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

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.

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

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.

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

Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

PubMed Central

Ogunnariwo, J A; Schryvers, A B

1990-01-01

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

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.

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

Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates.

PubMed Central

Bell, J; Grass, S; Jeanteur, D; Munson, R S

1994-01-01

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein. PMID:8188390

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.

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.

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.

Vascularization and Cellular Isolation Potential of a Novel Electrospun Cell Delivery Vehicle

PubMed Central

Krishnan, Laxminarayanan; Touroo, Jeremy; Reed, Robert; Boland, Eugene; Hoying, James B.; Williams, Stuart K.

2014-01-01

A clinical need exists for a cell delivery device that supports long term cell viability, cell retention within the device and retrieval of delivered cells if necessary. Previously, cell isolation devices have been based on hollow fiber membranes, porous polymer scaffolds, alginate systems, or micro-machined membranes. We present the development and characterization of a novel dual porosity electrospun membrane based device, which supports cellular infiltration and vascularization of its outer porous layer and maintains cellular isolation within a lumen bounded by an inner low porosity layer. Electrospinning conditions were initially established to support electrospun fiber deposition onto nonconductive silicone surfaces. With these parameters established, devices for in vivo evaluations were produced using nylon as a nonconductive scaffold for deposition of dual porosity electrospun fibers. The outer porous layer supported the development of a penetrating microcirculation and the membrane supported the transfer of insulin from encapsulated sustained release pellets for four weeks. Viable cells implanted within the device could be identified after two weeks of implantation. Through the successful demonstration of survival and cellular isolation of human epithelial cells within the implanted devices and the ability to use the device to deliver insulin, we have established the utility of this device toward localized cell transplantation. The Cell Delivery Device establishes a platform to test the feasibility of approaches to cell dose control and cell localization at the site of implantation in the clinical use of modified autologous or allogeneic cells. PMID:23913805

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

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

Characterization of the modified Hodge test-positive isolates of Enterobacteriaceae in Taiwan.

PubMed

Hung, Kuei-Hsiang; Yan, Jing-Jou; Lu, Jang-Jih; Chen, Hung-Mo; Wu, Jiunn-Jong

2013-02-01

The modified Hodge test is a phenotypic test to detect KPC-type carbapenemase producers among Enterobacteriaceae, as recommended by the Clinical Laboratory Standards Institute. However, false positive results were reported. In this study, we aimed to large-scale investigate the characterization of the modified Hodge test-positive isolates of Enterobacteriaceae collected between 2006 and 2010 in Taiwan. Fifty-six isolates, including 24 Enterobacter cloacae, 17 Escherichia coli, 10 Klebsiella pneumoniae, and 5 Citrobacter freundii, tested positive with the modified Hodge test. The in vitro activities of 10 antimicrobial agents were determined by the agar dilution method. Boronic acid combined-disk test was used to further confirm the KPC producers. Phenotype of ESBL, AmpC, class B carbapenemases, and profile of outer membrane proteins were investigated by the confirmatory test, boronic acid disk method, 2-mercaptopropionic acid double-disk method, and urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. β-lactamase genes were examined by PCR and sequencing. These isolates were resistant to ceftazidime (100%), aztreonam (82.1%), ertapenem (64.3%), gentamicin (53.6%), ciprofloxacin (50%), levofloxacin (48.2%), cefepime (19.6%), imipenem (16.1%), meropenem (12.5%), and amikacin (8.9%). Phenotypic testing among isolates revealed the production of ESBLs, metallo-β-lactamases (MBLs), and AmpC in 10 (17.9%), 16 (28.6%), and 12 (44.4%) isolates, respectively. Carbapenemase and non-carbapenemase β-lactamase genes bla(TEM-1), bla(SHV), bla(CTX-M), bla(IMP-8), bla(CMY-2), and bla(DHA-1) were found in 32 (57.1%), 19 (33.9%), 4 (7.1%), 16 (28.6%), 14 (25%), and 5 (8.9%) of the strains, respectively. No class A and D carbapenemase genes were detected. Outer membrane protein profile showed obviously decreased expression in 49 (87.5%) isolates with positive result of modified Hodge test. Our data show that ESBLs, AmpC, and imipenemase-8 (IMP-8) carbapenemase coupled with decreased expression of outer membrane protein were prevalent in Enterobacteriaceae isolates testing positive for the modified Hodge test in Taiwan. Copyright © 2012. Published by Elsevier B.V.

Using MOMP typing as an epidemiological tool to investigate outbreaks caused by milkborne Campylobacter jejuni isolates in California

USDA-ARS?s Scientific Manuscript database

We describe using major outer membrane protein (MOMP) typing as a screen to compare the C. jejuni porA gene sequences of clinical outbreak strains from human stool with the porA sequences of dairy farm strains isolated during two milkborne campylobacteriosis outbreak investigations in California. Th...

Mitochondrial Ceramide-Rich Macrodomains Functionalize Bax upon Irradiation

PubMed Central

Lee, Hyunmi; Rotolo, Jimmy A.; Mesicek, Judith; Penate-Medina, Tuula; Rimner, Andreas; Liao, Wen-Chieh; Yin, Xianglei; Ragupathi, Govind; Ehleiter, Desiree; Gulbins, Erich; Zhai, Dayong; Reed, John C.; Haimovitz-Friedman, Adriana; Fuks, Zvi; Kolesnick, Richard

2011-01-01

Background Evidence indicates that Bax functions as a “lipidic” pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates. Methodology/Principal Findings MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1–2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight “pore-forming” oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. Conclusions/Significance Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo. PMID:21695182

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.

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.

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

Resistant mechanisms and molecular epidemiology of imipenem-resistant Acinetobacter baumannii.

PubMed

Xiao, Shu-Zhen; Chu, Hai-Qing; Han, Li-Zhong; Zhang, Zhe-Min; Li, Bing; Zhao, Lan; Xu, Liyun

2016-09-01

The aim of the study was to investigate the resistant mechanisms and homology of imipenem-resistant Acinetobacter baumannii (A. baumannii). A total of 46 non-duplicate imipenem‑resistant A. baumannii clinical isolates were collected from three tertiary hospitals between July, 2011 and June, 2012. The minimal inhibitory concentrations (MICs) of antimicrobial agents were determined using the agar dilution method. Phenylalanine‑arginine β-naphthylamide was used to detect the presence of the efflux pump-mediated resistant mechanism. Polymerase chain reaction was employed to amplify genes associated with drug resistance, including β‑lactamase genes, efflux pump genes and outer membrane protein gene CarO. A few amplicons were randomly selected and sequenced. Multilocus sequence analysis (MLST) was employed in typing A. baumanni. A. baumannii was resistant to imipenem, simultaneously showing resistance to several other antimicrobials. In addtition, 13 A. baumannii were found to mediate drug resistance through operation of the efflux pump. Of the various drug resistance genes tested, blaOXA‑51 was present in 46 isolates, blaOXA‑23 gene was present in 44 isolates and blaNDM gene was found in only one strain. Other drug resistant‑associated genes, including blaKPC, blaIMP, blaOXA-24, blaOXA‑58, blaSHV, blaGIM and blaVIM were not detected. Mutation of adeS and outer membrane protein gene CarO were found in a few of the imipenem‑resistant isolates. The MLST analysis revealed that all 46 clinical isolates were clustered into 11 genotypes and the most frequent genotype was ST208. In conclusion, β‑lactamase genes, genes involved in efflux pump and mutation of outer membrane protein encoding gene may be important in mediating imipenem resistance in A. baumannii. Of the 11 different genotypes, ST11 was shared by the majority of A. baumannii, which may be due to horizontal transfer of patients from hospitals.

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

PubMed Central

Razin, Shmuel; Hasin, Miriam; Ne'eman, Zvi; Rottem, Shlomo

1973-01-01

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with “Stubborn” disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm3. Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 μg/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily. Images PMID:4127633

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.

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

Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection

NASA Astrophysics Data System (ADS)

Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing

2016-11-01

Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

Molecular cloning, sequencing, and expression of the outer membrane protein P2 gene of Haemophilus parasuis.

PubMed

Li, Peng; Bai, Juan; Li, Jun-xing; Zhang, Guo-long; Song, Yan-hua; Li, Yu-feng; Wang, Xian-wei; Jiang, Ping

2012-10-01

Haemophilus parasuis is the etiological agent of Glässer's disease characterized by fibrinous polyserositis, polyarthritis, and meningitis in young pigs. But it is difficult to develop universal serological diagnostic tools and effective vaccines against this disease because of the serovar diversity of the isolates. In this study, enterobacterial repetitive intergenic consensus-polymerase chain reaction, were performed to investigate the gene profile of 111 isolates of H. parasuis from China. And a specific common gene of H. parasuis was cloned and identified as the outer-membrane protein (OMP) P2 gene. Sequencing results of OMP P2 genes of 22 isolates showed that they had high homology and could be divided into 2 genetic types. Moreover, the OMPP2 protein was expressed in Escherichia coli expressing system. And the purified recombinant protein provided partial protection against H. parasuis infection in mice. It suggested the OMP P2 was an immunogenic protein and had great potential to serve as a vaccine and diagnostic antigen. Copyright © 2011 Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

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

In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

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

Bureau, T.E.; Brown, R.M. Jr.

1987-10-01

The cytoplasmic and outer membranes of Acetobacter xylinum were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme and trypsin were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase activity was assayed as the conversion of radioactivity from UDP-(/sup 14/C)glucose into an alkali-insoluble ..beta..-1,4-D-(/sup 14/C)glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, themore » weight-average and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product--namely, cellulose I.« less

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

PubMed

Radolf, J D; Robinson, E J; Bourell, K W; Akins, D R; Porcella, S F; Weigel, L M; Jones, J D; Norgard, M V

1995-11-01

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

PubMed Central

Mouriño, Susana; Rodríguez-Ares, Isabel; Osorio, Carlos R.; Lemos, Manuel L.

2005-01-01

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity. PMID:16332832

Serum antibodies to outer membrane proteins (OMPs) of Moraxella (Branhamella) catarrhalis in patients with bronchiectasis: identification of OMP B1 as an important antigen.

PubMed Central

Sethi, S; Hill, S L; Murphy, T F

1995-01-01

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium. PMID:7890418

Mitochondrial respiratory control is lost during growth factor deprivation

PubMed Central

Gottlieb, Eyal; Armour, Sean M.; Thompson, Craig B.

2002-01-01

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-xL, restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control. PMID:12228733

Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition.

PubMed

Petit, P X; Goubern, M; Diolez, P; Susin, S A; Zamzami, N; Kroemer, G

1998-04-10

Upon induction of permeability transition with different agents (Ca2+, tert-butyl hydroperoxide, atractyloside), mouse hepatocyte mitochondria manifest a disruption of outer membrane integrity leading to the release of cytochrome c and apoptosis-inducing factor (AIF), two proteins which are involved in programmed cell death (apoptosis). Chelation of Ca2+ shortly (within 2 min) after its addition to isolated mitochondria reestablished the mitochondrial transmembrane potential (deltapsi(m)), prevented induction of large amplitude swelling and release of both cytochrome c and AIF. In contrast, late Ca2+ chelation (10 min after addition of Ca2+) failed to affect these parameters. Cytochrome c appears to be released through a mechanically damaged outer mitochondrial membrane rather than via a specific release mechanism. These findings clarify the mechanisms through which irreversible permeability transition occurs with subsequent large amplitude swelling culminating in the release of intermembrane proteins from mitochondria. Moreover, they confirm the hypothesis formulated by Skulachev [FEBS Lett. 397 (1996) 7-10 and Q. Rev. Biophys. 29 (1996) 169-2021 linking permeability transition to activation of the apoptogenic catabolic enzymes.

Localization of cellulose synthase in Acetobacter xylinum

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

Bureau, T.E.

1987-01-01

The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme and trypsin were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxy-octulosonic acid was used to identify the outer membranes. Cellulose synthetase activity was assayed as the conversion of radioactivity from UDP-(/sup 14/C)glucose into an alkali-insoluble ..beta..-1,4-D-(/sup 14/C)glucan. The cellulosic nature of the product was demonstrated by enzymatic hydrolysis followed by thin-layer chromatography, and by methylation analysis followed by thin-layer chromatography and gas chromatography-mass spectroscopy. X-ray diffraction analysis indicated that themore » in vitro product is cellulose II which is in contrast to the in vivo product, namely cellulose I. In addition, no microfibrillar morphology could be observed from negative stained and metal shadowed preparations of the in vitro product.« less

[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).

Genetic locus (nmp-1) affecting the principal outer membrane protein of Neisseria gonorrhoeae.

PubMed Central

Cannon, J G; Klapper, D G; Blackman, E Y; Sparling, P F

1980-01-01

An increase in the apparent molecular weight of the principal outer membrane protein (POMP) of Neisseria gonorrhoeae is associated with introduction of the penB2 genetic marker, which results in low-level, relatively nonspecific antibiotic resistance. Limited proteolysis of the two forms of POMP showed that they had few if any peptides in common. The nonspecific antibiotic resistance of penB2 was separated from the change in POMP by genetic transformation and by isolation of spontaneous penB mutants that showed no change in POMP. The genetic locus involved in the change from one POMP to another, which we have designated nmp-1, is closely linked to, but not identical with, penB2. Images PMID:6782080

ICTV Virus taxonomy profile: Asfarviridae

USDA-ARS?s Scientific Manuscript database

The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virons have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope. Infection of domestic pigs and wild boar results i...

Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig.

PubMed

Zhang, Zhengyu; Uchida, Yasuo; Hirano, Satoshi; Ando, Daisuke; Kubo, Yoshiyuki; Auriola, Seppo; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi; Urtti, Arto; Terasaki, Tetsuya; Tachikawa, Masanori

2017-11-06

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/μg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/μg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/μg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/μg protein), and sodium-potassium adenosine triphosphatase (Na + /K + -ATPase; 53.7 fmol/μg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R 2 = 0.8906), followed by outer BRB (R 2 = 0.7988) and BCSFB (R 2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/μg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

Identification of minor inner-membrane components of the Shigella type III secretion system 'needle complex'.

PubMed

Zenk, Sebastian F; Stabat, David; Hodgkinson, Julie L; Veenendaal, Andreas K J; Johnson, Steven; Blocker, Ariel J

2007-08-01

Type III secretion systems (T3SSs or secretons) are central virulence factors of many Gram-negative bacteria, used to inject protein effectors of virulence into eukaryotic host cells. Their overall morphology, consisting of a cytoplasmic region, an inner- and outer-membrane section and an extracellular needle, is conserved in various species. A portion of the secreton, containing the transmembrane regions and needle, has been isolated biochemically and termed the 'needle complex' (NC). However, there are still unsolved questions concerning the nature and relative arrangement of the proteins assembling the NC. Until these are resolved, the mode of function of the NC cannot be clarified. This paper describes an affinity purification method that enables highly efficient purification of Shigella NCs under near-physiological conditions. Using this method, three new minor components of the NC were identified by mass spectrometry: IpaD, a known component of the needle tip complex, and two predicted components of its central inner-membrane export apparatus, Spa40 and Spa24. A further minor component of the NC, MxiM, is only detected by immunoblotting. MxiM is a 'pilotin'-type protein for the outer-membrane 'secretin' ring formed of MxiD. As expected, it localized to the outer rim of the upper ring of NCs, validating the other findings.

Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

DTIC Science & Technology

2004-10-01

malonyl-CoA-sensitive form of carnitine palmitoyltransferase is not local - ized exclusively in the outer membrane of rat liver mitochondria . J Biol...for the isolation of fresh mitochondria , both subsarcolemmal and interfibrillar. Analytic methods. Detailed analytic methods have been previously cited...populations of mitochondria , the subsarcolemmal and inter- fibrillar, were isolated from hearts of normal and HF dogs using the procedure of Palmer et al

Zinc-dependent multi-conductance channel activity in mitochondria isolated from ischemic brain.

PubMed

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M; Pypaert, Marc; Hardwick, J Marie; Sensi, Stefano L; Zukin, R Suzanne; Jonas, Elizabeth A

2006-06-21

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear. Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, deltaN-BCL-xL. The findings implicate deltaN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant deltaN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate deltaN-BCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Zinc-Dependent Multi-Conductance Channel Activity in Mitochondria Isolated from Ischemic Brain

PubMed Central

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J.; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M.; Pypaert, Marc; Hardwick, J. Marie; Sensi, Stefano L.; Zukin, R. Suzanne; Jonas, Elizabeth A.

2015-01-01

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear.Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, ΔN-BCL-xL. The findings implicate ΔN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant ΔN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ΔNBCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria. PMID:16793892

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

PubMed

Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

2017-01-01

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Identification and strain differentiation of Vibrio cholerae by using polyclonal antibodies against outer membrane proteins.

PubMed

Martínez-Govea, A; Ambrosio, J; Gutiérrez-Cogco, L; Flisser, A

2001-07-01

Cholera is caused only by O1 and O139 Vibrio cholerae strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from V. cholerae O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal Vibrio antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for V. cholerae detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.

Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis†

PubMed Central

Wang, Jin; Edmondson, Dale E.

2011-01-01

TEMPO-substituted pargyline analogues differentially inhibit recombinant human Monoamine Oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane (Upadhyay, A. and Edmondson, D.E., Biochemistry 48, 3928, 2009). This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the meta- and para-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an opposite inhibition pattern to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast on trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardio-protectants or neuro-protectants. PMID:21341713

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

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.

Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation.

PubMed

Hutsul, J A; Worobec, E

1997-08-01

Serratia marcescens is a nosocomial pathogen with a high incidence of beta-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to beta-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.

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.

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant

PubMed Central

Ma, Bing; Reynolds, C. Michael; Raetz, Christian R. H.

2008-01-01

The core-lipid A domain of Escherichia coli lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperature-sensitive mutant (MB1) harboring the S22C and Q111P substitutions in LptA. MB1 stops growing after 30 min at 42°C. 32Pi and [35S]methionine labeling show that export of newly synthesized phospholipids and proteins is not severely impaired, but export of LPS is defective. Using the lipid A 1-phosphatase LpxE as a periplasmic IM marker and the lipid A 3-O-deacylase PagL as an OM marker, we show that core-lipid A reaches the periplasmic side of the IM at 42°C in MB1 but not the outer surface of the OM. Electron microscopy of MB1 reveals dense periplasmic material and a smooth OM at 42°C, consistent with a role for LptA in shuttling LPS across the periplasm. PMID:18768814

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant.

PubMed

Ma, Bing; Reynolds, C Michael; Raetz, Christian R H

2008-09-16

The core-lipid A domain of Escherichia coli lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperature-sensitive mutant (MB1) harboring the S22C and Q111P substitutions in LptA. MB1 stops growing after 30 min at 42 degrees C. (32)P(i) and [(35)S]methionine labeling show that export of newly synthesized phospholipids and proteins is not severely impaired, but export of LPS is defective. Using the lipid A 1-phosphatase LpxE as a periplasmic IM marker and the lipid A 3-O-deacylase PagL as an OM marker, we show that core-lipid A reaches the periplasmic side of the IM at 42 degrees C in MB1 but not the outer surface of the OM. Electron microscopy of MB1 reveals dense periplasmic material and a smooth OM at 42 degrees C, consistent with a role for LptA in shuttling LPS across the periplasm.

Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

PubMed Central

Ortiz, V; Isibasi, A; García-Ortigoza, E; Kumate, J

1989-01-01

The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status. Images PMID:2768450

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

Haemophilus ducreyi Outer Membrane Determinants, Including DsrA, Define Two Clonal Populations

PubMed Central

White, Catherine Dinitra; Leduc, Isabelle; Olsen, Bonnie; Jeter, Chrystina; Harris, Chavala; Elkins, Christopher

2005-01-01

The Haemophilus ducreyi outer membrane component DsrA (for ducreyi serum resistance A) is necessary for complete resistance to normal human serum (NHS). When DsrA expression in 19 temporally and geographically diverse clinical isolates of H. ducreyi was examined by Western blotting, 5 of the strains expressed a different immunotype of the DsrA protein (DsrAII) than the well-characterized prototypical strain 35000HP (DsrAI). The predicted DsrA proteins expressed by the DsrAII strains were 100% identical to each other but only 48% identical to that of strain 35000HP. In addition to the DsrAII protein, class II strains also expressed variant forms of other outer membrane proteins (OMPs) including NcaA (necessary for collagen adhesion A), DltA (ducreyi lectin A), Hlp (H. ducreyi lipoprotein), major OMP, and/or OmpA2 (for OMP A2) and synthesized a distinct, faster-migrating lipooligosaccharide. Based on these data, strains expressing DsrAI were termed class I, and those expressing DsrAII were termed class II. Expression of dsrAII from strain CIP 542 ATCC in the class I dsrAI mutant FX517 (35000HP background), which does not express a DsrA protein, rendered this strain resistant to 50% NHS. This demonstrates that DsrAII protein is also critical to serum resistance. Taken together, these results indicate that there are two clonal populations of H. ducreyi. The implications of two classes of H. ducreyi strains differing in important antigenic outer membrane components are discussed. PMID:15784585

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

Clonality, outer-membrane proteins profile and efflux pump in KPC- producing Enterobacter sp. in Brazil.

