OMPs may notmore » be threaded through the barrel during biogenesis. Finally and further, conformational shifts in the barrel domain lead to opening of the exit pore and rearrangement at the lateral gate.« less

The structure of the β-barrel assembly machinery complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bakelar, Jeremy; Buchanan, Susan K.; Noinaj, Nicholas

β-Barrel outer membrane proteins (OMPs) are found in the outer membranes of Gram-negative bacteria and are essential for nutrient import, signaling, and adhesion. A 200-kilodalton five-component complex called the β-barrel assembly machinery (BAM) complex has been implicated in the biogenesis of OMPs. In this paper, we report the structure of the BAM complex from Escherichia coli, revealing that binding of BamCDE modulates the conformation of BamA, the central component, which may serve to regulate the BAM complex. The periplasmic domain of BamA was in a closed state that prevents access to the barrel lumen, which indicates substrate OMPs may notmore » be threaded through the barrel during biogenesis. Finally and further, conformational shifts in the barrel domain lead to opening of the exit pore and rearrangement at the lateral gate.« less

Weakening effect of cell permeabilizers on gram-negative bacteria causing biodeterioration.

PubMed

Alakomi, H-L; Paananen, A; Suihko, M-L; Helander, I M; Saarela, M

2006-07-01

Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion.

PubMed

Van Ommen Kloeke, F; Bryant, R D; Laishley, E J

1995-12-01

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.

The Xylella fastidiosa PD1063 Protein Is Secreted in Association with Outer Membrane Vesicles

PubMed Central

Pierce, Brittany K.; Voegel, Tanja; Kirkpatrick, Bruce C.

2014-01-01

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa. PMID:25426629

The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

PubMed

Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

2014-01-01

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

Wzi is an outer membrane lectin that underpins group 1 capsule assembly in Escherichia coli.

PubMed

Bushell, Simon R; Mainprize, Iain L; Wear, Martin A; Lou, Hubing; Whitfield, Chris; Naismith, James H

2013-05-07

Many pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides. Copyright © 2013 Elsevier Ltd. All rights reserved.

Scanning electron microscopy of the collodion membrane from a self-healing collodion baby*

PubMed Central

de Almeida Jr., Hiram Larangeira; Isaacsson, Henrique; Guarenti, Isabelle Maffei; Silva, Ricardo Marques e; de Castro, Luis Antônio Suita

2015-01-01

Abstract Self-healing collodion baby is a well-established subtype of this condition. We examined a male newborn, who was covered by a collodion membrane. The shed membrane was examined with scanning electron microscopy. The outer surface showed a very compact keratin without the normal elimination of corneocytes. The lateral view of the specimen revealed a very thick, horny layer. The inner surface showed the structure of lower corneocytes with polygonal contour. With higher magnifications villous projections were seen in the cell membrane. PMID:26375232

In silico local structure approach: a case study on outer membrane proteins.

PubMed

Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

2008-04-01

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

PubMed

Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

2007-04-20

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

[In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

PubMed

Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

2010-01-01

Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence.

PubMed

Rossi, Rachael M; Yum, Lauren; Agaisse, Hervé; Payne, Shelley M

2017-08-29

Cardiolipin, an anionic phospholipid that resides at the poles of the inner and outer membranes, is synthesized primarily by the putative cardiolipin synthase ClsA in Shigella flexneri An S. flexneri clsA mutant had no cardiolipin detected within its membrane, grew normally in vitro , and invaded cultured epithelial cells, but it failed to form plaques in epithelial cell monolayers, indicating that cardiolipin is required for virulence. The clsA mutant was initially motile within the host cell cytoplasm but formed filaments and lost motility during replication and failed to spread efficiently to neighboring cells. Mutation of pbgA , which encodes the transporter for cardiolipin from the inner membrane to the outer membrane, also resulted in loss of plaque formation. The S. flexneri pbgA mutant had normal levels of cardiolipin in the inner membrane, but no cardiolipin was detected in the outer membrane. The pbgA mutant invaded and replicated normally within cultured epithelial cells but failed to localize the actin polymerization protein IcsA properly on the bacterial surface and was unable to spread to neighboring cells. The clsA mutant, but not the pbgA mutant, had increased phosphatidylglycerol in the outer membrane. This appeared to compensate partially for the loss of cardiolipin in the outer membrane, allowing some IcsA localization in the outer membrane of the clsA mutant. We propose a dual function for cardiolipin in S. flexneri pathogenesis. In the inner membrane, cardiolipin is essential for proper cell division during intracellular growth. In the outer membrane, cardiolipin facilitates proper presentation of IcsA on the bacterial surface. IMPORTANCE The human pathogen Shigella flexneri causes bacterial dysentery by invading colonic epithelial cells, rapidly multiplying within their cytoplasm, and then spreading intercellularly to neighboring cells. Worldwide, Shigella spp. infect hundreds of millions of people annually, with fatality rates up to 15%. Antibiotic treatment of Shigella infections is compromised by increasing antibiotic resistance, and there is no approved vaccine to prevent future infections. This has created a growing need to understand Shigella pathogenesis and identify new targets for antimicrobial therapeutics. Here we show a previously unknown role of phospholipids in S. flexneri pathogenesis. We demonstrate that cardiolipin is required in the outer membrane for proper surface localization of IcsA and in the inner membrane for cell division during growth in the host cell cytoplasm. Copyright © 2017 Rossi et al.

A heterozygous putative null mutation in ROM1 without a mutation in peripherin/RDS in a family with retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakuma, Hitoshi; Inana, G.; Murakami, Akira

1995-05-20

ROM1 is a 351-amino-acid, 37-kDa outer segment membrane protein of rod photoreceptors. ROM1 is related to peripherin/RDS, another outer segment membrane protein found in both rods and cones. The precise function of ROM1 or peripherin/RDS is not known, but they have been suggested to play important roles in the function and/or structure of the rod photoreceptor outer segment disks. A recent report implicated ROM1 in disease by suggesting that RP can be caused by a heterozygous null mutation in ROM1 but only in combination with another heterozygous mutation in peripherin/RDS. Screening of the ROM1 gene using polymerase chain reaction amplification,more » denaturing gradient gel electrophoresis, and direct DNA sequencing identified the same heterozygous putative null mutation in a family with RP.« less

Two-Dimensional Cochlear Micromechanics Measured In Vivo Demonstrate Radial Tuning within the Mouse Organ of Corti

PubMed Central

Lee, Hee Yoon; Raphael, Patrick D.; Xia, Anping; Kim, Jinkyung; Grillet, Nicolas; Applegate, Brian E.; Ellerbee Bowden, Audrey K.

2016-01-01

The exquisite sensitivity and frequency discrimination of mammalian hearing underlie the ability to understand complex speech in noise. This requires force generation by cochlear outer hair cells (OHCs) to amplify the basilar membrane traveling wave; however, it is unclear how amplification is achieved with sharp frequency tuning. Here we investigated the origin of tuning by measuring sound-induced 2-D vibrations within the mouse organ of Corti in vivo. Our goal was to determine the transfer function relating the radial shear between the structures that deflect the OHC bundle, the tectorial membrane and reticular lamina, to the transverse motion of the basilar membrane. We found that, after normalizing their responses to the vibration of the basilar membrane, the radial vibrations of the tectorial membrane and reticular lamina were tuned. The radial tuning peaked at a higher frequency than transverse basilar membrane tuning in the passive, postmortem condition. The radial tuning was similar in dead mice, indicating that this reflected passive, not active, mechanics. These findings were exaggerated in TectaC1509G/C1509G mice, where the tectorial membrane is detached from OHC stereocilia, arguing that the tuning of radial vibrations within the hair cell epithelium is distinct from tectorial membrane tuning. Together, these results reveal a passive, frequency-dependent contribution to cochlear filtering that is independent of basilar membrane filtering. These data argue that passive mechanics within the organ of Corti sharpen frequency selectivity by defining which OHCs enhance the vibration of the basilar membrane, thereby tuning the gain of cochlear amplification. SIGNIFICANCE STATEMENT Outer hair cells amplify the traveling wave within the mammalian cochlea. The resultant gain and frequency sharpening are necessary for speech discrimination, particularly in the presence of background noise. Here we measured the 2-D motion of the organ of Corti in mice and found that the structures that stimulate the outer hair cell stereocilia, the tectorial membrane and reticular lamina, were sharply tuned in the radial direction. Radial tuning was similar in dead mice and in mice lacking a tectorial membrane. This suggests that radial tuning comes from passive mechanics within the hair cell epithelium, and that these mechanics, at least in part, may tune the gain of cochlear amplification. PMID:27488636

Release of outer membrane vesicles from Bordetella pertussis.

PubMed

Hozbor, D; Rodriguez, M E; Fernández, J; Lagares, A; Guiso, N; Yantorno, O

1999-05-01

The aim of the study reported here was to investigate the production of Bordetella pertussis outer membrane vesicles (OMVs). Numerous vesicles released from cells grown in Stainer-Scholte liquid medium were observed. The formation of similar vesicle-like structures could also be artificially induced by sonication of concentrated bacterial suspensions. Immunoblot analysis showed that OMVs contain adenylate cyclase-hemolysin (AC-Hly), among other polypeptides, as well as the lipopolysaccharide (LPS). Experiments carried out employing purified AC-Hly and OMVs isolated from B. pertussis AC-Hly- showed that AC-Hly is an integral component of the vesicles. OMVs reported here contain several protective immunogens and might be considered a possible basic material for the development of acellular pertussis vaccines.

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

PubMed

Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

2003-07-01

Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

PubMed Central

Campanacci, Valérie; Bishop, Russell E.; Blangy, Stéphanie; Tegoni, Mariella; Cambillau, Christian

2016-01-01

Lipocalins, a widespread multifunctional family of small proteins (15–25 kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183–188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar Kd range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar Kd range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane. PMID:16920109

ELECTRON MICROSCOPE STUDY OF MYCOBACTERIUM LEPRAE AND ITS ENVIRONMENT IN A VESICULAR LEPROUS LESION

PubMed Central

Imaeda, Tamotsu; Convit, Jacinto

1962-01-01

Imaeda, Tamotsu (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela) and Jacinto Convit. Electron microscope study of Mycobacterium leprae and its environment in a vesicular leprous lesion. J. Bacteriol. 83:43–52. 1962.—Biopsied specimens of a borderline leprosy lesion were observed with the electron microscope. In this lesion, the majority of Mycobacterium leprae were laden with cytoplasmic components. The bacilli were separated from the cytoplasm of host cells by an enclosing membrane, thus differing from the environment of well-developed lepra cells in lepromatous lesions. The cell wall is composed of a moderately dense layer. A diffuse layer is discernible outside the cell wall, separated from it by a low density space. It is suggested that the cell wall is further coated by a low density layer, although the nature of the outermost diffuse layer has not yet been determined. The plasma membrane consists of a double layer, i.e., dense inner and outer layers separated by a low density space. The outer layer is closely adjacent to the cell wall. In the region where the outer layer of the plasma membrane enters the cytoplasm and is transformed into a complex membranous structure, the inner layer encloses this membranous configuration. Together they form the intracytoplasmic membrane system. In the bacterial cytoplasm, moderately dense, presumably polyphosphate bodies are apparent. As neither these bodies nor the intracytoplasmic membrane system are visible in the degenerating bacilli, it seems probable that these two components represent indicators of the state of bacillary activity. Images PMID:16561926

Structural and mechanistic insights into phospholipid transfer by Ups1–Mdm35 in mitochondria

PubMed Central

Watanabe, Yasunori; Tamura, Yasushi; Kawano, Shin; Endo, Toshiya

2015-01-01

Eukaryotic cells are compartmentalized into membrane-bounded organelles whose functions rely on lipid trafficking to achieve membrane-specific compositions of lipids. Here we focused on the Ups1–Mdm35 system, which mediates phosphatidic acid (PA) transfer between the outer and inner mitochondrial membranes, and determined the X-ray structures of Mdm35 and Ups1–Mdm35 with and without PA. The Ups1–Mdm35 complex constitutes a single domain that has a deep pocket and flexible Ω-loop lid. Structure-based mutational analyses revealed that a basic residue at the pocket bottom and the Ω-loop lid are important for PA extraction from the membrane following Ups1 binding. Ups1 binding to the membrane is enhanced by the dissociation of Mdm35. We also show that basic residues around the pocket entrance are important for Ups1 binding to the membrane and PA extraction. These results provide a structural basis for understanding the mechanism of PA transfer between mitochondrial membranes. PMID:26235513

Structural engineering of a phage lysin that targets Gram-negative pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lukacik, Petra; Barnard, Travis J.; Keller, Paul W.

Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against Gram-positive organisms but not against Gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a {beta}-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and anothermore » that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.« less

Molecular Mechanism of Uptake of Cationic Photoantimicrobial Phthalocyanine across Bacterial Membranes Revealed by Molecular Dynamics Simulations.

PubMed

Orekhov, Philipp S; Kholina, Ekaterina G; Bozdaganyan, Marine E; Nesterenko, Alexey M; Kovalenko, Ilya B; Strakhovskaya, Marina G

2018-04-12

Phthalocyanines are aromatic macrocyclic compounds, which are structurally related to porphyrins. In clinical practice, phthalocyanines are used in fluorescence imaging and photodynamic therapy of cancer and noncancer lesions. Certain forms of the substituted polycationic metallophthalocyanines have been previously shown to be active in photodynamic inactivation of both Gram-negative and Gram-positive bacteria; one of them is zinc octakis(cholinyl)phthalocyanine (ZnPcChol 8+ ). However, the molecular details of how these compounds translocate across bacterial membranes still remain unclear. In the present work, we have developed a coarse-grained (CG) molecular model of ZnPcChol 8+ within the framework of the popular MARTINI CG force field. The obtained model was used to probe the solvation behavior of phthalocyanine molecules, which agreed with experimental results. Subsequently, it was used to investigate the molecular details of interactions between phthalocyanines and membranes of various compositions. The results demonstrate that ZnPcChol 8+ has high affinity to both the inner and the outer model membranes of Gram-negative bacteria, although this species does not show noticeable affinity to the 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine membrane. Furthermore, we found out that the process of ZnPcChol 8+ penetration toward the center of the outer bacterial membrane is energetically favorable and leads to its overall disturbance and formation of the aqueous pore. Such intramembrane localization of ZnPcChol 8+ suggests their twofold cytotoxic effect on bacterial cells: (1) via induction of lipid peroxidation by enhanced production of reactive oxygen species (i.e., photodynamic toxicity); (2) via rendering the bacterial membrane more permeable for additional Pc molecules as well as other compounds. We also found that the kinetics of penetration depends on the presence of phospholipid defects in the lipopolysaccharide leaflet of the outer membrane and the type of counterions, which stabilize it. Thus, the results of our simulations provide a detailed molecular view of ZnPcChol 8+ "self-promoted uptake", the pathway previously proposed for some small molecules crossing the outer bacterial membrane.

Visco-Elastic Membrane Tethers Extracted from Escherichia coli by Optical Tweezers

PubMed Central

Jauffred, Liselotte; Callisen, Thomas Hønger; Oddershede, Lene Broeng

2007-01-01

Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10–12 pN/μm describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6–7 pN/μm, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion. PMID:17704145

Refolding, crystallization and preliminary X-ray crystallographic studies of the β-barrel domain of BamA, a membrane protein essential for outer membrane protein biogenesis.

PubMed

Ni, Dongchun; Yang, Kun; Huang, Yihua

2014-03-01

In Gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a five-protein β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. Here, the refolding, crystallization and preliminary X-ray crystallographic characterization of the β-barrel domain of BamA from Escherichia coli (EcBamA) are reported. Native and selenomethionine-substituted EcBamA proteins were crystallized at 16°C and X-ray diffraction data were collected to 2.6 and 3.7 Å resolution, respectively. The native crystals belonged to space group P21212, with unit-cell parameters a = 118.492, b = 159.883, c = 56.000 Å and two molecules in one asymmetric unit; selenomethionine-substituted protein crystals belonged to space group P4322, with unit-cell parameters a = b = 163.162, c = 46.388 Å and one molecule in one asymmetric unit. Initial phases for EcBamA β-barrel domain were obtained from a SeMet SAD data set. These preliminary X-ray crystallographic studies paved the way for further structural determination of the β-barrel domain of EcBamA.

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

PubMed Central

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment. PMID:23350036

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

PubMed

Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

Superresolution Imaging Captures Carbohydrate Utilization Dynamics in Human Gut Symbionts

PubMed Central

Karunatilaka, Krishanthi S.; Cameron, Elizabeth A.; Martens, Eric C.; Koropatkin, Nicole M.

2014-01-01

ABSTRACT Gut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the model Bacteroides thetaiotaomicron starch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on the B. thetaiotaomicron cell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to control Bacteroidetes in the intestinal tract to enhance human health and treat disease. PMID:25389179

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

PubMed Central

Hernáez, Bruno; Guerra, Milagros; Salas, María L.

2016-01-01

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

PubMed

Caveney, Nathanael A; Li, Franco Kk; Strynadka, Natalie Cj

2018-06-06

The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Atomic force microscopy study of erythrocyte shape and membrane structure after treatment with a dihydropyridinic drug

NASA Astrophysics Data System (ADS)

Girasole, M.; Cricenti, A.; Generosi, R.; Congiu-Castellano, A.; Boffi, F.; Arcovito, A.; Boumis, G.; Amiconi, G.

2000-06-01

The overall shape and membrane surface of human erythrocytes (RBCs) in the presence of nifedipine (a dihydropyridinic drug used in the clinical treatment of hypertension and angina pectoris) were imaged by contact-mode atomic force microscopy. Nifedipine induces in RBCs relevant morphological changes the extent of which increases as a function of drug concentration and incubation time. The modifications have been interpreted as mainly due to insertion of nifedipine into the outer layer of the RBC membrane. The potential effect of nifedipine as a hemoglobin denaturant has been ruled out by x-ray absorption near-edge structure and optical spectroscopies.

Suppressor Analysis of the Fusogenic Lambda Spanins.

PubMed

Cahill, Jesse; Rajaure, Manoj; Holt, Ashley; Moreland, Russell; O'Leary, Chandler; Kulkarni, Aneesha; Sloan, Jordan; Young, Ry

2017-07-15

The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality. IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system. Copyright © 2017 American Society for Microbiology.

Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1.

PubMed

Yun, Sung Ho; Lee, Sang-Yeop; Choi, Chi-Won; Lee, Hayoung; Ro, Hyun-Joo; Jun, Sangmi; Kwon, Yong Min; Kwon, Kae Kyoung; Kim, Sang-Jin; Kim, Gun-Hwa; Kim, Seung Il

2017-01-01

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV Novo ) are spherical in shape, and the average diameter of OMV Novo is 25-70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV Novo . Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.

Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judd, R.C.; Caldwell, H.D.

1985-01-01

The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. Inmore » addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.« less

Study of zinc-induced changes in lymphocyte membranes using atomic force microscopy, luminescence, and light scattering methods

NASA Astrophysics Data System (ADS)

Filimonenko, D. S.; Khairullina, A. Ya.; Yasinskii, V. M.; Kozlova, N. M.; Zubritskaja, G. P.; Slobozhanina, E. I.

2011-07-01

Changes in the surface structure of lymphocyte membranes exposed to various concentrations of zinc ions are studied. It is found by atomic force microscopy that increasing the concentration of zinc ions leads to a reduction in the correlation length of the autocorrelation function of the roughness profile of a lymphocyte compared to control samples; this may indicate the existence of fine structure in the membrane surface. Fluorescence markers are used to observe a reduction in the microviscosity of the lipids in the outer monolayer of the lipid bilayer after lymphocytes are exposed to Zn ions, as well as the exposure of phosphatidylserine on the surface membrane, and the oxidation of HS-groups of membrane proteins. Calculations of the absorption coefficients of lymphocytes modified with zinc reveal the existence of absorption bands owing to the formation of metal-protein complexes and zinc oxide nanoparticles. These results indicate significant changes in the structural and functional state of lymphocyte membranes exposed to zinc ions.

A dominant sulfhydryl-containing protein in the outer membrane of Neisseria gonorrhoeae.

PubMed Central

Norrod, E P; Browne, S L; Feldweg, A; Leonard, J

1993-01-01

By using a method that labels sulfhydryl-containing proteins in situ, we have detected a major outer membrane protein of Neisseria gonorrhoeae at 41 kDa. A protein of this molecular mass has not previously been shown to be a major outer membrane protein in gonococci. In addition, a minor protein rich in cysteinyl residues was detected at 31.5 kDa. Images PMID:8432710

Can direct extracellular electron transfer occur in the absence of outer membrane cytochromes in Desulfovibrio vulgaris?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elias, Dwayne A; Zane, Mr. Grant M.; Auer, Dr. Manfred

2010-01-01

Extracellular electron transfer has been investigated over several decades via forms of soluble electron transfer proteins that are exported for extracellular reoxidation. More recently, several organisms have been shown to reduce extracellular metals via the direct transfer of electron through appendages; also known as nanowires. They have been reported most predominantly in Shewanella and Geobacter. While the relevancy and composition of these structures in each genus has been debated, both possess outer membrane cytochrome complexes that could theoretically come into direct contact with solid phase oxidized metals. Members of the genus Desulfovibrio apparently have no such cytochromes although similar appendagesmore » are present, are electrically conductive, and are different from flagella. Upon U(VI)-reduction, the structures in Desulfovibrio become coated with U(IV). Deletion of flagellar genes did not alter soluble or amorphous Fe(III) or U(VI) reduction, or appendage appearance. Removal of the chromosomal pilA gene hampered amorphous Fe(III)-reduction by ca. 25%, but cells lacking the native plasmid, pDV1, reduced soluble Fe(III) and U(VI) at ca. 50% of the wild type rate while amorphous Fe(III)-reduction slowed to ca. 20% of the wild type rate. Appendages were present in all deletions as well as pDV1, except pilA. Gene complementation restored all activities and morphologies to wild type levels. This suggests that pilA encodes the structural component, whereas genes within pDV1 may provide the reactive members. How such appendages function without outer membrane cytochromes is under investigation.« less

Anatomical correlates to the bands seen in the outer retina by optical coherence tomography: literature review and model.

PubMed

Spaide, Richard F; Curcio, Christine A

2011-09-01

To evaluate the validity of commonly used anatomical designations for the four hyperreflective outer retinal bands seen in current-generation optical coherence tomography, a scale model of outer retinal morphology was created using published information for direct comparison with optical coherence tomography scans. Articles and books concerning histology of the outer retina from 1900 until 2009 were evaluated, and data were used to create a scale model drawing. Boundaries between outer retinal tissue compartments described by the model were compared with intensity variations of representative spectral-domain optical coherence tomography scans using longitudinal reflectance profiles to determine the region of origin of the hyperreflective outer retinal bands. This analysis showed a high likelihood that the spectral-domain optical coherence tomography bands attributed to the external limiting membrane (the first, innermost band) and to the retinal pigment epithelium (the fourth, outermost band) are correctly attributed. Comparative analysis showed that the second band, often attributed to the boundary between inner and outer segments of the photoreceptors, actually aligns with the ellipsoid portion of the inner segments. The third band corresponded to an ensheathment of the cone outer segments by apical processes of the retinal pigment epithelium in a structure known as the contact cylinder. Anatomical attributions and subsequent pathophysiologic assessments pertaining to the second and third outer retinal hyperreflective bands may not be correct. This analysis has identified testable hypotheses for the actual correlates of the second and third bands. Nonretinal pigment epithelium contributions to the fourth band (e.g., Bruch membrane) remain to be determined.

Targeted Protein Degradation of Outer Membrane Decaheme Cytochrome MtrC Metal Reductase in Shewanella oneidensis MR-1 Measured Using Biarsenical Probe CrAsH-EDT2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yijia; Chen, Baowei; Shi, Liang

2011-10-14

Development of efficient microbial biofuel cells requires an ability to exploit interfacial electron transfer reactions to external electron acceptors, such as metal oxides; such reactions occur in the facultative anaerobic gram-negative bacterium Shewanella oneidensis MR-1 through the catalytic activity of the outer membrane decaheme c-type cytochrome MtrC. Central to the utility of this pathway to synthetic biology is an understanding of cellular mechanisms that maintain optimal MtrC function, cellular localization, and renewal by degradation and resynthesis. In order to monitor trafficking to the outer membrane, and the environmental sensitivity of MtrC, we have engineered a tetracysteine tag (i.e., CCPGCC) atmore » its C-terminus that permits labeling by the cell impermeable biarsenical fluorophore, carboxy-FlAsH (CrAsH) of MtrC at the surface of living Shewanella oneidensis MR-1 cells. In comparison, the cell permeable reagent FlAsH permits labeling of the entire population of MtrC, including proteolytic fragments resulting from incorrect maturation. We demonstrate specific labeling by CrAsH of engineered MtrC which is dependent on the presence of a functional type-2 secretion system (T2S), as evidenced by T2S system gspD or gspG deletion mutants which are incapable of CrAsH labeling. Under these latter conditions, MtrC undergoes proteolytic degradation to form a large 35-38 kDa fragment; this degradation product is also resolved during normal turnover of the CrAsH-labeled MtrC protein. No MtrC protein is released into the medium during turnover, suggesting the presence of cellular turnover systems involving MtrC reuptake and degradation. The mature MtrC localized on the outer membrane is a long-lived protein, with a turnover rate of 0.043 hr-1 that is insensitive to O2 concentration. Maturation of MtrC is relatively inefficient, with substantial rates of turnover of the immature protein prior to export to the outer membrane (i.e., 0.028 hr-1) that are consistent with the inherent complexity associated with correct heme insertion and acylation of MtrC that occurs in the periplasm prior to its targeting to the outer membrane. These latter results suggest that MtrC protein trafficking to the outer membrane and its subsequent degradation are tightly regulated, which is consistent with cellular processing pathways that target MtrC to extracellular structures and their possible role in promoting electron transfer from Shewanella to extracellular acceptors.« less

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

PubMed Central

Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

2011-01-01

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion.

PubMed

Cascales, Eric; Christie, Peter J

2004-12-07

Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB/D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 "bridge" between inner and outer membrane subassemblies of the VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope.

Identification of a cell epitope that is globally conserved among outer membrane proteins (OMPs) OMP7, OMP8, and OMP9 of anaplasma marginale strains and with OMP7 from the A. marginale subsp. centrale vaccine strain

USDA-ARS?s Scientific Manuscript database

Within the protective outer membrane fraction of Anaplasma marginale, several vaccine candidates have emerged, including a family of outer membrane proteins (OMPs) 7-9, which share sequence identity with each other and with the single protein OMP7 in the vaccine strain A. marginale subsp. centrale. ...

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

PubMed

Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2017-11-01

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

PubMed

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

PubMed Central

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Molecular analysis of interactions between dendrimers and asymmetric membranes at different transport stages.

PubMed

He, XiaoCong; Qu, ZhiGuo; Xu, Feng; Lin, Min; Wang, JiuLing; Shi, XingHua; Lu, TianJian

2014-01-07

Studying dendrimer-biomembrane interactions is important for understanding drug and gene delivery. In this study, coarse-grained molecular dynamics simulations were performed to investigate the behaviors of polyamidoamine (PAMAM) dendrimers (G4 and G5) as they interacted with asymmetric membranes from different sides of the bilayer, thus mimicking different dendrimer transport stages. The G4 dendrimer could insert into the membrane during an equilibrated state, and the G5 dendrimer could induce pore formation in the membrane when the dendrimers interacted with the outer side (outer interactions) of an asymmetric membrane [with 10% dipalmitoyl phosphatidylserine (DPPS) in the inner leaflet of the membrane]. During the interaction with the inner side of the asymmetric membrane (inner interactions), the G4 and G5 dendrimers only adsorbed onto the membrane. As the membrane asymmetry increased (e.g., increased DPPS percentage in the inner leaflet of the membrane), the G4 and G5 dendrimers penetrated deeper into the membrane during the outer interactions and the G4 and G5 dendrimers were adsorbed more tightly onto the membrane for the inner interactions. When the DPPS content reached 50%, the G4 dendrimer could completely penetrate through the membrane from the outer side to the inner side. Our study provides molecular understanding and reference information about different dendrimer transport stages during drug and gene delivery.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis

PubMed Central

Elgass, Kirstin D.; Gabriel, Kipros; Dougan, Gordon; Lithgow, Trevor; Heinz, Eva

2018-01-01

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity. PMID:29601598

Periplasmic quality control in biogenesis of outer membrane proteins.