PubMed

Rosa, Juliana Ferraz; Rizek, Camila; Marchi, Ana Paula; Guimaraes, Thais; Miranda, Lourdes; Carrilho, Claudia; Levin, Anna S; Costa, Silvia F

2017-03-17

Carbapenems resistance in Enterobacter spp. has increased in the last decade, few studies, however, described the mechanisms of resistance in this bacterium. This study evaluated clonality and mechanisms of carbapenems resistance in clinical isolates of Enterobacter spp. identified in three hospitals in Brazil (Hospital A, B and C) over 7-year. Antibiotics sensitivity, pulsed-field gel electrophoresis (PFGE), PCR for carbapenemase and efflux pump genes were performed for all carbapenems-resistant isolates. Outer-membrane protein (OMP) was evaluated based on PFGE profile. A total of 130 isolates of Enterobacter spp were analyzed, 44/105 (41, 9%) E. aerogenes and 8/25 (32,0%) E. cloacae were resistant to carbapenems. All isolates were susceptible to fosfomycin, polymyxin B and tigecycline. KPC was present in 88.6% of E. aerogenes and in all E. cloacae resistant to carbapenems. The carbapenems-resistant E. aerogenes identified in hospital A belonged to six clones, however, a predominant clone was identified in this hospital over the study period. There is a predominant clone in Hospital B and Hospital C as well. The mechanisms of resistance to carbapenems differ among subtypes. Most of the isolates co-harbored blaKPC, blaTEM and /or blaCTX associated with decreased or lost of 35-36KDa and or 39 KDa OMP. The efflux pump AcrAB-TolC gene was only identified in carbapenems-resistant E. cloacae. There was a predominant clone in each hospital suggesting that cross-transmission of carbapenems-resistant Enterobacter spp. was frequent. The isolates presented multiple mechanisms of resistance to carbapenems including OMP alteration.

The Salmonella dublin virulence plasmid mediates systemic but not enteric phases of salmonellosis in cattle.

PubMed Central

Wallis, T S; Paulin, S M; Plested, J S; Watson, P R; Jones, P W

1995-01-01

Plasmid-bearing and plasmid-free isolates and a plasmid-cured strain of Salmonella dublin were compared for virulence in calves. The plasmid-bearing strains were highly virulent, causing severe enteric and systemic disease with high mortality. In contrast, the plasmid-free strains caused diarrhea but only low mortality. The infection kinetics of a wild-type and a derivative plasmid-cured strain were compared. Both strains were isolated in high numbers from intestinal sites at 3 and 6 days after oral challenge and were isolated at comparable frequencies from systemic sites at 3 days, but not at 6 days, when the wild-type strain was predominant. The strains were equally invasive in intestinal epithelia with and without Peyer's patch and elicited comparable secretory and inflammatory responses and intestinal pathology in ligated ileal loops. The effect of the virulence plasmid on growth kinetics and on the outer membrane protein profile was assessed in an in vivo growth chamber. The virulence plasmid did not influence either extracellular growth or the expression of major outer membrane proteins. These observations demonstrate that the virulence plasmid is not involved in either the enteric phase of infection or the systemic dissemination of S. dublin but probably mediates the persistence of S. dublin at systemic sites. PMID:7790094

Purification, characterization and sequence analysis of Omp50,a new porin isolated from Campylobacter jejuni.

PubMed Central

Bolla, J M; Dé, E; Dorez, A; Pagès, J M

2000-01-01

A novel pore-forming protein identified in Campylobacter was purified by ion-exchange chromatography and named Omp50 according to both its molecular mass and its outer membrane localization. We observed a pore-forming ability of Omp50 after re-incorporation into artificial membranes. The protein induced cation-selective channels with major conductance values of 50-60 pS in 1 M NaCl. N-terminal sequencing allowed us to identify the predicted coding sequence Cj1170c from the Campylobacter jejuni genome database as the corresponding gene in the NCTC 11168 genome sequence. The gene, designated omp50, consists of a 1425 bp open reading frame encoding a deduced 453-amino acid protein with a calculated pI of 5.81 and a molecular mass of 51169.2 Da. The protein possessed a 20-amino acid leader sequence. No significant similarity was found between Omp50 and porin protein sequences already determined. Moreover, the protein showed only weak sequence identity with the major outer-membrane protein (MOMP) of Campylobacter, correlating with the absence of antigenic cross-reactivity between these two proteins. Omp50 is expressed in C. jejuni and Campylobacter lari but not in Campylobacter coli. The gene, however, was detected in all three species by PCR. According to its conformation and functional properties, the protein would belong to the family of outer-membrane monomeric porins. PMID:11104668

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

PubMed

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

2017-05-23

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis. Copyright © 2017 Diao et al.

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.

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.

Novel utilization of the outer membrane proteins for the identification and differentiation of pathogenic versus nonpathogenic microbial strains using mass spectrometry-based proteomics approach

NASA Astrophysics Data System (ADS)

Jabbour, Rabih E.; Wade, Mary; Deshpande, Samir V.; McCubbin, Patrick; Snyder, A. Peter; Bevilacqua, Vicky

2012-06-01

Mass spectrometry based proteomic approaches are showing promising capabilities in addressing various biological and biochemical issues. Outer membrane proteins (OMPs) are often associated with virulence in gram-negative pathogens and could prove to be excellent model biomarkers for strain level differentiation among bacteria. Whole cells and OMP extracts were isolated from pathogenic and non-pathogenic strains of Francisella tularensis, Burkholderia thailandensis, and Burkholderia mallei. OMP extracts were compared for their ability to differentiate and delineate the correct database organism to an experimental sample and for the degree of dissimilarity to the nearest-neighbor database strains. This study addresses the comparative experimental proteome analyses of OMPs vs. whole cell lysates on the strain-level discrimination among gram negative pathogenic and non-pathogenic strains.

The Role of OmpK35, OmpK36 Porins, and Production of β-Lactamases on Imipenem Susceptibility in Klebsiella pneumoniae Clinical Isolates, Cairo, Egypt.

PubMed

Wassef, Mona; Abdelhaleim, Mona; AbdulRahman, Eiman; Ghaith, Doaa

2015-12-01

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. We aimed to study the effect of combined porin loss and production of extended-spectrum β-lactamases (ESBLs) on imipenem susceptibility among K. pneumoniae clinical isolates. This study included 91 suspected ESBL-producing K. pneumoniae clinical isolates, isolated from different patient specimens at the Cairo University hospital from January to June 2010. All isolates were subjected to genotypic analysis of the outer membrane protein gene expression using reverse transcription-PCR (RT-PCR) and analysis of OmpK35/36 of 38 isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By RT-PCR, loss of Omp35 was detected in 78 (85.7%) isolates, loss of Omp36 was detected in 64 (70.32%), and loss of both porins was detected in 62 (68.1%). Out of 91 isolates, 45 (49.5%) were resistant to cefoxitin, and 17 (18.7%) were confirmed as derepressed AmpC producers. Omp35 was lost in all FOX-resistant isolates, whereas Omp36 was lost in 42 (93.3%) (p-value 0.002). The mean of ceftazidime inhibition zone diameter was significantly decreased among ESBL-producing isolates with loss of Omp35/36 (p-value 0.041 and 0.006), respectively. The mean of imipenem minimal inhibitory concentration (MIC) was markedly increased to 8.55 μg/ml among AmpC-producing isolates with Omp35/36 loss, while the mean of imipenem MIC among the 66 confirmed ESBL producers was 0.32 μg/ml. Imipenem MIC was markedly increased among K. pneumoniae isolates showing AmpC production with loss of both porins OmpK35/36. Meanwhile, the association of porin OmpK35/36 loss with ESBL production was not a direct cause of resistance to imipenem.

Global survey of Klebsiella pneumoniae major porins from ertapenem non-susceptible isolates lacking carbapenemases.

PubMed

Wise, Mark G; Horvath, Elizabeth; Young, Katherine; Sahm, Daniel F; Kazmierczak, Krystyna M

2018-03-01

To understand the diversity of porin disruption in Klebsiella pneumoniae, the major outer membrane protein (OMP) porins, OmpK35 and OmpK36, were examined in a set of isolates that did not harbour traditional carbapenem-hydrolysing enzymes, but nevertheless tested non-susceptible to ertapenem. A world-wide collection of Klebsiella pneumoniae isolates that were part of the Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance project over the years 2008-2014 were characterised with regard to their β-lactamase gene carriage and potential permeability defects. Four hundred and eighty-seven isolates that did not carry carbapenemase genes, but were non-susceptible to ertapenem, were investigated by sequence analysis of the genes encoding OmpK35 and OmpK36. Isolates without obvious genetic lesions in either major porin gene were further examined by outer membrane protein SDS-PAGE. The majority of isolates, 83.0 % (404/487), exhibited clear genetic disruption in either or both of the ompK35 and ompK36 genes. Among the proportion of the collection with the highest ertapenem MIC value (>4 mg l -1 ), 60.5 % (115/190) showed mutation in both porin genes. Isolates without obvious genetic mutations were examined by SDS-PAGE, and 90.4 % (75/83) were found to lack or show altered expression of at least one of the major OMPs when compared to an ertapenem sensitive control strain. This study illustrates that porin deficiency in Klebsiella pneumoniae is a widespread phenomenon, and in combination with ESBLs and/or AmpC enzymes, likely accounts for the elevated ertapenem MICs observed in this study.

Dynamics of long-term colonization of respiratory tract by Haemophilus influenzae in cystic fibrosis patients shows a marked increase in hypermutable strains.

PubMed

Román, Federico; Cantón, Rafael; Pérez-Vázquez, María; Baquero, Fernando; Campos, José

2004-04-01

The persistence and variability of 188 Haemophilus influenzae isolates in respiratory tract of 30 cystic fibrosis (CF) patients over the course of 7 years was studied. Antibiotic susceptibility testing, DNA fingerprinting, and analysis of outer membrane protein profiles were performed on all isolates. A total of 115 distinct pulsed-field gel electrophoresis profiles were identified. Ninety percent of patients were cocolonized with two or more clones over the studied period. A third of the patients were cross-colonized with one or two H. influenzae strains; 11% of the clones persisted for 3 or more months. Biotype, outer membrane protein profiles, and resistance profiles showed variation along the studied period, even in persisting clones. Four isolates (2.1%) recovered from 3 patients were type f capsulate, with three of them belonging to the same clone. beta-Lactamase production was detected in 23.9% of isolates while 7% of the beta-lactamase-negative isolates presented diminished susceptibility to ampicillin (beta-lactamase-negative ampicillin resistance phenotype). Remarkably, 21.3% of the H. influenzae isolates presented decreased susceptibility to ciprofloxacin, which was mainly observed in persisting clones. Of the H. influenzae isolates from CF patients, 18 (14.5%) were found to be hypermutable in comparison with 1 (1.4%) from non-CF patients (P < 0.0001). Ten patients (33.3%) were colonized by hypermutable strains over the study period. A multiresistance phenotype and long-term clonal persistence were significantly associated in some cases for up to 7 years. These results suggest that H. influenzae bronchial colonization in CF patients is a dynamic process, but better-adapted clones can persist for long periods of time.

Dynamics of Long-Term Colonization of Respiratory Tract by Haemophilus influenzae in Cystic Fibrosis Patients Shows a Marked Increase in Hypermutable Strains

PubMed Central

Román, Federico; Cantón, Rafael; Pérez-Vázquez, María; Baquero, Fernando; Campos, José

2004-01-01

The persistence and variability of 188 Haemophilus influenzae isolates in respiratory tract of 30 cystic fibrosis (CF) patients over the course of 7 years was studied. Antibiotic susceptibility testing, DNA fingerprinting, and analysis of outer membrane protein profiles were performed on all isolates. A total of 115 distinct pulsed-field gel electrophoresis profiles were identified. Ninety percent of patients were cocolonized with two or more clones over the studied period. A third of the patients were cross-colonized with one or two H. influenzae strains; 11% of the clones persisted for 3 or more months. Biotype, outer membrane protein profiles, and resistance profiles showed variation along the studied period, even in persisting clones. Four isolates (2.1%) recovered from 3 patients were type f capsulate, with three of them belonging to the same clone. β-Lactamase production was detected in 23.9% of isolates while 7% of the β-lactamase-negative isolates presented diminished susceptibility to ampicillin (β-lactamase-negative ampicillin resistance phenotype). Remarkably, 21.3% of the H. influenzae isolates presented decreased susceptibility to ciprofloxacin, which was mainly observed in persisting clones. Of the H. influenzae isolates from CF patients, 18 (14.5%) were found to be hypermutable in comparison with 1 (1.4%) from non-CF patients (P < 0.0001). Ten patients (33.3%) were colonized by hypermutable strains over the study period. A multiresistance phenotype and long-term clonal persistence were significantly associated in some cases for up to 7 years. These results suggest that H. influenzae bronchial colonization in CF patients is a dynamic process, but better-adapted clones can persist for long periods of time. PMID:15070988

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.

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.

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.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

PubMed

Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

2015-04-21

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale.

PubMed

G Junior, Daniel S; Araújo, Flábio R; Almeida Junior, Nalvo F; Adi, Said S; Cheung, Luciana M; Fragoso, Stenio P; Ramos, Carlos A N; Oliveira, Renato Henrique M de; Santos, Caroline S; Bacanelli, Gisele; Soares, Cleber O; Rosinha, Grácia M S; Fonseca, Adivaldo H

2010-11-01

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

PubMed Central

Haake, David A.; Matsunaga, James

2002-01-01

The outer membrane (OM) of the mammalian pathogen Leptospira kirschneri was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31LipL45. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components. PMID:12183539

Drug discovery strategies to outer membrane targets in Gram-negative pathogens.

PubMed

Brown, Dean G

2016-12-15

This review will cover selected recent examples of drug discovery strategies which target the outer membrane (OM) of Gram-negative bacteria either by disruption of outer membrane function or by inhibition of essential gene products necessary for outer membrane assembly. Significant advances in pathway elucidation, structural biology and molecular inhibitor designs have created new opportunities for drug discovery within this target-class space. Copyright © 2016 Elsevier Ltd. All rights reserved.

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

Antigenic analysis of Campylobacter species and an intracellular Campylobacter-like organism associated with porcine proliferative enteropathies.

PubMed Central

McOrist, S; Boid, R; Lawson, G H

1989-01-01

Whole-cell and outer membrane preparations of Campylobacter mucosalis, C. hyointestinalis, C. jejuni, and C. coli isolated from porcine intestines were compared with preparations of intracellular Campylobacter-like organisms extracted directly from the lesions of pigs with proliferative enteropathy. By gradient polyacrylamide gel electrophoresis, outer membrane and total protein profiles of C. mucosalis, C. hyointestinalis, C. jejuni, and C. coli were significantly different from each other and from those of the Campylobacter-like organisms. Immunoblotting of these preparations with rabbit antisera or monoclonal antibodies prepared against the intracellular Campylobacter-like organisms showed strong reactions only with a 25,000- to 27,000-molecular-weight component of preparations of the intracellular organisms. Antisera to cultivable Campylobacter species isolates did not react with preparations of intracellular organisms. Isoelectric focusing of sonicated preparations showed protein profile differences and an immune-reactive component in the intracellular organisms with a pI of 4.5. This study suggests that the intracellular Campylobacter-like organism associated with proliferative enteropathy may be a novel bacterium with significant antigenic differences from the Campylobacter species previously associated with the disease. Images PMID:2917794

Genome sequence of the phylogenetically isolated spirochete Leptonema illini type strain (3055T)

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

Huntemann, Marcel; Stackebrandt, Erko; Held, Brittany

2013-01-01

Leptonema illini Hovind-Hougen 1979 is the type species of the genus Leptonema, family Leptospiraceae, phylum Spirochaetes. Organisms of this family have a Gram-negative-like cell enve- lope consisting of a cytoplasmic membrane and an outer membrane. The peptidoglycan layer is as- sociated with the cytoplasmic rather than the outer membrane. The two flagella of members of Leptospiraceae extend from the cytoplasmic membrane at the ends of the bacteria into the periplasmic space and are necessary for their motility. Here we describe the features of the L. illini type strain, together with the complete genome sequence, and annotation. This is the firstmore » genome sequence (finished at the level of Improved High Quality Draft) to be reported from of a member of the genus Leptonema and a representative of the third genus of the family Leptospiraceae for which complete or draft genome sequences are now available. The three scaffolds of the 4,522,760 bp draft genome sequence reported here, and its 4,230 protein-coding and 47 RNA genes are part of the Ge- nomic Encyclopedia of Bacteria and Archaea project.« less

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

PubMed

Alvarez, Angel H; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

2015-10-01

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

PubMed Central

Alvarez, Angel H.; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

2015-01-01

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD. PMID:26424916

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.

Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

PubMed Central

Lazzaroni, J C; Portalier, R C

1981-01-01

Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein. Images PMID:7009581

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method

PubMed Central

1987-01-01

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes. PMID:2447095

Identification and Characterization of Outer Membrane Vesicle-Associated Proteins in Salmonella enterica Serovar Typhimurium

PubMed Central

Bai, Jaewoo; Kim, Seul I; Ryu, Sangryeol

2014-01-01

Salmonella enterica serovar Typhimurium is a primary cause of enteric diseases and has acquired a variety of virulence factors during its evolution into a pathogen. Secreted virulence factors interact with commensal flora and host cells and enable Salmonella to survive and thrive in hostile environments. Outer membrane vesicles (OMVs) released from many Gram-negative bacteria function as a mechanism for the secretion of complex mixtures, including virulence factors. We performed a proteomic analysis of OMVs that were isolated under standard laboratory and acidic minimal medium conditions and identified 14 OMV-associated proteins that were observed in the OMV fraction isolated only under the acidic minimal medium conditions, which reproduced the nutrient-deficient intracellular milieu. The inferred roles of these 14 proteins were diverse, including transporter, enzyme, and transcriptional regulator. The absence of these proteins influenced Salmonella survival inside murine macrophages. Eleven of these proteins were predicted to possess secretion signal sequences at their N termini, and three (HupA, GlnH, and PhoN) of the proteins were found to be translocated into the cytoplasm of host cells. The comparative proteomic profiling of OMVs performed in this study revealed different protein compositions in the OMVs isolated under the two different conditions, which indicates that the OMV cargo depends on the growth conditions and provides a deeper insight into how Salmonella utilizes OMVs to adapt to environmental changes. PMID:24935973

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

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

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

PubMed Central

Simonis, Alexander; Hebling, Sabrina; Gulbins, Erich; Schneider-Schaulies, Sibylle; Schubert-Unkmeir, Alexandra

2014-01-01

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. PMID:24945304

Methods to study the biogenesis of membrane proteins in yeast mitochondria.

PubMed

Weckbecker, Daniel; Herrmann, Johannes M

2013-01-01

The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to be inserted into the outer and the inner membranes of the organelle where many of them assemble into multisubunit complexes. In this chapter we provide established protocols to study these different processes experimentally using mitochondria of budding yeast. In particular, methods are described in detail to purify mitochondria, to study mitochondrial protein synthesis, to follow the import of radiolabeled preproteins into isolated mitochondria, and to assess membrane association and the aggregation of mitochondrial proteins by fractionation. These protocols and a list of dos and don'ts shall enable beginners and experienced scientists to address the targeting and assembly of mitochondrial membrane proteins.

Insertional inactivation of oprD in carbapenem-resistant Pseudomonas aeruginosa strains isolated from burn patients in Tehran, Iran.

PubMed

Shariati, A; Azimi, T; Ardebili, A; Chirani, A S; Bahramian, A; Pormohammad, A; Sadredinamin, M; Erfanimanesh, S; Bostanghadiri, N; Shams, S; Hashemi, A

2018-01-01

In this study, we report the insertion sequence IS Ppu 21 in the opr D porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS-PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the IS Ppu 21 insertion element in the opr D gene. The extended-spectrum β-lactamase-encoding gene in IS Ppu 21-carrying isolates was bla TEM . PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmt A, aad A, aad B and arm A genes were positive in all IS Ppu 21 harbouring isolates. The relative expression levels of the mex X, mex B, mex T and mex D genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS-PAGE suggested that opr D was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing IS Ppu 21 were shown to be clonally related. The present study describes a novel molecular mechanism, IS Ppu 21 insertion of the opr D gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

PubMed Central

Wenke, Jamie L.; McDonald, W. Hayes; Schey, Kevin L.