PubMed

Lyu, Zhi Xin; Zhao, Xin Sheng

2015-04-01

The β-barrel outer membrane proteins (OMPs) are integral membrane proteins that reside in the outer membrane of Gram-negative bacteria and perform a diverse range of biological functions. Synthesized in the cytoplasm, OMPs must be transported across the inner membrane and through the periplasmic space before they are assembled in the outer membrane. In Escherichia coli, Skp, SurA and DegP are the most prominent factors identified to guide OMPs across the periplasm and to play the role of quality control. Although extensive genetic and biochemical analyses have revealed many basic functions of these periplasmic proteins, the mechanism of their collaboration in assisting the folding and insertion of OMPs is much less understood. Recently, biophysical approaches have shed light on the identification of the intricate network. In the present review, we summarize recent advances in the characterization of these key factors, with a special emphasis on the multifunctional protein DegP. In addition, we present our proposed model on the periplasmic quality control in biogenesis of OMPs.

Evolution of the Translocation and Assembly Module (TAM)

PubMed Central

Heinz, Eva; Selkrig, Joel; Belousoff, Matthew J.; Lithgow, Trevor

2015-01-01

Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi. PMID:25994932

The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm

PubMed Central

Kaplan, Christopher W.; Lux, Renate; Haake, Susan Kinder; Shi, Wenyuan

2009-01-01

Summary A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation. PMID:19007407

The Sus operon: a model system for starch uptake by the human gut Bacteroidetes

PubMed Central

Foley, Matthew H.; Cockburn, Darrell W.; Koropatkin, Nicole M.

2016-01-01

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-up-take system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex. PMID:27137179

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex ▿ †

PubMed Central

Pinto-Santini, Delia M.; Salama, Nina R.

2009-01-01

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein. PMID:19801411

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valentine, Jenny L.; Chen, Linxiao; Perregaux, Emily C.

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced highmore » titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.« less

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

DOE PAGES

Valentine, Jenny L.; Chen, Linxiao; Perregaux, Emily C.; ...

2016-06-23

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatory activity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced highmore » titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, in a serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.« less

The role of "blebbing" in overcoming the hydrophobic barrier during biooxidation of elemental sulfur by Thiobacillus thiooxidans

USGS Publications Warehouse

Knickerbocker, C.; Nordstrom, D. Kirk; Southam, G.

2000-01-01

Brimstone Basin, in southeastern Yellowstone National Park, Wyoming is an ancient hydrothermal area containing solfataric alteration. Drainage waters flowing from Brimstone Basin had pH values as low as 1.23 and contained up to 1.7×106 MPN/ml acidophilic sulfur-oxidizing bacteria. Thiobacillus thiooxidans was the dominant sulfur-oxidizing bacterium recovered from an enrichment culture and was used in a structural examination of bacterial sulfur oxidation. Growth in these sulfur cultures occurred in two phases with cells in association with the macroscopic sulfur grains and in suspension above these grains. Colonization of sulfur grains by individual cells and microcolonies was facilitated by organic material that appeared to be responsible for bacterial adhesion. Transmission electron microscopy of negatively stained (2% [wt./vol.] uranyl acetate), sulfur-grown T. thiooxidans revealed extensive membrane blebbing (sloughing of outer membrane vesicles) and the presence of approximately 100 nm sized sulfur particles adsorbed to membrane material surrounding individual bacteria. Sulfite-grown bacteria did not possess membrane blebs. The amphipathic nature of these outer membrane vesicles appear to be responsible for overcoming the hydrophobic barrier necessary for the growth of T. thiooxidans on elemental sulfur.

Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

PubMed

Grancara, Silvia; Ohkubo, Shinji; Artico, Marco; Ciccariello, Mauro; Manente, Sabrina; Bragadin, Marcantonio; Toninello, Antonio; Agostinelli, Enzo

2016-10-01

Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria which distinguish them from other amines. Enzymatic oxidative deamination of spermine by amine oxidases in tumor cells may produce reactive oxygen species, leading to transition pore opening and apoptosis. This process could be exploited as a new therapeutic strategy to combat cancer.

A distance measurement between specific sites on the cytoplasmic surface of bovine rhodopsin in rod outer segment disk membranes.

PubMed

Albert, A D; Watts, A; Spooner, P; Groebner, G; Young, J; Yeagle, P L

1997-08-14

Structural information on mammalian integral membrane proteins is scarce. As part of work on an alternative approach to the structure of bovine rhodopsin, a method was devised to obtain an intramolecular distance between two specific sites on rhodopsin while in the rod outer segment disk membrane. In this report, the distance between the rhodopsin kinase phosphorylation site(s) on the carboxyl terminal and the top of the third transmembrane helix was measured on native rhodopsin. Rhodopsin was labeled with a nuclear spin label (31P) by limited phosphorylation with rhodopsin kinase. Major phosphorylation occurs at serines 343 and 338 on the carboxyl terminal. The phosphorylated rhodopsin was then specifically labeled on cysteine 140 with an electron spin label. Magic angle spinning 31P-nuclear magnetic resonance revealed the resonance arising from the phosphorylated protein. The enhancement of the transverse relaxation of this resonance by the paramagnetic spin label was observed. The strength of this perturbation was used to determine the through-space distance between the phosphorylation site(s) and the spin label position. A distance of 18 +/- 3 A was obtained.

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

PubMed

Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

2009-05-01

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

PubMed

Gornicka, Agnieszka; Bragoszewski, Piotr; Chroscicki, Piotr; Wenz, Lena-Sophie; Schulz, Christian; Rehling, Peter; Chacinska, Agnieszka

2014-12-15

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex. © 2014 Gornicka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structure of a Type-1 Secretion System ABC Transporter.

PubMed

Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

2017-03-07

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure and function of POTRA domains of Omp85/TPS superfamily.

PubMed

Simmerman, Richard F; Dave, Ashita M; Bruce, Barry D

2014-01-01

The Omp85/TPS (outer-membrane protein of 85 kDa/two-partner secretion) superfamily is a ubiquitous and major class of β-barrel proteins. This superfamily is restricted to the outer membranes of gram-negative bacteria, mitochondria, and chloroplasts. The common architecture, with an N-terminus consisting of repeats of soluble polypeptide-transport-associated (POTRA) domains and a C-terminal β-barrel pore is highly conserved. The structures of multiple POTRA domains and one full-length TPS protein have been solved, yet discovering roles of individual POTRA domains has been difficult. This review focuses on similarities and differences between POTRA structures, emphasizing POTRA domains in autotrophic organisms including plants and cyanobacteria. Unique roles, specific for certain POTRA domains, are examined in the context of POTRA location with respect to their attachment to the β-barrel pore, and their degree of biological dispensability. Finally, because many POTRA domains may have the ability to interact with thousands of partner proteins, possible modes of these interactions are also explored. © 2014 Elsevier Inc. All rights reserved.

Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG.

PubMed

Luo, Qingshan; Yang, Xu; Yu, Shan; Shi, Huigang; Wang, Kun; Xiao, Le; Zhu, Guangyu; Sun, Chuanqi; Li, Tingting; Li, Dianfan; Zhang, Xinzheng; Zhou, Min; Huang, Yihua

2017-05-01

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB 2 FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB 2 FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB 2 FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

PubMed Central

Korotkov, Konstantin V.; Pardon, Els

2009-01-01

Summary Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a “nanobody”, the VHH domain of a “heavy-chain” camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains with the second and third subdomains exhibiting the KH-fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible β-strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by β-strand complementation. PMID:19217396

Human dermal papilla cells and outer root sheath cells: no follicular differentiation in nude mice and chicken embryos.

PubMed

Chiu, H C; Chang, C H; Jee, S H; Chang, C C; Wu, Y C

1994-09-01

Human scalp specimens were incubated in 5 U/ml dispase solution at 4 degrees C overnight before the isolation of dermal papillae and follicle epithelium. This pretreatment not only facilitated the attachment and cell outgrowth of dermal papillae but also made it easier to pluck out hairs with intact follicle epithelium. The outer root sheath cells were released from the follicle epithelium and grown on a feeder layer of mitomycin C-treated human dermal fibroblasts. The subcultured outer root sheath cells were grown in a serum-free medium. When the mixtures of early-passage dermal papilla cells and outer root sheath cells were injected into the subcutis of nude mice, an epidermal cyst surrounded by layers of fibrous tissue was found in three weeks. No hair follicles were found when the mixtures were implanted onto the chorioallantoic membrane of nine-day-old chicken embryos. A keratinized mass lying on the chorionic epithelium with or without smaller similar masses in the chorioallantoic membrane was found in eight days. No hair follicle-like structure could be found. Possible factors contributing to the failure to undergo follicular differentiation in this study are discussed.

Cell Envelope of Corynebacteria: Structure and Influence on Pathogenicity

PubMed Central

Burkovski, Andreas

2013-01-01

To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper. PMID:23724339

Cell envelope of corynebacteria: structure and influence on pathogenicity.

PubMed

Burkovski, Andreas

2013-01-01

To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper.

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE.

PubMed

Phoenix, D A

1996-12-01

Within gram-negative bacteria such as Escherichia coli, the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.

Phytochemicals prevent mitochondrial membrane permeabilization and protect SH-SY5Y cells against apoptosis induced by PK11195, a ligand for outer membrane translocator protein.

PubMed

Wu, Yuqiu; Shamoto-Nagai, Masayo; Maruyama, Wakako; Osawa, Toshihiko; Naoi, Makoto

2017-01-01

Epidemiological studies present the beneficial effects of dietary habits on prevention of aging-associated decline of brain function. Phytochemicals, the second metabolites of food, protect neuronal cells from cell death in cellular models of neurodegenerative disorders, and the neuroprotective activity has been ascribed to the anti-oxidant and anti-inflammatory functions. In this paper, the cellular mechanism of neuroprotection by phytochemicals was investigated, using the cellular model of mitochondrial apoptosis induced by PK11195, a ligand of outer membrane translocator protein, in SH-SY5Y cells. PK11195 induced mitochondrial membrane permeabilization with rapid transit production of superoxide (superoxide flashes) and calcium release from mitochondria, and activated apoptosis signal pathway. Study on the structure-activity relationship of astaxanthin, ferulic acid derivatives, and sesame lignans revealed that these phytochemicals inhibited mitochondrial membrane permeabilization and protected cells from apoptosis. Ferulic acid derivatives and sesame lignans inhibited or enhanced the mitochondrial pore formation and cell death by PK11195 according to their amphiphilic properties, not directly depending on the antioxidant activity. Regulation of pore formation at mitochondrial membrane is discussed as a novel mechanism behind neuroprotective activity of phytochemicals in aging and age-associated neurodegenerative disorders, and also behind dual functions of phytochemicals in neuronal and cancer cells.

Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae.

PubMed

Velkov, Tony; Deris, Zakuan Z; Huang, Johnny X; Azad, Mohammad A K; Butler, Mark; Sivanesan, Sivashangarie; Kaminskas, Lisa M; Dong, Yao-Da; Boyd, Ben; Baker, Mark A; Cooper, Matthew A; Nation, Roger L; Li, Jian

2014-05-01

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

An inducible amphipathic helix within the intrinsically disordered C terminus can participate in membrane curvature generation by peripherin-2/rds.

PubMed

Milstein, Michelle L; Kimler, Victoria A; Ghatak, Chiranjib; Ladokhin, Alexey S; Goldberg, Andrew F X

2017-05-12

Peripherin-2/rds is required for biogenesis of vertebrate photoreceptor outer segment organelles. Its localization at the high-curvature rim domains of outer segment disk membranes suggests that it may act to shape these structures; however, the molecular function of this protein is not yet resolved. Here, we apply biochemical, biophysical, and imaging techniques to elucidate the role(s) played by the protein's intrinsically disordered C-terminal domain and an incipient amphipathic α-helix contained within it. We investigated a deletion mutant lacking only this α-helix in stable cell lines and Xenopus laevis photoreceptors. We also studied a soluble form of the full-length ∼7-kDa cytoplasmic C terminus in cultured cells and purified from Escherichia coli The α-helical motif was not required for protein biosynthesis, tetrameric subunit assembly, tetramer polymerization, localization at disk rims, interaction with GARP2, or the generation of membrane curvature. Interestingly, however, loss of the helical motif up-regulated membrane curvature generation in cellulo , introducing the possibility that it may regulate this activity in photoreceptors. Furthermore, the incipient α-helix (within the purified soluble C terminus) partitioned into membranes only when its acidic residues were neutralized by protonation. This suggests that within the context of full-length peripherin-2/rds, partitioning would most likely occur at a bilayer interfacial region, potentially adjacent to the protein's transmembrane domains. In sum, this study significantly strengthens the evidence that peripherin-2/rds functions directly to shape the high-curvature rim domains of the outer segment disk and suggests that the protein's C terminus may modulate membrane curvature-generating activity present in other protein domains. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson's disease.

PubMed

Fiskum, Gary; Starkov, Anatoly; Polster, Brian M; Chinopoulos, Christos

2003-06-01

Mitochondrial dysfunction, due to either environmental or genetic factors, can result in excessive production of reactive oxygen species, triggering the apoptotic death of dopaminergic cells in Parkinson's disease. Mitochondrial free radical production is promoted by the inhibition of electron transport at any point distal to the sites of superoxide production. Neurotoxins that induce parkinsonian neuropathology, such as MPP(+) and rotenone, stimulate superoxide production at complex I of the electron transport chain and also stimulate free radical production at proximal redox sites including mitochondrial matrix dehydrogenases. The oxidative stress caused by elevated mitochondrial production of reactive oxygen species promotes the expression and (or) intracellular distribution of the proapoptotic protein Bax to the mitochondrial outer membrane. Interactions between Bax and BH3 death domain proteins such as tBid result in Bax membrane integration, oligomerization, and permeabilization of the outer membrane to intermembrane proteins such as cytochrome c. Once released into the cytosol, cytochrome c together with other proteins activates the caspase cascade of protease activities that mediate the biochemical and morphological alterations characteristic of apoptosis. In addition, loss of mitochondrial cytochrome c stimulates mitochondrial free radical production, further promoting cell death pathways. Excessive mitochondrial Ca(2+) accumulation can also release cytochrome c and promote superoxide production through a mechanism distinctly different from that of Bax. Ca(2+) activates a mitochondrial inner membrane permeability transition causing osmotic swelling, rupture of the outer membrane, and complete loss of mitochondrial structural and functional integrity. While amphiphilic cations, such as dibucaine and propranolol, inhibit Bax-mediated cytochrome c release, transient receptor potential channel inhibitors inhibit mitochondrial swelling and cytochrome c release induced by the inner membrane permeability transition. These advances in the knowledge of mitochondrial cell death mechanisms and their inhibitors may lead to neuroprotective interventions applicable to Parkinsons's disease.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles*

PubMed Central

Haurat, M. Florencia; Aduse-Opoku, Joseph; Rangarajan, Minnie; Dorobantu, Loredana; Gray, Murray R.; Curtis, Michael A.; Feldman, Mario F.

2011-01-01

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions. PMID:21056982

Production of Recombinant Injectosome and Outer Membrane Proteins from Yersinia Pestis KIM5

DTIC Science & Technology

2009-06-01

Overview ........................................................................ 45 20. Test Ammonium Sulfate Precipitation Overview...52 24. LcrV and YscF Test Expressions .................................................................. 53...coli modify the proteins’ primary structure post - translationally? 1.5 Significance of Results The study of VHH as immunotherapeutics for combating

Structure of a bacterial cell surface decaheme electron conduit

USDA-ARS?s Scientific Manuscript database

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

The 3D structures of VDAC represent a native conformation

PubMed Central

Hiller, Sebastian; Abramson, Jeff; Mannella, Carmen; Wagner, Gerhard; Zeth, Kornelius

2010-01-01

The most abundant protein of the mitochondrial outer membrane is the voltage-dependent anion channel (VDAC), which facilitates the exchange of ions and molecules between mitochondria and cytosol and is regulated by interactions with other proteins and small molecules. VDAC has been extensively studied for more than three decades, and last year three independent investigations revealed a structure of VDAC-1 exhibiting 19 transmembrane β-strands, constituting a unique structural class of β-barrel membrane proteins. Here, we provide a historical perspective on VDAC research and give an overview of the experimental design used to obtain these structures. Furthermore, we validate the protein refolding approach and summarize biochemical and biophysical evidence that links the 19-stranded structure to the native form of VDAC. PMID:20708406

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties[S

PubMed Central

Sousa, Tânia; Castro, Rui E.; Pinto, Sandra N.; Coutinho, Ana; Lucas, Susana D.; Moreira, Rui; Rodrigues, Cecília M. P.; Prieto, Manuel; Fernandes, Fábio

2015-01-01

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure. PMID:26351365

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System.

PubMed

Vanderlinde, Elizabeth M; Strozen, Timothy G; Hernández, Sara B; Cava, Felipe; Howard, S Peter

2017-04-15

In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila , outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila In a heterologous assembly model in E. coli , expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly. Copyright © 2017 American Society for Microbiology.

The Free Energy of Small Solute Permeation through the Escherichia coli Outer Membrane Has a Distinctly Asymmetric Profile

DOE PAGES

Carpenter, Timothy S.; Parkin, Jamie; Khalid, Syma

2016-08-12

Permeation of small molecules across cell membranes is a ubiquitous process in biology and is dependent on the principles of physical chemistry at the molecular level. Here we use atomistic molecular dynamics simulations to calculate the free energy of permeation of a range of small molecules through a model of the outer membrane of Escherichia coli, an archetypical Gram-negative bacterium. The model membrane contains lipopolysaccharide (LPS) molecules in the outer leaflet and phospholipids in the inner leaflet. Our results show that the energetic barriers to permeation through the two leaflets of the membrane are distinctly asymmetric; the LPS headgroups providemore » a less energetically favorable environment for organic compounds than do phospholipids. In summary, we provide the first reported estimates of the relative free energies associated with the different chemical environments experienced by solutes as they attempt to cross the outer membrane of a Gram-negative bacterium. Furthermore, these results provide key insights for the development of novel antibiotics that target these bacteria.« less

The Free Energy of Small Solute Permeation through the Escherichia coli Outer Membrane Has a Distinctly Asymmetric Profile

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carpenter, Timothy S.; Parkin, Jamie; Khalid, Syma

Permeation of small molecules across cell membranes is a ubiquitous process in biology and is dependent on the principles of physical chemistry at the molecular level. Here we use atomistic molecular dynamics simulations to calculate the free energy of permeation of a range of small molecules through a model of the outer membrane of Escherichia coli, an archetypical Gram-negative bacterium. The model membrane contains lipopolysaccharide (LPS) molecules in the outer leaflet and phospholipids in the inner leaflet. Our results show that the energetic barriers to permeation through the two leaflets of the membrane are distinctly asymmetric; the LPS headgroups providemore » a less energetically favorable environment for organic compounds than do phospholipids. In summary, we provide the first reported estimates of the relative free energies associated with the different chemical environments experienced by solutes as they attempt to cross the outer membrane of a Gram-negative bacterium. Furthermore, these results provide key insights for the development of novel antibiotics that target these bacteria.« less

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins.

PubMed

Imai, Kenichiro; Fujita, Naoya; Gromiha, M Michael; Horton, Paul

2011-01-28

The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.

Identification of outer membrane proteins with emulsifying activity by prediction of beta-barrel regions.

PubMed

Walzer, Gil; Rosenberg, Eugene; Ron, Eliora Z

2009-01-01

Microbial bioemulsifiers are secreted by many bacteria and are important for bacterial interactions with hydrophobic substrates or nutrients and for a variety of biotechnological applications. We have recently shown that the OmpA protein in several members of the Acinetobacter family has emulsifying properties. These properties of OmpA depend on the amino acid composition of four putative extra-membrane loops, which in various strains of Acinetobacter, but not in E. coli, are highly hydrophobic. As many Acinetobacter strains can utilize hydrophobic carbon sources, such as oil, the emulsifying activity of their OmpA may be important for the utilization and uptake of hydrocarbons. We assumed that if outer membrane proteins with emulsifying activity are physiologically important, they may exist in additional oil degrading bacteria. In order to identify such proteins, it was necessary to obtain bioinformatics-based predictions for hydrophobic extra-membrane loops. Here we describe a method for using protein sequence data for predicting the hydrophobic properties of the extra-membrane loops of outer membrane proteins. The feasibility of this method is demonstrated by its use to identify a new microbial bioemulsifier - OprG - an outer membrane protein of the oil degrading Pseudomonas putida KT2440.

Mutation K42E in dehydrodolichol diphosphate synthase (DHDDS) causes recessive retinitis pigmentosa.

PubMed

Lam, Byron L; Züchner, Stephan L; Dallman, Julia; Wen, Rong; Alfonso, Eduardo C; Vance, Jeffery M; Peričak-Vance, Margaret A

2014-01-01

A single-nucleotide mutation in the gene that encodes DHDDS has been identified by whole exome sequencing as the cause of the non-syndromic recessive retinitis pigmentosa (RP) in a family of Ashkenazi Jewish origin in which three of the four siblings have early onset retinal degeneration. The peripheral retinal degeneration in the affected siblings was evident in the initial examination in 1992 and only one had detectable electroretinogram (ERG) that suggested cone-rod dysfunction. The pigmentary retinal degeneration subsequently progressed rapidly. The identified mutation changes the highly conserved residue Lys42 to Glu, resulting in lower catalytic efficiency. Patterns of plasma transferrin isoelectric focusing gel were normal in all family members, indicating no significant abnormality in protein glycosylation. Dolichols have been shown to influence the fluidity and of the membrane and promote vesicle fusion. Considering that photoreceptor outer segments contain stacks of membrane discs, we believe that the mutation may lead to low dolichol levels in photoreceptor outer segments, resulting in unstable membrane structure that leads to photoreceptor degeneration.

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

PubMed

Eisenbeis, Simone; Lohmiller, Stefanie; Valdebenito, Marianne; Leicht, Stefan; Braun, Volkmar

2008-08-01

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis.

PubMed

Godlewska, Renata; Wiśniewska, Katarzyna; Pietras, Zbigniew; Jagusztyn-Krynicka, Elzbieta Katarzyna

2009-09-01

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

Polymeric molecular sieve membranes for gas separation

DOEpatents

Dai, Sheng; Qiao, Zhenan; Chai, Songhai

2017-08-15

A porous polymer membrane useful in gas separation, the porous polymer membrane comprising a polymeric structure having crosslinked aromatic groups and a hierarchical porosity in which micropores having a pore size less than 2 nm are present at least in an outer layer of the porous polymer membrane, and macropores having a pore size of over 50 nm are present at least in an inner layer of the porous polymer membrane. Also described are methods for producing the porous polymer membrane in which a non-porous polymer membrane containing aromatic rings is subjected to a Friedel-Crafts crosslinking reaction in which a crosslinking molecule crosslinks the aromatic rings in the presence of a Friedel-Crafts catalyst and organic solvent under sufficiently elevated temperature, as well as methods for using the porous polymer membranes for gas or liquid separation, filtration, or purification.

Structural basis for catalysis at the membrane-water interface.

PubMed

Dufrisne, Meagan Belcher; Petrou, Vasileios I; Clarke, Oliver B; Mancia, Filippo

2017-11-01

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Toward Understanding the Outer Membrane Uptake of Small Molecules by Pseudomonas aeruginosa*

PubMed Central

Eren, Elif; Parkin, Jamie; Adelanwa, Ayodele; Cheneke, Belete; Movileanu, Liviu; Khalid, Syma; van den Berg, Bert

2013-01-01

Because small molecules enter Gram-negative bacteria via outer membrane (OM) channels, understanding OM transport is essential for the rational design of improved and new antibiotics. In the human pathogen Pseudomonas aeruginosa, most small molecules are taken up by outer membrane carboxylate channel (Occ) proteins, which can be divided into two distinct subfamilies, OccD and OccK. Here we characterize substrate transport mediated by Occ proteins belonging to both subfamilies. Based on the determination of the OccK2-glucuronate co-crystal structure, we identify the channel residues that are essential for substrate transport. We further show that the pore regions of the channels are rigid in the OccK subfamily and highly dynamic in the OccD subfamily. We also demonstrate that the substrate carboxylate group interacts with central residues of the basic ladder, a row of arginine and lysine residues that leads to and away from the binding site at the channel constriction. Moreover, the importance of the basic ladder residues corresponds to their degree of conservation. Finally, we apply the generated insights by converting the archetype of the entire family, OccD1, from a basic amino acid-specific channel into a channel with a preference for negatively charged amino acids. PMID:23467408

Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

PubMed

Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2014-08-25

Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli.

PubMed

Tran, An X; Dong, Changjiang; Whitfield, Chris

2010-10-22

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.

Subcellular Biological Effects of Nanosecond Pulsed Electric Fields

NASA Astrophysics Data System (ADS)

Kolb, Juergen F.; Stacey, Michael

Membranes of biological cells can be charged by exposure to pulsed electric fields. After the potential difference across the barrier reaches critical values on the order of 1 V, pores will form. For moderate pulse parameters of duration and amplitude, the effect is limited to the outer cell membrane. With the exposure to nanosecond pulses of several tens of kilovolts per centimeter, a similar effect is also expected for subcellular membranes and structures. Cells will respond to the disruption by different biochemical processes. This offers possibilities for the development of novel medical therapies, the manipulation of cells and microbiological decontamination.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

PubMed

Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

PubMed

Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

2016-03-01

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

PubMed

Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Photoreceptor change and visual outcome after idiopathic epiretinal membrane removal with or without additional internal limiting membrane peeling.

PubMed

Ahn, Seong Joon; Ahn, Jeeyun; Woo, Se Joon; Park, Kyu Hyung

2014-01-01

To compare the postoperative photoreceptor status and visual outcome after epiretinal membrane removal with or without additional internal limiting membrane (ILM) peeling. Medical records of 40 eyes from 37 patients undergoing epiretinal membrane removal with residual ILM peeling (additional ILM peeling group) and 69 eyes from 65 patients undergoing epiretinal membrane removal without additional ILM peeling (no additional peeling group) were reviewed. The length of defects in cone outer segment tips, inner segment/outer segment junction, and external limiting membrane line were measured using spectral domain optical coherence tomography images of the fovea before and at 1, 3, 6, and 12 months after the surgery. Cone outer segment tips and inner segment/outer segment junction line defects were most severe at postoperative 1 month and gradually restored at 12 months postoperatively. The cone outer segment tips line defect in the additional ILM peeling group was significantly greater than that in the no additional peeling group at postoperative 1 month (P = 0.006), and best-corrected visual acuity was significantly worse in the former group at the same month (P = 0.001). There was no significant difference in the defect size and best-corrected visual acuity at subsequent visits and recurrence rates between the two groups. Patients who received epiretinal membrane surgery without additional ILM peeling showed better visual and anatomical outcome than those with additional ILM peeling at postoperative 1 month. However, surgical outcomes were comparable between the two groups, thereafter. In terms of visual outcome and photoreceptor integrity, additional ILM peeling may not be an essential procedure.

Effects of CNT size on the desalination performance of an outer-wall CNT slit membrane.