2016-01-01

Purpose To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. Methods Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. Results Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. Conclusions Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ. PMID:27537260

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

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

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas.

PubMed

Valenti, Daniela; de Bari, Lidia; De Filippis, Bianca; Ricceri, Laura; Vacca, Rosa Anna

2014-01-01

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure.

PubMed

Pavez, Monica; Vieira, Camila; de Araujo, Maria Rita; Cerda, Alvaro; de Almeida, Lara Mendes; Lincopan, Nilton; Mamizuka, Elsa Masae

2016-07-01

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller-Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4.

PubMed

Casillo, Angela; Parrilli, Ermenegilda; Filomena, Sannino; Lindner, Buko; Lanzetta, Rosa; Parrilli, Michelangelo; Tutino, Maria Luisa; Corsaro, Maria Michela

2015-07-22

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by (1)H and (13)C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.

Assembly of β-barrel proteins in the mitochondrial outer membrane.

PubMed

Höhr, Alexandra I C; Straub, Sebastian P; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils

2015-01-01

Mitochondria evolved through endosymbiosis of a Gram-negative progenitor with a host cell to generate eukaryotes. Therefore, the outer membrane of mitochondria and Gram-negative bacteria contain pore proteins with β-barrel topology. After synthesis in the cytosol, β-barrel precursor proteins are first transported into the mitochondrial intermembrane space. Folding and membrane integration of β-barrel proteins depend on the mitochondrial sorting and assembly machinery (SAM) located in the outer membrane, which is related to the β-barrel assembly machinery (BAM) in bacteria. The SAM complex recognizes β-barrel proteins by a β-signal in the C-terminal β-strand that is required to initiate β-barrel protein insertion into the outer membrane. In addition, the SAM complex is crucial to form membrane contacts with the inner mitochondrial membrane by interacting with the mitochondrial contact site and cristae organizing system (MICOS) and shares a subunit with the endoplasmic reticulum-mitochondria encounter structure (ERMES) that links the outer mitochondrial membrane to the endoplasmic reticulum (ER). Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Use of human cancer cell lines mitochondria to explore the mechanisms of BH3 peptides and ABT-737-induced mitochondrial membrane permeabilization.

PubMed

Buron, Nelly; Porceddu, Mathieu; Brabant, Magali; Desgué, Diana; Racoeur, Cindy; Lassalle, Myriam; Péchoux, Christine; Rustin, Pierre; Jacotot, Etienne; Borgne-Sanchez, Annie

2010-03-31

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.

Antioxidant capacity of Ugni molinae fruit extract on human erythrocytes: an in vitro study.

PubMed

Suwalsky, Mario; Avello, Marcia

2014-08-01

Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile-Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions.

In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

PubMed

Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

2014-09-01

In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

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

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

PubMed

Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

2018-01-01

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

Sequencing of the Chlamydophila psittaci ompA Gene Reveals a New Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method

PubMed Central

Geens, Tom; Desplanques, Ann; Van Loock, Marnix; Bönner, Brigitte M.; Kaleta, Erhard F.; Magnino, Simone; Andersen, Arthur A.; Everett, Karin D. E.; Vanrompay, Daisy

2005-01-01

Twenty-one avian Chlamydophila psittaci isolates from different European countries were characterized using ompA restriction fragment length polymorphism, ompA sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method. PMID:15872282

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate

PubMed Central

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K.; Liu, Peter; Pantua, Homer; Abbas, Alexander R.; Nickerson, Nicholas N.; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min

2017-01-01

ABSTRACT Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. PMID:28536290

Characterization of airborne ice-nucleation-active bacteria and bacterial fragments

NASA Astrophysics Data System (ADS)

Šantl-Temkiv, Tina; Sahyoun, Maher; Finster, Kai; Hartmann, Susan; Augustin-Bauditz, Stefanie; Stratmann, Frank; Wex, Heike; Clauss, Tina; Nielsen, Niels Woetmann; Sørensen, Jens Havskov; Korsholm, Ulrik Smith; Wick, Lukas Y.; Karlson, Ulrich Gosewinkel

2015-05-01

Some bacteria have the unique capacity of synthesising ice-nucleation-active (INA) proteins and exposing them at their outer membrane surface. As INA bacteria enter the atmosphere, they may impact the formation of clouds and precipitation. We studied members of airborne bacterial communities for their capacity to catalyse ice formation and we report on the excretion of INA proteins by airborne Pseudomonas sp. We also observed for the first time that INA biological fragments <220 nm were present in precipitation samples (199 and 482 INA fragments per L of precipitation), which confirms the presence of submicron INA biological fragments in the atmosphere. During 14 precipitation events, strains affiliated with the genus Pseudomonas, which are known to carry ina genes, were dominant. A screening for INA properties revealed that ∼12% of the cultivable bacteria caused ice formation at ≤-7 °C. They had likely been emitted to the atmosphere from terrestrial surfaces, e.g. by convective transport. We tested the ability of isolated INA strains to produce outer membrane vesicles and found that two isolates could do so. However, only very few INA vesicles were released per INA cell. Thus, the source of the submicron INA proteinaceous particles that we detected in the atmosphere remains to be elucidated.

Dissecting Escherichia coli Outer Membrane Biogenesis Using Differential Proteomics

PubMed Central

Martorana, Alessandra M.; Motta, Sara; Di Silvestre, Dario; Falchi, Federica; Dehò, Gianni; Mauri, Pierluigi; Sperandeo, Paola; Polissi, Alessandra

2014-01-01

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. PMID:24967819

Detection of Rickettsia and Ehrlichia spp. in Ticks Associated with Exotic Reptiles and Amphibians Imported into Japan.

PubMed

Andoh, Masako; Sakata, Akiko; Takano, Ai; Kawabata, Hiroki; Fujita, Hiromi; Une, Yumi; Goka, Koichi; Kishimoto, Toshio; Ando, Shuji

2015-01-01

One of the major routes of transmission of rickettsial and ehrlichial diseases is via ticks that infest numerous host species, including humans. Besides mammals, reptiles and amphibians also carry ticks that may harbor Rickettsia and Ehrlichia strains that are pathogenic to humans. Furthermore, reptiles and amphibians are exempt from quarantine in Japan, thus facilitating the entry of parasites and pathogens to the country through import. Accordingly, in the current study, we examined the presence of Rickettsia and Ehrlichia spp. genes in ticks associated with reptiles and amphibians originating from outside Japan. Ninety-three ticks representing nine tick species (genera Amblyomma and Hyalomma) were isolated from at least 28 animals spanning 10 species and originating from 12 countries (Ghana, Jordan, Madagascar, Panama, Russia, Sri Lanka, Sudan, Suriname, Tanzania, Togo, Uzbekistan, and Zambia). None of the nine tick species are indigenous in Japan. The genes encoding the common rickettsial 17-kDa antigen, citrate synthase (gltA), and outer membrane protein A (ompA) were positively detected in 45.2% (42/93), 40.9% (38/93), and 23.7% (22/93) of the ticks, respectively, by polymerase chain reaction (PCR). The genes encoding ehrlichial heat shock protein (groEL) and major outer membrane protein (omp-1) were PCR-positive in 7.5% (7/93) and 2.2% (2/93) of the ticks, respectively. The p44 gene, which encodes the Anaplasma outer membrane protein, was not detected. Phylogenetic analysis showed that several of the rickettsial and ehrlichial sequences isolated in this study were highly similar to human pathogen genes, including agents not previously detected in Japan. These data demonstrate the global transportation of pathogenic Rickettsia and Ehrlichia through reptile- and amphibian-associated ticks. These imported animals have potential to transfer pathogens into human life. These results highlight the need to control the international transportation of known and potential pathogens carried by ticks in reptiles, amphibians, and other animals, in order to improve national and international public health.

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).

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

PubMed Central

Bradford, P A; Urban, C; Mariano, N; Projan, S J; Rahal, J J; Bush, K

1997-01-01

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein. PMID:9055993

Isolation of mitochondria from Saccharomyces cerevisiae using magnetic bead affinity purification

PubMed Central

Liao, Pin-Chao; Boldogh, Istvan R.; Siegmund, Stephanie E.

2018-01-01

Isolated mitochondria are widely used to study the function of the organelle. Typically, mitochondria are prepared using differential centrifugation alone or in conjunction with density gradient ultracentrifugation. However, mitochondria isolated using differential centrifugation contain membrane or organelle contaminants, and further purification of crude mitochondria by density gradient ultracentrifugation requires large amounts of starting material, and is time-consuming. Mitochondria have also been isolated by irreversible binding to antibody-coated magnetic beads. We developed a method to prepare mitochondria from budding yeast that overcomes many of the limitations of other methods. Mitochondria are tagged by insertion of 6 histidines (6xHis) into the TOM70 (Translocase of outer membrane 70) gene at its chromosomal locus, isolated using Ni-NTA (nickel (II) nitrilotriacetic acid) paramagnetic beads and released from the magnetic beads by washing with imidazole. Mitochondria prepared using this method contain fewer contaminants, and are similar in ultrastructure as well as protein import and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. Moreover, this isolation method is amenable to small samples, faster than purification by differential and density gradient centrifugation, and more cost-effective than purification using antibody-coated magnetic beads. Importantly, this method can be applied to any cell type where the genetic modification can be introduced by CRISPR or other methods. PMID:29698455

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.

Crystallization and preliminary X-ray diffraction analysis of ScrY, a specific bacterial outer membrane porin.

PubMed

Forst, D; Schülein, K; Wacker, T; Diederichs, K; Kreutz, W; Benz, R; Welte, W

1993-01-05

The sucrose-specific outer membrane porin ScrY of Salmonella typhimurium was isolated from Escherichia coli K-12 strain KS 26 containing the plasmid pPSO112. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2.3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112.85 A and c = 149.9 A. The crystal volume per unit of protein molecular weight was 3.47 A3/Da.

VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

PubMed

Lemeshko, Victor V

2016-07-01

Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death. Copyright © 2016 Elsevier B.V. All rights reserved.

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

PubMed

Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

2008-12-01

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

[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.

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.

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

PubMed

Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

2012-08-01

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

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

Pore forming properties of cecropin-melittin hybrid peptide in a natural membrane.

PubMed

Milani, Alberto; Benedusi, Mascia; Aquila, Marco; Rispoli, Giorgio

2009-12-11

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH(2); CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in approximately 50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was >or=1 G Omega, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

Neutron Reflectivity as a Tool for Physics-Based Studies of Model Bacterial Membranes.

PubMed

Barker, Robert D; McKinley, Laura E; Titmuss, Simon

2016-01-01

The principles of neutron reflectivity and its application as a tool to provide structural information at the (sub-) molecular unit length scale from models for bacterial membranes are described. The model membranes can take the form of a monolayer for a single leaflet spread at the air/water interface, or bilayers of increasing complexity at the solid/liquid interface. Solid-supported bilayers constrain the bilayer to 2D but can be used to characterize interactions with antimicrobial peptides and benchmark high throughput lab-based techniques. Floating bilayers allow for membrane fluctuations, making the phase behaviour more representative of native membranes. Bilayers of varying levels of compositional accuracy can now be constructed, facilitating studies with aims that range from characterizing the fundamental physical interactions, through to the characterization of accurate mimetics for the inner and outer membranes of Gram-negative bacteria. Studies of the interactions of antimicrobial peptides with monolayer and bilayer models for the inner and outer membranes have revealed information about the molecular control of the outer membrane permeability, and the mode of interaction of antimicrobials with both inner and outer membranes.

Identification and Characterization of an Immunodominant 28-Kilodalton Coxiella burnetii Outer Membrane Protein Specific to Isolates Associated with Acute Disease

PubMed Central

Zhang, Guoquan; To, Ho; Russell, Kasi E.; Hendrix, Laura R.; Yamaguchi, Tsuyoshi; Fukushi, Hideto; Hirai, Katsuya; Samuel, James E.

2005-01-01

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ∼28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a ∼28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever. PMID:15731054

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.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement.

PubMed

Renault, Thibaud T; Luna-Vargas, Mark P A; Chipuk, Jerry E

2016-08-05

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al ., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al ., 2015).

The asymmetry of avian egg-shape: an adaptation for reproduction on dry land

PubMed Central

Mao, Kun-Ming; Murakami, Ayako; Iwasawa, Atsushi; Yoshizaki, Norio

2007-01-01

The present study describes the biological meaning of the asymmetrical shape in avian reproduction using quail. During the incubation of eggs, water was gradually lost and the air chamber which appeared in between the inner and outer shell membranes at the blunt end expanded, so that the angle made by the long egg-axis and the horizontal line increased, presumably because the centre of gravity of the egg contents moved toward the sharp end. The increase in angle occurred in both fertile and infertile eggs, suggesting that this phenomenon occurs irrespective of fertility and is due to the asymmetrical shape. The increase in the volume of the air chamber resulted in an increase in the area of the inner shell membrane at the chamber to satisfy the amount of gas exchange needed by the developing embryo for better hatching. We isolated a 300-kDa protein from the inner shell membrane. It was produced by cells in the luminal epithelium of the oviductal isthmus and was found in the cortex of the fibres of shell membranes and a lining surrounding the air chamber. The lining comprised a medial layer between the inner and outer shell membranes in uterine eggs. The asymmetrical ellipsoid produces the air chamber at the blunt end of the avian egg during its sojourn in the oviductal isthmus, to maintain the blunt end up after oviposition and to raise that end during incubation in a dry environment, leading to high hatchability. PMID:17523938

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

PubMed Central

1983-01-01

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment. PMID:6411740

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

The presence of OMP inclusion bodies in a Escherichia coli K-12 mutated strain is not related to lipopolysaccharide structure.

PubMed

Corsaro, M Michela; Parrilli, Ermenegilda; Lanzetta, Rosa; Naldi, Teresa; Pieretti, Giuseppina; Lindner, Buko; Carpentieri, Andrea; Parrilli, Michelangelo; Tutino, M Luisa

2009-08-01

The role of lipopolysaccharides (LPSs) in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that LPS is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from LPS structure. In this article, we investigated the involvement of LPS structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(lambdapir)). To achieve this aim, the LPS structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that LPS of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a mutation in waaQ, a gene encoding the transferase for the branch heptose HepIII residue. The mutation was complemented to find out if the restoration of LPS structure influenced the observed outer membrane proteins aggregation. Data reported in this work demonstrated that, in E. coli S17-1(lambdapir) there is no influence of LPS structure on the outer membrane proteins inclusion bodies formation.

Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae

PubMed Central

Gouré, Julien; Findlay, Wendy A; Deslandes, Vincent; Bouevitch, Anne; Foote, Simon J; MacInnes, Janet I; Coulton, James W; Nash, John HE; Jacques, Mario

2009-01-01

Background Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines. Results Using hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements. Conclusion Although A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology. PMID:19239696

Battacin (Octapeptin B5), a New Cyclic Lipopeptide Antibiotic from Paenibacillus tianmuensis Active against Multidrug-Resistant Gram-Negative Bacteria

PubMed Central

Qian, Chao-Dong; Teng, Yi; Zhao, Wen-Peng; Li, Ou; Fang, Sheng-Guo; Huang, Zhao-Hui; Gao, Hai-Chun

2012-01-01

Hospital-acquired infections caused by drug-resistant bacteria are a significant challenge to patient safety. Numerous clinical isolates resistant to almost all commercially available antibiotics have emerged. Thus, novel antimicrobial agents, specifically those for multidrug-resistant Gram-negative bacteria, are urgently needed. In the current study, we report the isolation, structure elucidation, and preliminary biological characterization of a new cationic lipopeptide antibiotic, battacin or octapeptin B5, produced from a Paenibacillus tianmuensis soil isolate. Battacin kills bacteria in vitro and has potent activity against Gram-negative bacteria, including multidrug-resistant and extremely drug-resistant clinical isolates. Hospital strains of Escherichia coli and Pseudomonas aeruginosa are the pathogens most sensitive to battacin, with MICs of 2 to 4 μg/ml. The ability of battacin to disrupt the outer membrane of Gram-negative bacteria is comparable to that of polymyxin B, the last-line therapy for infections caused by antibiotic-resistant Gram-negative bacteria. However, the capacity of battacin to permeate bacterial plasma membranes is less extensive than that of polymyxin B. The bactericidal kinetics of battacin correlate with the depolarization of the cell membrane, suggesting that battacin kills bacteria by disrupting the cytoplasmic membrane. Other studies indicate that battacin is less acutely toxic than polymyxin B and has potent in vivo biological activity against E. coli. Based on the findings of the current study, battacin may be considered a potential therapeutic agent for the treatment of infections caused by antibiotic-resistant Gram-negative bacteria. PMID:22183171

Outer membrane vesicles from flagellin-deficient Salmonella enterica serovar Typhimurium induce cross-reactive immunity and provide cross-protection against heterologous Salmonella challenge.

PubMed

Liu, Qiong; Liu, Qing; Yi, Jie; Liang, Kang; Hu, Bo; Zhang, Xiangmin; Curtiss, Roy; Kong, Qingke

2016-10-04

Outer membrane vesicles (OMVs) isolated from Salmonella Typhimurium are potentially useful for developing subunit vaccines because of high immunogenicity and protective efficacy. However, flagella might remain in OMV pellets following OMV purification, resulting in non-essential immune responses and counteraction of bacterial protective immune responses when developing a vaccine against infection of multiple serotypes Salmonella. In this study, a flagellin-deficient S. Typhimurium mutant was constructed. Lipopolysaccharide profiles, protein profiles and cryo-electron microscopy revealed that there were no significant differences between the wild-type and mutant OMVs, with the exception of a large amount of flagellin in the wild-type OMVs. Neither the wild-type OMVs nor the non-flagellin OMVs were toxic to macrophages. Mice immunized with the non-flagellin OMVs produced high concentrations of IgG. The non-flagellin OMVs elicited strong mucosal antibody responses in mice when administered via the intranasal route in addition to provoking higher cross-reactive immune responses against OMPs isolated from S. Choleraesuis and S. Enteritidis. Both intranasal and intraperitoneal immunization with the non-flagellin OMVs provided efficient protection against heterologous S. Choleraesuis and S. Enteritidis challenge. Our results indicate that the flagellin-deficient OMVs may represent a new vaccine platform that could be exploited to facilitate the production of a broadly protective vaccine.

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

Profiling of Virulence Determinants in Cronobacter sakazakii Isolates from Different Plant and Environmental Commodities.

PubMed

Singh, Niharika; Raghav, Mamta; Narula, Shifa; Tandon, Simran; Goel, Gunjan

2017-05-01

Cronobacter sakazakii is an emerging pathogen causing meningitis, sepsis and necrotizing enterocolitis in neonates and immune-compromised adults. The present study describes the profiling of different virulence factors associated with C. sakazakii isolates derived from plant-based materials and environmental samples (soil, water, and vacuum dust). All the isolates exhibited β-hemolysis and chitinase activity, and were able to utilize inositol. Among the nine virulence-associated genes, hly gene coding for hemolysin was detected in all the isolates followed by ompA (outer membrane protein); however, plasmid-borne genes were detected at a level of 60% for both cpa (cronobacter plasminogen activator) and eitA (Ferric ion transporter protein) gene, respectively. Furthermore, the isolate C. sakazakii N81 showed cytotoxicity for Caco-2 cells. The presence of the virulence determinants investigated in this study indicates the pathogenic potential of C. sakazakii with their plausible connection with clinical manifestations.

Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement

PubMed Central

Renault, Thibaud T.; Luna-Vargas, Mark P.A.; Chipuk, Jerry E.

2016-01-01

The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is “Liver mitochondria isolation” in which the liver is dissected from mice to obtain mitochondria. “Mitochondria labeling with JC-1 and size fractionation” is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the “Real-time MOMP measurements” protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015). PMID:28093578

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. ...

Ignicoccus hospitalis sp. nov., the host of 'Nanoarchaeum equitans'.

PubMed

Paper, Walter; Jahn, Ulrike; Hohn, Michael J; Kronner, Michaela; Näther, Daniela J; Burghardt, Tillmann; Rachel, Reinhard; Stetter, Karl O; Huber, Harald

2007-04-01

A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for 'Nanoarchaeum equitans', the only cultivated member of the 'Nanoarchaeota'. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/I(T)=DSM 18386(T)=JCM 14125(T)).

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

An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.

PubMed

Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène; Bouziges, Nicole; Pagès, Jean-Marie; Davin-Regli, Anne

2013-02-01

Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for β-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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.

Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria.

PubMed

Majewski, Dorothy D; Worrall, Liam J; Strynadka, Natalie Cj

2018-03-23

The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action-including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space. Copyright © 2018. Published by Elsevier Ltd.

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.

Epoxide-mediated differential packaging of Cif and other virulence factors into outer membrane vesicles.

PubMed

Ballok, Alicia E; Filkins, Laura M; Bomberger, Jennifer M; Stanton, Bruce A; O'Toole, George A

2014-10-01

Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Iron Uptake Mechanisms in the Fish Pathogen Tenacibaculum maritimum

PubMed Central

Avendaño-Herrera, Ruben; Toranzo, Alicia E.; Romalde, Jesús L.; Lemos, Manuel L.; Magariños, Beatriz

2005-01-01

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding. PMID:16269729

Iron uptake mechanisms in the fish pathogen Tenacibaculum maritimum.

PubMed

Avendaño-Herrera, Ruben; Toranzo, Alicia E; Romalde, Jesús L; Lemos, Manuel L; Magariños, Beatriz

2005-11-01

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. Proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.

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.

Immunopotentiation of outer membrane protein through anti-idiotype Pasteurella multocida vaccine in rabbits.

PubMed

Arif, Javid; Rahman, Sajjad-Ur; Arshad, Muhammad; Akhtar, Pervez

2013-11-01

Pasteurella multocida was isolated from cattle affected with haemorrhagic septicaemia and characterized on the basis of morphological, cultural and biochemical tests. Bacterial outer membrane proteins (OMPs) were extracted with 1% Sarkosyl method. P. multocida anti-idiotype vaccine prepared from OMPs (21.3 mg per 100 ml), was evaluated and compared with bacterin supplemented with 10% OMPs and plain alum-adsorbed bacterin in rabbit models. It was observed that OMPs-anti-idiotype vaccine induced high levels of antibody titres (geomean titres -GMT) detected using indirect haemagglutination (IHA) test. The OMPs anti-idiotype antibody titres of 168.9 GMT were obtained to 42.2 GMT in OMPs supplemented bacterin on 21 days post vaccination, while the plain bacterin had the least titre of 27.9 GMT. The OMPs-anti-idiotype vaccine provoked better immunogenic response in terms of highest GMT titres and long lasting effect in rabbits and 100% protection against the challenge with homologous strain of P. multocida,while 88% protection was obtained in rabbits, given OMPs supplemented bacterin. Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Insight into mitochondrial structure and function from electron tomography.

PubMed

Frey, T G; Renken, C W; Perkins, G A

2002-09-10

In recent years, electron tomography has provided detailed three-dimensional models of mitochondria that have redefined our concept of mitochondrial structure. The models reveal an inner membrane consisting of two components, the inner boundary membrane (IBM) closely apposed to the outer membrane and the cristae membrane that projects into the matrix compartment. These two components are connected by tubular structures of relatively uniform size called crista junctions. The distribution of crista junction sizes and shapes is predicted by a thermodynamic model based upon the energy of membrane bending, but proteins likely also play a role in determining the conformation of the inner membrane. Results of structural studies of mitochondria during apoptosis demonstrate that cytochrome c is released without detectable disruption of the outer membrane or extensive swelling of the mitochondrial matrix, suggesting the formation of an outer membrane pore large enough to allow passage of holo-cytochrome c. The possible compartmentation of inner membrane function between the IBM and the cristae membrane is also discussed.

Leak test fixture and method for using same

DOEpatents

Hawk, Lawrence S.

1976-01-01

A method and apparatus are provided which are especially useful for leak testing seams such as an end closure or joint in an article. The test does not require an enclosed pressurized volume within the article or joint section to be leak checked. A flexible impervious membrane is disposed over an area of the seamed surfaces to be leak checked and sealed around the outer edges. A preselected vacuum is applied through an opening in the membrane to evacuate the area between the membrane and the surface being leak checked to essentially collapse the membrane to conform to the article surface or joined adjacent surfaces. A pressure differential is concentrated at the seam bounded by the membrane and only the seam experiences a pressure differential as air or helium molecules are drawn into the vacuum system through a leak in the seam. A helium detector may be placed in a vacuum exhaust line from the membrane to detect the helium. Alternatively, the vacuum system may be isolated at a preselected pressure and leaks may be detected by a subsequent pressure increase in the vacuum system.

Calcium homeostasis in the outer segments of retinal rods from the tiger salamander.

PubMed Central

Lagnado, L; Cervetto, L; McNaughton, P A

1992-01-01

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1282928

Detection of Rickettsia and Ehrlichia spp. in Ticks Associated with Exotic Reptiles and Amphibians Imported into Japan

PubMed Central

Andoh, Masako; Sakata, Akiko; Takano, Ai; Kawabata, Hiroki; Fujita, Hiromi; Une, Yumi; Goka, Koichi; Kishimoto, Toshio; Ando, Shuji

2015-01-01

One of the major routes of transmission of rickettsial and ehrlichial diseases is via ticks that infest numerous host species, including humans. Besides mammals, reptiles and amphibians also carry ticks that may harbor Rickettsia and Ehrlichia strains that are pathogenic to humans. Furthermore, reptiles and amphibians are exempt from quarantine in Japan, thus facilitating the entry of parasites and pathogens to the country through import. Accordingly, in the current study, we examined the presence of Rickettsia and Ehrlichia spp. genes in ticks associated with reptiles and amphibians originating from outside Japan. Ninety-three ticks representing nine tick species (genera Amblyomma and Hyalomma) were isolated from at least 28 animals spanning 10 species and originating from 12 countries (Ghana, Jordan, Madagascar, Panama, Russia, Sri Lanka, Sudan, Suriname, Tanzania, Togo, Uzbekistan, and Zambia). None of the nine tick species are indigenous in Japan. The genes encoding the common rickettsial 17-kDa antigen, citrate synthase (gltA), and outer membrane protein A (ompA) were positively detected in 45.2% (42/93), 40.9% (38/93), and 23.7% (22/93) of the ticks, respectively, by polymerase chain reaction (PCR). The genes encoding ehrlichial heat shock protein (groEL) and major outer membrane protein (omp-1) were PCR-positive in 7.5% (7/93) and 2.2% (2/93) of the ticks, respectively. The p44 gene, which encodes the Anaplasma outer membrane protein, was not detected. Phylogenetic analysis showed that several of the rickettsial and ehrlichial sequences isolated in this study were highly similar to human pathogen genes, including agents not previously detected in Japan. These data demonstrate the global transportation of pathogenic Rickettsia and Ehrlichia through reptile- and amphibian-associated ticks. These imported animals have potential to transfer pathogens into human life. These results highlight the need to control the international transportation of known and potential pathogens carried by ticks in reptiles, amphibians, and other animals, in order to improve national and international public health. PMID:26207382

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).

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis

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

Caldwell, H.D.; Kromhout, J.; Schachter, J.

1981-03-01

Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtainedmore » after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.« less

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.

Outer Hair Cell Electromotility in vivo

NASA Astrophysics Data System (ADS)

Ramamoorthy, Sripriya; Nuttall, Alfred L.

2011-11-01

The effectiveness of outer hair cell (OHC) electro-motility in vivo has been challenged by the expected low-pass filtering of the transmembrane potential due to the cell's own capacitance. The OHC electromotility is characterized here by an electromechanical ratio defined as the ratio of the OHC contraction to the transmembrane potential. This ratio has been measured in isolated cells to be approximately 26 nm/mV. We estimate the OHC electromechanical ratio in vivo from the recently measured displacements of the reticular lamina and the basilar membrane near the 19 kHz characteristic frequency in the basal region of guinea pig cochlea. Our analysis strongly suggests OHC electromotility process is effective for cochlear amplification in vivo at least around the characteristic frequency of the basal location in spite of the low-pass filtering.

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.

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

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 RNA isolation.

PubMed

Ares, Manuel

2012-09-01

In this bacterial RNA isolation protocol, an "RNA-protective" treatment is followed by lysozyme digestion of the peptidoglycan component of the cell wall. EDTA promotes the loss of the outer membrane of Gram-negative bacteria and allows the lysozyme better access to the peptidoglycan. Cells begin to lyse during digestion in hypotonic lysozyme buffer and lysis is completed by sodium dodecyl sulfate (SDS) and hot phenol:chloroform:isoamyl alcohol (PCA) extraction. SDS and hot phenol disrupt membranes, denature protein (including RNase), and strip proteins from RNA. The separation of the organic phase from the aqueous phase is achieved using Phase Lock Gel, an inert material with a density intermediate between the organic and aqueous samples. The sample is split into three phases: from bottom to top, these are phenol and chloroform (organic phase), the inert gel with the interface material, and the aqueous phase with the RNA. The gel acts as a physical barrier between the sample and the organic phase plus interface. Following organic extraction, the RNA is concentrated by ethanol precipitation.

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

Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

PubMed Central

Drynan, L; Quant, P A; Zammit, V A

1996-01-01

The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty oxidation [Moir and Zammit (1994) Trends Biochem. Sci. 19, 313-317], this is transferred to one or more steps proximal to acylcarnitine synthesis (e.g. decreased delivery of fatty acids to the liver) or to the reaction catalysed by mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase [Hegardt (1995) Biochem. Soc. Trans. 23, 486-490]. Therefore isolated hepatocytes were used in the present study to exclude the involvement of changes in the rate of delivery of non-esterified fatty acids (NEFA) to the liver, such as occur in vivo, and to ascertain whether, under conditions of constant supply of NEFA, CPT I retains control over the relevant fluxes of fatty acid oxidation to ketones and carbon dioxide, or whether control is transferred to another (intrahepatocytic) site. The results clearly show that the Flux Control Coefficients of CPT I with respect to overall beta-oxidation and ketogenesis are very high under all conditions investigated, indicating that control is not lost to another intrahepatic site during the metabolic transitions studied. The control of CPT I over tricarboxylic acid cycle activity was always very low. The significance of these findings for the integration of fatty acid and carbohydrate metabolism in the liver is discussed. PMID:8760364

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

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

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris.

PubMed

Kuddus, Md Ruhul; Rumi, Farhana; Tsutsumi, Motosuke; Takahashi, Rika; Yamano, Megumi; Kamiya, Masakatsu; Kikukawa, Takashi; Demura, Makoto; Aizawa, Tomoyasu

2016-06-01

Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes

PubMed Central

Hoogerheide, David P.; Noskov, Sergei Y.; Jacobs, Daniel; Bergdoll, Lucie; Silin, Vitalii; Worcester, David L.; Abramson, Jeff; Nanda, Hirsh; Rostovtseva, Tatiana K.; Bezrukov, Sergey M.

2017-01-01

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents. PMID:28420794

Acellular pertussis vaccine based on outer membrane vesicles capable of conferring both long-lasting immunity and protection against different strain genotypes.

PubMed

Gaillard, María Emilia; Bottero, Daniela; Errea, Agustina; Ormazábal, Maximiliano; Zurita, M Eugenia; Moreno, Griselda; Rumbo, Martin; Castuma, Celina; Bartel, Erika; Flores, Dario; van der Ley, Peter; van der Ark, Arno; F Hozbor, Daniela

2014-02-12

Despite high vaccination coverage rates, pertussis continues to be a global concern, with increased incidence widely noted. The current pertussis epidemiologic situation has been mainly attributed to waning immunity and pathogen adaptation. To improve the disease control, a new generation of vaccines capable to overcome those weaknesses associated to the current vaccines need to be developed. Previously we have demonstrated that the outer membrane vesicles obtained from the recombinant Bordetella pertussis strain expressing PagL enzyme (OMVs(BpPagL)) are good vaccine candidates to protect against pertussis. In this work the OMVs(BpPagL) formulated with diphtheria and tetanus toxoids (Tdap(OMVsBpPagL)) was used to evaluate its capacity to offer protection against Argentinean clinical isolates and to induce long-term immunity. To these aims BALB/c mice were immunized with Tdap(OMVsBpPagL) and challenged with sublethal doses of the clinical isolate Bp106 selected as a representative circulating isolate. Comparisons with a current commercial Tdap vaccine used at a dose in which pertussis toxin level was equivalent to that of Tdap(OMVsBpPagL) were performed. With the normalized doses of both vaccines we observed that Tdap(OMVsBpPagL) protected against the clinical isolate infection, whereas current commercial Tdap vaccine showed little protection against such pathogen. Regarding long-term immunity we observed that the Tdap(OMVsBpPagL) protective capacity against the recommended WHO reference strain persisted at least 9 months. In agreement with these results Tdap(OMVsBpPagL) induced Th1 and Th2 immune response. In contrast, commercial Tdap induced Th2 but weak Th1 responses. All results presented here showed that Tdap(OMVsBpPagL) is an interesting formulation to be considered for the development of novel acellular multi-antigen vaccine. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

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.

Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia.

PubMed

Damron, F Heath; Napper, Jennifer; Teter, M Allison; Yu, Hongwei D

2009-04-01

Chronic lung infection with P. aeruginosa and excessive neutrophil-associated inflammation are major causes of morbidity and mortality in patients with cystic fibrosis (CF). Overproduction of an exopolysaccharide known as alginate leads to the formation of mucoid biofilms that are resistant to antibiotics and host defences. Alginate overproduction or mucoidy is controlled by a stress-related ECF sigma factor AlgU/T. Mutation in the anti-sigma factor MucA is a known mechanism for conversion to mucoidy. Recently, we showed that inactivation of a kinase (KinB) in nonmucoid strain PAO1 results in overproduction of alginate. Here, we report the initial characterization of lipotoxin F (LptF, PA3692), an OmpA-like outer membrane protein that exhibited increased expression in the mucoid PAO1kinB mutant. The lipotoxin family of proteins has been previously shown to induce inflammation in lung epithelia, which may play a role in CF disease progression. Expression of LptF was observed to be AlgU-dependent and upregulated in CF isolates. Deletion of lptF from the kinB mutant had no effect on alginate production. Deletion of lptF from PAO1 caused a differential susceptibility to oxidants that can be generated by phagocytes. The lptF and algU mutants were more sensitive to hypochlorite than PAO1. However, the lptF mutant displayed increased resistance to hydrogen peroxide. LptF also contributed to adhesion to A549 human lung epithelial cells. Our data suggest that LptF is an outer membrane protein that may be important for P. aeruginosa survival in harsh environments, including lung colonization in CF.

Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia

PubMed Central

Damron, F. Heath; Napper, Jennifer; Teter, M. Allison; Yu, Hongwei D.

2009-01-01

Chronic lung infection with P. aeruginosa and excessive neutrophil-associated inflammation are major causes of morbidity and mortality in patients with cystic fibrosis (CF). Overproduction of an exopolysaccharide known as alginate leads to the formation of mucoid biofilms that are resistant to antibiotics and host defences. Alginate overproduction or mucoidy is controlled by a stress-related ECF sigma factor AlgU/T. Mutation in the anti-sigma factor MucA is a known mechanism for conversion to mucoidy. Recently, we showed that inactivation of a kinase (KinB) in nonmucoid strain PAO1 results in overproduction of alginate. Here, we report the initial characterization of lipotoxin F (LptF, PA3692), an OmpA-like outer membrane protein that exhibited increased expression in the mucoid PAO1kinB mutant. The lipotoxin family of proteins has been previously shown to induce inflammation in lung epithelia, which may play a role in CF disease progression. Expression of LptF was observed to be AlgU-dependent and upregulated in CF isolates. Deletion of lptF from the kinB mutant had no effect on alginate production. Deletion of lptF from PAO1 caused a differential susceptibility to oxidants that can be generated by phagocytes. The lptF and algU mutants were more sensitive to hypochlorite than PAO1. However, the lptF mutant displayed increased resistance to hydrogen peroxide. LptF also contributed to adhesion to A549 human lung epithelial cells. Our data suggest that LptF is an outer membrane protein that may be important for P. aeruginosa survival in harsh environments, including lung colonization in CF. PMID:19332805

FINE STRUCTURE OF CELLS ISOLATED FROM ADULT MOUSE LIVER

PubMed Central

Berry, M. N.; Simpson, F. O.

1962-01-01

Suspensions of isolated cells in various media were prepared from mouse liver which had been perfused via the portal vein with a buffered medium containing 0.40 M sucrose, and the cells were fixed with osmium tetroxide. Their fine structure was compared with that of cells from perfused and unperfused intact liver. Perfusion brought about some separation of the cells with little or no damage to cell membranes. When cells were dispersed in 0.40 M sucrose medium the plasma membranes partially broke down, and this disintegration was increased by transfer of the cells to media of lower osmolarity. This is presumed to account for the loss of permeability barriers which occurs in isolated liver cells. The mitochondria in cells of perfused liver and in isolated cells remained elongated, but the layers of many mitochondrial cristae became separated by clear spaces. When cells were transferred to a medium containing 0.20 M sucrose, the mitochondria swelled and became spherical, often with displacement of the swollen cristae to the periphery. In a medium containing 0.06 M sucrose and 0.08 M potassium chloride the outer chamber of many mitochondria became swollen with displacement of the mitochondrial body to one side to give a crescent-shaped appearance. These changes in mitochondrial morphology are discussed in relation to the metabolic activity of isolated liver cells. PMID:19866610

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.

Unique activity spectrum of colicin FY: all 110 characterized Yersinia enterocolitica isolates were colicin FY susceptible.

PubMed

Bosák, Juraj; Micenková, Lenka; Vrba, Martin; Ševčíková, Alena; Dědičová, Daniela; Garzetti, Debora; Šmajs, David

2013-01-01

Colicin FY is a plasmid encoded toxin that recognizes a yersinia-specific outer membrane protein (YiuR) as a receptor molecule. We have previously shown that the activity spectrum of colicin FY comprises strains of the genus Yersinia. In this study, we analyzed the activity of colicin FY against 110 Yersinia enterocolitica isolates differing in geographical origin and source. All isolates were characterized through analysis of 16S rRNA genes, serotyping, biotyping, restriction profiling of genomic DNA, detection of virulence markers and susceptibility to antibiotics. This confirmed the broad variability of the collection, in which all 110 Y. enterocolitica isolates, representing 77 various strains, were inhibited by colicin FY. Although isolates showed variable levels of susceptibility to colicin FY, it was not associated with any strain characteristic. The universal susceptibility of Y. enterocolitica strains to colicin FY together with the absence of activity towards strains outside the Yersinia genus suggests potential therapeutic applications for colicin FY.

Unique Activity Spectrum of Colicin FY: All 110 Characterized Yersinia enterocolitica Isolates Were Colicin FY Susceptible

PubMed Central

Bosák, Juraj; Micenková, Lenka; Vrba, Martin; Ševčíková, Alena; Dědičová, Daniela; Garzetti, Debora; Šmajs, David

2013-01-01

Colicin FY is a plasmid encoded toxin that recognizes a yersinia-specific outer membrane protein (YiuR) as a receptor molecule. We have previously shown that the activity spectrum of colicin FY comprises strains of the genus Yersinia. In this study, we analyzed the activity of colicin FY against 110 Yersinia enterocolitica isolates differing in geographical origin and source. All isolates were characterized through analysis of 16S rRNA genes, serotyping, biotyping, restriction profiling of genomic DNA, detection of virulence markers and susceptibility to antibiotics. This confirmed the broad variability of the collection, in which all 110 Y. enterocolitica isolates, representing 77 various strains, were inhibited by colicin FY. Although isolates showed variable levels of susceptibility to colicin FY, it was not associated with any strain characteristic. The universal susceptibility of Y. enterocolitica strains to colicin FY together with the absence of activity towards strains outside the Yersinia genus suggests potential therapeutic applications for colicin FY. PMID:24339971

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.

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.

16S rRNA and Omp31 Gene Based Molecular Characterization of Field Strains of B. melitensis from Aborted Foetus of Goats in India

PubMed Central

Singh, Ajay; Gupta, Vivek Kumar; Kumar, Amit; Singh, Vikas Kumar; Nayakwadi, Shivasharanappa

2013-01-01

Brucellosis is a reemerging infectious zoonotic disease of worldwide importance. In human, it is mainly caused by Brucella melitensis, a natural pathogen for goats. In India, a large number of goats are reared in semi-intensive to intensive system within the close vicinity of human being. At present, there is no vaccination and control strategy for caprine brucellosis in the country. Thus, to formulate an effective control strategy, the status of etiological agent is essential. To cope up with these, the present study was conducted to isolate and identify the prevalent Brucella species in caprine brucellosis in India. The 30 samples (fetal membrane, fetal stomach content and vaginal swabs) collected throughout India from the aborted fetus of goats revealed the isolation of 05 isolates all belonging to Brucella melitensis biovars 3. All the isolates produced amplification products of 1412 and 720 bp in polymerase chain reaction with genus and species specific 16S rRNA and omp31 gene based primers, respectively. Moreover, the amplification of omp31 gene in all the isolates confirmed the presence of immuno dominant outer membrane protein (31 kDa omp) in all the field isolates of B. melitensis in aborted foetus of goats in India. These findings can support the development of omp31 based specific serodiagnostic test as well as vaccine for the control of caprine brucellosis in India. PMID:24453799

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

Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.