PubMed

Ang, Elisa Y M; Ng, Teng Yong; Yeo, Jingjie; Lin, Rongming; Liu, Zishun; Geethalakshmi, K R

2018-05-23

We investigate the effect of varying carbon nanotube (CNT) size on the desalination performance through slit confinements formed by horizontally aligned CNTs stacked on top of one another. By increasing the CNT size, the results obtained from this study indicate a corresponding increase in the water flow rate, accompanied by a slight reduction in salt rejection performance. However, due to the increase in the membrane area with CNT size, the permeability performance is observed to reduce as the CNT size increases. Nevertheless, a comparison with nanoporous 2D membranes shows that the permeability of an outer-wall CNT slit membrane remains significantly higher for all CNT sizes considered. This indicates that precise dimensions of the CNTs are not highly crucial for achieving ultra-high permeability performance in such membranes, as long as the critical slit size is maintained. In-depth analytical studies were further conducted to correlate the influence of curvature effects due to increasing CNT size on the flow characteristcis of the outer-wall CNT membrane. These include the analysis of the measured velocity profiles, oxygen density mapping, potential of mean force profile and friction profile. The present numerical results demonstrate the superb desalination performance of the outer-wall CNT slit membrane, regardless of the size of CNTs used. In addition, an extensive analysis conducted provides detailed characterization of how the curvature affects flow across outer-wall CNTs, and can be used to guide future design and fabrication for experimental testing.

In situ structural analysis of the Yersinia enterocolitica injectisome

PubMed Central

Kudryashev, Mikhail; Stenta, Marco; Schmelz, Stefan; Amstutz, Marlise; Wiesand, Ulrich; Castaño-Díez, Daniel; Degiacomi, Matteo T; Münnich, Stefan; Bleck, Christopher KE; Kowal, Julia; Diepold, Andreas; Heinz, Dirk W; Dal Peraro, Matteo; Cornelis, Guy R; Stahlberg, Henning

2013-01-01

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI: http://dx.doi.org/10.7554/eLife.00792.001 PMID:23908767

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells.

PubMed

Wahlin, Karl J; Maruotti, Julien A; Sripathi, Srinivasa R; Ball, John; Angueyra, Juan M; Kim, Catherine; Grebe, Rhonda; Li, Wei; Jones, Bryan W; Zack, Donald J

2017-04-10

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suwalsky, Mario, E-mail: msuwalsk@udec.cl; Zambrano, Pablo; Mennickent, Sigrid

Research highlights: {yields} PPA is a common ingredient in cough-cold medication and appetite suppressants. {yields} Reports on its effects on human erythrocytes are very scarce. {yields} We found that PPA induced in vitro morphological changes to human erythrocytes. {yields} PPA interacted with isolated unsealed human erythrocyte membranes. {yields} PPA interacted with class of lipid present in the erythrocyte membrane outer monolayer. -- Abstract: Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada.more » Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 {mu}M; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.« less

A trans-outer membrane porin-cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

DOE PAGES

Liu, Yimo; Wang, Zheming; Liu, Juan; ...

2014-09-24

The multiheme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC), and an outer membrane c-Cyt (OmcB/OmcC), respectively. Here we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pccmore » protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate, but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Finally, complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.« less

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili.

PubMed

Dougan, G; Dowd, G; Kehoe, M

1983-01-01

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.

Transient sensitivity to nisin in cold-shocked Gram negatives.

PubMed

Boziaris, I S; Adams, M R

2000-09-01

Rapid chilling in the presence of nisin caused a dose-dependent reduction in the populations of several Gram-negative bacteria, despite the fact that appreciable structural injury to the outer membrane was not detected. Pseudomonas aeruginosa was most affected, followed by Pseudomonas fragi, Salmonella enteritidis PT4, PT7 and Escherichia coli, respectively. Addition of nisin after the chilling treatment had no effect. The results are ascribed to a transient susceptibility caused by phase changes in the lipids associated with the outer membrane, which are rapidly reversed when the cells return to higher temperatures. Combinations of chilling shock, nisin and EDTA gave much lower reductions of Salmonella and Pseudomonas on chicken skin in comparison with broths. This is attributed to a buffering of the temperature shock experienced by adherent bacteria and binding of the nisin by food particles.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Straatsma, TP

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium with high metabolic versatility and an exceptional ability to adapt to a wide range of ecological environments, including soil, marches, coastal habitats, plant and animal tissues. Gram-negative microbes are characterized by the asymmetric lipopolysaccharide outer membrane, the study of which is important for a number of applications. The adhesion to mineral surfaces plays a central role in characterizing their contribution to the fate of contaminants in complex environmental systems by effecting microbial transport through soils, respiration redox chemistry, and ion mobility. Another important application stems from the fact that it is alsomore » a major opportunistic human pathogen that can result in life-threatening infections in many immunocompromised patients, such as lung infections in children with cystic fibrosis, bacteraemia in burn victims, urinary-tract infections in catheterized patients, hospital-acquired pneumonia in patients on respirators, infections in cancer patients receiving chemotherapy, and keratitis and corneal ulcers in users of extended-wear soft contact lenses. The inherent resistance against antibiotics which has been linked with the specific interactions in the outer membrane of P. aeruginosa makes these infections difficult to treat. Developments in simulation methodologies as well as computer hardware have enabled the molecular simulation of biological systems of increasing size and with increasing accuracy, providing detail that is difficult or impossible to obtain experimentally. Computer simulation studies contribute to our understanding of the behavior of proteins, protein-protein and protein-DNA complexes. In recent years, a number of research groups have made significant progress in applying these methods to the study of biological membranes. However, these applications have been focused exclusively on lipid bilayer membranes and on membrane proteins in lipid bilayers. A few simulation studies of outer membrane proteins of Gram-negative bacteria have been reported using simple lipid bilayers, even though this is not a realistic representation of the outer membrane environment. This contribution describes our recent molecular simulation studies of the rough lipopolysaccharide membrane of P. aeruginosa, which are the first and only reported studies to date for a complete, periodic lipopolysaccharide outer membrane. This also includes our current efforts in building on our initial and unique experience simulating the lipopolysaccharide membrane in the development and application of novel computational procedures and tools that allow molecular simulation studies of outer membrane proteins of Gram-negative bacteria to be carried out in realistic membrane models.« less

Bacterial decolorization of textile dyes is an extracellular process requiring a multicomponent electron transfer pathway

PubMed Central

Brigé, Ann; Motte, Bart; Borloo, Jimmy; Buysschaert, Géraldine; Devreese, Bart; Van Beeumen, Jozef J.

2008-01-01

Summary Many studies have reported microorganisms as efficient biocatalysts for colour removal of dye‐containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR‐1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox‐active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin. PMID:21261820

Electrically evoked reticular lamina and basilar membrane vibrations in mice with alpha tectorin C1509G mutation

NASA Astrophysics Data System (ADS)

Ren, Tianying; He, Wenxuan

2015-12-01

Mechanical coupling between the tectorial membrane and the hair bundles of outer hair cells is crucial for stimulating mechanoelectrical transduction channels, which convert sound-induced vibrations into electrical signal, and for transmitting outer hair cell-generated force back to the basilar membrane to boost hearing sensitivity. It has been demonstrated that the detached tectorial membrane in mice with C1509G alpha tectorin mutation caused hearing loss, but enhanced electrically evoked otoacoustic emissions. To understand how the mutated cochlea emits sounds, the reticular lamina and basilar membrane vibrations were measured in the electrically stimulated cochlea in this study. The results showed that the electrically evoked basilar membrane vibration decreased dramatically while the reticular lamina vibration and otoacoustic emissions exhibited no significant change in C1509G mutation mice. This result indicates that a functional cochlear amplifier and a normal basilar membrane vibration are not required for the outer hair cell-generated sound to exit the cochlea.

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner.

PubMed

Uzcanga, Graciela L; Becerra, Aniuska R; Perdomo, Deisy; Bubis, José

2011-03-15

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1. Copyright © 2010 Elsevier Inc. All rights reserved.

Poliovirus hybrids expressing neutralization epitopes from variable domains I and IV of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies.

PubMed Central

Murdin, A D; Su, H; Klein, M H; Caldwell, H D

1995-01-01

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes from the variable domains of the major outer membrane protein are candidates for vaccine development. We have constructed hybrid polioviruses expressing sequences from major outer membrane protein variable domains I and IV. Antisera to the hybrids could, in combination, strongly neutralize 8 of the 12 C. trachomatis serovars most commonly associated with oculogenital infections and weakly neutralize the others. PMID:7532625

The structure of some cytoplasmic components of plant cells in relation to the biochemical properties of isolated particles.

PubMed

HODGE, A J; MARTIN, E M; MORTON, R K

1957-01-25

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported.

THE STRUCTURE OF SOME CYTOPLASMIC COMPONENTS OF PLANT CELLS IN RELATION TO THE BIOCHEMICAL PROPERTIES OF ISOLATED PARTICLES

PubMed Central

Hodge, A. J.; Martin, E. M.; Morton, R. K.

1957-01-01

1. Electron micrographs of thin sections of material fixed with buffered osmium tetroxide have been used for comparison of the fine structure of isolated cytoplasmic particles from silver beet petioles and roots of germinating wheat with that of the cytoplasm of the intact cells. 2. Mitochondria of wheat roots have an external double membrane and poorly oriented internal double membranes. As compared with the structures seen in situ, the isolated mitochondria showed evidence of some disorganisation of the fine internal structure, probably due to osmotic effects. The possible influence of such changes on the enzymic properties of the isolated mitochondria is discussed. 3. The isolated plant microsomes are mainly spherical vesicular structures consisting of (a) an outer membrane enclosing (b) either an homogeneous slightly dense material (wheat root microsomes) or some granular dense material (silver beet microsomes) and (c) small dense particles, mostly associated with the vesicle membranes. 4. The cytoplasm of the wheat root cells does not contain any structures similar to the isolated microsomes but has a very dense reticular network, consisting of membranes with associated small dense particles, here called the endoplasmic reticulum. The observations indicate that the isolated microsomes arise mainly by rupture and transformation of the membranes of this structure. The effects of such extensive changes in the lipoprotein membranes on the enzymic activities of the endoplasmic reticulum, as studied in isolated microsomes, is discussed. 5. Meristematic wheat root cells contain structures which consist of smooth membranes with associated vacuoles and are similar to the Golgi zones of animal cells. The membranes of these zones probably contribute to the microsomal fraction under the conditions of preparation used for the enzymic and chemical studies previously reported. PMID:13416311

ARL2BP, a protein linked to Retinitis Pigmentosa, is needed for normal photoreceptor cilia doublets and outer segment structure.

PubMed

Moye, Abigail R; Singh, Ratnesh; Kimler, Victoria A; Dilan, Tanya L; Munezero, Daniella; Saravanan, Thamaraiselvi; Goldberg, Andrew F X; Ramamurthy, Visvanathan

2018-05-02

The outer segment (OS) of photoreceptor cells is an elaboration of a primary cilium with organized stacks of membranous discs that contain the proteins needed for phototransduction and vision. Though cilia formation and function has been well characterized, little is known about the role of cilia in the development of photoreceptor OS. Nevertheless, progress has been made by studying mutations in ciliary proteins which often result in malformed outer segments and lead to blinding diseases. To investigate how ciliary proteins contribute to outer segment formation, we generated a knockout mouse model for ARL2BP, a ciliary protein linked to Retinitis Pigmentosa. The knockout mice display an early and progressive reduction in visual response. Prior to photoreceptor degeneration we observed disorganization of the photoreceptor OS, with vertically aligned discs and shortened axonemes. Interestingly, ciliary doublet microtubule structure was also impaired, displaying open B-tubule doublets, paired with loss of singlet microtubules. Based on results from this study, we conclude that ARL2BP is necessary for photoreceptor cilia doublet formation and axoneme elongation, which is required for outer segment morphogenesis and vision.

Protection of ATP-Depleted Cells by Impermeant Strychnine Derivatives

PubMed Central

Dong, Zheng; Venkatachalam, Manjeri A.; Weinberg, Joel M.; Saikumar, Pothana; Patel, Yogendra

2001-01-01

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine. PMID:11238050

Protection of ATP-depleted cells by impermeant strychnine derivatives: implications for glycine cytoprotection.

PubMed

Dong, Z; Venkatachalam, M A; Weinberg, J M; Saikumar, P; Patel, Y

2001-03-01

Glycine and structurally related amino acids with activities at chloride channel receptors in the central nervous system also have robust protective effects against cell injury by ATP depletion. The glycine receptor antagonist strychnine shares this protective activity. An essential step toward identification of the molecular targets for these compounds is to determine whether they protect cells through interactions with intracellular targets or with molecules on the outer surface of plasma membranes. Here we report cytoprotection by a cell-impermeant derivative of strychnine. A strychnine-fluorescein conjugate (SF) was synthesized, and impermeability of plasma membranes to this compound was verified by fluorescence confocal microscopy. In an injury model of Madin-Darby canine kidney cells, ATP depletion led to lactate dehydrogenase release. SF prevented lactate dehydrogenase leakage without ameliorating ATP depletion. This was accompanied by preservation of cellular ultrastructure and exclusion of vital dyes. SF protection was also shown for ATP-depleted rat hepatocytes. On the other hand, when a key structural motif in the active site of strychnine was chemically blocked, the SF lost its protective effect, establishing strychnine-related specificity for SF protection. Cytoprotective effects of the cell-impermeant strychnine derivative provide compelling evidence suggesting that molecular targets on the outer surface of plasma membranes may mediate cytoprotection by strychnine and glycine.

Signal transfer through three compartments: transcription initiation of the Escherichia coli ferric citrate transport system from the cell surface.

PubMed

Härle, C; Kim, I; Angerer, A; Braun, V

1995-04-03

Transport of ferric citrate into cells of Escherichia coli K-12 involves two energy-coupled transport systems, one across the outer membrane and one across the cytoplasmic membrane. Previously, we have shown that ferric citrate does not have to enter the cytoplasm of E. coli K-12 to induce transcription of the fec ferric citrate transport genes. Here we demonstrate that ferric citrate uptake into the periplasmic space between the outer and the cytoplasmic membranes is not required for fec gene induction. Rather, FecA and the TonB, ExbB and ExbD proteins are involved in induction of the fec transport genes independent of their role in ferric citrate transport across the outer membrane. The uptake of ferric citrate into the periplasmic space of fecA and tonB mutants via diffusion through the porin channels did not induce transcription of fec transport genes. Point mutants in FecA displayed the constitutive expression of fec transport genes in the absence of ferric citrate but still required TonB, with the exception of one FecA mutant which showed a TonB-independent induction. The phenotype of the FecA mutants suggests a signal transduction mechanism across three compartments: the outer membrane, the periplasmic space and the cytoplasmic membrane. The signal is triggered upon the interaction of ferric citrate with FecA protein. It is postulated that FecA, TonB, ExbB and ExbD transfer the signal across the outer membrane, while the regulatory protein FecR transmits the signal across the cytoplasmic membrane to FecI in the cytoplasm. FecI serves as a sigma factor which facilitates binding of the RNA polymerase to the fec transport gene promoter upstream of fecA.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40*

PubMed Central

Kuszak, Adam J.; Jacobs, Daniel; Gurnev, Philip A.; Shiota, Takuya; Louis, John M.; Lithgow, Trevor; Bezrukov, Sergey M.; Rostovtseva, Tatiana K.; Buchanan, Susan K.

2015-01-01

Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function. PMID:26336107

Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

PubMed Central

2011-01-01

Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism. PMID:21272379

Structural adaptations of proteins to different biological membranes

PubMed Central

Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

2013-01-01

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin

PubMed Central

Bae, Chanhyung; Anselmi, Claudio; Kalia, Jeet; Jara-Oseguera, Andres; Schwieters, Charles D; Krepkiy, Dmitriy; Won Lee, Chul; Kim, Eun-Hee; Kim, Jae Il; Faraldo-Gómez, José D; Swartz, Kenton J

2016-01-01

Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation. DOI: http://dx.doi.org/10.7554/eLife.11273.001 PMID:26880553

Role of subunit assembly in autosomal dominant retinitis pigmentosa linked to mutations in peripherin 2.

PubMed

Molday, Robert S; Molday, Laurie L; Loewen, Christopher J R

2004-01-01

Peripherin 2 is a photoreceptor-specific membrane protein implicated in outer segment disk morphogenesis and linked to various retinopathies including autosomal dominant retinitis pigmentosa (ADRP). Peripherin 2 and ROM1 assemble as a mixture of core noncovalent homomeric and heteromeric tetramers that further link together through disulfide bonds to form higher order oligomers. These complexes are critical for disk rim formation and outer segment structure through interaction with the cGMP-gated channel and other photoreceptor proteins. We have examined the role of subunit assembly in peripherin 2 targeting to disks, outer segment structure, and photoreceptor degeneration by examining molecular and cellular properties of peripherin 2 mutants in COS-1 cells and transgenic Xenopus laevis rod photoreceptors. Wild-type (WT) and the ADRP-linked P216L mutant were transported and incorporated into newly formed outer segment disks of transgenic X. laevis. The P216L mutant, however, induced progressive outer segment instability and photoreceptor degeneration possibly through the introduction of a new N-linked oligosaccharide chain. In contrast, the C214S and L185P disease-linked, tetramerization-defective mutants, were retained in the inner segment, but did not affect outer segment structure or induce photoreceptor degeneration. Together, these results indicate that peripherin 2 mutations can cause ADRP either through a deficiency in WT peripherin 2 (C214S, 1.185P) or by a dominant negative effect on disk stability (P216L).

Outer membrane lipoprotein VacJ is required for the membrane integrity, serum resistance and biofilm formation of Actinobacillus pleuropneumoniae.

PubMed

Xie, Fang; Li, Gang; Zhang, Wanjiang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

2016-02-01

The outer membrane proteins of Actinobacillus pleuropneumoniae are mediators of infection, acting as targets for the host's defense system. The outer membrane lipoprotein VacJ is involved in serum resistance and intercellular spreading in several pathogenic bacteria. To investigate the role of VacJ in the pathogenicity of Actinobacillus pleuropneumoniae, the vacJ gene-deletion mutant MD12 ΔvacJ was constructed. The increased susceptibility to KCl, SDS plus EDTA, and several antibiotics in the MD12ΔvacJ mutant suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. The increased NPN fluorescence and significant cellular morphological variation in the MD12ΔvacJ mutant further demonstrated the crucial role of the VacJ lipoprotein in maintaining the outer membrane integrity of A. pleuropneumoniae. In addition, the MD12ΔvacJ mutant exhibited decreased survival from the serum and complement killing compared to the wild-type strain. Interestingly, the MD12ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. To our knowledge, this is the first description of the VacJ lipoprotein contributing to bacterial biofilm formation. The data presented in this study illustrate the important role of the VacJ lipoprotein in the maintenance of cellular integrity, serum resistance, and biofilm formation in A. pleuropneumoniae. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage.

PubMed

Garmiri, Penelope; Coles, Karen E; Humphrey, Tom J; Cogan, Tristan A

2008-04-01

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

PubMed

Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy.

PubMed

Sass, H J; Büldt, G; Beckmann, E; Zemlin, F; van Heel, M; Zeitler, E; Rosenbusch, J P; Dorset, D L; Massalski, A

1989-09-05

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

ELECTRON MICROSCOPIC OBSERVATIONS OF AMOEBA PROTEUS IN GROWTH AND INANITION

PubMed Central

Cohen, Adolph I.

1957-01-01

Electron microscopic observations have been made on growing and dividing specimens of Amoeba proteus and also on starving animals. Structures presumably corresponding to the mitochondria, alpha particles, vacuoles, and Golgi material are described. A new entity, designated as a foamy particle, is noted. Descriptions are given of the cytoplasmic and nuclear membranes. During division the inner, thick nuclear membrane component is seen to vanish and the outer membrane persist. Measurements suggest a gradual reappearance of the inner component with growth. Starving animals show a loss of cytoplasmic granularity and an increase in the electron density of mitochondria, presumably due to lipide accumulation. PMID:13481020

Electron microscopic observations of amoeba proteus in growth and inanition.

PubMed

COHEN, A I

1957-11-25

Electron microscopic observations have been made on growing and dividing specimens of Amoeba proteus and also on starving animals. Structures presumably corresponding to the mitochondria, alpha particles, vacuoles, and Golgi material are described. A new entity, designated as a foamy particle, is noted. Descriptions are given of the cytoplasmic and nuclear membranes. During division the inner, thick nuclear membrane component is seen to vanish and the outer membrane persist. Measurements suggest a gradual reappearance of the inner component with growth. Starving animals show a loss of cytoplasmic granularity and an increase in the electron density of mitochondria, presumably due to lipide accumulation.

Protein Composition of Trypanosoma brucei Mitochondrial Membranes

PubMed Central

Acestor, Nathalie; Panigrahi, Aswini K.; Ogata, Yuko; Anupama, Atashi; Stuart, Kenneth D.

2010-01-01

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane and matrix; each harboring specific functions and structures. In this study, we used mass spectrometry (LC-MS/MS) to characterize the protein composition of Trypanosoma brucei mitochondrial membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mitochondrial membranes with high confidence, and 106 with moderate to low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mitochondrial membrane have putative roles in metabolic, energy generating, and transport processes, ~50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mitochondrial functions. PMID:19834910

Morphological evidence of neurotoxicity in retina after methylmercury exposure.

PubMed

Mela, Maritana; Grötzner, Sonia Regina; Legeay, Alexia; Mesmer-Dudons, Nathalie; Massabuau, Jean-Charles; Ventura, Dora Fix; de Oliveira Ribeiro, Ciro Alberto

2012-06-01

The visual system is particularly sensitive to methylmercury (MeHg) exposure and, therefore, provides a useful model for investigating the fundamental mechanisms that direct toxic effects. During a period of 70 days, adult of a freshwater fish species Hoplias malabaricus were fed with fish prey previously labeled with two different doses of methylmercury (0.075 and 0.75 μgg(-1)) to determine the mercury distribution and morphological changes in the retina. Mercury deposits were found in the photoreceptor layer, in the inner plexiform layer and in the outer plexiform layer, demonstrating a dose-dependent bioaccumulation. The ultrastructure analysis of retina revealed a cellular deterioration in the photoreceptor layer, morphological changes in the inner and outer segments of rods, structural changes in the plasma membrane of rods and double cones, changes in the process of removal of membranous discs and a structural discontinuity. These results lead to the conclusion that methylmercury is able to cross the blood-retina barrier, accumulate in the cells and layers of retina and induce changes in photoreceptors of H. malabaricus even under subchronic exposure. Copyright © 2012 Elsevier Inc. All rights reserved.

Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.

PubMed

Kitagaki, Hiroshi

2009-05-29

Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.

A novel crosslinking strategy for preparing poly(vinyl alcohol)-based proton-conducting membranes with high sulfonation

NASA Astrophysics Data System (ADS)

Tsai, Chun-En; Lin, Chi-Wen; Hwang, Bing-Joe

This study synthesizes poly(vinyl alcohol) (PVA)-based polymer electrolyte membranes by a two-step crosslinking process involving esterization and acetal ring formation reactions. This work also uses sulfosuccinic acid (SSA) as the first crosslinking agent to form an inter-crosslinked structure and a promoting sulfonating agent. Glutaraldehyde (GA) as the second crosslinking agent, reacts with the spare OH group of PVA and forms, not only a dense structure at the outer membrane surface, but also a hydrophobic protective layer. Compared with membranes prepared by a traditional one-step crosslinking process, membranes prepared by the two-step crosslinking process exhibit excellent dissolution resistance in water. The membranes become water-insoluble even at a molar ratio of SO 3H/PVA-OH as high as 0.45. Moreover, the synthesized membranes also exhibit high proton conductivities and high methanol permeability resistance. The current study measures highest proton conductivity of 5.3 × 10 -2 S cm -1 at room temperature from one of the synthesized membranes, higher than that of the Nafion ® membrane. Methanol permeability of the synthesized membranes measures about 1 × 10 -7 cm 2 S -1, about one order of magnitude lower than that of the Nafion ® membrane.

IroN, a Novel Outer Membrane Siderophore Receptor Characteristic of Salmonella enterica

PubMed Central

Bäumler, Andreas J.; Norris, Tracy L.; Lasco, Todd; Voigt, Wolfgang; Reissbrodt, Rolf; Rabsch, Wolfgang; Heffron, Fred

1998-01-01

Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize iroN, a gene located downstream of the iroBC operon in the iroA locus of Salmonella enterica serotype Typhi. Like iroBC, the iroN gene is present in all phylogenetic lineages of S. enterica but is absent from closely related species such as Salmonella bongori or Escherichia coli. Comparison of the deduced amino acid sequence of iroN with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. When introduced into an E. coli fepA cir fiu aroB mutant on a cosmid, iroN mediated utilization of structurally related catecholate siderophores, including N-(2,3-dihydroxybenzoyl)-l-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-l-lysine amide, and enterochelin. These results suggest that the iroA locus functions in iron acquisition in S. enterica. PMID:9515912

Protein secretion through autotransporter and two-partner pathways.

PubMed

Jacob-Dubuisson, Françoise; Fernandez, Rachel; Coutte, Loic

2004-11-11

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.

Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana.

PubMed

Kelly, Amélie A; Kalisch, Barbara; Hölzl, Georg; Schulze, Sandra; Thiele, Juliane; Melzer, Michael; Roston, Rebecca L; Benning, Christoph; Dörmann, Peter

2016-09-20

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

PubMed Central

Schwechheimer, Carmen; Kuehn, Meta J.

2017-01-01

Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles. PMID:26373371

Proteins required for lipopolysaccharide assembly in Escherichia coli form a transenvelope complex.

PubMed

Chng, Shu-Sin; Gronenberg, Luisa S; Kahne, Daniel

2010-06-08

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential lipopolysaccharide transport (Lpt) proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes and that they copurify. This constitutes the first evidence that the Lpt proteins form a transenvelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope.

Proteins required for lipopolysaccharide assembly in Escherichia coli form a trans-envelope complex†

PubMed Central

Chng, Shu-Sin; Gronenberg, Luisa S.; Kahne, Daniel

2010-01-01

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope. PMID:20446753

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

NASA Astrophysics Data System (ADS)

Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

2011-12-01

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

Current State of Theoretical and Experimental Studies of the Voltage-Dependent Anion Channel (VDAC)

PubMed Central

Noskov, Sergei Yu.; Rostovtseva, Tatiana K.; Chamberlin, Adam C.; Teijido, Oscar; Jiang, Wei; Bezrukov, Sergey M.

2016-01-01

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane provides a controlled pathway for respiratory metabolites in and out of the mitochondria. In spite of the wealth of experimental data from structural, biochemical, and biophysical investigations, the exact mechanisms governing selective ion and metabolite transport, especially the role of titratable charged residues and interactions with soluble cytosolic proteins, remain hotly debated in the field. The computational advances hold a promise to provide a much sought-after solution to many of the scientific disputes around solute and ion transport through VDAC and hence, across the mitochondrial outer membrane. In this review, we examine how Molecular Dynamics, Free Energy, and Brownian Dynamics simulations of the large β-barrel channel, VDAC, advanced our understanding. We will provide a short overview of non-conventional techniques and also discuss examples of how the modeling excursions into VDAC biophysics prospectively aid experimental efforts. PMID:26940625

Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2.