PubMed

Ben-Hail, Danya; Palty, Raz; Shoshan-Barmatz, Varda

2014-02-01

Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.

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.

Mitochondrial filaments and clusters as intracellular power-transmitting cables.

PubMed

Skulachev, V P

2001-01-01

Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.

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)

Synthesis of protein in host-free reticulate bodies of Chlamydia psittaci and Chlamydia trachomatis

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

Hatch, T.P.; Miceli, M.; Silverman, J.A.

1985-06-01

Synthesis of protein by the obligate intracellular parasitic bacteria Chlamydia psittaci (6BC) and Chlamydia trachomatis (serovar L2) isolated from host cells (host-free chlamydiae) was demonstrated for the first time. Incorporation of (/sup 35/S)methionine and (/sup 35/S)cysteine into trichloroacetic acid-precipitable material by reticulate bodies of chlamydiae persisted for 2 h and was dependent upon a exogenous source of ATP, an ATP-regenerating system, and potassium or sodium ions. Magnesium ions and amino acids stimulated synthesis; chloramphenicol, rifampin, oligomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a proton ionophore) inhibited incorporation. Ribonucleoside triphosphates (other than ATP) had little stimulatory effect. The optimum pH for host-free synthesismore » was between 7.0 and 7.5. The molecular weights of proteins synthesized by host-free reticulate bodies closely resembled the molecular weights of proteins synthesized by reticulate bodies in an intracellular environment, and included outer membrane proteins. Elementary bodies of chlamydiae were unable to synthesize protein even when incubated in the presence of 10 mM dithiothreitol, a reducing agent which converted the highly disulfide bond cross-linked major outer membrane protein to monomeric form.« less

Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex*

PubMed Central

Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.

2011-01-01

CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

PubMed

Bong, Ji-Hong; Song, Hyun-Woo; Kim, Tae-Hun; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2018-04-15

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

PubMed Central

Summer, Elizabeth J.; Cline, Kenneth

1999-01-01

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

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.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

PubMed

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

2017-09-01

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-X L , 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-X L 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. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

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.

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

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

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

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.

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

PubMed Central

Chaudhuri, K; Bhadra, R K; Das, J

1992-01-01

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations. Images PMID:1282793

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

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.

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

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

Solid-Supported Lipid Membranes: Formation, Stability and Applications

NASA Astrophysics Data System (ADS)

Goh, Haw Zan

This thesis presents a comprehensive investigation of the formation of supported lipid membranes with vesicle hemifusion, their stability under detergents and organic solvents and their applications in molecular biology. In Chapter 3, we describe how isolated patches of DOPC bilayers supported on glass surfaces are dissolved by various detergents (decyl maltoside, dodecyl maltoside, CHAPS, CTAB, SDS, TritonX-100 and Tween20) at their CMC, as investigated by fluorescence video microscopy. In general, detergents partition into distal leaflets of bilayers and lead to the expansion of the bilayers through a rolling motion of the distal over the proximal leaflets, in agreement with the first stage of the established 3-stage model of lipid vesicle solubilization by detergents. Subsequently, we study the partitioning of organic solvents (methanol, ethanol, isopropanol, propanol, acetone and chloroform) into isolated bilayer patches on glass in Chapter 4 with fluorescence microscopy. The area expansion of bilayers due to the partitioning of organic solvents is measured. From the titration of organic solvents, we measured the rate of area expansion as a function of the volume fraction of organic solvents, which is proposed to be a measure of strength of interactions between solvents and membranes. From the same experiments, we also measure the maximum expansion of bilayers (or the maximum binding stoichiometry between organic solvents and lipids) before structural breakdown, which depends on the depth of penetration of solvents to the membranes. In Chapter 5, we investigate the formation of sparsely-tethered bilayer lipid membranes (stBLMs) with vesicle hemifusion. In vesicle hemifusion, lipid vesicles in contact with a hydrophobic alkyl-terminated self-assembled monolayer (SAM) deposit a lipid monolayer to the SAM surface, thus completing the bilayer. Electrical Impedance Spectroscopy and Neutron Reflectivity are used to probe the integrity of stBLMs in terms of their insulating and structural properties. Preparation conditions are screened for those that are optimal for stBLM formation. Concentrations of lipid vesicles, hydrophobicity of SAMs, the presence of calcium and high concentrations of salt are identified as the key parameters. We show that stBLMs can be formed with vesicles of different compositions. Vesicle hemifusion opens up a new route in preserving the chemical compositions of stBLMs and facilitating membrane proteins incorporation. In Chapter 6, we visualize the hemifusion pathway of giant unilamellar vesicles (GUVs) with planar hydrophobic surfaces at the single vesicle level with fluorescence video microscopy. When a GUV hemifuses to a surface, its outer leaflet breaks apart and remains connected to the surface presumably through a hemifusion diaphragm. Lipids from the outer leaflet are transferred to the surface as a lipid monolayer that expands radially outward from the hemifusion diaphragm, thereby forming the loosely packed outer hemifusion zone. In Chapter 7, we develop an in vitro assay employing stBLMs and lipid vesicles to examine the functionality of GRASP in membrane tethering. Membrane-bound GRASP on opposing membranes dimerizes and tethers fluorescently-labeled vesicles to stBLMs. The fluorescence intensity of images taken at stBLM surfaces is used to quantify the tethering activity. Both wild type and mutant proteins were studied to shed light on the molecular mechanism of tethering. We show that the GRASP domain is sufficient and necessary for membrane tethering. In addition, the tethering capability of GRASP is impaired when the internal ligands and the binding pockets participating in dimerization are deleted and mutated. Membrane anchors, sizes of vesicles and membrane compositions are explored for their influence on the outcomes of the assay. Furthermore, preliminary analysis from neutron reflectivity measurements shows that both the internal ligands and binding pockets are exposed instead of buried toward the membrane surface. In summary, we establish a functional assay for studying GRASP activity in vitro. (Abstract shortened by UMI.)

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.

Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials.

PubMed

Zhang, Xiao-Ping; Wang, Wei-Hong; Tian, Yu; Gao, Wen; Li, Jiang

2009-02-28

To investigate the mechanisms of aspirin increasing the susceptibility of Helicobacter pylori (H pylori) to metronidazole. H pylori reference strain 26695 and two metronidazole-resistant isolates of H pylori were included in this study. Strains were incubated in Brucella broth with or without aspirin (1 mmol/L). The rdxA gene of H pylori was amplified by PCR and sequenced. The permeability of H pylori to antimicrobials was determined by analyzing the endocellular radioactivity of the cells after incubated with [7-(3)H]-tetracycline. The outer membrane proteins (OMPs) of H pylori 26695 were depurated and analyzed by SDS-PAGE. The expression of 5 porins (hopA, hopB, hopC, hopD and hopE) and the putative RND efflux system (hefABC) of H pylori were analyzed using real-time quantitative PCR. The mutations in rdxA gene did not change in metronidazole resistant isolates treated with aspirin. The radioactivity of H pylori increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori. However, the expression of two OMP bands between 55 kDa and 72 kDa altered in the presence of aspirin. The expression of the mRNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC of H pylori did not change when treated with aspirin. Although aspirin increases the susceptibility of H pylori to metronidazole, it has no effect on the mutations of rdxA gene of H pylori. Aspirin increases endocellular concentrations of antimicrobials probably by altering the OMP expression.

Mechanical Properties of Nanoscopic Lipid Domains

DOE PAGES

Nickels, Jonathan D.; Cheng, Xiaolin; Mostofian, Barmak; ...

2015-09-28

We found that the lipid raft hypothesis presents insight into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. Thus, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approachesmore » with inelastic neutron scattering, we isolate the bending modulus of ~13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. Moreover, from additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.« less

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.

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

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...

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...

The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica.

PubMed

Cho, Jaeyong; Choi, Hyemin; Lee, Juneyoung; Kim, Mi-Sun; Sohn, Ho-Yong; Lee, Dong Gun

2013-03-01

Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3'-dipropylthiadicarbocyanine iodide [DiSC(3)(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Metal Accumulation and Vanadium-Induced Multidrug Resistance by Environmental Isolates of Escherichia hermannii and Enterobacter cloacae

PubMed Central

Hernández, Alicia; Mellado, Rafael P.; Martínez, José L.

1998-01-01

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family Enterobacteriaceae were selected. Two of them were Escherichia hermannii strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an Enterobacter cloacae strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in E. hermannii and E. cloacae. Incubation with this metal changed the OMP profile of E. hermannii but did not produce variations in the expression of the major OMPs of E. cloacae. PMID:9797283

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

NASA Astrophysics Data System (ADS)

Heuvingh, J.; Pincet, F.; Cribier, S.

2004-07-01

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

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.

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.

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.

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.

Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum.

PubMed Central

Cobbs, W H; Pugh, E N

1987-01-01

1. Membrane currents initiated by intense, 20 microseconds flashes (photocurrents) were recorded from isolated salamander rods by combined extracellular suction electrodes and intracellular tight-seal electrodes either in current or voltage clamp mode. The magnitudes (mean +/- 2 S.E.M.) of the maximal photoresponses recorded by the suction and by the intracellular electrode respectively were 40 +/- 5 pA (n = 18) and 35 +/- 7 mV (n = 8) for current clamp at zero current; 43 +/- 9 pA and 66 +/- 13 (n = 11) pA for voltage clamp at the zero-current holding potential, -24 +/- 3 mV. 2. Photocurrents initiated by flashes isomerizing 0.1% or more of the outer segment's rhodopsin achieved a saturated velocity and were 95% complete in less than 50 ms. The effect of incrementing flash intensity above 0.1% isomerization can be described as a translation of the photocurrent along the time axis towards the origin. Within the interval 0-50 ms the latter two-thirds of the velocity-saturated photocurrent is well described as a single-exponential decay. The decay was much faster in voltage clamp (2.8 +/- 1.2 ms, n = 11) than in current clamp mode (17 +/- 5 ms, n = 17). 3. The initial third of the velocity-saturated photocurrent, occurring over the interval from the flash to the onset of exponential decay, followed about the same time course in current and voltage clamp. The time interval occupied by this initial 'latent' phase decreased with increasing flash intensity and attained an apparent minimum of about 7 ms in response to flashes isomerizing 10% or more of the rhodopsin at ca. 22 degrees C. 4. The hypothesis that the decay of outer segment light-sensitive membrane current is the same in current and voltage clamp was supported by an analysis of the difference between outer segment currents measured successively in the two recording modes. First, the tail of the difference current decayed exponentially with a time constant approximately equal to R x C, where R and C are independently estimated slope resistance and capacitance of the rod. Secondly, the integral of the difference current, when divided by outer segment capacitance, closely approximated the hyperpolarizing light response measured under current clamp. Thus, displacement current accounted for the difference between photocurrents measured in current and voltage clamp.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2832596

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

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.

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

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.

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.

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

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.

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout.

PubMed

Sokolova, Niina; Vendelin, Marko; Birkedal, Rikke

2009-12-17

Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20 degrees C in the absence and presence of creatine. Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts.

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

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

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.

Quantum changes in Helicobacter pylori gene expression accompany host-adaptation

PubMed Central

Wise, Michael J.; Khosravi, Yalda; Seow, Shih-Wee; Amoyo, Arlaine A.; Pettersson, Sven; Peters, Fanny; Tay, Chin-Yen; Perkins, Timothy T.; Loke, Mun-Fai; Marshall, Barry J.; Vadivelu, Jamuna

2017-01-01

Abstract Helicobacter pylori is a highly successful gastric pathogen. High genomic plasticity allows its adaptation to changing host environments. Complete genomes of H. pylori clinical isolate UM032 and its mice-adapted serial derivatives 298 and 299, generated using both PacBio RS and Illumina MiSeq sequencing technologies, were compared to identify novel elements responsible for host-adaptation. The acquisition of a jhp0562-like allele, which encodes for a galactosyltransferase, was identified in the mice-adapted strains. Our analysis implies a new β-1,4-galactosyltransferase role for this enzyme, essential for Ley antigen expression. Intragenomic recombination between babA and babB genes was also observed. Further, we expanded on the list of candidate genes whose expression patterns have been mediated by upstream homopolymer-length alterations to facilitate host adaption. Importantly, greater than four-fold reduction of mRNA levels was demonstrated in five genes. Among the down-regulated genes, three encode for outer membrane proteins, including BabA, BabB and HopD. As expected, a substantial reduction in BabA protein abundance was detected in mice-adapted strains 298 and 299 via Western analysis. Our results suggest that the expression of Ley antigen and reduced outer membrane protein expressions may facilitate H. pylori colonisation of mouse gastric epithelium. PMID:27803027

Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

PubMed Central

Lück, P. Christian; Schmitt, Jürgen W.; Hengerer, Arne; Helbig, Jürgen H.

1998-01-01

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected. PMID:9797218

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...

Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

PubMed

Gruss, Fabian; Hiller, Sebastian; Maier, Timm

2015-01-01

TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Particle size effect and the mechanism of hematite reduction by the outer membrane cytochrome OmcA of Shewanella oneidensis MR-1

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

Liu, Juan; Pearce, Carolyn I.; Shi, Liang

The cycling of iron at the Earth’s near surface is profoundly influenced by dissimilatory metal reducing microorganisms, and many studies have focused on unraveling electron transfer mechanisms between these bacteria and Fe(III)-(oxyhydr)oxides. However, these efforts have been complicated by the fact that these minerals often occur in the micro- to nanosize regime, and in relevant natural environments as well as in the laboratory are subject to aggregation. The nature of the physical interface between the cellular envelope, the outer-membrane cytochromes responsible for facilitating the interfacial electron transfer step, and these complex mineral particulates is thus difficult to probe. Previous studiesmore » using whole cells have reported reduction rates that do not correlate with particle size. In the present study we isolate the interaction between the decaheme outer-membrane cytochrome OmcA of Shewanella oneidensis and nanoparticulate hematite, examining the reduction rate as a function of particle size and reaction products through detailed characterization of the electron balance and the structure and valence of iron at particle surfaces. By comparison with abiotic reduction via the smaller molecule ascorbic acid, we show that the reduction rate is systematically controlled by the sterically accessible interfacial contact area between OmcA and hematite in particle aggregates; rates increase once pore throat sizes in aggregates become as large as OmcA. Simultaneous measure of OmcA oxidation against Fe(II) release shows a ratio of 1:10, consistent with a cascade OmcA oxidation mechanism heme by heme. X-ray absorption spectroscopies reveal incipient magnetite on the reacted surfaces of the hematite nanoparticles after reaction. The collective findings establish the importance of accessibility of physical contact between the terminal reductases and iron oxide surfaces, and through apparent consistency of observations help reconcile behavior reported at the larger more complex scale of whole cell studies.« less

Particle size effect and the mechanism of hematite reduction by the outer membrane cytochrome OmcA of Shewanella oneidensis MR-1

NASA Astrophysics Data System (ADS)

Liu, Juan; Pearce, Carolyn I.; Shi, Liang; Wang, Zheming; Shi, Zhi; Arenholz, Elke; Rosso, Kevin M.

2016-11-01

The cycling of iron at the Earth's near surface is profoundly influenced by dissimilatory metal reducing microorganisms, and many studies have focused on unraveling electron transfer mechanisms between these bacteria and Fe(III)-(oxyhydr)oxides. However, these efforts have been complicated by the fact that these minerals often occur in the micro- to nanosize regime, and in relevant natural environments as well as in the laboratory are subject to aggregation. The nature of the physical interface between the cellular envelope, the outer-membrane cytochromes responsible for facilitating the interfacial electron transfer step, and these complex mineral particulates is thus difficult to probe. Previous studies using whole cells have reported reduction rates that do not correlate with particle size. In the present study we isolate the interaction between the decaheme outer-membrane cytochrome OmcA of Shewanella oneidensis and nanoparticulate hematite, examining the reduction rate as a function of particle size and reaction products through detailed characterization of the electron balance and the structure and valence of iron at particle surfaces. By comparison with abiotic reduction via the smaller molecule ascorbic acid, we show that the reduction rate is systematically controlled by the sterically accessible interfacial contact area between OmcA and hematite in particle aggregates; rates increase once pore throat sizes in aggregates become as large as OmcA. Simultaneous measure of OmcA oxidation against Fe(II) release shows a ratio of 1:10, consistent with a cascade OmcA oxidation mechanism heme by heme. X-ray absorption spectroscopies reveal incipient magnetite on the reacted surfaces of the hematite nanoparticles after reaction. The collective findings establish the importance of accessibility of physical contact between the terminal reductases and iron oxide surfaces, and through apparent consistency of observations help reconcile behavior reported at the larger more complex scale of whole cell studies.

Vacuum-isolation vessel and method for measurement of thermal noise in microphones

NASA Technical Reports Server (NTRS)

Zuckerwar, Allan J. (Inventor); Ngo, Kim Chi T. (Inventor)

1992-01-01

The vacuum isolation vessel and method in accordance with the present invention are used to accurately measure thermal noise in microphones. The apparatus and method could be used in a microphone calibration facility or any facility used for testing microphones. Thermal noise is measured to determine the minimum detectable sound pressure by the microphone. Conventional isolation apparatus and methods have been unable to provide an acoustically quiet and substantially vibration free environment for accurately measuring thermal noise. In the present invention, an isolation vessel assembly comprises a vacuum sealed outer vessel, a vacuum sealed inner vessel, and an interior suspension assembly coupled between the outer and inner vessels for suspending the inner vessel within the outer vessel. A noise measurement system records thermal noise data from the isolation vessel assembly. A vacuum system creates a vacuum between an internal surface of the outer vessel and an external surface of the inner vessel. The present invention thus provides an acoustically quiet environment due to the vacuum created between the inner and outer vessels and a substantially vibration free environment due to the suspension assembly suspending the inner vessel within the outer vessel. The thermal noise in the microphone, effectively isolated according to the invention, can be accurately measured.

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.

Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials

PubMed Central

Zhang, Xiao-Ping; Wang, Wei-Hong; Tian, Yu; Gao, Wen; Li, Jiang

2009-01-01

AIM: To investigate the mechanisms of aspirin increasing the susceptibility of Helicobacter pylori (H pylori) to metronidazole. METHODS: H pylori reference strain 26 695 and two metronidazole-resistant isolates of H pylori were included in this study. Strains were incubated in Brucella broth with or without aspirin (1 mmol/L). The rdxA gene of H pylori was amplified by PCR and sequenced. The permeability of H pylori to antimicrobials was determined by analyzing the endocellular radioactivity of the cells after incubated with [7-3H]-tetracycline. The outer membrane proteins (OMPs) of H pylori 26 695 were depurated and analyzed by SDS-PAGE. The expression of 5 porins (hopA, hopB, hopC, hopD and hopE) and the putative RND efflux system (hefABC) of H pylori were analyzed using real-time quantitative PCR. RESULTS: The mutations in rdxA gene did not change in metronidazole resistant isolates treated with aspirin. The radioactivity of H pylori increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori. However, the expression of two OMP bands between 55 kDa and 72 kDa altered in the presence of aspirin. The expression of the mRNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC of H pylori did not change when treated with aspirin. CONCLUSION: Although aspirin increases the susceptibility of H pylori to metronidazole, it has no effect on the mutations of rdxA gene of H pylori. Aspirin increases endocellular concentrations of antimicrobials probably by altering the OMP expression. PMID:19248190

Outer membrane vesicles of Gallibacterium anatis induce protective immunity in egg-laying hens.

PubMed

Pors, Susanne E; Pedersen, Ida J; Skjerning, Ragnhild Bager; Thøfner, Ida C N; Persson, Gry; Bojesen, Anders M

2016-11-15

Gallibacterium anatis causes infections in the reproductive tract of egg-laying hens and induce increased mortality and decreased egg production. New prophylactic measures are needed in order to improve animal welfare and production efficiency. Bacterial outer membrane vesicles (OMVs) have previously shown promising results in protection against infections and we hypothesized that OMVs could serve as an immunogen to protect egg-laying hens against G. anatis. To investigate the immunogenic potential of G. anatis OMVs, two in vivo studies in egg-laying hens were made. The trials assessedthe degree of protection provided by immunization with G. anatis OMV against challenge and the IgY responses in serum after immunization and challenge, respectively. A total of 64 egg-laying hens were included in the trials. OMVs for immunization were produced and purified from a high-producing G. anatis ΔtolR mutant. Challenge was done with G. anatis 12656-12 and evaluated by scoring lesions and bacterial re-isolation rates from peritoneum. Finally, levels of OMV-specific IgY in sera were assayed by ELISA. Immunization with OMVs decreased the lesions scores significantly, while the bacterial re-isolation remained unchanged. Furthermore, a high OMV-specific IgY response was induced by immunization and subsequent challenge of the hens. The results strongly indicate that immunization with G. anatis OMVs provides significant protection against G. anatis challenge and induces specific antibody responses with high titers of OMV-specific IgY in serum. The results therefore show great promise for OMV based vaccines aiming at providing protecting against G. anatis in egg-laying hens. Copyright © 2016 Elsevier B.V. All rights reserved.

Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes.

PubMed

Nyholm, Spencer V; Stewart, Jennifer J; Ruby, Edward G; McFall-Ngai, Margaret J

2009-02-01

The light organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis, but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like haemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host's environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by haemocytes from uncured animals to the level observed for haemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting that they produce a factor that complements the mutant's defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting indicated that once binding to haemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of haemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host haemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association.

Recognition between symbiotic Vibrio fischeri and the hemocytes of Euprymna scolopes

PubMed Central

Nyholm, Spencer V.; Stewart, Jennifer J.; Ruby, Edward G.; McFall-Ngai, Margaret J.

2008-01-01

Summary The light-organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like hemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host’s environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five-times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by hemocytes from uncured animals to the level observed for hemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting they produce a factor that complements the mutant’s defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting (FACS) indicated that, once binding to hemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of hemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and, (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host hemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association. PMID:19196278

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

Detection of carbapenemases and other mechanisms of enzymatic resistance to β-lactams in Enterobacteriaceae with diminished susceptibility to carbapenems in a tertiary care hospital.

PubMed

Gómara, Marta; López-Calleja, Ana Isabel; Iglesia, Berta María Pilar Vela; Cerón, Isabel Ferrer; López, Antonio Rezusta; Pinilla, María José Revillo

2018-05-01

Our objective was to characterize the enzymatic β-lactam resistance in clinical Enterobacteriaceae isolates with diminished susceptibility to carbapenems from 2013 to 2014 at Hospital Universitario Miguel Servet. A total of 63 clinical isolates were analyzed for the presence of carbapenemases (KPC, OXA-48 and MBL), ESBLs and AmpC enzymes by combined disk methods and PCR detection of carbapenemase-encoding and beta-lactamase-encoding genes. Fifteen isolates had a phenotypic test compatible with carbapenemase production; two of these were confirmed by PCR as OXA-48 producers. ESBL detection was positive in 27 isolates (43%); plasmid-mediated AmpC was detected in nine isolates (14.2%) and derepressed AmpC β-lactamase was present in 18 isolates (28%). During the study period, the decreased susceptibility to carbapenems in Enterobacteriaceae in our area was not due to true carbapenemases but rather to β-lactamase activity (82.5% were ESBL or AmpC producers), probably in combination with decreased permeability of the outer membrane. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Polymerase chain reaction-based identification of clinically relevant Pasteurellaceae isolated from cats and dogs in Poland.

PubMed

Król, Jaroslaw; Bania, Jacek; Florek, Magdalena; Pliszczak-Król, Aleksandra; Staroniewicz, Zdzislaw

2011-05-01

A set of polymerase chain reaction (PCR) assays for identification of the most important Pasteurellaceae species encountered in cats and dogs were developed. Primers for Pasteurella multocida were designed to detect a fragment of the kmt, a gene encoding the outer-membrane protein. Primers specific to Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis were based on the manganese-dependent superoxide dismutase gene (sodA) and those specific to [Haemophilus] haemoglobinophilus on species-specific sequences of the 16S ribosomal RNA gene. All the primers were tested on respective reference and control strains and applied to the identification of 47 canine and feline field isolates of Pasteurellaceae. The PCR assays were shown to be species specific, providing a valuable supplement to phenotypic identification of species within this group of bacteria. © 2011 The Author(s)

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.

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content.

PubMed

Abi-Rizk, Georges; Besson, Françoise

2008-10-15

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.

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.

LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacterium tuberculosis

PubMed Central

Sulzenbacher, Gerlind; Canaan, Stéphane; Bordat, Yann; Neyrolles, Olivier; Stadthagen, Gustavo; Roig-Zamboni, Véronique; Rauzier, Jean; Maurin, Damien; Laval, Françoise; Daffé, Mamadou; Cambillau, Christian; Gicquel, Brigitte; Bourne, Yves; Jackson, Mary

2006-01-01

Cell envelope lipids play an important role in the pathogenicity of mycobacteria, but the mechanisms by which they are transported to the outer membrane of these prokaryotes are largely unknown. Here, we provide evidence that LppX is a lipoprotein required for the translocation of complex lipids, the phthiocerol dimycocerosates (DIM), to the outer membrane of Mycobacterium tuberculosis. Abolition of DIM transport following disruption of the lppX gene is accompanied by an important attenuation of the virulence of the tubercle bacillus. The crystal structure of LppX unveils an U-shaped β-half-barrel dominated by a large hydrophobic cavity suitable to accommodate a single DIM molecule. LppX shares a similar fold with the periplasmic molecular chaperone LolA and the outer membrane lipoprotein LolB, which are involved in the localization of lipoproteins to the outer membrane of Gram-negative bacteria. Based on the structure and although an indirect participation of LppX in DIM transport cannot yet be ruled out, we propose LppX to be the first characterized member of a family of structurally related lipoproteins that carry lipophilic molecules across the mycobacterial cell envelope. PMID:16541102

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.

The roles of mutations in gyrA, parC, and ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae.

PubMed

Chen, Feng-Jui; Lauderdale, Tsai-Ling; Ho, Monto; Lo, Hsiu-Jung

2003-01-01

In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters of < or = 15 mm, 16-26 mm, and > or = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.

A Hyalinized Trichilemmoma of the Eyelid in a Teenager.

PubMed

Jakobiec, Frederick A; Stagner, Anna M; Sassoon, Jodi; Goldstein, Scott; Mihm, Martin C

2016-01-01

A 16-year-old African American male, the youngest patient to date, presented with a well-circumscribed upper eyelid lesion. On excision, the dermal nodule was contiguous with the epidermis, displayed trichohyalin-like bodies in an expanded outer root sheath, and was composed chiefly of small cellular clusters separated by a prominent network of periodic acid Schiff -positive hyaline bands of basement membrane material. The tumor cells were positive for high molecular weight cytokeratins (CK) 5/6, CK14, and CK34βE12 and were negative for CK7, carcinoembryonic antigen and epithelial membrane antigen. Negative S100, glial fibrillary acidic protein, and smooth muscle actin immunoreactions ruled out a myoepithelial lesion. The Ki-67 proliferation index was <10%. The diagnosis was a hyalinized trichilemmoma, contrasting with the more common lobular type. As an isolated lesion, trichilemmoma does not portend Cowden syndrome.

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.

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

PubMed Central

Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

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.

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.

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.

Loss of VPS13C Function in Autosomal-Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy

PubMed Central

Lesage, Suzanne; Drouet, Valérie; Majounie, Elisa; Deramecourt, Vincent; Jacoupy, Maxime; Nicolas, Aude; Cormier-Dequaire, Florence; Hassoun, Sidi Mohamed; Pujol, Claire; Ciura, Sorana; Erpapazoglou, Zoi; Usenko, Tatiana; Maurage, Claude-Alain; Sahbatou, Mourad; Liebau, Stefan; Ding, Jinhui; Bilgic, Basar; Emre, Murat; Erginel-Unaltuna, Nihan; Guven, Gamze; Tison, François; Tranchant, Christine; Vidailhet, Marie; Corvol, Jean-Christophe; Krack, Paul; Leutenegger, Anne-Louise; Nalls, Michael A.; Hernandez, Dena G.; Heutink, Peter; Gibbs, J. Raphael; Hardy, John; Wood, Nicholas W.; Gasser, Thomas; Durr, Alexandra; Deleuze, Jean-François; Tazir, Meriem; Destée, Alain; Lohmann, Ebba; Kabashi, Edor; Singleton, Andrew; Corti, Olga; Brice, Alexis; Lesage, Suzanne; Tison, François; Vidailhet, Marie; Corvol, Jean-Christophe; Agid, Yves; Anheim, Mathieu; Bonnet, Anne-Marie; Borg, Michel; Broussolle, Emmanuel; Damier, Philippe; Destée, Alain; Dürr, Alexandra; Durif, Franck; Krack, Paul; Klebe, Stephan; Lohmann, Ebba; Martinez, Maria; Pollak, Pierre; Rascol, Olivier; Tranchant, Christine; Vérin, Marc; Viallet, François; Brice, Alexis; Lesage, Suzanne; Majounie, Elisa; Tison, François; Vidailhet, Marie; Corvol, Jean Christophe; Nalls, Michael A.; Hernandez, Dena G.; Gibbs, J. Raphael; Dürr, Alexandra; Arepalli, Sampath; Barker, Roger A.; Ben-Shlomo, Yoav; Berg, Daniela; Bettella, Francesco; Bhatia, Kailash; de Bie, Rob M.A.; Biffi, Alessandro; Bloem, Bastiaan R.; Bochdanovits, Zoltan; Bonin, Michael; Lesage, Suzanne; Tison, François; Vidailhet, Marie; Corvol, Jean-Christophe; Agid, Yves; Anheim, Mathieu; Bonnet, Anne-Marie; Borg, Michel; Broussolle, Emmanuel; Damier, Philippe; Destée, Alain; Dürr, Alexandra; Durif, Franck; Krack, Paul; Klebe, Stephan; Lohmann, Ebba; Martinez, Maria; Pollak, Pierre; Rascol, Olivier; Tranchant, Christine; Vérin, Marc; Bras, Jose M.; Brockmann, Kathrin; Brooks, Janet; Burn, David J.; Charlesworth, Gavin; Chen, Honglei; Chinnery, Patrick F.; Chong, Sean; Clarke, Carl E.; Cookson, Mark R.; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T.; van Dijk, Karin D.; Dillman, Allissa; Dong, Jing; Durif, Frank; Edkins, Sarah; Escott-Price, Valentina; Evans, Jonathan R.; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Goate, Alison; Gray, Emma; Guerreiro, Rita; Harris, Clare; van Hilten, Jacobus J.; Hofman, Albert; Hollenbeck, Albert; Holmans, Peter; Holton, Janice; Hu, Michèle; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E.; Huttenlocher, Johanna; Illig, Thomas; Jónsson, Pálmi V.; Kilarski, Laura L.; Jansen, Iris E.; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; Lubbe, Steven; Lungu, Codrin; Martinez, María; Mätzler, Walter; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morrison, Karen E.; Mudanohwo, Ese; O’Sullivan, Sean S.; Owen, Michael J.; Pearson, Justin; Perlmutter, Joel S.; Pétursson, Hjörvar; Plagnol, Vincent; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Saad, Mohamad; Simón-Sánchez, Javier; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Schulte, Claudia; Sharma, Manu; Shaw, Karen; Sheerin, Una-Marie; Shoulson, Ira; Shulman, Joshua; Sidransky, Ellen; Spencer, Chris C.A.; Stefánsson, Hreinn; Stefánsson, Kári; Stockton, Joanna D.; Strange, Amy; Talbot, Kevin; Tanner, Carlie M.; Tashakkori-Ghanbaria, Avazeh; Trabzuni, Daniah; Traynor, Bryan J.; Uitterlinden, André G.; Velseboer, Daan; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams-Gray, Caroline H.; Winder-Rhodes, Sophie; Wurster, Isabel; Williams, Nigel; Morris, Huw R.; Heutink, Peter; Hardy, John; Wood, Nicholas W.; Gasser, Thomas; Singleton, Andrew B.; Brice, Alexis

2016-01-01

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression. PMID:26942284

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.

Carbapenem-resistant Serratia marcescens isolates producing Bush group 2f beta-lactamase (SME-1) in the United States: results from the MYSTIC Programme.

PubMed

Gales, A C; Biedenbach, D J; Winokur, P; Hacek, D M; Pfaller, M A; Jones, R N

2001-02-01

Two carbapenem (imipenem, meropenem)-resistant Serratia marcescens strains were isolated in the United States (Chicago, IL) through the 1999 MYSTIC (Meropenem Yearly Susceptibility Test Information Collection) Programme. The S. marcescens antimicrobial susceptible patterns were: susceptible to ceftriaxone, ceftazidime, and cefepime (MICs, < or = 0.25 microg/ml), and resistance to the carbapenems (imipenem and meropenem; MIC, > 32 microg/ml) and aztreonam (MIC, > = 16 microg/ml). Each S. marcescens isolate shared an identical epidemiologic type (ribotype and PFGE) and the outer membrane protein profile was also identical to those of the wild type susceptible strains from the same medical center. The PCR utilizing bla(sme-1) primers amplified a gene product that was identified as consistent with SME-1 after DNA sequencing. Imipenem and meropenem resistance due to production of carbapenem-hydrolyzing enzymes among clinical isolates is still very rare, but microbiology laboratories should be aware of these chromosomally encoded enzymes among class C beta-lactamases producing enteric bacilli such as S. marcescens and Enterobacter cloacae.

Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins.

PubMed

Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Krzyżewska, Eva; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-07-11

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg ( S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.

Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins

PubMed Central

Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-01-01

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins’ patterns. The strategy of bacterial membrane proteins’ analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species. PMID:28696348

Gating modifier toxins isolated from spider venom: modulation of voltage-gated sodium channels and the role of lipid membranes.

PubMed

Agwa, Akello J; Peigneur, Steve; Chow, Chun Yuen; Lawrence, Nicole; Craik, David J; Tytgat, Jan; King, Glenn F; Henriques, Sonia Troeira; Schroeder, Christina I

2018-04-27

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures that modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here we examined whether there is a relationship among spider GMT amphipathicity, membrane binding and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

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.

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.

Dissection of the Mechanical Impedance Components of the Outer Hair Cell Using a Chloride-Channel Blocker

NASA Astrophysics Data System (ADS)

Harasztosi, Csaba; Gummer, Anthony W.

2011-11-01

The voltage-dependent chloride-channel blocker anthracene-9-carboxylic acid (9AC) has been found to reduce the imaginary but not the real part of the mechanical impedance of the organ of Corti, suggesting that the effective stiffness of outer hair cells (OHCs) is reduced by 9AC. To examine whether 9AC interacts directly with the motor protein prestin to reduce the membrane component of the impedance, the patch-clamp technique in whole-cell configuration was used to measure the nonlinear capacitance (NLC) of isolated OHCs and, as control, prestin-transfected human embryonic kidney 293 (HEK293) cells. Extracellular application of 9AC significantly reduced the NLC of both OHCs and HEK293 cells. Intracellular 9AC did not influence the blocking effect of the extracellular applied drug. These results suggest that 9AC interacts directly with prestin, reducing the effective stiffness of the motor, and that the interaction is extracellular.

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

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.

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.

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

Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models.

PubMed

Askarian, Fatemeh; Lapek, John D; Dongre, Mitesh; Tsai, Chih-Ming; Kumaraswamy, Monika; Kousha, Armin; Valderrama, J Andrés; Ludviksen, Judith A; Cavanagh, Jorunn P; Uchiyama, Satoshi; Mollnes, Tom E; Gonzalez, David J; Wai, Sun N; Nizet, Victor; Johannessen, Mona

2018-01-01

Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus -derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo . Finally, immunization of mice with S. aureus -derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial-host interactions during systemic infections and provide protective immunity in murine models of infection.

Avidin/PSS membrane microcapsules with biotin-binding activity.

PubMed

Endo, Yoshihiro; Sato, Katsuhiko; Sugimoto, Kentaro; Anzai, Jun-ichi

2011-08-15

Polyelectrolyte microcapsules with avidin-poly(styrene sulfonate) (PSS) membrane were prepared by a layer-by-layer deposition technique. The uptake and release of biotin-labeled fluorescein (b-FITC) as well as immobilization of biotin-labeled glucose oxidase (b-GOx) to the microcapsule were studied. The polyelectrolyte microcapsules were prepared by coating the surface of calcium carbonate (CaCO(3)) microparticles with an avidin/PSS multilayer membrane, followed by dissolution of CaCO(3) core in an ethylenediaminetetraacetic acid solution. Inner and outer poly(allylamine)/PSS films were required to isolate the microcapsules, whereas microcapsules could not be formed without the support. The uptake of b-FITC into the microcapsule was highly enhanced through a strong binding of b-FITC to avidin as compared with the uptake of biotin-free FITC. Release of b-FITC from the microcapsule was accelerated upon addition of biotin due to a competitive binding of the added biotin to the binding site of avidin. Similarly, the surface of microcapsule was modified with b-GOx with retaining its catalytic activity. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

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.

Most Enterobacter aerogenes Strains in France Belong to a Prevalent Clone

PubMed Central

Bosi, Claude; Davin-Regli, Anne; Bornet, Charleric; Mallea, Monique; Pages, Jean-Marie; Bollet, Claude

1999-01-01

The aim of this study was to determine the distribution in France of the Enterobacter aerogenes prevalent clone isolated in the hospitals of the Marseille area (A. Davin-Regli, D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bollet, J. Clin. Microbiol. 34:1474–1480, 1996). A total of 123 E. aerogenes isolates were collected from 23 hospital laboratories and analyzed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR to determine their epidemiological relatedness. Molecular typing revealed that 21 of the 23 laboratories had isolated this prevalent clone harboring the plasmid encoding for extended-spectrum β-lactamase of the TEM-24 type. Most isolates were susceptible only to imipenem and gentamicin. Their dissemination seems to be clonal and was probably the result of the general use of broad-spectrum cephalosporins and quinolones. Four isolates showed an alteration of their outer membrane proteins, causing decrease of susceptibility to third-generation cephalosporins and imipenem and leading to the critical situation of having no alternative therapeutic. The large dissemination of the E. aerogenes prevalent clone probably results from its good adaptation to the antibiotics administered in France and the hospital environment, particularly in intensive care units. PMID:10364580

Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods

PubMed Central

1991-01-01

The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual-wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)- indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described. PMID:1722239

Calcium and magnesium fluxes across the plasma membrane of the toad rod outer segment.

PubMed Central

Nakatani, K; Yau, K W

1988-01-01

1. Membrane current was recorded from an isolated, dark-adapted toad rod by sucking either its inner segment or outer segment into a tight-fitting glass pipette containing Ringer solution. The remainder of the cell was exposed to bath solution which could be changed rapidly. 2. In normal Ringer solution the current response of a cell to a saturating flash or step of light showed a small secondary rise at its initial peak. The profile of this secondary rise (i.e. amplitude and time course) was independent of both the intensity and the duration of illumination once the light response had reached a plateau level. 3. This secondary rise disappeared when external Na+ around the outer segment was replaced by Li+ or guanidinium, suggesting that it represented an electrogenic Na+-dependent Ca2+ efflux which was declining after the onset of light. 4. This Na+-Ca2+ exchange activity showed a roughly exponential decline, with a time constant of about 0.5 s. Exponential extrapolation of the exchange current to the time at half-height of the light response gave an initial amplitude of about 2 pA. Using La3+ as a blocker, we did not detect any steady exchange current after the initial exponential decline. 5. An intense flash superposed on a just-saturating steady background light failed to produce any incremental exchange current transient. 6. Our interpretation of the above results is that in darkness there are counterbalancing levels of Ca2+ influx (through the light-sensitive conductance) and efflux (through the Na+-Ca2+ exchange) across the plasma membrane of the rod outer segment. The exchange current transient at the onset of light merely represents the unidirectional Ca2+ efflux which becomes revealed as a result of the stoppage of the Ca2+ influx, rather than a de novo Ca2+ efflux triggered by light. 7. Consistent with this interpretation, a test light delivered soon after a saturating, conditioning light elicited little exchange current, which then gradually recovered to control value with a time course parallel to the restoration of the dark current. Conversely, when the dark current was increased above its physiological level by IBMX (isobutylmethylxanthine) the exchange current transient became larger than control.(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 8 PMID:2457685

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

Rod outer segment retinol formation is independent of Abca4, arrestin, rhodopsin kinase, and rhodopsin palmitylation.