PubMed

Wang, Jiarong; Li, Yan; Wang, Xiaoming; Chen, Wei; Sun, Hongbin; Wang, Junfeng

2014-11-01

Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane and protects the cell against bactericidal agents, including antimicrobial peptides. However, the mechanisms of interaction between antimicrobial peptides and LPS are not clearly understood. Halictines-2 (HAL-2), one of the novel antimicrobial peptides, was isolated from the venom of the eusocial bee Halictus sexcinctus. HAL-2 has exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria and even against cancer cells. Here, we studied the interactions between HAL-2 and LPS to elucidate the antibacterial mechanism of HAL-2 in vitro. Our results show that HAL-2 adopts a significant degree of β-strand structure in the presence of LPS. LPS is capable of inducing HAL-2 amyloid formation, which may play a vital role in its antimicrobial activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Understanding Mircrobial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2016-10-01

limitation, we have developed an expanded metabolic labeling approach that chemically tags lipopolysaccharide, capsular polysaccharide , and peptidoglycan...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory...bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory bowel disease, microbiome, microbiota

2,4-Dichlorophenoxyacetic Acid Inhibits the Outer Membrane NADH Dehydrogenase of Plant Mitochondria 1

PubMed Central

Mannella, Carmen A.; Bonner, Walter D.

1978-01-01

The NADH dehydrogenase of potato (Solanum tuberosum) and mung bean (Phaseolus aureus) outer mitochondrial membranes is specifically inhibited by both 2,4-dichlorophenoxyacetic and 2,4,5-trichlorophenoxyacetic acids but not by the natural auxin indole-3-acetic acid. PMID:16660539

Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

DOE PAGES

McCaig, William D.; Loving, Crystal L.; Hughes, Holly R.; ...

2016-03-01

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structuresmore » has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Lastly, vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.« less

Characterization and vaccine potential of outer membrane vesicles produced by Haemophilus parasuis

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCaig, William D.; Loving, Crystal L.; Hughes, Holly R.

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structuresmore » has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Lastly, vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.« less

The lethal cargo of Myxococcus xanthus outer membrane vesicles.

PubMed

Berleman, James E; Allen, Simon; Danielewicz, Megan A; Remis, Jonathan P; Gorur, Amita; Cunha, Jack; Hadi, Masood Z; Zusman, David R; Northen, Trent R; Witkowska, H Ewa; Auer, Manfred

2014-01-01

Myxococcus xanthus is a bacterial micro-predator known for hunting other microbes in a wolf pack-like manner. Outer membrane vesicles (OMVs) are produced in large quantities by M. xanthus and have a highly organized structure in the extracellular milieu, sometimes occurring in chains that link neighboring cells within a biofilm. OMVs may be a vehicle for mediating wolf pack activity by delivering hydrolytic enzymes and antibiotics aimed at killing prey microbes. Here, both the protein and small molecule cargo of the OMV and membrane fractions of M. xanthus were characterized and compared. Our analysis indicates a number of proteins that are OMV-specific or OMV-enriched, including several with putative hydrolytic function. Secondary metabolite profiling of OMVs identifies 16 molecules, many associated with antibiotic activities. Several hydrolytic enzyme homologs were identified, including the protein encoded by MXAN_3564 (mepA), an M36 protease homolog. Genetic disruption of mepA leads to a significant reduction in extracellular protease activity suggesting MepA is part of the long-predicted (yet to date undetermined) extracellular protease suite of M. xanthus.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

PubMed Central

Gray, Andrew N; Egan, Alexander JF; van't Veer, Inge L; Verheul, Jolanda; Colavin, Alexandre; Koumoutsi, Alexandra; Biboy, Jacob; Altelaar, A F Maarten; Damen, Mirjam J; Huang, Kerwyn Casey; Simorre, Jean-Pierre; Breukink, Eefjan; den Blaauwen, Tanneke; Typas, Athanasios; Gross, Carol A; Vollmer, Waldemar

2015-01-01

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001 PMID:25951518

Micro-scale NMR Screening of New Detergents for Membrane Protein Structural Biology

PubMed Central

Zhang, Qinghai; Horst, Reto; Geralt, Michael; Ma, Xingquan; Hong, Wen-Xu; Finn, M. G.; Stevens, Raymond C.; Wüthrich, Kurt

2008-01-01

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that micro-coil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E.coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with μg-amounts of uniformly 2H,15N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [15N,1H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX. PMID:18479092

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

PubMed

Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

2016-04-15

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

Membrane-spacer assembly for flow-electrode capacitive deionization

NASA Astrophysics Data System (ADS)

Lee, Ki Sook; Cho, Younghyun; Choo, Ko Yeon; Yang, SeungCheol; Han, Moon Hee; Kim, Dong Kook

2018-03-01

Flow-electrode capacitive deionization (FCDI) is a desalination process designed to overcome the limited desalination capacity of conventional CDI systems due to their fixed electrodes. Such a FCDI cell system is comprised of a current collector, freestanding ion-exchange membrane (IEM), gasket, and spacer for flowing saline water. To simplify the cell system, in this study we combined the membrane and spacer into a single unit, by coating the IEM on a porous ceramic structure that acts as the spacer. The combination of membrane with the porous structure avoids the use of costly freestanding IEM. Furthermore, the FCDI system can be readily scaled up by simply inserting the IEM-coated porous structures in between the channels for flow electrodes. However, coating the IEM on such porous ceramic structures can cause a sudden drop in the treatment capacity, if the coated IEM penetrates the ceramic pores and prevents these pores from acting as saline flow channels. To address this issue, we blocked the larger microscale pores on the outer surface with SiO2 and polymeric multilayers. Thus, the IEM is coated only onto the top surface of the porous structure, while the internal pores remain empty to function as water channels.

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

PubMed

Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

2008-01-04

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

PubMed

Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

2016-06-21

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Light-induced import of the chromoprotein, phytochrome, into mitochondria

NASA Technical Reports Server (NTRS)

Serlin, B. S.; Roux, S. J.

1986-01-01

Mitochondria extracted from plants that were irradiated with actinic light in vivo have associated with them the chromoprotein, phytochrome. This phytochrome retains its native subunit size of 124 kDa after proteolytic treatment of the mitochondria with trypsin and chymotrypsin. This result suggests that phytochrome is not exposed on the outer surface of the outer mitochondrial membrane. Phytochrome, so protected, is not found to be associated with mitochondria derived from unirradiated plants. The possibility that the photoactivation of phytochrome induces a conformational change in its structure which facilitates its transport into the mitochondrion is discussed.

Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress.

PubMed

Klupp, Barbara G; Hellberg, Teresa; Granzow, Harald; Franzke, Kati; Dominguez Gonzalez, Beatriz; Goodchild, Rose E; Mettenleiter, Thomas C

2017-10-01

Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking. Copyright © 2017 American Society for Microbiology.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

Linkage between anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

USDA-ARS?s Scientific Manuscript database

Prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective; they are difficult and expensive to isolate and standardize, and thus often impractical for development and implementation in vaccination programs. In contrast, ind...

Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

USDA-ARS?s Scientific Manuscript database

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

PubMed

Misra, Rajeev; Morrison, Keith D; Cho, Hyun Jae; Khuu, Thanh

2015-08-01

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cryogenic Insulation System

NASA Technical Reports Server (NTRS)

Davis, Randall C. (Inventor); Taylor, Allan H. (Inventor); Jackson, L. Robert (Inventor); Mcauliffe, Patrick S. (Inventor)

1988-01-01

This invention relates to reusable, low density, high temperature cryogenic foam insulation systems and the process for their manufacture. A pacing technology for liquid hydrogen fueled, high speed aircraft is the development of a fully reusable, flight weight cryogenic insulation system for propellant tank structures. In the invention cryogenic foam insulation is adhesively bonded to the outer wall of the fuel tank structure. The cryogenic insulation consists of square sheets fabricated from an array of abutting square blocks. Each block consists of a sheet of glass cloth adhesively bonded between two layers of polymethacrylimide foam. Each block is wrapped in a vapor impermeable membrane, such as Kapton(R) aluminum Kapton(R), to provide a vapor barrier. Very beneficial results can be obtained by employing the present invention in conjunction with fibrous insulation and an outer aeroshell, a hot fuselage structure with an internal thermal protection system.

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhongshan; College of Life Sciences, Sichuan University, Chengdu 610065; Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST

2014-09-26

Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane bymore » seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.« less

Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eicher, Barbara; Heberle, Frederick A.; Marquardt, Drew T.

2017-02-28

Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effortmore » but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ~120 nm diameter palmitoyloleoyl phosphatidylcholine (POPC) vesicles, compared to the inner leaflet. Here, analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K, i.e.above the melting transition temperature of the two lipids.« less

The Acrosome Reaction: A Historical Perspective.

PubMed

Okabe, Masaru

2016-01-01

Acrosome reaction is often referred to as acrosomal exocytosis, but it differs significantly from normal exocytosis. While the vesicle membrane initially holding excreting molecules remains on the cell surface during exocytosis, the outer acrosomal membrane and plasma membrane are lost by forming vesicles during acrosome reaction. In this context, the latter process resembles a release of exosome. However, recent experimental data indicate that the most important roles of acrosome reaction lie not in the release of acrosomal contents (or "vesiculated" plasma and outer acrosomal membrane complexes) but rather in changes in sperm membrane. This review describes the mechanism of fertilization vis-a-vis sperm membrane change, with a brief historical overview of the half-century study of acrosome reaction.

Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes

DOE PAGES

Smith, Jeremy C.; Cheng, Xiaolin; Nickels, Jonathan D.

2015-07-29

Understanding of cell membrane organization has evolved significantly from the classic fluid mosaic model. It is now recognized that biological membranes are highly organized structures, with differences in lipid compositions between inner and outer leaflets and in lateral structures within the bilayer plane, known as lipid rafts. These organizing principles are important for protein localization and function as well as cellular signaling. However, the mechanisms and biophysical basis of lipid raft formation, structure, dynamics and function are not clearly understood. One key question, which we focus on in this review, is how lateral organization and leaflet compositional asymmetry are coupled.more » Detailed information elucidating this question has been sparse because of the small size and transient nature of rafts and the experimental challenges in constructing asymmetric bilayers. Resolving this mystery will require advances in both experimentation and modeling. We discuss here the preparation of model systems along with experimental and computational approaches that have been applied in efforts to address this key question in membrane biology. Furthermore, we seek to place recent and future advances in experimental and computational techniques in context, providing insight into in-plane and transverse organization of biological membranes.« less

Nanoporous Pirani sensor based on anodic aluminum oxide

NASA Astrophysics Data System (ADS)

Jeon, Gwang-Jae; Kim, Woo Young; Shim, Hyun Bin; Lee, Hee Chul

2016-09-01

A nanoporous Pirani sensor based on anodic aluminum oxide (AAO) is proposed, and the quantitative relationship between the performance of the sensor and the porosity of the AAO membrane is characterized with a theoretical model. The proposed Pirani sensor is composed of a metallic resistor on a suspended nanoporous membrane, which simultaneously serves as the sensing area and the supporting structure. The AAO membrane has numerous vertically-tufted nanopores, resulting in a lower measurable pressure limit due to both the increased effective sensing area and the decreased effective thermal loss through the supporting structure. Additionally, the suspended AAO membrane structure, with its outer periphery anchored to the substrate, known as a closed-type design, is demonstrated using nanopores of AAO as an etch hole without a bulk micromachining process used on the substrate. In a CMOS-compatible process, a 200 μm × 200 μm nanoporous Pirani sensor with porosity of 25% was capable of measuring the pressure from 0.1 mTorr to 760 Torr. With adjustment of the porosity of the AAO, the measurable range could be extended toward lower pressures of more than one decade compared to a non-porous membrane with an identical footprint.

Morphology of one-time coated palladium-alumina composite membrane prepared by sol-gel process and electroless plating technique

NASA Astrophysics Data System (ADS)

Sari, R.; Dewi, R.; Pardi; Hakim, L.; Diana, S.

2018-03-01

Palladium coated porous alumina ceramic membrane tube was obtained using a combination of sol-gel process and electroless plating technique. The thickness, structure and composition of palladium-alumina composite membrane were analyzed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and atomic force microscopy (AFM). Palladium particle size was 6.18 to 7.64 nm. Palladium membrane with thickness of approximately 301.5 to 815.1 nm was formed at the outer surface of the alumina layer. EDX data confirmed the formation of palladium-alumina membrane containing 45% of palladium. From this research it shows the combination of sol-gel process and electroless plating technique with one-time coating can produce a homogeneous and smoother palladium nano layer film on alumina substrate.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Jeremy C.; Cheng, Xiaolin; Nickels, Jonathan D.

Understanding of cell membrane organization has evolved significantly from the classic fluid mosaic model. It is now recognized that biological membranes are highly organized structures, with differences in lipid compositions between inner and outer leaflets and in lateral structures within the bilayer plane, known as lipid rafts. These organizing principles are important for protein localization and function as well as cellular signaling. However, the mechanisms and biophysical basis of lipid raft formation, structure, dynamics and function are not clearly understood. One key question, which we focus on in this review, is how lateral organization and leaflet compositional asymmetry are coupled.more » Detailed information elucidating this question has been sparse because of the small size and transient nature of rafts and the experimental challenges in constructing asymmetric bilayers. Resolving this mystery will require advances in both experimentation and modeling. We discuss here the preparation of model systems along with experimental and computational approaches that have been applied in efforts to address this key question in membrane biology. Furthermore, we seek to place recent and future advances in experimental and computational techniques in context, providing insight into in-plane and transverse organization of biological membranes.« less

Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobre, Thatyane M.; Martynowycz, Michael W.; Andreev, Konstantin

Small hydrophilic antibiotics traverse the outer membrane of Gram-negative bacteria through porin channels. Large lipophilic agents traverse the outer membrane through its bilayer, containing a majority of lipopolysaccharides in its outer leaflet. Genes controlled by the two-component regulatory system PhoPQ modify lipopolysaccharides. We isolate lipopolysaccharides from isogenic mutants of Salmonella sp., one lacking the modification, the other fully modified. These lipopolysaccharides were reconstituted asmonolayers at the air-water interface, and their properties, aswell as their interaction with a large lipophilic drug, novobiocin, was studied. X-ray reflectivity showed that the drug penetrated the monolayer of the unmodified lipopolysaccharides reaching the hydrophobic region,butwasmore » prevented fromthis penetration intothemodified lipopolysaccharides.Results correlatewith behavior of bacterial cells, which become resistant to antibiotics after PhoPQ-regulated modifications. Grazing incidence x-ray diffraction showed that novobiocin produced a striking increase in crystalline coherence length, and the size of the near-crystalline domains.« less

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

PubMed

Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

2008-02-01

Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments.

PubMed

Cheng, Christiana L; Molday, Robert S

2013-12-15

In photoreceptors, the assembly of signaling molecules into macromolecular complexes is important for phototransduction and maintaining the structural integrity of rod outer segments (ROSs). However, the molecular composition and formation of these complexes are poorly understood. Using immunoprecipitation and mass spectrometry, 4.1G was identified as a new interacting partner for the cyclic-nucleotide gated (CNG) channels in ROSs. 4.1G is a widely expressed multifunctional protein that plays a role in the assembly and stability of membrane protein complexes. Multiple splice variants of 4.1G were cloned from bovine retina. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with peripherin-2-CNG channel complex. A combination of truncation studies and domain-binding assays demonstrated that CNG channels selectively interacted with 4.1G through their FERM and CTD domains. Using immunofluorescence, labeling of 4.1G was seen to be punctate and partially colocalized with CNG channels in the ROS. Our studies indicate that 4.1G interacts with a subset of CNG channels in the ROS and implicate this protein-protein interaction in organizing the spatial arrangement of CNG channels in the plasma membrane of outer segments.

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

Tob38, a novel essential component in the biogenesis of β-barrel proteins of mitochondria

PubMed Central

Waizenegger, Thomas; Habib, Shukry J; Lech, Maciej; Mokranjac, Dejana; Paschen, Stefan A; Hell, Kai; Neupert, Walter; Rapaport, Doron

2004-01-01

Insertion of β-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of β-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. PMID:15205677

An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export.

PubMed

Freudl, R; Schwarz, H; Stierhof, Y D; Gamon, K; Hindennach, I; Henning, U

1986-08-25

Pulse-chase experiments were performed to follow the export of the Escherichia coli outer membrane protein OmpA. Besides the pro-OmpA protein, which carries a 21-residue signal sequence, three species of ompA gene products were distinguishable. One probably represented an incomplete nascent chain, another the mature protein in the outer membrane, and the third, designated imp-OmpA (immature processed), a protein which was already processed but apparently was still associated with the plasma membrane. The pro- and imp-OmpA proteins could be characterized more fully by using a strain overproducing the ompA gene products; pro- and imp-OmpA accumulated in large amounts. It could be shown that the imp- and pro-OmpA proteins differ markedly in conformation from the OmpA protein. The imp-OmpA, but not the pro-OmpA, underwent a conformational change and gained phage receptor activity upon addition of lipopolysaccharide. Utilizing a difference in detergent solubility between the two polypeptides and employing immunoelectron microscopy, it could be demonstrated that the pro-OmpA protein accumulated in the cytoplasm while the imp-OmpA was present in the periplasmic space. The results suggest that the pro-OmpA protein, bound to the plasma membrane, is processed, and the resulting imp-OmpA, still associated with the plasma membrane, recognizes the lipid A moiety of the lipopolysaccharide. The resulting conformational change may then force the protein into the outer membrane.

Studies of Water Diffusion on Single-Supported Bilayer Lipid Membranes by Quasielastic Neutron Scattering

NASA Astrophysics Data System (ADS)

Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Hansen, F. Y.

2010-03-01

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently demonstrated the feasibility of using quasielastic neutron scattering to study on a ˜1 ns time scale the diffusion of water bound to single-supported bilayer lipid membranes. Two different membrane samples characterized by AFM were investigated: protonated DMPC + D2O and tail-deuterated DMPC + H2O. Both fully hydrated membranes were deposited onto SiO2-coated Si(100) substrates. Measurements of elastic neutron intensity as a function of temperature on the High Flux Backscattering Spectrometer at NIST reveal features in the diffusive motion of water that have not been observed previously using multilayer membrane stacks. On slow cooling, the elastic intensity shows sharp step-like increases in the temperature range 265 to 272 K that we tentatively interpret as successive mobile-to-immobile transitions of water bound to the membrane.

Membrane curvature generation by a C-terminal amphipathic helix in peripherin-2/rds, a tetraspanin required for photoreceptor sensory cilium morphogenesis

PubMed Central

Khattree, Nidhi; Ritter, Linda M.; Goldberg, Andrew F. X.

2013-01-01

Summary Vertebrate vision requires photon absorption by photoreceptor outer segments (OSs), structurally elaborate membranous organelles derived from non-motile sensory cilia. The structure and function of OSs depends on a precise stacking of hundreds of membranous disks. Each disk is fully (as in rods) or partially (as in cones) bounded by a rim, at which the membrane is distorted into an energetically unfavorable high-curvature bend; however, the mechanism(s) underlying disk rim structure is (are) not established. Here, we demonstrate that the intrinsically disordered cytoplasmic C-terminus of the photoreceptor tetraspanin peripherin-2/rds (P/rds) can directly generate membrane curvature. A P/rds C-terminal domain and a peptide mimetic of an amphipathic helix contained within it each generated curvature in liposomes with a composition similar to that of OS disks and in liposomes generated from native OS lipids. Association of the C-terminal domain with liposomes required conical phospholipids, and was promoted by membrane curvature and anionic surface charge, results suggesting that the P/rds C-terminal amphipathic helix can partition into the cytosolic membrane leaflet to generate curvature by a hydrophobic insertion (wedging) mechanism. This activity was evidenced in full-length P/rds by its induction of small-diameter tubulovesicular membrane foci in cultured cells. In sum, the findings suggest that curvature generation by the P/rds C-terminus contributes to the distinctive structure of OS disk rims, and provide insight into how inherited defects in P/rds can disrupt organelle structure to cause retinal disease. They also raise the possibility that tethered amphipathic helices can function for shaping cellular membranes more generally. PMID:23886945

Simulation of controllable permeation in PNIPAAm coated membranes

NASA Astrophysics Data System (ADS)

Ehrenhofer, Adrian; Wallmersperger, Thomas; Richter, Andreas

2016-04-01

Membranes separate fluid compartments and can comprise transport structures for selective permeation. In biology, channel proteins are specialized in their atomic structure to allow transport of specific compounds (selectivity). Conformational changes in protein structure allow the control of the permeation abilities by outer stimuli (gating). In polymeric membranes, the selectivity is due to electrostatic or size-exclusion. It can thus be controlled by size variation or electric charges. Controllable permeation can be useful to determine particle-size distributions in continuous flow, e.g. in microfluidics and biomedicine to gain cell diameter profiles in blood. The present approach uses patterned polyethylene terephthalate (PET) membranes with hydrogel surface coating for permeation control by size-exclusion. The thermosensitive hydrogel poly(N-isopropylacrylamide) (PNIPAAm) is structured with a cross-shaped pore geometry. A change in the temperature of the water flow through the membrane leads to a pore shape variation. The temperature dependent behavior of PNIPAAm can be numerically modeled with a temperature expansion model, where the swelling and deswelling is depicted by temperature dependent expansion coefficients. In the present study, the free swelling behavior was implemented to the Finite Element tool ABAQUS for the complex composite structure of the permeation control membrane. Experimental values of the geometry characteristics were derived from microscopy images with the tool Image J and compared to simulation results. Numerical simulations using the derived thermo-mechanical model for different pore geometries (circular, rectangle, cross and triangle) were performed. With this study, we show that the temperature expansion model with values from the free swelling behavior can be used to adequately predict the deformation behavior of the complex membrane system. The predictions can be used to optimize the behavior of the membrane pores and the overall performance of the smart membrane.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

PubMed

Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

2016-08-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

PubMed Central

Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

2016-01-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

The voltage-dependent anion channel as a biological transistor: theoretical considerations.

PubMed

Lemeshko, V V; Lemeshko, S V

2004-07-01

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.

Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

PubMed Central

Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

2014-01-01

Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

Solution Structure of Homology Region (HR) Domain of Type II Secretion System*

PubMed Central

Gu, Shuang; Kelly, Geoff; Wang, Xiaohui; Frenkiel, Tom; Shevchik, Vladimir E.; Pickersgill, Richard W.

2012-01-01

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system. PMID:22253442

Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

PubMed

Martínez, Eriel; Estupiñán, Mónica; Pastor, F I Javier; Busquets, Montserrat; Díaz, Pilar; Manresa, Angeles

2013-02-01

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex.

PubMed

Goldberg, Martin W

2016-01-01

Scanning electron microscopy (SEM) is a technique used to image surfaces. Field emission SEMs (feSEMs) can resolve structures that are ~0.5-1.5 nm apart. FeSEM, therefore is a useful technique for imaging molecular structures that exist at surfaces such as membranes. The nuclear envelope consists of four membrane surfaces, all of which may be accessible for imaging. Imaging of the cytoplasmic face of the outer membrane gives information about ribosomes and cytoskeletal attachments, as well as details of the cytoplasmic peripheral components of the nuclear pore complex, and is the most easily accessed surface. The nucleoplasmic face of the inner membrane is easily accessible in some cells, such as amphibian oocytes, giving valuable details about the organization of the nuclear lamina and how it interacts with the nuclear pore complexes. The luminal faces of both membranes are difficult to access, but may be exposed by various fracturing techniques. Protocols are presented here for the preparation, labeling, and feSEM imaging of Xenopus laevis oocyte nuclear envelopes.

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography.

PubMed

Höhn, Katharina; Sailer, Michaela; Wang, Li; Lorenz, Myriam; Schneider, Marion E; Walther, Paul

2011-01-01

Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.

Prestin modulates mechanics and electromechanical force of the plasma membrane.

PubMed

Zhang, Rui; Qian, Feng; Rajagopalan, Lavanya; Pereira, Fred A; Brownell, William E; Anvari, Bahman

2007-07-01

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.

Prestin Modulates Mechanics and Electromechanical Force of the Plasma Membrane

PubMed Central

Zhang, Rui; Qian, Feng; Rajagopalan, Lavanya; Pereira, Fred A.; Brownell, William E.; Anvari, Bahman

2007-01-01

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane. PMID:17468166

Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms.

PubMed

Couto, Narciso; Schooling, Sarah R; Dutcher, John R; Barber, Jill

2015-10-02

In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physicochemical characteristics of these subproteomes, the two strategies showed complementarity, and thus, the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analyzed in terms of their biological context, namely, a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analyzing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.

A preliminary study of aquaporin 1 immunolocalization in chronic subdural hematoma membranes.

PubMed

Basaldella, Luca; Perin, Alessandro; Orvieto, Enrico; Marton, Elisabetta; Itskevich, David; Dei Tos, Angelo Paolo; Longatti, Pierluigi

2010-07-01

Aquaporin 1 (AQP1) is a molecular water channel expressed in many anatomical locations, particularly in epithelial barriers specialized in water transport. The aim of this study was to investigate AQP1 expression in chronic subdural hematoma (CSDH) membranes. In this preliminary study, 11 patients with CSDH underwent burr hole craniectomy and drainage. Membrane specimens were stained with a monoclonal antibody targeting AQP1 for immunohistochemical analysis. The endothelial cells of the sinusoid capillaries of the outer membranes exhibited an elevated immunoreactivity to AQP1 antibody compared to the staining intensity of specimens from the inner membrane and normal dura. These findings suggest that the outer membrane might be the source of the increased fluid accumulation responsible for chronic hematoma enlargement.

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

PubMed Central

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197. Images PMID:1447140

Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis.

PubMed

Fulsundar, Shweta; Kulkarni, Heramb M; Jagannadham, Medicharla V; Nair, Rashmi; Keerthi, Sravani; Sant, Pooja; Pardesi, Karishma; Bellare, Jayesh; Chopade, Balu Ananda

2015-01-01

Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undetermined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sam37 is crucial for formation of the mitochondrial TOM-SAM supercomplex, thereby promoting β-barrel biogenesis.

PubMed

Wenz, Lena-Sophie; Ellenrieder, Lars; Qiu, Jian; Bohnert, Maria; Zufall, Nicole; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

2015-09-28

Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM-SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane. © 2015 Wenz et al.

Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2.

PubMed

Kumar, Abhinendra; Yogisharadhya, Revanaiah; Ramakrishnan, Muthannan A; Viswas, K N; Shivachandra, Sathish B

2013-12-01

Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal β-barrel domain (β1- β16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock. Copyright © 2013 Elsevier Ltd. All rights reserved.

Host cell interactions of outer membrane vesicle-associated virulence factors of Enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury

USDA-ARS?s Scientific Manuscript database

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, confocal laser...

Cooperation of Pd-1 and LAG-3 contributes to T-cell exhaustion in anaplasma marginale-infected cattle

USDA-ARS?s Scientific Manuscript database

The CD4+ T-cell response is central for control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4+ T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OM), which presumably facilit...

Synthesis of Various Metal/TiO2 Core/shell Nanorod Arrays

NASA Astrophysics Data System (ADS)

Zhu, Wei; Wang, Guan-zhong; Hong, Xun; Shen, Xiao-shuang

2011-02-01

We present a general approach to fabricate metal/TiO2 core/shell nanorod structures by two-step electrodeposition. Firstly, TiO2 nanotubes with uniform wall thickness are prepared in anodic aluminum oxide (AAO) membranes by electrodeposition. The wall thickness of the nanotubes could be easily controlled by modulating the deposition time, and their outer diameter and length are only limited by the channel diameter and the thickness of the AAO membranes, respectively. The nanotubes' tops prepared by this method are open, while the bottoms are connected directly with the Au film at the back of the AAO membranes. Secondly, Pd, Cu, and Fe elements are filled into the TiO2 nanotubes to form core/shell structures. The core/shell nanorods prepared by this two-step process are high density and free-standing, and their length is dependent on the deposition time.

Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD.

PubMed

Iyer, Ramkumar; Sylvester, Mark A; Velez-Vega, Camilo; Tommasi, Ruben; Durand-Reville, Thomas F; Miller, Alita A

2017-04-14

The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin-permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria.

Insights into PG-binding, conformational change, and dimerization of the OmpA C-terminal domains from Salmonella enterica serovar Typhimurium and Borrelia burgdorferi: Characterization of OmpA C-Terminal Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Kemin; Deatherage Kaiser, Brooke L.; Wu, Ruiying

S. Typhimurium can induce both humoral and cell-mediated responses when establishing itself in the host. These responses are primarily stimulated against the lipopolysaccharide and major outer membrane (OM) proteins of the bacterium. OmpA is one of these major OM proteins. It comprises a N-terminal eight-stranded -barrel membrane domain and a C-terminal so-called OmpA C-terminal domain (OmpACTD). The OmpACTD and its homologs are believed to bind to peptidoglycan (PG) within the periplasm, maintaining bacterial osmotic homeostasis and modulating the permeability and integrity of the outer membrane. Here we present the structures of two forms of the OmpACTD of S. Typhimurium (STOmpACTD)more » and one structure of the less-studied OmpACTD of Borrelia burgdorferi (BbOmpACTD). In the open form of STOmpACTD, an aspartic acid residue from a long 2-3 loop points into the binding pocket, suggesting that an anion group such as a carboxylate group from PG is favored at the binding site. In the closed form of STOmpACTD and in the structure of BbOmpACTD, a sulfate group from the crystallization buffer is tightly bound at the equivalent site. The differences between the closed and open forms of STOmpACTD, suggest a large conformational change that includes an extension of 3 helix by ordering a part of 2-3 loop. We suggest that the sulfate anion observed in these structures mimics the carboxylate group of PG when bound to STOmpACTD. In addition, the binding of PG or a ligand mimic may enhance dimerization of STOmpACTD, or possibly that of full length STOmpA.« less

Planar ceramic membrane assembly and oxidation reactor system

DOEpatents

Carolan, Michael Francis; Dyer, legal representative, Kathryn Beverly; Wilson, Merrill Anderson; Ohm, Ted R.; Kneidel, Kurt E.; Peterson, David; Chen, Christopher M.; Rackers, Keith Gerard; Dyer, deceased, Paul Nigel

2007-10-09

Planar ceramic membrane assembly comprising a dense layer of mixed-conducting multi-component metal oxide material, wherein the dense layer has a first side and a second side, a porous layer of mixed-conducting multi-component metal oxide material in contact with the first side of the dense layer, and a ceramic channeled support layer in contact with the second side of the dense layer. The planar ceramic membrane assembly can be used in a ceramic wafer assembly comprising a planar ceramic channeled support layer having a first side and a second side; a first dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the first side of the ceramic channeled support layer; a first outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the first dense layer; a second dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the second side of the ceramic channeled layer; and a second outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the second dense layer.

Planar ceramic membrane assembly and oxidation reactor system

DOEpatents

Carolan, Michael Francis; Dyer, legal representative, Kathryn Beverly; Wilson, Merrill Anderson; Ohrn, Ted R.; Kneidel, Kurt E.; Peterson, David; Chen, Christopher M.; Rackers, Keith Gerard; Dyer, Paul Nigel

2009-04-07

Planar ceramic membrane assembly comprising a dense layer of mixed-conducting multi-component metal oxide material, wherein the dense layer has a first side and a second side, a porous layer of mixed-conducting multi-component metal oxide material in contact with the first side of the dense layer, and a ceramic channeled support layer in contact with the second side of the dense layer. The planar ceramic membrane assembly can be used in a ceramic wafer assembly comprising a planar ceramic channeled support layer having a first side and a second side; a first dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the first side of the ceramic channeled support layer; a first outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the first dense layer; a second dense layer of mixed-conducting multi-component metal oxide material having an inner side and an outer side, wherein the inner side is in contact with the second side of the ceramic channeled layer; and a second outer support layer comprising porous mixed-conducting multi-component metal oxide material and having an inner side and an outer side, wherein the inner side is in contact with the outer side of the second dense layer.

Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

PubMed Central

Guillemin, Yannis; Lopez, Jonathan; Gimenez, Diana; Fuertes, Gustavo; Valero, Juan Garcia; Blum, Loïc; Gonzalo, Philippe; Salgado, Jesùs; Girard-Egrot, Agnès; Aouacheria, Abdel

2010-01-01

Background The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. Methodology/Principal Findings Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. Conclusion/Significance BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains. PMID:20140092

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

PubMed

Maraviglia, B; Herring, F G; Weeks, G; Godin, D V

1979-01-01

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism*

PubMed Central

Cameron, Elizabeth A.; Maynard, Mallory A.; Smith, Christopher J.; Smith, Thomas J.; Koropatkin, Nicole M.; Martens, Eric C.

2012-01-01

Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their Kd values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem. PMID:22910908

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.

PubMed

Sahly, Iman; Dufour, Eric; Schietroma, Cataldo; Michel, Vincent; Bahloul, Amel; Perfettini, Isabelle; Pepermans, Elise; Estivalet, Amrit; Carette, Diane; Aghaie, Asadollah; Ebermann, Inga; Lelli, Andrea; Iribarne, Maria; Hardelin, Jean-Pierre; Weil, Dominique; Sahel, José-Alain; El-Amraoui, Aziz; Petit, Christine

2012-10-15

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice

PubMed Central

Sahly, Iman; Dufour, Eric; Schietroma, Cataldo; Michel, Vincent; Bahloul, Amel; Perfettini, Isabelle; Pepermans, Elise; Estivalet, Amrit; Carette, Diane; Aghaie, Asadollah; Ebermann, Inga; Lelli, Andrea; Iribarne, Maria; Hardelin, Jean-Pierre; Weil, Dominique; Sahel, José-Alain

2012-01-01

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins—myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans—do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner–outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients. PMID:23045546

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis.

PubMed

Ramírez-Guadiana, Fernando H; Meeske, Alexander J; Rodrigues, Christopher D A; Barajas-Ornelas, Rocío Del Carmen; Kruse, Andrew C; Rudner, David Z

2017-09-01

One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

Natural Diterpenoid Compound Elevates Expression of Bim Protein, Which Interacts with Antiapoptotic Protein Bcl-2, Converting It to Proapoptotic Bax-like Molecule*

PubMed Central

Zhao, Lixia; He, Feng; Liu, Haiyang; Zhu, Yushan; Tian, Weili; Gao, Ping; He, Hongping; Yue, Wen; Lei, Xiaobo; Ni, Biyun; Wang, Xiaohui; Jin, Haijing; Hao, Xiaojiang; Lin, Jialing; Chen, Quan

2012-01-01

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics. PMID:22065578

A Trimeric Lipoprotein Assists in Trimeric Autotransporter Biogenesis in Enterobacteria*

PubMed Central

Grin, Iwan; Hartmann, Marcus D.; Sauer, Guido; Hernandez Alvarez, Birte; Schütz, Monika; Wagner, Samuel; Madlung, Johannes; Macek, Boris; Felipe-Lopez, Alfonso; Hensel, Michael; Lupas, Andrei; Linke, Dirk

2014-01-01

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of β-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins. PMID:24369174

CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin

PubMed Central

van Pee, Katharina; Neuhaus, Alexander; D'Imprima, Edoardo; Mills, Deryck J; Kühlbrandt, Werner; Yildiz, Özkan

2017-01-01

Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane. DOI: http://dx.doi.org/10.7554/eLife.23644.001 PMID:28323617

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel.

PubMed

Freinkman, Elizaveta; Chng, Shu-Sin; Kahne, Daniel

2011-02-08

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.

The FapF amyloid secretion transporter possesses an atypical asymmetric coiled coil.

PubMed

Rouse, Sarah L; Stylianou, Fisentzos; Grace Wu, H Y; Berry, Jamie-Lee; Sewell, Lee; Morgan, R Marc L; Sauerwein, Andrea C; Matthews, Steve

2018-06-07

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits, the fibres of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialised outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8 Å resolution. This domain forms a novel asymmetric trimeric coiled-coil that possesses a single buried tyrosine residue as well as a extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail. Copyright © 2018. Published by Elsevier Ltd.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

LE COMPLEXE MEMBRANAIRE SUPERFICIEL ET SON EVOLUTION LORS DE L'ELABORATION DES INDIVIDUS-FILS CHEZ TOXOPLASMA GONDII

PubMed Central

Vivier, Emile; Petitprez, André

1969-01-01

The parasitic protozoan Toxoplasma gondii has been examined with the electron microscope in order to study the fine structure and the formation of the membranes surrounding the cell. The study of the ultrastructure of the membranes covering the parasite shows the existence of a three-membraned complex. Only the outer membrane is considered to be the plasma membrane; the two membranes below it form an inseparable whole of changeable molecular architecture (modifications in appearance depending on the methods of fixation, local differentiation). During reproduction, which takes place by fission or more often by endogeny, the membranes of the daughter individuals are formed from the membranes of the parent. At first the middle and inner membranes of the parent extend, separating the cytoplasm of the daughter cells from that of the parent. The three-membrane complex of the endozoites is completed at the time of their liberation; the external membrane of the parent covers the leaving endozoites; thus, the plasma membrane of the daughter cells derives also from that of the parent. These findings on the origin and role of limiting membranes during reproduction differ entirely from those described so far for other cells. PMID:5344151

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

PubMed

Sperandeo, Paola; Cescutti, Rachele; Villa, Riccardo; Di Benedetto, Cristiano; Candia, Daniela; Dehò, Gianni; Polissi, Alessandra

2007-01-01

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.

Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli▿

PubMed Central

Sperandeo, Paola; Cescutti, Rachele; Villa, Riccardo; Di Benedetto, Cristiano; Candia, Daniela; Dehò, Gianni; Polissi, Alessandra

2007-01-01

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 σ 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. PMID:17056748

Current status of meningococcal group B vaccine candidates: capsular or noncapsular?

PubMed

Diaz Romero, J; Outschoorn, I M

1994-10-01

Meningococcal meningitis is a severe, life-threatening infection for which no adequate vaccine exists. Current vaccines, based on the group-specific capsular polysaccharides, provide short-term protection in adults against serogroups A and C but are ineffective in infants and do not induce protection against group B strains, the predominant cause of infection in western countries, because the purified serogroup B polysaccharide fails to elicit human bactericidal antibodies. Because of the poor immunogenicity of group B capsular polysaccharide, different noncapsular antigens have been considered for inclusion in a vaccine against this serogroup: outer membrane proteins, lipooligosaccharides, iron-regulated proteins, Lip, pili, CtrA, and the immunoglobulin A proteases. Alternatively, attempts to increase the immunogenicity of the capsular polysaccharide have been made by using noncovalent complexes with outer membrane proteins, chemical modifications, and structural analogs. Here, we review the strategies employed for the development of a vaccine for Neisseria meningitidis serogroup B; the difficulties associated with the different approaches are discussed.

Current status of meningococcal group B vaccine candidates: capsular or noncapsular?

PubMed Central

Diaz Romero, J; Outschoorn, I M

1994-01-01

Meningococcal meningitis is a severe, life-threatening infection for which no adequate vaccine exists. Current vaccines, based on the group-specific capsular polysaccharides, provide short-term protection in adults against serogroups A and C but are ineffective in infants and do not induce protection against group B strains, the predominant cause of infection in western countries, because the purified serogroup B polysaccharide fails to elicit human bactericidal antibodies. Because of the poor immunogenicity of group B capsular polysaccharide, different noncapsular antigens have been considered for inclusion in a vaccine against this serogroup: outer membrane proteins, lipooligosaccharides, iron-regulated proteins, Lip, pili, CtrA, and the immunoglobulin A proteases. Alternatively, attempts to increase the immunogenicity of the capsular polysaccharide have been made by using noncovalent complexes with outer membrane proteins, chemical modifications, and structural analogs. Here, we review the strategies employed for the development of a vaccine for Neisseria meningitidis serogroup B; the difficulties associated with the different approaches are discussed. PMID:7834605

Toward the rational design of carbapenem uptake in Pseudomonas aeruginosa.

PubMed

Isabella, Vincent M; Campbell, Arthur J; Manchester, John; Sylvester, Mark; Nayar, Asha S; Ferguson, Keith E; Tommasi, Ruben; Miller, Alita A

2015-04-23

Understanding how compound penetration occurs across the complex cell walls of Gram-negative bacteria is one of the greatest challenges in discovering new drugs to treat the infections they cause. A combination of next-generation transposon sequencing, computational metadynamics simulations (CMDS), and medicinal chemistry was used to define genetic and structural elements involved in facilitated carbapenem entry into Pseudomonas aeruginosa. Here we show for the first time that these compounds are taken up not only by the major outer membrane channel OccD1 (also called OprD or PA0958) but also by a closely related channel OccD3 (OpdP or PA4501). Transport-mediating molecular interactions predicted by CMDS for these channels were first confirmed genetically, then used to guide the design of carbapenem analogs with altered uptake properties. These results bring us closer to the rational design of channel transmissibility and may ultimately lead to improved permeability of compounds across bacterial outer membranes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

DOE PAGES

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; ...

2016-07-27

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different beta-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin alpha-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels aremore » consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. In conclusion, the obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.« less

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

PubMed Central

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; Gurnev, Philip A.; Muthukumar, Murugappan; Parsegian, V. Adrian

2016-01-01

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

Mechanism of protein import across the chloroplast envelope.

PubMed

Chen, K; Chen, X; Schnell, D J

2000-01-01

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

Identification and characterization of Vibrio cholerae surface proteins by radioiodination.

PubMed Central

Richardson, K; Parker, C D

1985-01-01

Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides. Images PMID:3980099

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

PubMed

Horobin, R W; Stockert, J C; Rashid-Doubell, F

2015-05-01

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium.

PubMed

Fu, Xiang-Yang

2010-09-01

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.

Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids.

PubMed

Gumí-Audenis, Berta; Costa, Luca; Carlá, Francesco; Comin, Fabio; Sanz, Fausto; Giannotti, Marina I

2016-12-19

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information.

Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids

PubMed Central

Gumí-Audenis, Berta; Costa, Luca; Carlá, Francesco; Comin, Fabio; Sanz, Fausto; Giannotti, Marina I.

2016-01-01

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information. PMID:27999368

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria.

PubMed

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-08-05

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12.

PubMed Central

Kawaji, H; Mizuno, T; Mizushima, S

1979-01-01

Supplementation of the growth medium with high concentrations of sugars or low-molecular-weight dextrans results in a drastic change in the ratio of outer membrane proteins O-8 and O-9, due to induction of O-8 synthesis and suppression of O-9 synthesis. Sugars and dextrans of molecular weights greater than 600 to 700 switched the synthesis of O-9 to that of O-8 more effectively than those of lower molecular weight, although the effect was almost the same within each of the two groups irrespective of the differences in molecular weight within the group. Proteins O-8 or O-9, or both, are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than about 600 (T. Nakae, Biochem. Biophys. Res. Commun. 71:877-884, 1976). The results indicate that substances that cannot pass through the outer membrane switch the synthesis of O-9 to that of O-8 more effectively than those that can penetrate this membrane with the aid of O-8, O-9, or both. It is suggested that the osmotic pressure exerted on the outer membrane plays an important role in the regulation of synthesis of the two proteins. PMID:391802

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria*

PubMed Central

Wang, Yan; Wang, Rui; Jin, Feng; Liu, Yang; Yu, Jiayu; Fu, Xinmiao; Chang, Zengyi

2016-01-01

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis. PMID:27298319

Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes

PubMed Central

Shi, Liang; Squier, Thomas C; Zachara, John M; Fredrickson, James K

2007-01-01

Dissimilatory reduction of metal (e.g. Fe, Mn) (hydr)oxides represents a challenge for microorganisms, as their cell envelopes are impermeable to metal (hydr)oxides that are poorly soluble in water. To overcome this physical barrier, the Gram-negative bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens have developed electron transfer (ET) strategies that require multihaem c-type cytochromes (c-Cyts). In S. oneidensis MR-1, multihaem c-Cyts CymA and MtrA are believed to transfer electrons from the inner membrane quinone/quinol pool through the periplasm to the outer membrane. The type II secretion system of S. oneidensis MR-1 has been implicated in the reduction of metal (hydr)oxides, most likely by translocating decahaem c-Cyts MtrC and OmcA across outer membrane to the surface of bacterial cells where they form a protein complex. The extracellular MtrC and OmcA can directly reduce solid metal (hydr)oxides. Likewise, outer membrane multihaem c-Cyts OmcE and OmcS of G. sulfurreducens are suggested to transfer electrons from outer membrane to type IV pili that are hypothesized to relay the electrons to solid metal (hydr)oxides. Thus, multihaem c-Cyts play critical roles in S. oneidensis MR-1- and G. sulfurreducens-mediated dissimilatory reduction of solid metal (hydr)oxides by facilitating ET across the bacterial cell envelope. PMID:17581116

Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides.

PubMed

Narita, Shin-ichiro; Tokuda, Hajime

2009-07-07

Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits. To examine this possibility, LptB was expressed with LptF and LptG. We report here that both LptF and LptG formed a complex with LptB. Furthermore, an inner membrane protein, LptC, which had been implicated in lipopolysaccharide transport, was also included in this complex.

Probing the mechanism of fusion in a two-dimensional computer simulation.

PubMed Central

Chanturiya, Alexandr; Scaria, Puthurapamil; Kuksenok, Oleksandr; Woodle, Martin C

2002-01-01

A two-dimensional (2D) model of lipid bilayers was developed and used to investigate a possible role of membrane lateral tension in membrane fusion. We found that an increase of lateral tension in contacting monolayers of 2D analogs of liposomes and planar membranes could cause not only hemifusion, but also complete fusion when internal pressure is introduced in the model. With a certain set of model parameters it was possible to induce hemifusion-like structural changes by a tension increase in only one of the two contacting bilayers. The effect of lysolipids was modeled as an insertion of a small number of extra molecules into the cis or trans side of the interacting bilayers at different stages of simulation. It was found that cis insertion arrests fusion and trans insertion has no inhibitory effect on fusion. The possibility of protein participation in tension-driven fusion was tested in simulation, with one of two model liposomes containing a number of structures capable of reducing the area occupied by them in the outer monolayer. It was found that condensation of these structures was sufficient to produce membrane reorganization similar to that observed in simulations with "protein-free" bilayers. These data support the hypothesis that changes in membrane lateral tension may be responsible for fusion in both model phospholipid membranes and in biological protein-mediated fusion. PMID:12023230

Method for determining the three-dimensional structure of a protein

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor)

2004-01-01

Microcapsules prepared by encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane by are disclosed. The microcapsules are formed by interfacial coacervation under conditions where the shear forces are limited to 0-100 dynes/cm.sup.2 at the interface. By placing the microcapsules in a high osmotic dewatering solution, the protein solution is gradually made saturated and then supersaturated, and the controlled nucleation and crystallization of the protein is achieved. The crystal-filled microcapsules prepared by this method can be conveniently harvested and stored while keeping the encapsulated crystals in essentially pristine condition due to the rugged, protective membrane. Because the membrane components themselves are x-ray transparent, large crystal-containing microcapsules can be individually selected, mounted in x-ray capillary tubes and subjected to high energy x-ray diffraction studies to determine the 3-D structure of the protein molecules. Certain embodiments of the microcapsules of the invention have composite polymeric outer membranes which are somewhat elastic, water insoluble, permeable only to water, salts, and low molecular weight molecules and are structurally stable in fluid shear forces typically encountered in the human vascular system.

Structure and membrane remodeling activity of ESCRT-III helical polymers

PubMed Central

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; Hanson, Phyllis I.; Frost, Adam

2015-01-01

The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 Å resolution cryo-EM reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, CHMP1B and IST1. The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture, and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively-curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature. PMID:26634441

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes

NASA Astrophysics Data System (ADS)

Shen, Hsin-Hui; Leyton, Denisse L.; Shiota, Takuya; Belousoff, Matthew J.; Noinaj, Nicholas; Lu, Jingxiong; Holt, Stephen A.; Tan, Khershing; Selkrig, Joel; Webb, Chaille T.; Buchanan, Susan K.; Martin, Lisandra L.; Lithgow, Trevor

2014-10-01

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

Studies of molecular diffusion in single-supported bilayer lipid membranes at high hydration by quasielastic neutron scattering

NASA Astrophysics Data System (ADS)

Bai, M.; Miskowiec, A.; Wang, S.-K.; Taub, H.; Hansen, F. Y.; Jenkins, T.; Tyagi, M.; Neumann, D. A.; Diallo, S. O.; Mamontov, E.; Herwig, K. W.

2011-03-01

Bilayer lipid membranes supported on a solid surface are attractive model systems for understanding the structure and dynamics of more complex biological membranes that form the outer boundary of living cells. We have recently obtained quasielastic neutron spectra from single-supported bilayer lipid membranes using the backscattering spectrometer BASIS at the Spallation Neutron Source. Protonated DMPC membranes were deposited onto Si O2 -coated Si(100) substrates and characterized by AFM. Analysis of their neutron spectra shows evidence of a relatively broad Lorentzian component that we associate with bulk-like water above a freezing temperature of ~ 267 K. At lower temperatures, the spectra differ qualitatively from that of bulk supercooled water, a behavior that we attribute to water bound to the membrane. We also find evidence of a narrow Lorentzian component that we tentatively identify with a slower motion (time scale ~ 1 ns) associated with conformational changes of the alkyl tails of the lipid molecules. Supported by NSF Grant No. DMR-0705974.

Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli.

PubMed Central

Asai, Y; Katayose, Y; Hikita, C; Ohta, A; Shibuya, I

1989-01-01

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation. Images PMID:2556377

Resolving the negative potential side (n-side) water-accessible proton pathway of F-type ATP synthase by molecular dynamics simulations.

PubMed

Gohlke, Holger; Schlieper, Daniel; Groth, Georg

2012-10-19

The rotation of F(1)F(o)-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane F(o) domain, which is coupled to the ATP-producing F(1) domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the "locked" and "open" conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F(1)F(o)-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode.

Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli

PubMed Central

Giri, Janhavi; Tang, John M.; Wirth, Christophe; Peneff, Caroline M.

2012-01-01

NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (Vreversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer. PMID:22246445

Theoretical analysis of the performance of glucose sensors with layer-by-layer assembled outer membranes.

PubMed

Croce, Robert A; Vaddiraju, Santhisagar; Papadimitrakopoulos, Fotios; Jain, Faquir C

2012-10-01

The performance of implantable electrochemical glucose sensors is highly dependent on the flux-limiting (glucose, H(2)O(2), O(2)) properties of their outer membranes. A careful understanding of the diffusion profiles of the participating species throughout the sensor architecture (enzyme and membrane layer) plays a crucial role in designing a robust sensor for both in vitro and in vivo operation. This paper reports the results from the mathematical modeling of Clark's first generation amperometric glucose sensor coated with layer-by-layer assembled outer membranes in order to obtain and compare the diffusion profiles of various participating species and their effect on sensor performance. Devices coated with highly glucose permeable (HAs/Fe(3+)) membranes were compared with devices coated with PSS/PDDA membranes, which have an order of magnitude lower permeability. The simulation showed that the low glucose permeable membrane (PSS/PDDA) sensors exhibited a 27% higher amperometric response than the high glucose permeable (HAs/Fe(3+)) sensors. Upon closer inspection of H(2)O(2)diffusion profiles, this non-typical higher response from PSS/PDDA is not due to either a larger glucose flux or comparatively larger O(2) concentrations within the sensor geometry, but rather is attributed to a 48% higher H(2)O(2) concentration in the glucose oxidase enzyme layer of PSS/PDDA coated sensors as compared to HAs/Fe(3+) coated ones. These simulated results corroborate our experimental findings reported previously. The high concentration of H(2)O(2) in the PSS/PDDA coated sensors is due to the low permeability of H(2)O(2) through the PSS/PDDA membrane, which also led to an undesired increase in sensor response time. Additionally, it was found that this phenomenon occurs for all enzyme thicknesses investigated (15, 20 and 25 nm), signifying the need for a holistic approach in designing outer membranes for amperometric biosensors.

[Ultrastructure and cytochemistry of the pellicle and apical complexes of the kinete of Babesia bigemina and Babesia ovis in the hemolymph and oavry of the tick].

PubMed

Weber, G

1980-02-01

The term kinete is used in this paper for the cigar-shaped, motile development stages (VERMICULE") OF Babesia occurring intra- and extracellularly in hemolymph and overy (including oocytes) of vectors, hard ticks (Ixodoidea). The structure of, and cytochemical activities of hydrolases (acid phosphatase, nonspecific esterase) in the pellicle and the apical complex was studied at the fine-structural level in kinetes of Babesia bigemina Smith & Kilborne, in hemolympho of female Boophilus microplus Canestrini. The cytochemistry of acid hydrolases was studied also in kinetes of Babesia ovis (Babès) Starcovici, in hemolymph and ovary of Rhipicephalus bursa Canestrini & Fanzago. The pellicle of the B. bigemina kinetes is composted of 3 membranes (pellicular complex): an outer membrane, approximately 8 nm thick (the plasmalemma) and 2 innder ones, each approximately nm thick, lying closely together. The outer membrane appears to be covered by a structureless coat, 3 nm thick. The space between the inner double membrane and the plasmalemma is 7.5 nm. The whole pellicular complex is 30 nm in diameter. The 2 inner pellicular membranes appear to be derived from the endoplasmic reticulum (ER) for the following reasons: (a) a layer of hydrolase-active material is enclosed by these membranes; (b) in the spheroid parasite stages which transform from kinetes inside hemocytes, the inner double membrane is apparently replaced by an ER cisterna; (c) the thickness of each of the inner pellicular membranes is approximately the same as that of the ER membrane. There are circular openings in the pellicular double membrane with average diameters of 100 nm; despite some similarity to micropores, they have a specific structure. The term Intrapellikularfenster (IPF) (intrapellicular windows) or pseudomicropores is proposed for these pellicular differentiations. The margin of an IPF is formed by the 2 inner membranes folding into each other; cytoplasmic, electron-dense material is accumulated alongside this edge. Unlike that of micropores, the plasmalemma of the IPF is not invaginated. The IPF appears as a single, dark ring in tangential sections. At times, rhoptry-like bodies are associated with the openings. The function of the IPF is not known. An intrapellicular opening similar to the IPF, although wider, is present at the apex of the parasite. Its margin coincides with the inners edge of the apical ring. Typical subpellicular microtubuli were not observed in the Babesia kinetes. The apical complex of the B. bigemina kinetes consists of an Apikalschirm (apical umbrella), a crown of microtubuli beneath it, and rhoptries: micronemes are also present in large numbers. The Apikalschirm is located beneath the pellicle of the apical pole of the parasite. It is a wheel-like structure composed of spokes radiating from a wide, hub=like central ring (apical ring). It should be stressed that the apical ring is not identical with the polar ring described as an integral part of the pellicular complex in other Apicomplexa...

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic β Taco Fold.

PubMed

Josts, Inokentijs; Stubenrauch, Christopher James; Vadlamani, Grishma; Mosbahi, Khedidja; Walker, Daniel; Lithgow, Trevor; Grinter, Rhys

2017-12-05

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB 963-1138 consists of a concave, taco-shaped β sheet with a hydrophobic interior. This β taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic β strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB 963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes.

PubMed

Suwalsky, Mario; Belmar, Jessica; Villena, Fernando; Gallardo, María José; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2013-11-01

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures. Copyright © 2013 Elsevier Inc. All rights reserved.

Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay.

PubMed

Okamoto, K; Gotoh, N; Nishino, T

2001-07-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

Pseudomonas aeruginosa Reveals High Intrinsic Resistance to Penem Antibiotics: Penem Resistance Mechanisms and Their Interplay

PubMed Central

Okamoto, Kiyomi; Gotoh, Naomasa; Nishino, Takeshi

2001-01-01

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC β-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC β-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC β-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC β-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC β-lactamase but not from the lower affinity of PBPs for penems. PMID:11408209

Serum antibodies in mares and foals to Actinobacillus equuli whole cells, outer membrane proteins, and Aqx toxin.