PubMed

Blakeley, Lorie R; Chen, Chunhe; Chen, Ching-Kang; Chen, Jeannie; Crouch, Rosalie K; Travis, Gabriel H; Koutalos, Yiannis

2011-06-01

The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal. Experiments were performed in isolated retinas and single living rods derived from 129/sv wild-type mice and Abca4-, arrestin-, and rhodopsin kinase-deficient mice and in genetically modified mice containing unpalmitylated rhodopsin. Formation of all-trans retinol was measured by imaging its fluorescence and by HPLC of retina extracts. The release of all-trans retinal from photoactivated rhodopsin was measured in purified rod outer segment membranes according to the increase in tryptophan fluorescence. All experiments were performed at 37°C. The kinetics of all-trans retinol formation in the different types of genetically modified mice are in reasonable agreement with those in wild-type animals. The kinetics of all-trans retinol formation in 129/sv mice are similar to those in C57BL/6, although the latter are known to regenerate rhodopsin much more slowly. The release of all-trans retinal from rhodopsin in purified membranes is significantly faster than the formation of all-trans retinol in intact cells and is independent of the presence of the palmitate groups. The regeneration of rhodopsin and the recycling of its chromophore are not strongly coupled. Neither the activities of Abca4, rhodopsin kinase, and arrestin, nor the palmitylation of rhodopsin affects the formation of all-trans retinol.

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.

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...

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

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.

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

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.

Microbead-based immunoassay using the outer membrane layer of Escherichia coli combined with autodisplayed Z-domains

NASA Astrophysics Data System (ADS)

Kim, Do-Hoon; Bong, Ji-Hong; Yoo, Gu; Chang, Seo-Yoon; Park, Min; Chang, Young Wook; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2016-01-01

The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml).

Characterization of outer membrane vesicles from a neonatal meningitic strain of Cronobacter sakazakii.

PubMed

Alzahrani, Hayat; Winter, Jody; Boocock, David; De Girolamo, Luigi; Forsythe, Stephen J

2015-06-01

Cronobacter sakazakii is associated with severe and often fatal cases of infant meningitis and necrotizing enterocolitis. The form of meningitis differs from that due to Neisseria meningitidis and Streptococcus spp., in that it is highly invasive and destructive towards human brain cells. However, there is relatively little understanding of the cytopathogenic interaction of C. sakazakii with host cells which results in stimulation of an inflammatory immune response. The production of Cronobacter outer membrane vesicles (OMV) and their potential pathogenic functions have not yet been elucidated. This study is the first to show that C. sakazakii produce OMV, which may play a role in the activation of cytopathogenic and host cell responses on human intestinal epithelial cells. Cronobacter sakazakii strain 767 was used which had been isolated from a fatal outbreak of neonatal meningitis and necrotizing enterocolitis. Cronobacter sakazakii OMV were internalized by Caco-2 cells, increased cell proliferation and stimulated the host's innate proinflammatory response without inducing overt toxicity. A total of 18 OMV-associated proteins were identified by mass spectrometry and their potential pathogenicity roles were evaluated. Collectively, these data indicate that C. sakazakii OMV could play a role in pathogenesis by delivering bacterial toxins into host epithelial cells, driving proliferative and proinflammatory responses. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Outer Membrane Vesicle Vaccines from Biosafe Surrogates Prevent Acute Lethal Glanders in Mice

PubMed Central

Khan, Mohammad S. R.; Chirakul, Sunisa; Schweizer, Herbert P.; Tuanyok, Apichai

2018-01-01

Burkholderia mallei is a host-adapted Gram-negative mammalian pathogen that causes the severe disease glanders. Glanders can manifest as a rapid acute progression or a chronic debilitating syndrome primarily affecting solipeds and humans in close association with infected animals. In USA, B. mallei is classified as one of the most important bacterial biothreat agents. Presently, there is no licensed glanders vaccine available for humans or animals. In this work, outer membrane vesicles (OMVs) were isolated from three attenuated biosafe bacterial strains, Burkholderia pseudomallei Bp82, B. thailandensis E555, and B. thailandensis TxDOH and used to vaccinate mice. B. thailandensis OMVs induced significantly higher antibody responses that were investigated. B. mallei specific serum antibody responses were of higher magnitude in mice vaccinated with B. thailandensis OMVs compared to levels in mice vaccinated with B. pseudomallei OMVs. OMVs derived from biosafe strains protected mice from acute lethal glanders with vesicles from the two B. thailandensis strains affording significant protection (>90%) up to 35 days post-infection with some up to 60 days. Organ loads from 35-day survivors indicated bacteria colonization of the lungs, liver, and spleen while those from 60 days had high CFUs in the spleens. The highest antibody producing vaccine (B. thailandensis E555 OMVs) also protected C57BL/6 mice from acute inhalational glanders with evidence of full protection. PMID:29320408

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.

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.

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.

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

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

Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification.

PubMed

Koncsos, Gábor; Varga, Zoltán V; Baranyai, Tamás; Ferdinandy, Péter; Schulz, Rainer; Giricz, Zoltán; Boengler, Kerstin

In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.

PubMed

Worrall, L J; Hong, C; Vuckovic, M; Deng, W; Bergeron, J R C; Majewski, D D; Huang, R K; Spreter, T; Finlay, B B; Yu, Z; Strynadka, N C J

2016-12-14

The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.

Enterobacter gergoviae membrane modifications are involved in the adaptive response to preservatives used in cosmetic industry.

PubMed

Périamé, Marina; Pagès, Jean-Marie; Davin-Regli, Anne

2015-01-01

The objective of this study was to understand the adaptive mechanisms in Enterobacter gergoviae which are involved in recurrent contaminations in cosmetic products that are incorporated with preservatives. Bacterial strains from two backgrounds were examined for a profound understanding of the mechanisms of adaptation against preservatives. It included a series of Ent. gergoviae strain-ATCC 33028 derivatives, isolated using increasing methylisothiazolinone-chloromethylisothiazolinone (MIT-CMIT) and triclosan concentrations. The other series was of Ent. gergoviae isolates from cosmetic products exhibiting MIT-CMIT and triclosan resistance. We evaluated the outer membrane protein modifications and efflux mechanisms activities responsible for the resistant trait via immunoblotting assays. Additionally, for understanding the efflux activity real-time efflux, experiments were performed. A cross-insusceptibility between preservatives and some disinfectants was observed in MIT-CMIT-resistant derivative isolates, but antibiotics susceptibility was not altered. Resistance to EDTA was significant in all preservatives insusceptible derivative strains, indicating modifications in the LPS layer. Furthermore, an array of real-time efflux assays indicated different activity levels while no variations were detected in porins and AcrAB-TolC pumps production. Overexpression of a specific flagellin-type protein was observed in one of the MIT-CMIT- and triclosan-resistant strains. Another candidate, a 25-kDa peroxiredoxin enzyme involved in oxidative detoxification, was identified to be overexpressed in MIT-CMIT derivative. A similar profile was also observed among strains isolated from cosmetic products. Our study highlights the existence of adaptive mechanisms such as overexpression of detoxifying enzymes, flagellin, modification of membrane structure/function in Ent. gergoviae. They might be involved in recurrent episodes of contaminations occurring in the cosmetic production lines. No cross-resistance could be observed with antibiotics when MICs to preservatives were increased; however, a decrease in the disinfectants bactericidal effects was confirmed in preservative-tolerant strains. This will impact industry disinfection strategies treatment against bacteria. © 2014 The Society for Applied Microbiology.

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.

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.

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

Triton Hodge Test: Improved Protocol for Modified Hodge Test for Enhanced Detection of NDM and Other Carbapenemase Producers

PubMed Central

Pasteran, Fernando; Gonzalez, Lisandro J.; Albornoz, Ezequiel; Bahr, Guillermo; Vila, Alejandro J.

2015-01-01

Accurate detection of carbapenemase-producing Gram-negative bacilli is of utmost importance for the control of nosocomial spread and the initiation of appropriate antimicrobial therapy. The modified Hodge test (MHT), a carbapenem inactivation assay, has shown poor sensitivity in detecting the worldwide spread of New Delhi metallo-β-lactamase (NDM). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases. Here we report that membrane anchoring of β-lactamases precludes detection of carbapenemase activity by the MHT. We also show that this limitation can be overcome by the addition of Triton X-100 during the test, which allows detection of NDM. We propose an improved version of the assay, called the Triton Hodge test (THT), which allows detection of membrane-bound carbapenemases with the addition of this nonionic surfactant. This test was challenged with a panel of 185 clinical isolates (145 carrying known carbapenemase-encoding genes and 40 carbapenemase nonproducers). The THT displayed test sensitivity of >90% against NDM-producing clinical isolates, while improving performance against other carbapenemases. Ertapenem provided the highest sensitivity (97 to 100%, depending on the type of carbapenemase), followed by meropenem (92.5 to 100%). Test specificity was not affected by the addition of Triton (87.5% and 92.5% with ertapenem and meropenem, respectively). This simple inexpensive test confers a large improvement to the sensitivity of the MHT for the detection of NDM and other carbapenemases. PMID:26719442

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 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.

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.

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

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

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

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.

Intracellular and membrane-damaging activities of methyl gallate isolated from Terminalia chebula against multidrug-resistant Shigella spp.

PubMed

Acharyya, Saurabh; Sarkar, Prodipta; Saha, Dhira R; Patra, Amarendra; Ramamurthy, T; Bag, Prasanta K

2015-08-01

Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24  h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20  h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.

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

Hossain, Quazi Sohel; Department of Biochemistry, School of Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215; Ulziikhishig, Enkhbaatar

We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated withmore » GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.« less

Motuporamine Derivatives as Antimicrobial Agents and Antibiotic Enhancers against Resistant Gram-Negative Bacteria.

PubMed

Borselli, Diane; Blanchet, Marine; Bolla, Jean-Michel; Muth, Aaron; Skruber, Kristen; Phanstiel, Otto; Brunel, Jean Michel

2017-02-01

Dihydromotuporamine C and its derivatives were evaluated for their in vitro antimicrobial activities and antibiotic enhancement properties against Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives, MOTU-N44, was investigated against Enterobacter aerogenes by using fluorescent dyes to evaluate outer-membrane depolarization and permeabilization. Its efficiency correlated with inhibition of dye transport, thus suggesting that these molecules inhibit drug transporters by de-energization of the efflux pump rather than by direct interaction of the molecule with the pump. This suggests that depowering the efflux pump provides another strategy to address antibiotic resistance. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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.

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.

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

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.

Synergistic effect of EDTA and boric acid on corneal penetration of CS-088.

PubMed

Kikuchi, Takayuki; Suzuki, Masahiko; Kusai, Akira; Iseki, Ken; Sasaki, Hitoshi

2005-02-16

In order to investigate the effects of EDTA and boric acid (EDTA/boric acid) on the corneal penetration of CS-088, an ophthalmic agent, the apparent permeability coefficient of CS-088 in the presence of EDTA/boric acid across the isolated corneal membranes of rabbits was measured using an in vitro penetration chamber system. FITC-dextran (M.W. 4400) and an electrical method based on membrane resistance were used to provide a quantitative assessment of the enhancing effect of EDTA/boric acid. The corneal penetration of CS-088 was significantly enhanced in the presence of EDTA/boric acid by approximately 1.6-fold. The permeability-enhancing effect of EDTA/boric acid was apparently synergistic and concentration-dependent on both EDTA and boric acid. The penetration of FITC-dextran, a paracellular marker, and electrical resistance of corneal membranes were not affected in the presence of EDTA/boric acid. Furthermore, no enhancing effect of EDTA/boric acid was observed in de-epithelialized corneas, although de-epithelialized corneas exhibited a markedly higher permeability of CS-088 that was 24-fold greater than that for intact corneas. In conclusion, EDTA/boric acid synergistically enhances the transcellular permeability of CS-088 in the outer layer but not in the inner layers of the corneal membrane.

A novel technique to study pore-forming peptides in a natural membrane.

PubMed

Vedovato, Natascia; Rispoli, Giorgio

2007-09-01

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

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.

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

PubMed Central

McLean, Jeff; Beveridge, Terry J.

2001-01-01

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter−1 and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a Km of 23 mg liter−1 (437 μM) and a Vmax of 0.98 mg of Cr h−1 mg of protein−1 (317 nmol min−1 mg of protein−1). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments. PMID:11229894

Loss of VPS13C Function in Autosomal-Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy.

PubMed

Lesage, Suzanne; Drouet, Valérie; Majounie, Elisa; Deramecourt, Vincent; Jacoupy, Maxime; Nicolas, Aude; Cormier-Dequaire, Florence; Hassoun, Sidi Mohamed; Pujol, Claire; Ciura, Sorana; Erpapazoglou, Zoi; Usenko, Tatiana; Maurage, Claude-Alain; Sahbatou, Mourad; Liebau, Stefan; Ding, Jinhui; Bilgic, Basar; Emre, Murat; Erginel-Unaltuna, Nihan; Guven, Gamze; Tison, François; Tranchant, Christine; Vidailhet, Marie; Corvol, Jean-Christophe; Krack, Paul; Leutenegger, Anne-Louise; Nalls, Michael A; Hernandez, Dena G; Heutink, Peter; Gibbs, J Raphael; Hardy, John; Wood, Nicholas W; Gasser, Thomas; Durr, Alexandra; Deleuze, Jean-François; Tazir, Meriem; Destée, Alain; Lohmann, Ebba; Kabashi, Edor; Singleton, Andrew; Corti, Olga; Brice, Alexis

2016-03-03

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

PubMed Central

Echenique, J R; Arienti, H; Tolmasky, M E; Read, R R; Staneloni, R J; Crosa, J H; Actis, L A

1992-01-01

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Images PMID:1447137

Genomic comparison of Escherichia coli K1 strains isolated from the cerebrospinal fluid of patients with meningitis.

PubMed

Yao, Yufeng; Xie, Yi; Kim, Kwang Sik

2006-04-01

Escherichia coli is a major cause of enteric/diarrheal diseases, urinary tract infections, and sepsis. E. coli K1 is the leading gram-negative organism causing neonatal meningitis, but the microbial basis of E. coli K1 meningitis is incompletely understood. Here we employed comparative genomic hybridization to investigate 11 strains of E. coli K1 isolated from the cerebrospinal fluid (CSF) of patients with meningitis. These 11 strains cover the majority of common O serotypes in E. coli K1 isolates from CSF. Our data demonstrated that these 11 strains of E. coli K1 can be categorized into two groups based on their profile for putative virulence factors, lipoproteins, proteases, and outer membrane proteins. Of interest, we showed that some open reading frames (ORFs) encoding the type III secretion system apparatus were found in group 2 strains but not in group 1 strains, while ORFs encoding the general secretory pathway are predominant in group 1 strains. These findings suggest that E. coli K1 strains isolated from CSF can be divided into two groups and these two groups of E. coli K1 may utilize different mechanisms to induce meningitis.

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 responsible for imipenem resistance among Pseudomonas aeruginosa clinical isolates exposed to imipenem concentrations within the mutant selection window.

PubMed

Vassilara, Foula; Galani, Irene; Souli, Maria; Papanikolaou, Konstantinos; Giamarellou, Helen; Papadopoulos, Antonios

2017-07-01

The aim of this study was to determine the propensities of imipenem to select for resistant Pseudomonas aeruginosa mutants by determining the mutant prevention concentrations (MPCs) for 9 unrelated clinical isolates and the accession of any relationship with mechanisms of resistance development. The MPC/MIC ratios ranged from 4 to 16. Detection of resistance mechanisms in the mutant derivatives of the nine isolates mainly revealed inactivating mutations in the gene coding for outer membrane protein OprD. Point mutations leading to premature stop codons or amino acid substitution S278P, ≥1bp deletion leading to frameshift mutations and interruption of the oprD by an insertion sequence, were observed. MPC and mutant selection window (MSW) are unique parameters that may guide the implementation of antimicrobial treatment, providing useful information about the necessary imipenem concentration needed in the infection area, in order to avoid the emergence of resistance, especially in clinical situations with high bacterial load. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile.

PubMed

Bastardo, A; Bohle, H; Ravelo, C; Toranzo, A E; Romalde, J L

2011-02-22

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.

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.

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition.

PubMed

Ishida, Hiroaki; Garcia-Herrero, Alicia; Vogel, Hans J

2014-12-01

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1-171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180-325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA(180-325)). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA(180-325) with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA(1-325)) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the periplasmic domain of OmpA above physiological temperatures, which may induce dimerization and play a role in triggering the previously reported larger pore formation. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

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.

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.

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

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.

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences.

PubMed

Zhou, Z X; Wei, D F; Guan, Y; Zheng, A N; Zhong, J J

2010-03-01

The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). The bactericidal effect of PHMG on E. coli was investigated based on beta-galactosidase activity assay, fluorescein-5-isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 microg ml(-1)) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 microg ml(-1)) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Dose-dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 microg ml(-1)) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.

Pulmonary inflammation induced by bacteria-free outer membrane vesicles from Pseudomonas aeruginosa.

PubMed

Park, Kyong-Su; Lee, Jaewook; Jang, Su Chul; Kim, Sae Rom; Jang, Myoung Ho; Lötvall, Jan; Kim, Yoon-Keun; Gho, Yong Song

2013-10-01

Pseudomonas aeruginosa is often involved in lung diseases such as cystic fibrosis. These bacteria can release outer membrane vesicles (OMVs), which are bilayered proteolipids with diameters of approximately 20 to 250 nm. In vitro, these OMVs activate macrophages and airway epithelial cells. The aim of this study was to determine whether OMVs from P. aeruginosa can induce pulmonary inflammation in vivo and to elucidate the mechanisms involved. Bacteria-free OMVs were isolated from P. aeruginosa cultures. Wild-type, Toll-like receptor (TLR)2 and TLR4 knockout mice were exposed to OMVs by the airway, and inflammation in the lung was assessed using differential counts, histology, and quantification of chemokines and cytokines. The involvement of the TLR2 and TLR4 pathways was studied in human cells using transfection. OMVs given to the mouse lung caused dose- and time-dependent pulmonary cellular inflammation. Furthermore, OMVs increased concentrations of several chemokines and cytokines in the mouse lungs and mouse alveolar macrophages. The inflammatory responses to OMVs were comparable to those of live bacteria and were only partly regulated by the TLR2 and TLR4 pathways, according to studies in knockout mice. This study shows that OMVs from P. aeruginosa cause pulmonary inflammation without live bacteria in vivo. This effect is only partly controlled by TLR2 and TLR4. The role of OMVs in clinical disease warrants further studies because targeting of OMVs in addition to live bacteria may add clinical benefit compared with treating with antibiotics alone.

Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

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

Thomson, M.S.

1988-01-01

Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture andmore » used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.« less

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

PubMed Central

Falkenberg, Shollie M.; Briggs, Robert E.; Tatum, Fred M.; Sacco, Randy E.

2017-01-01

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2–5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10–30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates. PMID:28827826

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni.

PubMed

Dassanayake, Rohana P; Falkenberg, Shollie M; Briggs, Robert E; Tatum, Fred M; Sacco, Randy E

2017-01-01

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2-5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10-30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates.

A characteristic of polymorphic membrane protein F of Chlamydia trachomatis isolated from male urogenital tracts in Japan.

PubMed

Yamazaki, Tomohiro; Matsuo, Junji; Takahashi, Satoshi; Kumagai, Shouta; Shimoda, Tomoko; Abe, Kiyotaka; Minami, Kunihiro; Yamaguchi, Hiroyuki

2015-12-01

Although sexually transmitted disease due to Chlamydia trachomatis occurs similarly in both men and women, the female urogenital tract differs from that of males anatomically and physiologically, possibly leading to specific polymorphisms of the bacterial surface molecules. In the present study, we therefore characterized polymorphic features in a high-definition phylogenetic marker, polymorphic outer membrane protein (Pmp) F of C. trachomatis strains isolated from male urogenital tracts in Japan (Category: Japan-males, n = 12), when compared with those isolated from female cervical ducts in Japan (Category: Japan-females, n = 11), female cervical ducts in the other country (Category: Ref-females, n = 12) or homosexual male rectums in the other country (Category: Ref-males, n = 7), by general bioinformatics analysis tool with MAFFT software. As a result, phylogenetic reconstruction of the PmpF amino acid sequences showing three distinct clusters revealed that the Japan-males were limited into cluster 1 and 2, although there were only four clusters even though including an outgroup. Meanwhile, the phylogenetic distance values of PmpF passenger domain without hinge region, but not its full-length sequence, showed that the Japan-males were more stable and displayed less diversity when compared with the other categories, supported by the sequence conservation features. Thus, PmpF passenger domain is a useful phylogenetic maker, and the phylogenic features indicate that C. trachomatis strains isolated from male urogenital tracts in Japan may be unique, suggesting an adaptation depending on selective pressure, such as the presence or absence of microbial flora, furthermore possibly connecting to sexual differentiation. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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.

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

PubMed Central

Zook, James D.; Molugu, Trivikram R.; Jacobsen, Neil E.; Lin, Guangxin; Soll, Jürgen; Cherry, Brian R.; Brown, Michael F.; Fromme, Petra

2013-01-01

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein. PMID:24205117

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.