PubMed

Holyoak, G R; Smith, C M; Boyette, R; Montelongo, M; Wray, J H; Ayalew, S; Duggan, V E; Confer, A W

2007-08-15

Actinobacillus equuli is carried in the alimentary tract of mares and can cause severe septicemia of neonatal foals. A hemolytic subspecies, A. equuli subsp. haemolyticus, and a non-hemolytic subspecies, A. equuli subsp. equuli, have been identified. Hemolytic strains produce the RTX toxin Aqx. The purpose of this study was to demonstrate sequentially in two sets of mare-foal pairs antibodies to A. equuli whole bacterial cells, outer membrane proteins, and recombinant Aqx and to compare the transfer of antibodies to these antigens between mares and their foals. Two mare/foal sets of sera were evaluated. Cohort A consisted of 18 mare-foal pairs obtained in the spring of 2005. Cohort B consisted of 10 mare-foal pairs obtained in the spring of 2006. For both sets, mare and foal sera were obtained immediately after foaling and prior to nursing (time 0) as well as at 12 and 24h and daily thereafter for 7 days. For Cohort B, sera were also obtained 30 days after birth. At parturition all mares had detectable antibodies to A. equuli whole cells and outer membranes; however, of those mares, two in Cohort A had undetectable antibodies to Aqx and their foals likewise had undetectable anti-Aqx antibodies. Antibodies against whole cells, outer membrane proteins, and Aqx were readily transferred from mares to foals. In most cases, there were significant correlations (p<0.05) between antibodies against whole cells, outer membrane proteins, and Aqx in mares' sera at the time of parturition and foal sera 24 after birth. Antibodies against the three antigen preparations had declined insignificantly (p>0.05) by day 30.

Free-radicals and advanced chemistries involved in cell membrane organization influence oxygen diffusion and pathology treatment.

PubMed

Petersen, Richard C

2017-01-01

A breakthrough has been discovered in pathology chemistry related to increasing molecular structure that can interfere with oxygen diffusion through cell membranes. Free radicals can crosslink unsaturated low-viscosity fatty acid oils by chain-growth polymerization into more viscous liquids and even solids. Free radicals are released by mitochondria in response to intermittent hypoxia that can increase membrane molecular organization to reduce fluidity and oxygen diffusion in a possible continuing vicious cycle toward pathological disease. Alternate computational chemistry demonstrates molecular bond dynamics in free energy for cell membrane physiologic movements. Paired electrons in oxygen and nitrogen atoms require that oxygen bonds rotate and nitrogen bonds invert to seek polar nano-environments and hide from nonpolar nano-environments thus creating fluctuating instability at a nonpolar membrane and polar biologic fluid interface. Subsequent mechanomolecular movements provide free energy to increase diffusion by membrane transport of molecules and oxygen into the cell, cell-membrane signaling/recognition/defense in addition to protein movements for enzyme mixing. In other chemistry calcium bonds to membrane phosphates primarily on the outer plasma cell membrane surface to influence the membrane firing threshold for excitability and better seal out water permeation. Because calcium is an excellent metal conductor and membrane phosphate headgroups form a semiconductor at the biologic fluid interface, excess electrons released by mitochondria may have more broad dissipation potential by safe conduction through calcium atomic-sized circuits on the outer membrane surface. Regarding medical conditions, free radicals are known to produce pathology especially in age-related disease in addition to aging. Because cancer cell membranes develop extreme polymorphism that has been extensively followed in research, accentuated easily-visualized free-radical models are developed. In terms of treatment, use of vitamin nutrient supplements purported to be antioxidants that remove free radicals has not proved worthwhile in clinical trials presumably due to errors with early antioxidant measurements based on inaccurate colorimetry tests. However, newer covalent-bond shrinkage tests now provide accurate measurements for free-radical inhibitor hydroquinone and other molecules toward drug therapy.

Free-radicals and advanced chemistries involved in cell membrane organization influence oxygen diffusion and pathology treatment

PubMed Central

Petersen, Richard C

2017-01-01

A breakthrough has been discovered in pathology chemistry related to increasing molecular structure that can interfere with oxygen diffusion through cell membranes. Free radicals can crosslink unsaturated low-viscosity fatty acid oils by chain-growth polymerization into more viscous liquids and even solids. Free radicals are released by mitochondria in response to intermittent hypoxia that can increase membrane molecular organization to reduce fluidity and oxygen diffusion in a possible continuing vicious cycle toward pathological disease. Alternate computational chemistry demonstrates molecular bond dynamics in free energy for cell membrane physiologic movements. Paired electrons in oxygen and nitrogen atoms require that oxygen bonds rotate and nitrogen bonds invert to seek polar nano-environments and hide from nonpolar nano-environments thus creating fluctuating instability at a nonpolar membrane and polar biologic fluid interface. Subsequent mechanomolecular movements provide free energy to increase diffusion by membrane transport of molecules and oxygen into the cell, cell-membrane signaling/recognition/defense in addition to protein movements for enzyme mixing. In other chemistry calcium bonds to membrane phosphates primarily on the outer plasma cell membrane surface to influence the membrane firing threshold for excitability and better seal out water permeation. Because calcium is an excellent metal conductor and membrane phosphate headgroups form a semiconductor at the biologic fluid interface, excess electrons released by mitochondria may have more broad dissipation potential by safe conduction through calcium atomic-sized circuits on the outer membrane surface. Regarding medical conditions, free radicals are known to produce pathology especially in age-related disease in addition to aging. Because cancer cell membranes develop extreme polymorphism that has been extensively followed in research, accentuated easily-visualized free-radical models are developed. In terms of treatment, use of vitamin nutrient supplements purported to be antioxidants that remove free radicals has not proved worthwhile in clinical trials presumably due to errors with early antioxidant measurements based on inaccurate colorimetry tests. However, newer covalent-bond shrinkage tests now provide accurate measurements for free-radical inhibitor hydroquinone and other molecules toward drug therapy. PMID:29202036

Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

PubMed

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

PubMed Central

de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

2016-01-01

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

Colicin Killing: Foiled Cell Defense and Hijacked Cell Functions

NASA Astrophysics Data System (ADS)

de Zamaroczy, Miklos; Chauleau, Mathieu

The study of bacteriocins, notably those produced by E. coli (and named colicins), was initiated in 1925 by Gratia, who first discovered "un remarquable exemple d'antagonisme entre deux souches de colibacilles". Since this innovating observation, the production of toxic exoproteins has been widely reported in all major lineages of Eubacteria and in Archaebacteria. Bacteriocins belong to the most abundant and most diverse group of these bacterial defense systems. Paradoxically, these antimicrobial cytotoxins are actually powerful weapons in the intense battle for bacterial survival. They are also biotechnologically useful since several bacteriocins are used as preservatives in the food industry or as antibiotics or as potential antitumor agents in human health care. Most colicins kill bacteria in one of two ways. The first type is those that form pores in the phospholipid bilayer of the inner membrane. They are active immediately after their translocation across the outer membrane. The translocation pathway requires generally either the BtuB receptor and the Tol (OmpF/TolABQR) complex, or the FepA, FhuA, or Cir receptor and the Ton (TonB/ExbBD) system. The second type of colicins encodes specific endonuclease activities that target DNA, rRNA, or tRNAs in the cytoplasm. To be active, these colicins require translocation across both the outer and inner membranes. The molecular mechanisms implicated in the complex cascade of interactions, required for the transfers of colicin molecules from the extracellular medium through the different "cellular compartments" (outer membrane, periplasm, inner membrane, and cytoplasm), are still incompletely understood. It is clear, however, that the colicins "hijack" specific cellular functions to facilitate access to their target. In this chapter, following a general presentation of colicin biology, we describe, compare, and update several of the concepts related to colicin toxicity and discuss recent, often unexpected findings, which help to advance our understanding of the molecular events governing colicin import. In particular, our review includes the following: (1) Structural data on the tripartite interaction of a colicin with the outer membrane receptor and the translocation machinery, (2) Comparison of the normal cellular functions of the Tol and Ton systems of the inner membrane with their "hijacked" roles during colicin import, (3) An analysis of the interaction of a nuclease-type colicin with its cognate immunity protein in the context of the immunity of producer cells, and of the dissociation of this complex in the context of the attack of the colicin on target cells, (4) Information on the endoproteolytic cleavage, which presumably accompanies the penetration of nuclease-type colicins into the cytoplasm. The new data presented here provides further insight into cellular functions "hijacked" or "borrowed" by colicins to permit their entry into target cells.

Extra-mitochondrial aerobic metabolism in retinal rod outer segments: new perspectives in retinopathies.

PubMed

Panfoli, I; Calzia, D; Ravera, S; Morelli, A M; Traverso, C E

2012-04-01

Vertebrate retinal rods are photoreceptors for dim-light vision. They display extreme sensitivity to light thanks to a specialized subcellular organelle, the rod outer segment. This is filled with a stack of membranous disks, expressing the proteins involved in visual transduction, a very energy demanding process. Our previous proteomic and biochemical studies have shed new light on the chemical energy processes that supply ATP to the outer segment, suggesting the presence of an extra-mitochondrial aerobic metabolism in rod outer segment, devoid of mitochondria, which would account for a quantitatively adequate ATP supply for phototransduction. Here the functional presence of an oxidative phosphorylation in the rod outer limb is examined for its relationship to many physiological and pathological data on the rod outer segment. We hypothesize that the rod outer limb is at risk of oxidative stress, in any case of impairment in the respiratory chain functioning, or of blood supply. In fact, the electron transfer chain is a major source of reactive O(2) species, known to produce severe alteration to the membrane lipids, especially those of the outer segment that are rich in polyunsaturated fatty acids. We propose that the disk membrane may become the target of reactive oxygen species that may be released by the electron transport chain under pathologic conditions. For example, during aging reactive oxygen species production increases, while cellular antioxidant capacity decreases. Also the apoptosis of the rod observed after exposure to bright or continuous illumination can be explained considering that an overfunctioning of phototransduction may damage the disk membrane to a point at which cytochrome c escapes from the intradiskal space, where it is presently supposed to be, activating a putative caspase 9 and the apoptosome. A pathogenic mechanism for many inherited and acquired retinal degenerations, representing a major problem in clinical ophthalmology, is proposed: a number of rod pathologies would be promoted by impairment of energy supply and/or oxidative stress in the rod outer segment. In conclusion we suppose that the damaging role of oxygen, be it hypoxia or hyperoxia invoked in most of the blinding diseases, acquired and even hereditary is to be seeked for inside the photoreceptor outer segment that would conceal a potential for cell death that is still to be recognized. Copyright © 2012 Elsevier Ltd. All rights reserved.

Electrophysiological and Genetic Analysis of Chemosensory Mechanisms in Spirochaeta Aurantia

DTIC Science & Technology

1988-05-01

Ghiorse, and E. P. Greenberg. 1987. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta... proteins which at least in part constitute the flagella. Three proteins in the 60-65 kdaltons range predominate in preparations consisting of HBBs...the filaments can be solubilized by acid treatment) and thus, these proteins have been assigned as HBB components. Six proteins in the 30-38 kdalton

Role of outer membrane c-type cytochromes MtrC and OmcA in Shewanella oneidensis MR-1 cell production, accumulation and detachment during respiration on hematite

USDA-ARS?s Scientific Manuscript database

The iron-reducing bacterium Shewanella oneidensis MR-1 has the capacity to contribute to iron cycling over the long term by respiring on crystalline iron oxides such as hematite when poorly crystalline phases are depleted. The ability of outer membrane cytochromes OmcA and MtrC of MR-1 to bind to an...

Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniae Strains

PubMed Central

Martínez-Martínez, Luis; García, Isabel; Ballesta, Sofía; Benedí, Vicente Javier; Hernández-Allés, Santiago; Pascual, Alvaro

1998-01-01

The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents. PMID:9661034

Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

PubMed Central

Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican

2014-01-01

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii. PMID:24837387

The participation of outer membrane proteins in the bacterial sensitivity to nanosilver.

PubMed

Kędziora, Anna; Krzyżewska, Eva; Dudek, Bartłomiej; Bugla-Płoskońska, Gabriela

2016-06-13

The presented study is to analyze the participation of outer membrane proteins of Gram- negative bacteria in sensitivity to silver nanomaterials. The mechanism of interaction of silver with the bacterial cell is best described in this group of microorganisms. There are several theories regarding the effectiveness of antimicrobial ions and nanosilver, and at the indicated differences in the way they work. Outer membrane proteins of Gram-negative bacteria are involved in the procurement of silver from the environment and contribute to the development mechanisms of resistance to nanometals. They are measurable parameter in the field of cell phenotypic response to the presence of Gram-negative bacteria in the environment silver nanoforms: its properties, chemical composition, content or times of action. Proteomic methods (including two dimensional electrophoresis and MALDI‑TOF MS) are therefore relevant techniques for determining the susceptibility of bacteria to silver and the changes taking place in the outer membrane under the influence: uptime/exposure and physical and chemical parameters of silver nanomaterials. Many products containing nanosilver is still in the research phase in terms of physico‑chemical characteristics and biological activity, others have been already implemented in many industries. During the very fast nanotechnology developing and introduction to the market products based on the nanosilver the bacterial answer to nanosilver is needed.

Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.

PubMed

Newton, S M; Klebba, P E; Michel, V; Hofnung, M; Charbit, A

1996-06-01

We previously developed a genetic approach to study, with a single antibody, the topology of the outer membrane protein LamB, an Escherichia coli porin with specificity towards maltodextrins and a receptor for bacteriophage lambda. Our initial procedure consisted of inserting at random the same reporter epitope (the C3 neutralization epitope from poliovirus) into permissive sites of LamB (i.e., sites which tolerate insertions without deleterious effects on the protein activities or the cell). A specific monoclonal antibody was then used to examine the position of the inserted epitope with respect to the protein and the membrane. In the present work, we set up a site-directed procedure to insert the C3 epitope at new sites in order to distinguish between two-dimensional folding models. This allowed us to identify two new surface loops of LamB and to predict another periplasmic exposed region. The results obtained by random and directed epitope tagging are analyzed in light of the recently published X-ray structure of the LamB protein. Study of 23 hybrid LamB-C3 proteins led to the direct identification of five of the nine external loops (L4, L5, L6, L7, and L9) and led to the prediction of four periplasmic loops (I1, I4, I5, and I8) of LamB. Nine of the hybrid proteins did not lead to topological conclusions, and none led to the wrong predictions or conclusions. The comparison indicates that parts of models based on secondary structure predictions alone are not reliable and points to the importance of experimental data in the establishment of outer membrane protein topological models. The advantages and limitations of genetic foreign epitope insertion for the study of integral membrane proteins are discussed.

Some Observations on the Fine Structure of the Giant Nerve Fibers of the Earthworm, Eisenia foetida

PubMed Central

Hama, Kiyoshi

1959-01-01

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa. PMID:13673048

Light-Induced Thickening of Photoreceptor Outer Segment Layer Detected by Ultra-High Resolution OCT Imaging.

PubMed

Li, Yichao; Fariss, Robert N; Qian, Jennifer W; Cohen, Ethan D; Qian, Haohua

2016-07-01

We examined if light induces changes in the retinal structure that can be observed using optical coherence tomography (OCT). Normal C57BL/6J mice (age 3-6 months) adapted to either room light (15 minutes to ∼5 hours, 50-500 lux) or darkness (overnight) were imaged using a Bioptigen UHR-OCT system. Confocal histologic images were obtained from mice killed under light- or dark-adapted conditions. The OCT image of eyes adapted to room light exhibited significant increases (6.1 ± 0.8 μm, n = 13) in total retina thickness compared to the same eyes after overnight dark adaptation. These light-adapted retinal thickness changes occurred mainly in the outer retina, with the development of a hyporeflective band between the RPE and photoreceptor-tip layers. Histologic analysis revealed a light-evoked elongation between the outer limiting membrane and Bruch's membrane from 45.8 ± 1.7 μm in the dark (n = 5) to 52.1 ± 3.7 μm (n = 5) in the light. Light-adapted retinas showed an increase of actin staining in RPE apical microvilli at the same location as the hyporeflective band observed in OCT images. Elongation of the outer retina could be detected even with brief light exposures, increasing 2.1 ± 0.3 μm after 15 minutes (n = 9), and 4.1 ± 1.0 μm after 2 hours (n = 6). Conversely, dark-adaptation caused outer retinal shortening of 1.4 ± 0.4 μm (n = 7) and 3.0 ± 0.5 μm (n = 8) after 15 minutes and 2 hours, respectively. Light-adaption induces an increase in the thickness of the outer retina and the appearance of a hyporeflective band in the OCT image. This is consistent with previous reports of light-induced fluid accumulation in the subretinal space.

Optimization design combined with coupled structural-electrostatic analysis for the electrostatically controlled deployable membrane reflector

NASA Astrophysics Data System (ADS)

Liu, Chao; Yang, Guigeng; Zhang, Yiqun

2015-01-01

The electrostatically controlled deployable membrane reflector (ECDMR) is a promising scheme to construct large size and high precision space deployable reflector antennas. This paper presents a novel design method for the large size and small F/D ECDMR considering the coupled structure-electrostatic problem. First, the fully coupled structural-electrostatic system is described by a three field formulation, in which the structure and passive electrical field is modeled by finite element method, and the deformation of the electrostatic domain is predicted by a finite element formulation of a fictitious elastic structure. A residual formulation of the structural-electrostatic field finite element model is established and solved by Newton-Raphson method. The coupled structural-electrostatic analysis procedure is summarized. Then, with the aid of this coupled analysis procedure, an integrated optimization method of membrane shape accuracy and stress uniformity is proposed, which is divided into inner and outer iterative loops. The initial state of relatively high shape accuracy and uniform stress distribution is achieved by applying the uniform prestress on the membrane design shape and optimizing the voltages, in which the optimal voltage is computed by a sensitivity analysis. The shape accuracy is further improved by the iterative prestress modification using the reposition balance method. Finally, the results of the uncoupled and coupled methods are compared and the proposed optimization method is applied to design an ECDMR. The results validate the effectiveness of this proposed methods.

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli*

PubMed Central

Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H.; Pessi, Gabriella; Eberl, Leo; Robinson, John A.

2016-01-01

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

Auditory Mechanics of the Tectorial Membrane and the Cochlear Spiral

PubMed Central

Gavara, Núria; Manoussaki, Daphne; Chadwick, Richard S.

2012-01-01

Purpose of review This review is timely and relevant since new experimental and theoretical findings suggest that cochlear mechanics from the nanoscale to the macroscale are affected by mechanical properties of the tectorial membrane and the spiral shape. Recent findings Main tectorial membrane themes covered are i) composition and morphology, ii) nanoscale mechanical interactions with the outer hair cell bundle, iii) macroscale longitudinal coupling, iv) fluid interaction with inner hair cell bundles, v) macroscale dynamics and waves. Main cochlear spiral themes are macroscale low-frequency energy focusing and microscale organ of Corti shear gain. Implications Findings from new experimental and theoretical models reveal exquisite sensitivity of cochlear mechanical performance to tectorial membrane structural organization, mechanics, and its positioning with respect to hair bundles. The cochlear spiral geometry is a major determinant of low frequency hearing. Suggestions are made for future research directions. PMID:21785353

Cavin Family: New Players in the Biology of Caveolae.

PubMed

Nassar, Zeyad D; Parat, Marie-Odile

2015-01-01

Caveolae are specialized small plasma-membrane invaginations that play crucial cellular functions. Two essential protein families are required for caveola formation: membrane caveolin proteins and cytoplasmic cavin proteins. Each family includes members with specific tissue distribution, and their expression is altered under physiological and pathological conditions, implying highly specialized functions. Cavins not only stabilize caveolae, but modulate their morphology and functions as well. Before association with the plasma membrane, cavins form homo- and hetero-oligomers with strikingly strict stoichiometry in the cytosol. At the plasma membrane, they provide an outer peripheral cytosolic layer, necessary for caveola stability. Interestingly, upon stimulation, cavins can be released from caveolae into the cytoplasm in distinct subcomplexes, providing a rapid dynamic link between caveolae and cellular organelles including the nucleus. In this review, we detail the biology of cavins, their structural and functional roles, and their implication in pathophysiology. Copyright © 2015 Elsevier Inc. All rights reserved.

Changes in the lipid composition of Bradyrhizobium cell envelope reveal a rapid response to water deficit involving lysophosphatidylethanolamine synthesis from phosphatidylethanolamine in outer membrane.

PubMed

Cesari, Adriana B; Paulucci, Natalia S; Biasutti, María A; Morales, Gustavo M; Dardanelli, Marta S

2018-06-02

We evaluate the behavior of the membrane of Bradyrhizobium sp. SEMIA6144 during adaptation to polyethylene glycol (PEG). A dehydrating effect on the morphology of the cell surface, as well as a fluidizing effect on the membrane was observed 10 min after PEG shock; however, the bacteria were able to restore optimal membrane fluidity. Shock for 1 h caused an increase of lysophosphatidylethanolamine in the outer membrane at the expense of phosphatidylcholine and phosphatidylethanolamine (PE), through an increase in phospholipase activity. The amount of lysophosphatidylethanolamine did not remain constant during PEG shock, but after 24 h the outer membrane was composed of large amounts of phosphatidylcholine and less amount of lysophosphatidylethanolamine similar to the control. The inner membrane composition was also modified after 1 h of shock, observing an increase of phosphatidylcholine at the expense of PE, the proportions of these phospholipids were then modified to reach 24 h of shock values similar to the control. Vesicles prepared with the lipids of cells exposed to 1 h shock presented higher rigidity compared to the control, indicating that changes in the composition of phospholipids after 1 h of shock restoring fluidity after the PEG effect and would allow cells to maintain surface morphology. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

PubMed

Vasiljev, Andreja; Ahting, Uwe; Nargang, Frank E; Go, Nancy E; Habib, Shukry J; Kozany, Christian; Panneels, Valérie; Sinning, Irmgard; Prokisch, Holger; Neupert, Walter; Nussberger, Stephan; Rapaport, Doron

2004-03-01

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia.

PubMed

Crouser, Elliott D; Julian, Mark W; Huff, Jennifer E; Joshi, Mandar S; Bauer, John A; Gadd, Martha E; Wewers, Mark D; Pfeiffer, Douglas R

2004-02-01

This study was designed to determine the role played by the mitochondrial permeability transition in the pathogenesis of mitochondrial damage and dysfunction in a representative systemic organ during the acute phase of endotoxemia. A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporine A, a potent inhibitor of the mitochondrial permeability transition, normalizes mitochondrial ultrastructural injury and dysfunction in the liver during acute endotoxemia. The Ohio State University Medical Center research laboratory. Random source, adult, male conditioned cats. Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Treatment groups received isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 8), or cyclosporine A (6.0 mg/kg, intravenously; n = 6) or tacrolimus (FK506, 0.1 mg/kg, intravenously; n = 4) followed in 30 mins by lipopolysaccharide (3.0 mg/kg, intravenously). Liver samples were obtained 4 hrs posttreatment, and mitochondrial ultrastructure, function, and cytochrome c, Bax, and ceramide contents were assessed. As expected, significant mitochondrial injury was apparent in the liver 4 hrs after lipopolysaccharide treatment, despite maintenance of regional tissue oxygen availability. Namely, mitochondria demonstrated high-amplitude swelling and exhibited altered respiratory function. Cyclosporine A pretreatment attenuated lipopolysaccharide-induced mitochondrial ultrastructural abnormalities and normalized mitochondrial respiratory control, reflecting protection against inner mitochondrial membrane damage. However, an abnormal permeability of outer mitochondrial membranes to cytochrome c was observed in all lipopolysaccharide-treated groups and was associated with increased mitochondrial concentrations of Bax and ceramide. These studies confirm that liver mitochondria are early targets of injury during endotoxemia and that inner and outer mitochondrial membrane damage occurs through different mechanisms. Inner mitochondrial membrane damage appears to relate to the mitochondrial permeability transition, whereas outer mitochondrial membrane damage can occur independent of the mitochondrial permeability transition. Preliminary evidence suggests that Bax may participate in lipopolysaccharide-induced outer mitochondrial membrane damage, but further investigations are needed to confirm this.

Uptake and trans-membrane transport of petroleum hydrocarbons by microorganisms

PubMed Central

Hua, Fei; Wang, Hong Qi

2014-01-01

Petroleum-based products are a primary energy source in the industry and daily life. During the exploration, processing, transport and storage of petroleum and petroleum products, water or soil pollution occurs regularly. Biodegradation of the hydrocarbon pollutants by indigenous microorganisms is one of the primary mechanisms of removal of petroleum compounds from the environment. However, the physical contact between microorganisms and hydrophobic hydrocarbons limits the biodegradation rate. This paper presents an updated review of the petroleum hydrocarbon uptake and transport across the outer membrane of microorganisms with the help of outer membrane proteins. PMID:26740752

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

PubMed Central

Watts, T H; Worobec, E A; Paranchych, W

1982-01-01

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain. Images PMID:6813311

Water and solute permeability of rat lung caveolae: high permeabilities explained by acyl chain unsaturation.

PubMed

Hill, Warren G; Almasri, Eyad; Ruiz, W Giovanni; Apodaca, Gerard; Zeidel, Mark L

2005-07-01

Caveolae are invaginated membrane structures with high levels of cholesterol, sphingomyelin, and caveolin protein that are predicted to exist as liquid-ordered domains with low water permeability. We isolated a caveolae-enriched membrane fraction without detergents from rat lung and characterized its permeability properties to nonelectrolytes and protons. Membrane permeability to water was 2.85 +/- 0.41 x 10(-3) cm/s, a value 5-10 times higher than expected based on comparisons with other cholesterol and sphingolipid-enriched membranes. Permeabilities to urea, ammonia, and protons were measured and found to be moderately high for urea and ammonia at 8.85 +/- 2.40 x 10(-7)and 6.84 +/- 1.03 x 10(-2) respectively and high for protons at 8.84 +/- 3.06 x 10(-2) cm/s. To examine whether caveolin or other integral membrane proteins were responsible for high permeabilities, liposomes designed to mimic the lipids of the inner and outer leaflets of the caveolar membrane were made. Osmotic water permeability to both liposome compositions were determined and a combined inner/outer leaflet water permeability was calculated and found to be close to that of native caveolae at 1.58 +/- 1.1 x 10(-3) cm/s. In caveolae, activation energy for water flux was high (19.4 kcal/mol) and water permeability was not inhibited by HgCl2; however, aquaporin 1 was detectable by immunoblotting. Immunostaining of rat lung with AQP1 and caveolin antisera revealed very low levels of colocalization. We conclude that aquaporin water channels do not contribute significantly to the observed water flux and that caveolae have relatively high water and solute permeabilities due to the high degree of unsaturation in their fatty acyl chains.

Recovery of cesium

DOEpatents

Izatt, Reed M.; Christensen, James J.; Hawkins, Richard T.

1984-01-01

A process of recovering cesium ions from mixtures of ions containing them and other ions, e.g., a solution of nuclear waste materials, which comprises establishing a separate source phase containing such a mixture of ions, establishing a separate recipient phase, establishing a liquid membrane phase in interfacial contact with said source and recipient phases, said membrane phase containing a ligand, preferably a selected calixarene as depicted in the drawing, maintaining said interfacial contact for a period of time long enough to transport by said ligand a substantial portion of the cesium ion from the source phase to the recipient phase, and recovering the cesium ion from the recipient phase. The separation of the source and recipient phases may be by the membrane phase only, e.g., where these aqueous phases are emulsified as dispersed phases in a continuous membrane phase, or may include a physical barrier as well, e.g., an open-top outer container with an inner open-ended container of smaller cross-section mounted in the outer container with its open bottom end spaced from and above the closed bottom of the outer container so that the membrane phase may fill the outer container to a level above the bottom of the inner container and have floating on its upper surface a source phase and a recipient phase separated by the wall of the inner container as a physical barrier. A preferred solvent for the ligand is a mixture of methylene chloride and carbon tetrachloride.

The Effect of Lipopolysaccharide Core Oligosaccharide Size on the Electrostatic Binding of Antimicrobial Proteins to Models of the Gram Negative Bacterial Outer Membrane

PubMed Central

2016-01-01

Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate-rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different-sized core oligosaccharide regions we demonstrate the role of uncharged sugars in blocking short-range electrostatic interactions between the cationic antibiotics and the vulnerable anionic phosphate groups. PMID:27003358

Comprehensive Analysis of Transport Proteins Encoded Within the Genome of Bdellovibrio bacteriovorus

PubMed Central

Barabote, Ravi D.; Rendulic, Snjezana; Schuster, Stephan C.; Saier, Milton H.

2012-01-01

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the transporter classification database (TCDB). A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least 4 types of inner membrane secretion systems and 5 types for outer membrane secretion are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer membrane transport and motility than does E. coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes were also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses. PMID:17706914

Lipids and ions traverse the membrane by the same physical pathway in the nhTMEM16 scramblase

PubMed Central

Jiang, Tao; Yu, Kuai

2017-01-01

From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca2+ binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl- channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca2+-activated Cl- channel convert it into a robust scramblase. PMID:28917060

Initial assembly steps of a translocase for folded proteins

PubMed Central

Blümmel, Anne-Sophie; Haag, Laura A.; Eimer, Ekaterina; Müller, Matthias; Fröbel, Julia

2015-01-01

The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors. PMID:26068441

LINCing complex functions at the nuclear envelope

PubMed Central

Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

2013-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

Identification of the structural proteins of an ATP-driven potassium transport system in Escherichia coli.

PubMed Central

Laimins, L A; Rhoads, D B; Altendorf, K; Epstein, W

1978-01-01

The three structural proteins of the ATP-driven Kdp potassium transport system of Escherichia coli [Rhoads, D. B., Waters, F. B. & Epstein, W. (1976) J. Gen. Physiol. 67, 325-341] have been identified and found to be located in the inner membrane. The high-affinity repressible Kdp system in one of four potassium transport systems in E. coli. The Kdp proteins were identified both in growing cells as well as in heavily UV-irradiated cells infected with transducing phages carrying the kdp operon. Although all previously identified ATP-driven transport systems of Gram-negative bacteria have been shown to contain a periplasmic protein component, no evidence was found for such a component or for an outer membrane component of the Kdp system. The molecular weights of the three inner membrane proteins, KdpA, KdpB, and KdpC, were determined to be 47,000, 90,000 and 22,000, respectively. Images PMID:356049

Crystallization of Proteins from Crude Bovine Rod Outer Segments☆

PubMed Central

Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

2015-01-01

Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

Biophysical characterization of the outer membrane polysaccharide export protein and the polysaccharide co-polymerase protein from Xanthomonas campestris.

PubMed

Bianco, M I; Jacobs, M; Salinas, S R; Salvay, A G; Ielmini, M V; Ielpi, L

2014-09-01

This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action. Copyright © 2014 Elsevier Inc. All rights reserved.

Mn2+ exerts stronger structural effects than the Mn-citrate complex on the human erythrocyte membrane and molecular models.

PubMed

Suwalsky, M; Villena, F; Sotomayor, C P

2010-01-01

While traces of manganese (Mn) take part in important and essential functions in biology, elevated exposures have been shown to cause significant toxicity. Chronic exposure to the metal leads to manganese neurotoxicity (or manganism), a brain disorder that resembles Parkinsonism. Toxic effect mechanisms of Mn is not understood, toxic concentrations of manganese are not well defined and blood manganese concentration at which neurotoxicity occurs has not been identified. There are reports indicating that the most abundant Mn-species in Mn carriers within blood is the Mn-citrate complex. Despite the well-documented information about the toxic effects of Mn, there are scarce reports concerning the effects of manganese compounds on both structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of Mn with cell membranes, MnCl(2), and the Mn-citrate complex were incubated with intact erythrocytes, isolated unsealead human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of the Mn compounds to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). In all these systems it was found that Mn(2+) exerted considerable higher structural perturbations than the Mn-citrate complex.

Membrane Remodeling by the Double-Barrel Scaffolding Protein of Poxvirus

PubMed Central

Hijnen, Marcel; Schult, Philipp; Pettikiriarachchi, Anne; Mitra, Alok K.; Coulibaly, Fasséli

2011-01-01

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors. PMID:21931553

Imaging Dot Patterns for Measuring Gossamer Space Structures

NASA Technical Reports Server (NTRS)

Dorrington, A. A.; Danehy, P. M.; Jones, T. W.; Pappa, R. S.; Connell, J. W.

2005-01-01

A paper describes a photogrammetric method for measuring the changing shape of a gossamer (membrane) structure deployed in outer space. Such a structure is typified by a solar sail comprising a transparent polymeric membrane aluminized on its Sun-facing side and coated black on the opposite side. Unlike some prior photogrammetric methods, this method does not require an artificial light source or the attachment of retroreflectors to the gossamer structure. In a basic version of the method, the membrane contains a fluorescent dye, and the front and back coats are removed in matching patterns of dots. The dye in the dots absorbs some sunlight and fluoresces at a longer wavelength in all directions, thereby enabling acquisition of high-contrast images from almost any viewing angle. The fluorescent dots are observed by one or more electronic camera(s) on the Sun side, the shade side, or both sides. Filters that pass the fluorescent light and suppress most of the solar spectrum are placed in front of the camera(s) to increase the contrast of the dots against the background. The dot image(s) in the camera(s) are digitized, then processed by use of commercially available photogrammetric software.

How to Design a Biosensor

PubMed Central

Ward, W. Kenneth

2007-01-01

Amperometric sensors for continuous glucose monitoring could prevent acute and chronic complications of diabetes, but research is needed to improve accuracy and stability. In designing sensors, interference from non-glucose analytes can be minimized by use of filtration membranes or electron transfer mediators that allow polarization at low potentials. If oxygen is required for the enzymatic reaction with glucose, then the outer permselective membrane must have substantial oxygen permeability. For this reason, during development of permselective membranes, permeability studies (such as performed by Tipnis and colleagues in this issue) can be used to measure transport of glucose and oxygen and optimize membrane structure. Tipnis and colleagues present a novel biosensor based with separate layers for glucose-oxygen permselectivity, enzymatic conversion, and avoidance of interference. They also address sensor stability, in part by comparing sensor function during ascending vs descending glucose levels. By measuring the difference, they were able to minimize this aspect of instability (hysterisis), which assisted them in selecting a promising permselective membrane based on iron and humic acid. PMID:19888407

Evolution and development of fetal membranes and placentation in amniote vertebrates.

PubMed

Ferner, Kirsten; Mess, Andrea

2011-08-31

We review aspects of fetal membrane evolution and patterns of placentation within amniotes, the most successful land vertebrates. Special reference is given to embryonic gas supply. The evolution of fetal membranes is a prerequisite for reproduction independent from aquatic environments. Starting from a basically similar repertoire of fetal membranes - the amnion, chorion, allantois and yolk sac, which form the cleidoic egg - different structural solutions for embryonic development have evolved. In oviparous amniotes the chorioallantoic membrane is the major site for the exchange of respiratory gases between fetus and outer environment. The richly vascularised yolk sac and allantois in concert with the chorion play an important role in the evolution of placentation in various viviparous amniotes. Highly complex placentas have evolved independently among squamate sauropsids and in marsupial and placental mammals. In conclusion, there seems to be a natural force to improve gas exchange processes in intrauterine environments by reducing the barrier between the blood systems and optimising the exchange areas. Copyright © 2011 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hee Yoon; Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, California; Raphael, Patrick D.

Cochlear amplification has been most commonly investigated by measuring the vibrations of the basilar membrane in animal models. Several different techniques have been used for measuring these vibrations such as laser Doppler vibrometry, miniature pressure sensors, low coherence interferometry, and spectral-domain optical coherence tomography (SD-OCT). We have built a swept-source OCT (SS-OCT) system, which is similar to SD-OCT in that it is capable of performing both imaging and vibration measurements within the mouse cochlea in vivo without having to open the bone. In vivo 3D images of a mouse cochlea were obtained, and the basilar membrane, tectorial membrane, Reissner’s membrane,more » tunnel of Corti, and reticular lamina could all be resolved. We measured vibrations of multiple structures within the mouse cochlea to sound stimuli. As well, we measured the radial deflections of the reticular lamina and tectorial membrane to estimate the displacement of the outer hair cell stereocilia. These measurements have the potential to more clearly define the mechanisms underlying the linear and non-linear processes within the mammalian cochlea.« less

How to design a biosensor.

PubMed

Ward, W Kenneth

2007-03-01

Amperometric sensors for continuous glucose monitoring could prevent acute and chronic complications of diabetes, but research is needed to improve accuracy and stability. In designing sensors, interference from non-glucose analytes can be minimized by use of filtration membranes or electron transfer mediators that allow polarization at low potentials. If oxygen is required for the enzymatic reaction with glucose, then the outer permselective membrane must have substantial oxygen permeability. For this reason, during development of permselective membranes, permeability studies (such as performed by Tipnis and colleagues in this issue) can be used to measure transport of glucose and oxygen and optimize membrane structure. Tipnis and colleagues present a novel biosensor based with separate layers for glucose-oxygen permselectivity, enzymatic conversion, and avoidance of interference. They also address sensor stability, in part by comparing sensor function during ascending vs descending glucose levels. By measuring the difference, they were able to minimize this aspect of instability (hysterisis), which assisted them in selecting a promising permselective membrane based on iron and humic acid.

The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

PubMed

Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

1997-09-01

Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

Engineering multi-functional bacterial outer membrane vesicles as modular nanodevices for biosensing and bioimaging.

PubMed

Chen, Qi; Rozovsky, Sharon; Chen, Wilfred

2017-07-04

Outer membrane vesicles (OMVs) are proteoliposomes derived from the outer membrane and periplasmic space of many Gram-negative bacteria including E. coli as part of their natural growth cycle. Inspired by the natural ability of E. coli to sort proteins to both the exterior and interior of OMVs, we reported here a one-pot synthesis approach to engineer multi-functionalized OMV-based sensors for both antigen binding and signal generation. SlyB, a native lipoprotein, was used a fusion partner to package nanoluciferase (Nluc) within OMVs, while a previously developed INP-Scaf3 surface scaffold was fused to the Z-domain for antibody recruiting. The multi-functionalized OMVs were used for thrombin detection with a detection limit of 0.5 nM, comparable to other detection methods. Using the cohesin domains inserted between the Z-domain and INP, these engineered OMVs were further functionalized with a dockerin-tagged GFP for cancer cell imaging.

Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Ungar, Eugene K.; Almlie, Jay C.

2010-01-01

A water membrane evaporator (WME) has been conceived and tested as an alternative to the contamination-sensitive and corrosion-prone evaporators currently used for dissipating heat from space vehicles. The WME consists mainly of the following components: An outer stainless-steel screen that provides structural support for the components mentioned next; Inside and in contact with the stainless-steel screen, a hydrophobic membrane that is permeable to water vapor; Inside and in contact with the hydrophobic membrane, a hydrophilic membrane that transports the liquid feedwater to the inner surface of the hydrophobic membrane; Inside and in contact with the hydrophilic membrane, an annular array of tubes through which flows the spacecraft coolant carrying the heat to be dissipated; and An inner exclusion tube that limits the volume of feedwater in the WME. In operation, a pressurized feedwater reservoir is connected to the volume between the exclusion tube and the coolant tubes. Feedwater fills the volume, saturates the hydrophilic membrane, and is retained by the hydrophobic membrane. The outside of the WME is exposed to space vacuum. Heat from the spacecraft coolant is conducted through the tube walls and the water-saturated hydrophilic membrane to the liquid/vapor interface at the hydrophobic membrane, causing water to evaporate to space. Makeup water flows into the hydrophilic membrane through gaps between the coolant tubes.

Primate cathelicidin orthologues display different structures and membrane interactions.

PubMed

Morgera, Francesca; Vaccari, Lisa; Antcheva, Nikolinka; Scaini, Denis; Pacor, Sabrina; Tossi, Alessandro

2009-02-01

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Other Fabric Structures

NASA Technical Reports Server (NTRS)

1985-01-01

There are two kinds of fabric structures - tension, supported by cables and pylons, and those supported by air pressure within an enclosed fabric envelope. They are becoming increasingly popular with architects, engineers, etc., because of their aesthetic appeal, low cost and maintenance, energy efficiency and good space utilization. The Structo-Fab roof weighs only 1/30 as much as a conventional roof of that size. Giant fans are used to blow air into the envelope between the roof's outer membrane and its inner liner automatically maintaining the pressure differential necessary for roof rigidity.

The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12.

PubMed

Freudl, R; Schwarz, H; Klose, M; Movva, N R; Henning, U

1985-12-16

Information, in addition to that provided by signal sequences, for translocation across the plasma membrane is thought to be present in exported proteins of Escherichia coli. Such information must also exist for the localization of such proteins. To determine the nature of this information, overlapping inframe deletions have been constructed in the ompA gene which codes for a 325-residue major outer membrane protein. In addition, one deletion, encoding only the NH2-terminal part of the protein up to residue 160, was prepared. The location of each product was determined by immunoelectron microscopy. Proteins missing residues 4-45, 43-84, 46-227, 86-227 or 160-325 of the mature protein were all efficiently translocated across the plasma membrane. The first two proteins were found in the outer membrane, the others in the periplasmic space. It has been proposed that export and sorting signals consist of relatively small amino acid sequences near the NH2 terminus of an outer membrane protein. On the basis of sequence homologies it has also been suggested that such proteins possess a common sorting signal. The locations of the partially deleted proteins described here show that a unique export signal does not exist in the OmpA protein. The proposed common sorting signal spans residues 1-14 of OmpA. Since this region is not essential for routing the protein, the existence of a common sorting signal is doubtful. It is suggested that information both for export (if existent) and localization lies within protein conformation which for the former process should be present repeatedly in the polypeptide.

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

PubMed Central

Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

2012-01-01

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

Polyester polymer alloy as a high-performance membrane.

PubMed

Igoshi, Tadaaki; Tomisawa, Narumi; Hori, Yoshinori; Jinbo, Yoichi

2011-01-01

Polyester polymer alloy (PEPA) membrane is developed as a synthetic polymermembrane. It consists of two polymers - polyethersulfone (PES) and polyarylate (PAR).The pore size in membrane can be controlled by a blend ratio of PES and PAR. One unique characteristic is that PEPA membrane has three layers of a skin layer on the inner surface, a porous layer in the membrane, and a skin layer on the outer surface, respectively. The permeability of water and substances is controlled by the skin layer on the inner surface. PEPA membrane dialyzer can be adequately considered as a high-performance dialyzer. Furthermore, the skin layer on the outer surface can block endotoxin from the dialysis fluid side. PEPA membrane can therefore be used as an endotoxin-retentive filter. The other unique characteristic is that each amount of albumin loss or β2-microglobulin removal can be controlled by an additive amount of polyvinylpyrrolidone. This means that the PEPA dialyzer can be clinically used to meet the conditions of the patient. Copyright © 2011 S. Karger AG, Basel.

Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

PubMed

Bahar, Ofir; Mordukhovich, Gideon; Luu, Dee Dee; Schwessinger, Benjamin; Daudi, Arsalan; Jehle, Anna Kristina; Felix, Georg; Ronald, Pamela C

2016-05-01

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

Dissimilatory Reduction of Extracellular Electron Acceptors in Anaerobic Respiration

PubMed Central

Richter, Katrin; Schicklberger, Marcus

2012-01-01

An extension of the respiratory chain to the cell surface is necessary to reduce extracellular electron acceptors like ferric iron or manganese oxides. In the past few years, more and more compounds were revealed to be reduced at the surface of the outer membrane of Gram-negative bacteria, and the list does not seem to have an end so far. Shewanella as well as Geobacter strains are model organisms to discover the biochemistry that enables the dissimilatory reduction of extracellular electron acceptors. In both cases, c-type cytochromes are essential electron-transferring proteins. They make the journey of respiratory electrons from the cytoplasmic membrane through periplasm and over the outer membrane possible. Outer membrane cytochromes have the ability to catalyze the last step of the respiratory chains. Still, recent discoveries provided evidence that they are accompanied by further factors that allow or at least facilitate extracellular reduction. This review gives a condensed overview of our current knowledge of extracellular respiration, highlights recent discoveries, and discusses critically the influence of different strategies for terminal electron transfer reactions. PMID:22179232

Three-dimensional visualization of the Autographa californica multiple nucleopolyhedrovirus occlusion-derived virion envelopment process gives new clues as to its mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yang; Li, Kunpeng; Tang, Peiping

2015-02-15

Baculoviruses produce two virion phenotypes, occlusion-derived virion (ODV) and budded virion (BV). ODV envelopment occurs in the nucleus. Morphogenesis of the ODV has been studied extensively; however, the mechanisms underlying microvesicle formation and ODV envelopment in nuclei remain unclear. In this study, we used electron tomography (ET) together with the conventional electron microscopy to study the envelopment of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ODV. Our results demonstrate that not only the inner but also the outer nuclear membrane can invaginate and vesiculate into microvesicles and that intranuclear microvesicles are the direct source of the ODV membrane. Five main events inmore » the ODV envelopment process are summarized, from which we propose a model to explain this process. - Highlights: • Both the inner and outer nuclear membranes could invaginate. • Both the inner and outer nuclear membranes could vesiculate into microvesicles. • Five main events in the ODV envelopment process are summarized. • A model is proposed to explain this ODV envelopment.« less

Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

PubMed

Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

2012-02-14

Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

PubMed

Gunasinghe, Sachith D; Shiota, Takuya; Stubenrauch, Christopher J; Schulze, Keith E; Webb, Chaille T; Fulcher, Alex J; Dunstan, Rhys A; Hay, Iain D; Naderer, Thomas; Whelan, Donna R; Bell, Toby D M; Elgass, Kirstin D; Strugnell, Richard A; Lithgow, Trevor

2018-05-29

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

PubMed Central

Piek, Susannah; Kahler, Charlene M.

2012-01-01

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism. PMID:23267440

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes

PubMed Central

Gillies, Laura A; Du, Han; Peters, Bjoern; Knudson, C. Michael; Newmeyer, Donald D.; Kuwana, Tomomi

2015-01-01

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress. PMID:25411335

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Legionella dumoffii Utilizes Exogenous Choline for Phosphatidylcholine Synthesis

PubMed Central

Palusinska-Szysz, Marta; Szuster-Ciesielska, Agnieszka; Kania, Magdalena; Janczarek, Monika; Chmiel, Elżbieta; Danikiewicz, Witold

2014-01-01

Phosphatidycholine (PC) is the major membrane-forming phospholipid in eukaryotes but it has been found in only a limited number of prokaryotes. Bacteria synthesize PC via the phospholipid N-methylation pathway (Pmt) or via the phosphatidylcholine synthase pathway (Pcs) or both. Here, we demonstrated that Legionella dumoffii has the ability to utilize exogenous choline for phosphatidylcholine (PC) synthesis when bacteria grow in the presence of choline. The Pcs seems to be a primary pathway for synthesis of this phospholipid in L. dumoffii. Structurally different PC species were distributed in the outer and inner membranes. As shown by the LC/ESI-MS analyses, PC15:0/15:0, PC16:0/15:0, and PC17:0/17:1 were identified in the outer membrane and PC14:0/16:0, PC16:0/17:1, and PC20:0/15:0 in the inner membrane. L. dumoffii pcsA gene encoding phosphatidylcholine synthase revealed the highest sequence identity to pcsA of L. bozemanae (82%) and L. longbeachae (81%) and lower identity to pcsA of L. drancourtii (78%) and L. pneumophila (71%). The level of TNF-α in THP1-differentiated cells induced by live and temperature-killed L. dumoffii cultured on a medium supplemented with choline was assessed. Live L. dumoffii bacteria cultured on the choline-supplemented medium induced TNF-α three-fold less efficiently than cells grown on the non-supplemented medium. There is an evident effect of PC modification, which impairs the macrophage inflammatory response. PMID:24821544

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump

PubMed Central

Llabrés, Salomé; Neuberger, Arthur; Blaza, James N.; Bai, Xiao-chen; Okada, Ui; Murakami, Satoshi; van Veen, Hendrik W.; Zachariae, Ulrich; Scheres, Sjors H.W.; Luisi, Ben F.

2017-01-01

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain (TMD) with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism. PMID:28504659

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Haiyan, E-mail: zhaohy@ku.ed; Sequeira, Reuben D., E-mail: sequen@ku.ed; Galeva, Nadezhda A., E-mail: galeva@ku.ed

2011-01-20

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus maymore » be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.« less

Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes

PubMed Central

Dimmer, Kai S

2018-01-01

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged. PMID:29923829

Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport

PubMed Central

Sherman, David J.; Lazarus, Michael B.; Murphy, Lea; Liu, Charles; Walker, Suzanne; Ruiz, Natividad; Kahne, Daniel

2014-01-01

The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site. PMID:24639492

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

PubMed

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

[Conversion of sound into auditory nerve action potentials].

PubMed

Encke, J; Kreh, J; Völk, F; Hemmert, W

2016-11-01

Outer hair cells play a major role in the hearing process: they amplify the motion of the basilar membrane up to a 1000-fold and at the same time sharpen the excitation patterns. These patterns are converted by inner hair cells into action potentials of the auditory nerve. Outer hair cells are delicate structures and easily damaged, e. g., by overexposure to noise. Hearing aids can amplify the amplitude of the excitation patterns, but they cannot restore their degraded frequency selectivity. Noise overexposure also leads to delayed degeneration of auditory nerve fibers, particularly those with low a spontaneous rate, which are important for the coding of sound in noise. However, this loss cannot be diagnosed by pure-tone audiometry.

The Escherichia coli Phospholipase PldA Regulates Outer Membrane Homeostasis via Lipid Signaling.

PubMed

May, Kerrie L; Silhavy, Thomas J

2018-03-20

The outer membrane (OM) bilayer of Gram-negative bacteria is biologically unique in its asymmetrical organization of lipids, with an inner leaflet composed of glycerophospholipids (PLs) and a surface-exposed outer leaflet composed of lipopolysaccharide (LPS). This lipid organization is integral to the OM's barrier properties. Perturbations of the outer leaflet by antimicrobial peptides or defects in LPS biosynthesis or transport to the OM cause a compensatory flipping of PLs to the outer leaflet. As a result, lipid asymmetry is disrupted and OM integrity is compromised. Recently, we identified an Escherichia coli mutant that exhibits aberrant accumulation of surface PLs accompanied by a cellular increase in LPS production. Remarkably, the observed hyperproduction of LPS is PldA dependent. Here we provide evidence that the fatty acids generated by PldA at the OM are transported into the cytoplasm and simultaneously activated by thioesterification to coenzyme A (CoA) by FadD. The acyl-CoAs produced ultimately inhibit LpxC degradation by FtsH. The increased levels of LpxC, the enzyme that catalyzes the first committed step in LPS biosynthesis, increases the amount of LPS produced. Our data suggest that PldA acts as a sensor for lipid asymmetry in the OM. PldA protects the OM barrier by both degrading mislocalized PLs and generating lipid second messengers that enable long-distance signaling that prompts the cell to restore homeostasis at a distant organelle. IMPORTANCE The outer membrane of Gram-negative bacteria is an effective permeability barrier that protects the cell from toxic agents, including antibiotics. Barrier defects are often manifested by phospholipids present in the outer leaflet of this membrane that take up space normally occupied by lipopolysaccharide. We have discovered a signaling mechanism that operates across the entire cell envelope used by the cell to detect these outer membrane defects. A phospholipase, PldA, that functions to degrade these mislocalized phospholipids has a second, equally important function as a sensor. The fatty acids produced by hydrolysis of the phospholipids act as second messengers to signal the cell that more lipopolysaccharide is needed. These fatty acids diffuse across the periplasm and are transported into the cytoplasm by a process that attaches coenzyme A. The acyl-CoA molecule produces signals to inhibit the degradation of the critical enzyme LpxC by the ATP-dependent protease FtsH, increasing lipopolysaccharide production. Copyright © 2018 May and Silhavy.

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

PubMed

Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

2018-04-05

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

PubMed Central

Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

2013-01-01

Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved. PMID:24343586

Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

NASA Astrophysics Data System (ADS)

Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

2013-12-01

Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved.

Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

DTIC Science & Technology

2005-01-01

13-09-20061 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cloning, expression and purification of Brucella suis outer membrane proteins 5b. GRANT NUMBER...attractive for this purpose. In this study, we cloned, expressed and purified seven predicted OMPs of Brucella suis . The recombinant proteins were...fused with 6-his and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based

Evaluation of a real-time polymerase chain reaction assay of the outer membrane protein P2 gene for the detection of Haemophilus parasuis in clinical samples.

PubMed

McDowall, Rebeccah; Slavic, Durda; MacInnes, Janet I; Cai, Hugh Y

2014-04-01

A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively.

Evaluation of a real-time polymerase chain reaction assay of the outer membrane protein P2 gene for the detection of Haemophilus parasuis in clinical samples

PubMed Central

McDowall, Rebeccah; Slavic, Durda; MacInnes, Janet I.; Cai, Hugh Y.

2014-01-01

A real-time polymerase chain reaction (PCR) assay of the outer membrane protein (OMP) P2 gene was developed and used to test 97 putative Haemophilus parasuis pure cultures and 175 clinical tissue samples. With standard culture isolation as the gold standard, the diagnostic sensitivity and specificity of the PCR assay were determined to be 83% and 80%, respectively. PMID:24688178

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes.

PubMed

Niemann, Moritz; Harsman, Anke; Mani, Jan; Peikert, Christian D; Oeljeklaus, Silke; Warscheid, Bettina; Wagner, Richard; Schneider, André

2017-09-12

Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.

Detection of Iss and Bor on the surface of Escherichia coli.

PubMed

Lynne, A M; Skyberg, J A; Logue, C M; Nolan, L K

2007-03-01

To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

PubMed Central

Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

2017-01-01

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Static charge outside chamber induces dielectric breakdown of solid-state nanopore membranes

NASA Astrophysics Data System (ADS)

Matsui, Kazuma; Goto, Yusuke; Yanagi, Itaru; Yanagawa, Yoshimitsu; Ishige, Yu; Takeda, Ken-ichi

2018-04-01

Reducing device capacitance is effective for decreasing current noise observed in a solid-state nanopore-based DNA sequencer. On the other hand, we have recently found that voltage stress causes pinhole-like defects in such low-capacitance devices. The origin of voltage stress, however, has not been determined. In this research, we identified that a dominant origin is static charge on the outer surface of a flow cell. Even though the outer surface was not in direct contact with electrolytes in the flow cell, the charge induces high voltage stress on a membrane according to the capacitance coupling ratio of the flow cell to the membrane.

S-layer and cytoplasmic membrane - exceptions from the typical archaeal cell wall with a focus on double membranes.

PubMed

Klingl, Andreas

2014-01-01

The common idea of typical cell wall architecture in archaea consists of a pseudo-crystalline proteinaceous surface layer (S-layer), situated upon the cytoplasmic membrane. This is true for the majority of described archaea, hitherto. Within the crenarchaea, the S-layer often represents the only cell wall component, but there are various exceptions from this wall architecture. Beside (glycosylated) S-layers in (hyper)thermophilic cren- and euryarchaea as well as halophilic archaea, one can find a great variety of other cell wall structures like proteoglycan-like S-layers (Halobacteria), glutaminylglycan (Natronococci), methanochondroitin (Methanosarcina) or double layered cell walls with pseudomurein (Methanothermus and Methanopyrus). The presence of an outermost cellular membrane in the crenarchaeal species Ignicoccus hospitalis already gave indications for an outer membrane similar to Gram-negative bacteria. Although there is just limited data concerning their biochemistry and ultrastructure, recent studies on the euryarchaeal methanogen Methanomassiliicoccus luminyensis, cells of the ARMAN group, and the SM1 euryarchaeon delivered further examples for this exceptional cell envelope type consisting of two membranes.

MICOS and phospholipid transfer by Ups2-Mdm35 organize membrane lipid synthesis in mitochondria.

PubMed

Aaltonen, Mari J; Friedman, Jonathan R; Osman, Christof; Salin, Bénédicte; di Rago, Jean-Paul; Nunnari, Jodi; Langer, Thomas; Tatsuta, Takashi

2016-06-06

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function. © 2016 Aaltonen et al.

Structure and membrane remodeling activity of ESCRT-III helical polymers

DOE PAGES

McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; ...

2015-12-18

The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecturemore » and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. In conclusion, our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.« less

Electrodeionization Using Microseparated Bipolar Membranes

NASA Technical Reports Server (NTRS)

Lyons, Donald; Jackson, George; Andrews, Craig C.; Tennakoon, Charles L, K.; Singh, Waheguru; Hitchens, G. Duncan; Jabs, Harry; Chepin, James F.; Archer, Shivaun; Gonzalez-Martinez, Anukia;

Mechanism regulating nuclear calcium signaling.

PubMed

Malviya, Anant N; Klein, Christian

2006-01-01

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.

Protein profiles of hatchery egg shell membrane

USDA-ARS?s Scientific Manuscript database

Background: Eggshells, which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of m...

Origin, differentiation and functional ultrastructure of egg envelopes in the cestode Echinococcus multilocularis Leuckart, 1863 (Cyclophyllidea: Taeniidae).

PubMed

Świderski, Zdzisław; Miquel, Jordi; Azzouz-Maache, Samira; Pétavy, Anne-Françoise

2017-07-01

The origin, differentiation and functional ultrastructure of oncospheral or egg envelopes in Echinococcus multilocularis Leuckart, 1863 were studied by transmission electron microscopy (TEM) and cytochemistry. The purpose of our study is to describe the formation of the four primary embryonic envelopes, namely vitelline capsule, outer envelope, inner envelope and oncospheral membrane, and their transformation into the oncospheral or egg envelopes surrounding the mature hexacanth. This transformation takes place in the preoncospheral phase of embryonic development. The vitelline capsule and oncospheral membrane are thin membranes, while the outer and inner envelopes are thick cytoplasmic layers formed by two specific types of blastomeres: the outer envelope by cytoplasmic fusion of two macromeres and the inner envelope by cytoplasmic fusion of three mesomeres. Both outer and inner envelopes are therefore cellular in origin and syncytial in nature. During the advanced phase of embryonic development, the outer and inner envelopes undergo great modifications. The outer envelope remains as a metabolically active layer involved in the storage of glycogen and lipids for the final stages of egg development and survival. The inner envelope is the most important protective layer because of its thick layer of embryophoric blocks that assures oncospheral protection and survival. This embryophore is the principal layer of mature eggs, affording physical and physiological protection for the differentiated embryo or oncosphere, since the outer envelope is stripped from the egg before it is liberated. The embryophore is very thick and impermeable, consisting of polygonal blocks of an inert keratin-like protein held together by a cementing substance. The embryophore therefore assures extreme resistance of eggs, enabling them to withstand a wide range of environmental temperatures and physicochemical conditions.

Mechanisms of membrane toxicity of hydrocarbons.

PubMed Central

Sikkema, J; de Bont, J A; Poolman, B

1995-01-01

Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated. PMID:7603409

Oxidative Stress and Maxi Calcium-Activated Potassium (BK) Channels

PubMed Central

Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

2015-01-01

All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

Engineering monolayer poration for rapid exfoliation of microbial membranes.

PubMed

Pyne, Alice; Pfeil, Marc-Philipp; Bennett, Isabel; Ravi, Jascindra; Iavicoli, Patrizia; Lamarre, Baptiste; Roethke, Anita; Ray, Santanu; Jiang, Haibo; Bella, Angelo; Reisinger, Bernd; Yin, Daniel; Little, Benjamin; Muñoz-García, Juan C; Cerasoli, Eleonora; Judge, Peter J; Faruqui, Nilofar; Calzolai, Luigi; Henrion, Andre; Martyna, Glenn J; Grovenor, Chris R M; Crain, Jason; Hoogenboom, Bart W; Watts, Anthony; Ryadnov, Maxim G

2017-02-01

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

PubMed Central

Frank, Daniel O.; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. PMID:25875815

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

PubMed

Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant.

PubMed

Maezawa, S; Hayashi, Y; Nakae, T; Ishii, J; Kameyama, K; Takagi, T

1983-09-28

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.

Membrane recycling at the infranuclear pole of the outer hair cell

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Harasztosi, Emese; Gummer, Anthony W.

2015-12-01

Rapid endocytic activity of outer hair cells (OHCs) in the guinea-pig cochlea has been already studied using the fluorescent membrane marker FM1-43. It was demonstrated that vesicles were endocytosed at the apical pole of OHCs and transcytosed to the basolateral membrane and through a central strand towards the nucleus. The significance of endocytic activity in the infranuclear region is still not clear. Therefore, in this study endocytic activity at the synaptic pole of OHCs was investigated. Confocal laser scanning microscopy was used to visualize dye uptake of OHCs isolated from the guinea-pig cochlea. Signal intensity changes were quantified in the apical and basal poles relative to the signal at the membrane. Data showed no significant difference in fluorescent signal intensity changes between the opposite poles of the OHC. These results suggest that endocytic activities in both the basal and the apical poles contribute equally to the membrane recycling of OHCs.

Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

PubMed Central

Clifton, Luke A.; Skoda, Maximilian W. A.; Daulton, Emma L.; Hughes, Arwel V.; Le Brun, Anton P.; Lakey, Jeremy H.; Holt, Stephen A.

2013-01-01

The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions. PMID:24132206

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

PubMed

Mamat, Uwe; Wilke, Kathleen; Bramhill, David; Schromm, Andra Beate; Lindner, Buko; Kohl, Thomas Andreas; Corchero, José Luis; Villaverde, Antonio; Schaffer, Lana; Head, Steven Robert; Souvignier, Chad; Meredith, Timothy Charles; Woodard, Ronald Wesley

2015-04-16

Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

An outer membrane protein (porin) as an eliciting antigen for delayed-type hypersensitivity in murine salmonellosis.

PubMed Central

Udhayakumar, V; Muthukkaruppan, V R

1987-01-01

The porin, an outer membrane protein of Salmonella typhimurium, was found to be a suitable antigen for eliciting delayed-type hypersensitivity in mouse salmonellosis. Histological examination of the reaction site revealed that the porin was superior to other antigenic preparations in eliciting a typical delayed-type hypersensitivity reaction consisting of mononuclear cell infiltration without polymorphonuclear cell contamination. This study indicates the importance of using a suitable protein antigen from S. typhi for human application. Images PMID:3028963

Functional advantages conferred by extracellular prokaryotic membrane vesicles.

PubMed

Manning, Andrew J; Kuehn, Meta J

2013-01-01

The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane-derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials and ridding the cell of toxic envelope proteins. Here, we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane-bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world. Copyright © 2013 S. Karger AG, Basel.

Molecular basis for photoreceptor outer segment architecture

PubMed Central

Goldberg, Andrew F. X.; Moritz, Orson L.; Williams, David S.

2016-01-01

To serve vision, vertebrate rod and cone photoreceptors must detect photons, convert the light stimuli into cellular signals, and then convey the encoded information to downstream neurons. Rods and cones are sensory neurons that each rely on specialized ciliary organelles to detect light. These organelles, called outer segments, possess elaborate architectures that include many hundreds of light-sensitive membranous disks arrayed one atop another in precise register. These stacked disks capture light and initiate the chain of molecular and cellular events that underlie normal vision. Outer segment organization is challenged by an inherently dynamic nature; these organelles are subject to a renewal process that replaces a significant fraction of their disks (up to ~10%) on a daily basis. In addition, a broad range of environmental and genetic insults can disrupt outer segment morphology to impair photoreceptor function and viability. In this chapter, we survey the major progress that has been made for understanding the molecular basis of outer segment architecture. We also discuss key aspects of organelle lipid and protein composition, and highlight distributions, interactions, and potential structural functions of key OS-resident molecules, including: kinesin-2, actin, RP1, prominin-1, protocadherin 21, peripherin-2/rds, rom-1, glutamic acid-rich proteins, and rhodopsin. Finally, we identify key knowledge gaps and challenges that remain for understanding how normal outer segment architecture is established and maintained. PMID:27260426

Arrestin-rhodopsin binding stoichiometry in isolated rod outer segment membranes depends on the percentage of activated receptors.

PubMed

Sommer, Martha E; Hofmann, Klaus Peter; Heck, Martin

2011-03-04

In the rod cell of the retina, arrestin is responsible for blocking signaling of the G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that arrestin must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would be generated at physiologically relevant low light levels. However, the elongated bi-lobed structure of arrestin suggests that it might be able to accommodate two rhodopsin molecules. In this study, we directly addressed the question of binding stoichiometry by quantifying arrestin binding to Rho*P in isolated rod outer segment membranes. We manipulated the "photoactivation density," i.e. the percentage of active receptors in the membrane, with the use of a light flash or by partially regenerating membranes containing phosphorylated opsin with 11-cis-retinal. Curiously, we found that the apparent arrestin-Rho*P binding stoichiometry was linearly dependent on the photoactivation density, with one-to-one binding at low photoactivation density and one-to-two binding at high photoactivation density. We also observed that, irrespective of the photoactivation density, a single arrestin molecule was able to stabilize the active metarhodopsin II conformation of only a single Rho*P. We hypothesize that, although arrestin requires at least a single Rho*P to bind the membrane, a single arrestin can actually interact with a pair of receptors. The ability of arrestin to interact with heterogeneous receptor pairs composed of two different photo-intermediate states would be well suited to the rod cell, which functions at low light intensity but is routinely exposed to several orders of magnitude more light.

Isolation, folding and structural investigations of the amino acid transporter OEP16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ni, Da Qun; Zook, James; Klewer, Douglas A.

2011-12-01

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on amore » Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of a-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.« less

Isolation, folding and structural investigations of the amino acid transporter OEP16.

PubMed

Ni, Da Qun; Zook, James; Klewer, Douglas A; Nieman, Ronald A; Soll, J; Fromme, Petra

2011-12-01

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹⁵N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection. Copyright © 2011. Published by Elsevier Inc.

Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

PubMed Central

2011-01-01

Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

Anticancer β-hairpin peptides: membrane-induced folding triggers activity

PubMed Central

Sinthuvanich, Chomdao; Veiga, Ana Salomé; Gupta, Kshitij; Gaspar, Diana; Blumenthal, Robert; Schneider, Joel P.

2012-01-01

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells relatively electronegative relative to non-cancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charge membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma) and MDA-MB-436 (breast carcinoma). However, the cytotoxicity towards non-cancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells. PMID:22413859

Scanning electron microscopy of echinoid podia.

PubMed

Florey, E; Cahill, M A

1982-01-01

Tube feet of the sea urchin Strongylocentrotus franciscanus were studied with the scanning electron microscope (SEM). By use of fractured preparations it was possible to obtain views of all components of the layered tube-foot wall. The outer epithelium was found to bear tufts of cilia possibly belonging to sensory cells. The nerve plexus was clearly revealed as being composed of bundles of varicose axons. The basal lamina, which covers the outer and inner surfaces of the connective tissue layer, was found to be a mechanically resistant and elastic membrane. The connective tissue appears as dense bundles of (collagen) fibers. The luminal epithelium (coelothelium) is a single layer of flagellated collar cells. There is no indication that the muscle fibers, which insert on the inner basal lamina of the connective tissue layer are innervated by axons from the basi-epithelial nerve plexus. The results agree with previous conclusions concerning tube-foot structure based on transmission electron microscopy, and provide additional information, particularly with regard to the outer and inner epithelia.

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

The membrane current of single rod outer segments.

PubMed

Baylor, D A; Lamb, T D; Yau, K W

1979-03-01

1. Outer segments of individual rods in the retina of the toad, Bufo marinus, were drawn into a glass pipette to record the membrane current. 2. Light flashes evoked transient outward currents. The peak response amplitude was related to flash intensity by a Michaelis equation with half-saturating intensity about 1 photon mum-2. 3. The saturating response amplitude ranged up to 27 pA and corresponded closely to complete suppression of the steady inward current present in darkness. 4. For a given cell the saturating response amplitude varied linearly with the length of outer segment within the pipette. This is consistent with a uniform density of light-sensitive channels and negligible gradient of membrane potential along the outer segment. 5. Responses to bright flashes never showed the relaxation from an initial peak seen previously in intracellular voltage recordings, suggesting that the conductance change responsible for the relaxation does not occur in the outer segment. 6. Responses to local illumination of only the recorded outer segment were very similar to those obtained with diffuse light at the same intensity, indicating that peripheral rods made little contribution to the responses. 7. The spectral sensitivity of 'red' rods was consistent with a retinal1-based pigment with lambda max = 498 +/- 2 nm. 8. The kinetics of the response were consistent with four stages of delay affecting action of the internal transmitter. Responses were faster at the basal end of the outer segment than at the distal tip. 9. Background light reduced the sensitivity to a superposed dim test flash and shortened the time course of the response, indicating that adapting light modifies the kinetics and gain of the transduction mechanism within the outer segment. 10. Responses to dim lights exhibited pronounced fluctuations which are attributed in the succeeding paper (Baylor, Lamb & Yau, 1979) to the quantal nature of light.

Simulations of Living Cell Origins Using a Cellular Automata Model

NASA Astrophysics Data System (ADS)

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

Simulations of living cell origins using a cellular automata model.

PubMed

Ishida, Takeshi

2014-04-01

Understanding the generalized mechanisms of cell self-assembly is fundamental for applications in various fields, such as mass producing molecular machines in nanotechnology. Thus, the details of real cellular reaction networks and the necessary conditions for self-organized cells must be elucidated. We constructed a 2-dimensional cellular automata model to investigate the emergence of biological cell formation, which incorporated a looped membrane and a membrane-bound information system (akin to a genetic code and gene expression system). In particular, with an artificial reaction system coupled with a thermal system, the simultaneous formation of a looped membrane and an inner reaction process resulted in a more stable structure. These double structures inspired the primitive biological cell formation process from chemical evolution stage. With a model to simulate cellular self-organization in a 2-dimensional cellular automata model, 3 phenomena could be realized: (1) an inner reaction system developed as an information carrier precursor (akin to DNA); (2) a cell border emerged (akin to a cell membrane); and (3) these cell structures could divide into 2. This double-structured cell was considered to be a primary biological cell. The outer loop evolved toward a lipid bilayer membrane, and inner polymeric particles evolved toward precursor information carriers (evolved toward DNA). This model did not completely clarify all the necessary and sufficient conditions for biological cell self-organization. Further, our virtual cells remained unstable and fragile. However, the "garbage bag model" of Dyson proposed that the first living cells were deficient; thus, it would be reasonable that the earliest cells were more unstable and fragile than the simplest current unicellular organisms.

The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core.

PubMed

Bergal, Hans Thor; Hopkins, Alex Hunt; Metzner, Sandra Ines; Sousa, Marcelo Carlos

2016-02-02

The β-barrel assembly machine (BAM) mediates folding and insertion of integral β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its tetratricopeptide repeats 3 and 4. The interface structural elements are conserved in the Escherichia coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore, the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Piracetam inhibits the lipid-destabilising effect of the amyloid peptide Abeta C-terminal fragment.

PubMed

Mingeot-Leclercq, Marie-Paule; Lins, Laurence; Bensliman, Mariam; Thomas, Annick; Van Bambeke, Françoise; Peuvot, Jacques; Schanck, André; Brasseur, Robert

2003-01-10

Amyloid peptide (Abeta) is a 40/42-residue proteolytic fragment of a precursor protein (APP), implicated in the pathogenesis of Alzheimer's disease. The hypothesis that interactions between Abeta aggregates and neuronal membranes play an important role in toxicity has gained some acceptance. Previously, we showed that the C-terminal domain (e.g. amino acids 29-42) of Abeta induces membrane permeabilisation and fusion, an effect which is related to the appearance of non-bilayer structures. Conformational studies showed that this peptide has properties similar to those of the fusion peptide of viral proteins i.e. a tilted penetration into membranes. Since piracetam interacts with lipids and has beneficial effects on several symptoms of Alzheimer's disease, we investigated in model membranes the ability of piracetam to hinder the destabilising effect of the Abeta 29-42 peptide. Using fluorescence studies and 31P and 2H NMR spectroscopy, we have shown that piracetam was able to significantly decrease the fusogenic and destabilising effect of Abeta 29-42, in a concentration-dependent manner. While the peptide induced lipid disorganisation and subsequent negative curvature at the membrane-water interface, the conformational analysis showed that piracetam, when preincubated with lipids, coats the phospholipid headgroups. Calculations suggest that this prevents appearance of the peptide-induced curvature. In addition, insertion of molecules with an inverted cone shape, like piracetam, into the outer membrane leaflet should make the formation of such structures energetically less favourable and therefore decrease the likelihood of membrane fusion.

Life at the border: Adaptation of proteins to anisotropic membrane environment

PubMed Central

Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

2014-01-01

This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

Membrane Fission: Model for Intermediate Structures

PubMed Central

Kozlovsky, Yonathan; Kozlov, Michael M.

2003-01-01

Membrane budding-fission is a fundamental process generating intracellular carriers of proteins. Earlier works were focused only on formation of coated buds connected to the initial membrane by narrow membrane necks. We present the theoretical analysis of the whole pathway of budding-fission, including the crucial stage where the membrane neck undergoes fission and the carrier separates from the donor membrane. We consider two successive intermediates of the reaction: 1), a constricted membrane neck coming out of aperture of the assembling protein coat, and 2), hemifission intermediate resulting from self-fusion of the inner monolayer of the neck, while its outer monolayer remains continuous. Transformation of the constricted neck into the hemifission intermediate is driven by the membrane stress produced in the neck by the protein coat. Although apparently similar to hemifusion, the fission is predicted to have an opposite dependence on the monolayer spontaneous curvature. Analysis of the further stages of the process demonstrates that in all practically important cases the hemifission intermediate decays spontaneously into two separate membranes, thereby completing the fission process. We formulate the “job description” for fission proteins by calculating the energy they have to deliver and the radii of the protein coat aperture which have to be reached to drive the fission process. PMID:12829467

Comparative proteome analysis reveals pathogen specific outer membrane proteins of Leptospira.

PubMed

Dhandapani, Gunasekaran; Sikha, Thoduvayil; Rana, Aarti; Brahma, Rahul; Akhter, Yusuf; Gopalakrishnan Madanan, Madathiparambil

2018-04-10

Proteomes of pathogenic Leptospira interrogans and L. borgpetersenii and the saprophytic L. biflexa were filtered through computational tools to identify Outer Membrane Proteins (OMPs) that satisfy the required biophysical parameters for their presence on the outer membrane. A total of 133, 130, and 144 OMPs were identified in L. interrogans, L. borgpetersenii, and L. biflexa, respectively, which forms approximately 4% of proteomes. A holistic analysis of transporting and pathogenic characteristics of OMPs together with Clusters of Orthologous Groups (COGs) among the OMPs and their distribution across 3 species was made and put forward a set of 21 candidate OMPs specific to pathogenic leptospires. It is also found that proteins homologous to the candidate OMPs were also present in other pathogenic species of leptospires. Six OMPs from L. interrogans and 2 from L. borgpetersenii observed to have similar COGs while those were not found in any intermediate or saprophytic forms. These OMPs appears to have role in infection and pathogenesis and useful for anti-leptospiral strategies. © 2018 Wiley Periodicals, Inc.

Mitochondrial respiratory control is lost during growth factor deprivation.

PubMed

Gottlieb, Eyal; Armour, Sean M; Thompson, Craig B

2002-10-01

The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x(L), restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

PubMed

Wu, Si; Ge, Xi; Lv, Zhixin; Zhi, Zeyong; Chang, Zengyi; Zhao, Xin Sheng

2011-09-15

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

PubMed Central

Williamson, Yulanda M.; Moura, Hercules; Simmons, Kaneatra; Whitmon, Jennifer; Melnick, Nikkol; Rees, Jon; Woolfitt, Adrian; Schieltz, David M.; Tondella, Maria L.; Ades, Edwin; Sampson, Jacquelyn; Carlone, George; Barr, John R.

2017-01-01

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria. PMID:22537821

Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus

PubMed Central

Datta, Shreya

2011-01-01

Vibrio vulnificus, the cause of septicemia and serious wound infection in humans and fishes, require iron for its pathogenesis. Hemin uptake through the outer membrane receptor, HupA, is one of its many mechanisms by which it acquires iron. We report here the identification of an additional TonB-dependent hemin receptor HvtA, that is needed in conjunction with the HupA protein for optimal hemin utilization. The HvtA protein is significantly homologous to other outer membrane hemin receptors and its expression in trans restored the uptake of hemin and hemoglobin, the latter to a weaker extent, in a mutant strain that was defective in both receptors. Quantitative RT-PCR suggested that transcription of the hvtA gene was iron regulated. The operon containing the hvtA gene is homologous to the operon in V. cholerae containing the hemin receptor gene hutR suggesting a vertical transmission of the hvtA cluster from V. cholerae to V. vulnificus. PMID:22015545

Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge.

PubMed

Choi, Hyun-Il; Kim, Moonjeong; Jeon, Jinseong; Han, Jin Kwan; Kim, Kwang-Sun

2017-08-26

Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines. Copyright © 2017 Elsevier Inc. All rights reserved.

OmpA: A Flexible Clamp for Bacterial Cell Wall Attachment.

PubMed

Samsudin, Firdaus; Ortiz-Suarez, Maite L; Piggot, Thomas J; Bond, Peter J; Khalid, Syma

2016-12-06

The envelope of Gram-negative bacteria is highly complex, containing separate outer and inner membranes and an intervening periplasmic space encompassing a peptidoglycan (PGN) cell wall. The PGN scaffold is anchored non-covalently to the outer membrane via globular OmpA-like domains of various proteins. We report atomically detailed simulations of PGN bound to OmpA in three different states, including the isolated C-terminal domain (CTD), the full-length monomer, or the complete full-length dimeric form. Comparative analysis of dynamics of OmpA CTD from different bacteria helped to identify a conserved PGN-binding mode. The dynamics of full-length OmpA, embedded within a realistic representation of the outer membrane containing full-rough (Ra) lipopolysaccharide, phospholipids, and cardiolipin, suggested how the protein may provide flexible mechanical support to the cell wall. An accurate model of the heterogeneous bacterial cell envelope should facilitate future efforts to develop antibacterial agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Thermotropic phase transitions in model membranes of the outer skin layer based on ceramide 6

NASA Astrophysics Data System (ADS)

Gruzinov, A. Yu.; Kiselev, M. A.; Ermakova, E. V.; Zabelin, A. V.

2014-01-01

The lipid intercellular matrix stratum corneum of the outer skin layer is a multilayer membrane consisting of a complex mixture of different lipids: ceramides, fatty acids, cholesterol, and its derivatives. The basis of the multilayer membrane is the lipid bilayer, i.e., a two-dimensional liquid crystal. Currently, it is known that the main way of substance penetration through the skin is the lipid matrix. The complexity of the actual biological system does not allow reliable direct study of its properties; therefore, system modeling is often used. Phase transitions in the lipid system whose composition simulates the native lipid matrix are studied by the X-ray synchrotron radiation diffraction method.

Müller glial cells of the primate foveola: An electron microscopical study.

PubMed

Syrbe, Steffen; Kuhrt, Heidrun; Gärtner, Ulrich; Habermann, Gunnar; Wiedemann, Peter; Bringmann, Andreas; Reichenbach, Andreas

2018-02-01

Previous studies on the ultrastructure of the primate foveola suggested the presence of an inverted cone-like structure which is formed by 25-35 specialized Müller cells overlying the area of high photoreceptor density. We investigated the ultrastructure of the Müller cells in the foveola of a human and macaque retina. Sections through the posterior poles of an eye of a 40 years-old human donor and an eye of an adult cynomolgus monkey (Macaca fascicularis) were investigated with transmission electron microscopy. The foveola consisted of an inner layer (thickness, 5.5-12 μm) which mainly contained somata (including nuclei) and inner processes of Müller cells; this layer overlaid the central Henle fibers and outer nuclear layer. The inner layer contained numerous watery cysts and thin lamelliform and tubular Müller cell processes which spread along the inner limiting membrane (ILM). The cytoplasm of the outer Müller cell processes became increasingly dispersed and electron-lucent in the course towards the outer limiting membrane. The ILM of the foveola was formed by a very thin basal lamina (thickness, <40 nm) while the basal lamina of the parafovea was thick (0.9-1 μm). The data show that there are various conspicuous features of foveolar Müller cells. The numerous thin Müller cell processes below the ILM may smooth the inner surface of the foveola (to minimize image distortion resulting from varying light refraction angles at an uneven retinal surface), create additional barriers to the vitreous cavity (compensating the thinness of the ILM), and provide mechanical stability to the tissue. The decreasing density of the outer process cytoplasm may support the optical function of the foveola. Copyright © 2017. Published by Elsevier Ltd.

Protein secretion and membrane insertion systems in gram-negative bacteria.

PubMed

Saier, Milton H

2006-01-01

In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.

Disruption of gel phase lipid packing efficiency by sucralose studied with merocyanine 540.

PubMed

Barker, Morgan; Kennedy, Anthony

2017-04-01

Sucralose, an artificial sweetener, displays very different behavior towards membranes than its synthetic precursor sucrose. The impact of both sugars on model dipalmitoylphosphatidylcholine model membranes was investigated using absorbance and flourescence spectroscopy and the membrane probe merocyanine 540. This probe molecule is highly sensitive to changes in membrane packing, microviscosity and polarity. This work focuses on the impact of sugars on the outer leaflet of unilamellar dipalmitoyl phosphatidylcholine model membranes. The choice of lipid permits access to the gel phase at room temperature and incorporation of the dye after liposome formation allows us to examine the direct impact of the sugar on the outer leaflet while maximizing the response of the dye to changes in the bilayer. The results demonstrate that sucrose has no impact on the packing efficiency of lipids in unilamellar DPPC vesicles in the gel phase. Conversely sucralose decreases the packing efficiency of lipids in the gel phase and results in decreased microviscosity and increased membrane fluidity, which may be as a result of water disruption at the membrane water interface. Copyright © 2017 Elsevier B.V. All rights reserved.

Excess plasma membrane and effects of ionic amphipaths on mechanics of outer hair cell lateral wall.

PubMed

Morimoto, Noriko; Raphael, Robert M; Nygren, Anders; Brownell, William E

2002-05-01

The interaction between the outer hair cell (OHC) lateral wall plasma membrane and the underlying cortical lattice was examined by a morphometric analysis of cell images during cell deformation. Vesiculation of the plasma membrane was produced by micropipette aspiration in control cells and cells exposed to ionic amphipaths that alter membrane mechanics. An increase of total cell and vesicle surface area suggests that the plasma membrane possesses a membrane reservoir. Chlorpromazine (CPZ) decreased the pressure required for vesiculation, whereas salicylate (Sal) had no effect. The time required for vesiculation was decreased by CPZ, indicating that CPZ decreases the energy barrier required for vesiculation. An increase in total volume is observed during micropipette aspiration. A deformation-induced increase in hydraulic conductivity is also seen in response to micropipette-applied fluid jet deformation of the lateral wall. Application of CPZ and/or Sal decreased this strain-induced hydraulic conductivity. The impact of ionic amphipaths on OHC plasma membrane and lateral wall mechanics may contribute to their effects on OHC electromotility and hearing.