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

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

Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane

PubMed Central

Llamas, María A.; Rodríguez-Herva, José J.; Hancock, Robert E. W.; Bitter, Wilbert; Tommassen, Jan; Ramos, Juan L.

2003-01-01

Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria. Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane. Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain. Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain. The pattern and amount of outer membrane protein in the P. putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane. Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P. putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level. Generation of a proton motive force appeared to be unaffected in these mutants. To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants. These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane. PMID:12896989

Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

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

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less

Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

PubMed

Rogers, Jason V; Rose, Mark D

2014-12-02

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

Kar5p Is Required for Multiple Functions in Both Inner and Outer Nuclear Envelope Fusion in Saccharomyces cerevisiae

PubMed Central

Rogers, Jason V.; Rose, Mark D.

2014-01-01

During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide–sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p’s functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. PMID:25467943

Modelling motions within the organ of Corti

NASA Astrophysics Data System (ADS)

Ni, Guangjian; Baumgart, Johannes; Elliott, Stephen

2015-12-01

Most cochlear models used to describe the basilar membrane vibration along the cochlea are concerned with macromechanics, and often assume that the organ of Corti moves as a single unit, ignoring the individual motion of different components. New experimental technologies provide the opportunity to measure the dynamic behaviour of different components within the organ of Corti, but only for certain types of excitation. It is thus still difficult to directly measure every aspect of cochlear dynamics, particularly for acoustic excitation of the fully active cochlea. The present work studies the dynamic response of a model of the cross-section of the cochlea, at the microscopic level, using the finite element method. The elastic components are modelled with plate elements and the perilymph and endolymph are modelled with inviscid fluid elements. The individual motion of each component within the organ of Corti is calculated with dynamic pressure loading on the basilar membrane and the motions of the experimentally accessible parts are compared with measurements. The reticular lamina moves as a stiff plate, without much bending, and is pivoting around a point close to the region of the inner hair cells, as observed experimentally. The basilar membrane shows a slightly asymmetric mode shape, with maximum displacement occurring between the second-row and the third-row of the outer hair cells. The dynamics responses is also calculated, and compared with experiments, when driven by the outer hair cells. The receptance of the basilar membrane motion and of the deflection of the hair bundles of the outer hair cells is thus obtained, when driven either acoustically or electrically. In this way, the fully active linear response of the basilar membrane to acoustic excitation can be predicted by using a linear superposition of the calculated receptances and a defined gain function for the outer hair cell feedback.

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

Triton Hodge Test: Improved Protocol for Modified Hodge Test for Enhanced Detection of NDM and Other Carbapenemase Producers.

PubMed

Pasteran, Fernando; Gonzalez, Lisandro J; Albornoz, Ezequiel; Bahr, Guillermo; Vila, Alejandro J; Corso, Alejandra

2016-03-01

Accurate detection of carbapenemase-producing Gram-negative bacilli is of utmost importance for the control of nosocomial spread and the initiation of appropriate antimicrobial therapy. The modified Hodge test (MHT), a carbapenem inactivation assay, has shown poor sensitivity in detecting the worldwide spread of New Delhi metallo-β-lactamase (NDM). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases. Here we report that membrane anchoring of β-lactamases precludes detection of carbapenemase activity by the MHT. We also show that this limitation can be overcome by the addition of Triton X-100 during the test, which allows detection of NDM. We propose an improved version of the assay, called the Triton Hodge test (THT), which allows detection of membrane-bound carbapenemases with the addition of this nonionic surfactant. This test was challenged with a panel of 185 clinical isolates (145 carrying known carbapenemase-encoding genes and 40 carbapenemase nonproducers). The THT displayed test sensitivity of >90% against NDM-producing clinical isolates, while improving performance against other carbapenemases. Ertapenem provided the highest sensitivity (97 to 100%, depending on the type of carbapenemase), followed by meropenem (92.5 to 100%). Test specificity was not affected by the addition of Triton (87.5% and 92.5% with ertapenem and meropenem, respectively). This simple inexpensive test confers a large improvement to the sensitivity of the MHT for the detection of NDM and other carbapenemases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation.

PubMed Central

Chang, C N; Inouye, H; Model, P; Beckwith, J

1980-01-01

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer. Images PMID:6991486

Membrane Electromechanics at Hair-Cell Synapses

NASA Astrophysics Data System (ADS)

Brownell, W. E.; Farrell, B.; Raphael, R. M.

2003-02-01

Both outer hair cell electromotility and neurotransmission at the inner hair cell synapse are rapid mechanical events that are synchronized to the hair-cell receptor potential. We analyze whether the forces and potentials resulting from membrane flexoelectricity could affect synaptic vesicle fusion. The results suggest that the coupling of membrane curvature with membrane potential is of sufficient magnitude to influence neurotransmitter release.

Electron microscopy of the nuclear membrane of Amoeba proteus.

PubMed

FRAJOLA, W J; GREIDER, M H; KOSTIR, W J

1956-07-25

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

PubMed

Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

2009-01-01

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Oxygen isotope reservoirs in the outer asteroid belt inferred from oxygen isotope systematics of chondrule olivines and isolated forsterite and olivine grains in Tagish Lake-type carbonaceous chondrites, WIS 91600 and MET 00432

NASA Astrophysics Data System (ADS)

Yamanobe, Masakuni; Nakamura, Tomoki; Nakashima, Daisuke

2018-03-01

To understand oxygen isotope ratios and redox conditions of the chondrule formation environments of the outer rigions of the asteroid belt, we analyzed major element concentrations and oxygen isotope ratios of olivine grains in chondrules, isolated forsterite, and isolated olivine from the WIS 91600 and MET 00432 carbonaceous chondrites, which are thought to have originated from D-type asteroids located in the outer asteroid belt. The oxygen isotope ratios of individual chondrules and isolated grains show a wide variation in δ18O from -9.9‰ to +9.1‰ along the carbonaceous chondrite anhydrous mineral (CCAM) and primitive chondrule mineral (PCM) lines. The Δ17O (= δ17O - 0.52 × δ18O) values of the measured objects increase with decreasing Mg#; i.e., FeO-poor objects (Mg# > 90; type I chondrules and isolated forsterites) mainly have Δ17O values of ca. -6‰, and FeO-rich objects (Mg# < 90; type II chondrules and isolated olivines) have Δ17O values ranging from -3‰ to +2‰. Similar trends are observed for ferromagnesian silicate particles from comet Wild2 and CR chondrite chondrules, particularly in terms of FeO-rich objects with Δ17O values ranging from -3‰ to +2‰. It is suggested that FeO-rich objects formed in the outer regions of the asteroid belt and were transported to the outer solar nebular regions where comet Wild2 formed.

Effect of extrusion rate on morphology of Kaolin/PolyEtherSulfone (PESf) membrane precursor

NASA Astrophysics Data System (ADS)

Misaran, M. S.; Sarbatly, R.; Bono, A.; Rahman, M. M.

2016-11-01

This study aims to investigate the influence of apparent viscosity induced by spinneret geometry and extrusion rate on morphology of Kaolin/PESf hollow fiber membranes. Different extrusion rates at two different rheology properties were introduced on a straight and conical spinneret resulting in various shear rates. The hollow fiber membrane precursors were spun using the wet spinning method to decouple the effect of shear and elongation stress due to gravity stretched drawing. The morphology of the spun hollow fiber was observed under Scanning Electron Microscope (SEM) and the overall porosity were measured using mercury intrusion porosimeter. Shear rate and apparent viscosity at the tip of the spinneret annulus were simulated using a computational fluid dynamics package; solidworks floworks. Simulation data shows that extrusion rate increment increases the shear rate at the spinneret wall which in turn reduce the apparent viscosity; consistent with a non Newtonian shear thinning fluid behavior. Thus, the outer finger-like region grows as the shear rate increases. Also, overall porosity of hollow fiber membrane decreases with extrusion rate increment which is caused by better molecular orientation; resulting in denser hollow fiber membrane. Thin outer finger-like region is achieved at low shear experience of 109.55 s-1 via a straight spinneret. Increasing the extrusion rate; thus shear rate will cause outer finger-like region growth which is not desirable in a separation process.

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

Characteristics of Mitochondrial Transformation into Human Cells

PubMed Central

Kesner, E. E.; Saada-Reich, A.; Lorberboum-Galski, H.

2016-01-01

Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process. PMID:27184109

The Tom Core Complex

PubMed Central

Ahting, Uwe; Thun, Clemens; Hegerl, Reiner; Typke, Dieter; Nargang, Frank E.; Neupert, Walter; Nussberger, Stephan

1999-01-01

Translocation of nuclear-encoded preproteins across the outer membrane of mitochondria is mediated by the multicomponent transmembrane TOM complex. We have isolated the TOM core complex of Neurospora crassa by removing the receptors Tom70 and Tom20 from the isolated TOM holo complex by treatment with the detergent dodecyl maltoside. It consists of Tom40, Tom22, and the small Tom components, Tom6 and Tom7. This core complex was also purified directly from mitochondria after solubilization with dodecyl maltoside. The TOM core complex has the characteristics of the general insertion pore; it contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. It forms a double ring structure that, in contrast to the holo complex, lacks the third density seen in the latter particles. Three-dimensional reconstruction by electron tomography exhibits two open pores traversing the complex with a diameter of ∼2.1 nm and a height of ∼7 nm. Tom40 is the key structural element of the TOM core complex. PMID:10579717

Plasma membrane isolation using immobilized concanavalin A magnetic beads.

PubMed

Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

2012-01-01

Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

Physical and Electronic Isolation of Carbon Nanotube Conductors

NASA Technical Reports Server (NTRS)

OKeeffe, James; Biegel, Bryan (Technical Monitor)

2001-01-01

Multi-walled nanotubes are proposed as a method to electrically and physically isolate nanoscale conductors from their surroundings. We use tight binding (TB) and density functional theory (DFT) to simulate the effects of an external electric field on multi-wall nanotubes. Two categories of multi-wall nanotube are investigated, those with metallic and semiconducting outer shells. In the metallic case, simulations show that the outer wall effectively screens the inner core from an applied electric field. This offers the ability to reduce crosstalk between nanotube conductors. A semiconducting outer shell is found not to perturb an electric field incident on the inner core, thereby providing physical isolation while allowing the tube to remain electrically coupled to its surroundings.

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

PubMed

Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

2007-06-12

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377.

PubMed

Teng, Da; Wang, Xiumin; Xi, Di; Mao, Ruoyu; Zhang, Yong; Guan, Qingfeng; Zhang, Jun; Wang, Jianhua

2014-10-01

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models.

PubMed

Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

2017-01-29

Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40-50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. Copyright © 2016 Elsevier Inc. All rights reserved.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

PubMed

Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.

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.

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.

Operational Experience with the Internal Thermal Control System Dual-Membrane Gas Trap

NASA Technical Reports Server (NTRS)

Leimkuehler, Thomas O.; Lukens, Clark; Reeves, Daniel R.; Holt, James M.

2003-01-01

A dual-membrane gas trap is currently used to remove non-condensed gases (NCG) from the Internal Thermal Control System (ITCS) coolant on board the International Space Station. The gas trap consists of concentric tube membrane pairs, comprised of outer hydrophilic tubes and inner hydrophobic fibers. Liquid coolant passes through the outer hydrophilic membrane, which traps the NCG. The inner hydrophobic fiber allows the trapped NCG to pass through and vent to the ambient atmosphere in the cabin. The purpose of the gas trap is to prevent gas bubbles from causing depriming, overspeed, and shutdown of the ITCS pump, and the current gas trap has performed flawlessly in this regard. However, because of actual operational conditions on-orbit, its gas removal performance and operational lifetime have been affected. This paper discusses experiences with several of these dual- membrane gas traps, including on-orbit gas venting rate, effects due to the presence of nickel in the ITCS coolant, and subsequent refurbishing to remove the nickel from the gas trap.

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.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

PubMed

Renault, Thibaud T; Floros, Konstantinos V; Elkholi, Rana; Corrigan, Kelly-Ann; Kushnareva, Yulia; Wieder, Shira Y; Lindtner, Claudia; Serasinghe, Madhavika N; Asciolla, James J; Buettner, Christoph; Newmeyer, Donald D; Chipuk, Jerry E

2015-01-08

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Lateral microheterogeneity of diphenylhexatriene-labeled choline phospholipids in the erythrocyte ghost membrane as determined by time-resolved fluorescence spectroscopy.

PubMed

Prenner, E; Sommer, A; Maurer, N; Glatter, O; Gorges, R; Paltauf, F; Hermetter, A

2000-04-01

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.

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.

On the role of VDAC in apoptosis: fact and fiction.

PubMed

Rostovtseva, Tatiana K; Tan, Wenzhi; Colombini, Marco

2005-06-01

Research on VDAC has accelerated as evidence grows of its importance in mitochondrial function and in apoptosis. New investigators entering the field are often confounded by the VDAC literature and its many apparent conflicts and contradictions. This review is an effort to shed light on the situation and identify reliable information from more questionable claims. Our views on the most important controversial issues are as follows: VDAC is only present in the mitochondrial outer membrane. VDAC functions as a monomer. VDAC functions normally with or without Ca(2+). It does not form channels that mediate the flux of proteins through membranes (peptides and unfolded proteins are excluded from this statement). Closure of VDAC, not VDAC opening, leads to mitochondria outer membrane permeabilization and apoptosis.

Subminimal Inhibitory Concentrations of the Disinfectant Benzalkonium Chloride Select for a Tolerant Subpopulation of Escherichia coli with Inheritable Characteristics

PubMed Central

Moen, Birgitte; Rudi, Knut; Bore, Erlend; Langsrud, Solveig

2012-01-01

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1–5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture. PMID:22605968

Subminimal inhibitory concentrations of the disinfectant benzalkonium chloride select for a tolerant subpopulation of Escherichia coli with inheritable characteristics.

PubMed

Moen, Birgitte; Rudi, Knut; Bore, Erlend; Langsrud, Solveig

2012-01-01

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1-5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.

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.

Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

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

Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul, E-mail: hnc@pusan.ac.kr

2008-05-01

A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange andmore » gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.« less

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

PubMed

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans.

PubMed

Holzweber, Markus; Lippitz, Andreas; Krueger, Katharina; Jankowski, Joachim; Unger, Wolfgang E S

2015-03-24

The surfaces of polymeric dialyzer membranes consisting of polysulfone and polyvinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied.

ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

PubMed Central

Neugebauer, Heidi; Herrmann, Christina; Kammer, Winfried; Schwarz, Gerold; Nordheim, Alfred; Braun, Volkmar

2005-01-01

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe3+ and vitamin B12—the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [14C]maltodextrins from [14C]maltose to [14C]maltopentaose. [14C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a Kd of 0.2 μM, while the second transport had a Kd of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [14C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe3+-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with Kd values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B12 and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B12 has been demonstrated. PMID:16321934

Disruption the Outer Membrane of Enteropathogenic and Enterotoxigenic Escherichia coli using Proanthocyanidins

USDA-ARS?s Scientific Manuscript database

American cranberry (Vaccinium macrocarpon) proanthocyanidins (PACs) have been reported as a natural antibacterial agent to suppress the growth of pathogenic Escherichia coli. The objective of this study was to investigate the efficacy of cranberry-derived proanthocyanidins on destabilizing the outer...

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.

Recent Operational Experience with the Internal Thermal Control System Dual-Membrane Gas Trap

NASA Technical Reports Server (NTRS)

Leimkuehler, Thomas O.; Lukens, Clark; Reeves, Daniel R.; Holt, James M.

2004-01-01

A dual-membrane gas trap is currently used to remove gas bubbles from the Internal Thermal Control System (ITCS) coolant on board the International Space Station. The gas trap consists of concentric tube membrane pairs, comprised of outer hydrophilic tubes and inner hydrophobic fibers. Liquid coolant passes through the outer hydrophilic membrane, which traps the gas bubbles. The inner hydrophobic fiber allows the trapped gas bubbles to pass through and vent to the ambient atmosphere in the cabin. The gas removal performance and operational lifetime of the gas trap have been affected by contamination in the ITCS coolant. However, the gas trap has performed flawlessly with regard to its purpose of preventing gas bubbles from causing depriming, overspeed, and shutdown of the ITCS pump. This paper discusses on-orbit events over the course of the last year related to the performance and functioning of the gas trap.

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.

Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

PubMed

Lins, Roberto D; Vorpagel, Erich R; Guglielmi, Matteo; Straatsma, T P

2008-01-01

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.

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

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

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

PubMed Central

2006-01-01

Abstract Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis. PMID:16548331

Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization.

PubMed

Cao, Bin; Ahmed, Bulbul; Kennedy, David W; Wang, Zheming; Shi, Liang; Marshall, Matthew J; Fredrickson, Jim K; Isern, Nancy G; Majors, Paul D; Beyenal, Haluk

2011-07-01

The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.

Contribution of Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms to U(VI) Immobilization

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

Cao, Bin; Ahmed, B.; Kennedy, David W.

2011-06-05

The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) in U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells without EPS, we showed that i) bEPS from Shewanella sp. HRCR-1 biofilms contributed significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; ii) bEPS could be considered as a functional extension of the cells for U(VI) immobilization and they likely play more important roles at initial U(VI) concentrations; and iii) U(VI) reduction efficiency was found to be dependent uponmore » initial U(VI) concentration and the efficiency decreased at lower concentrations. To quantify relative contribution of sorption and reduction in U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(V). We found that, when in reduced form, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated reactivity of laEPS while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, might facilitate U(VI) reduction.« less

Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog.

PubMed

Piras, Cristian; Soggiu, Alessio; Greco, Viviana; Martino, Piera Anna; Del Chierico, Federica; Putignani, Lorenza; Urbani, Andrea; Nally, Jarlath E; Bonizzi, Luigi; Roncada, Paola

2015-09-08

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. The isolated E. coli has been induced with enrofloxacin and studied through 2D DIGE and shotgun MS. Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover the mechanism of this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane protein W, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanism of E. coli defense against this antibiotic. All 2D DIGE and MS data have been deposited into the ProteomeXchange Consortium with identifier PXD002000 and DOI http://dx.doi.org/10.6019/PXD002000. This article is part of a Special Issue entitled: HUPO 2014. Copyright © 2015 Elsevier B.V. All rights reserved.

Ectopic expression of TaOEP16-2-5B, a wheat plastid outer envelope protein gene, enhances heat and drought stress tolerance in transgenic Arabidopsis plants.

PubMed

Zang, Xinshan; Geng, Xiaoli; Liu, Kelu; Wang, Fei; Liu, Zhenshan; Zhang, Liyuan; Zhao, Yue; Tian, Xuejun; Hu, Zhaorong; Yao, Yingyin; Ni, Zhongfu; Xin, Mingming; Sun, Qixin; Peng, Huiru

2017-05-01

Abiotic stresses, such as heat and drought, are major environmental factors restricting crop productivity and quality worldwide. A plastid outer envelope protein gene, TaOEP16-2, was identified from our previous transcriptome analysis [1,2]. In this study, the isolation and functional characterization of the TaOEP16-2 gene was reported. Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat, which were localized on the chromosomes 5A, 5B and 5D, respectively. These three homoeologues exhibited different expression patterns under heat stress conditions, TaOEP16-2-5B was the dominant one, and TaOEP16-2-5B was selected for further analysis. Compared with wild type (WT) plants, transgenic Arabidopsis plants overexpressing the TaOEP16-2-5B gene exhibited enhanced tolerance to heat stress, which was supported by improved survival rate, strengthened cell membrane stability and increased sucrose content. It was also found that TaOEP16-2 was induced by drought stress and involved in drought stress tolerance. TaOEP16-2-5B has the same function in ABA-controlled seed germination as AtOEP16-2. Our results suggest that TaOEP16-2-5B plays an important role in heat and drought stress tolerance, and could be utilized in transgenic breeding of wheat and other crop plants. Copyright © 2017 Elsevier B.V. All rights reserved.

VDAC electronics: 1. VDAC-hexo(gluco)kinase generator of the mitochondrial outer membrane potential.

PubMed

Lemeshko, Victor V

2014-05-01

The simplest mechanism of the generation of the mitochondrial outer membrane potential (OMP) by the VDAC (voltage-dependent anion channel)-hexokinase complex (VHC), suggested earlier, and by the VDAC-glucokinase complex (VGC), was computationally analyzed. Even at less than 4% of VDACs bound to hexokinase, the calculated OMP is high enough to trigger the electrical closure of VDACs beyond the complexes at threshold concentrations of glucose. These results confirmed our previous hypothesis that the Warburg effect is caused by the electrical closure of VDACs, leading to global restriction of the outer membrane permeability coupled to aerobic glycolysis. The model showed that the inhibition of the conductance and/or an increase in the voltage sensitivity of a relatively small fraction of VDACs by factors like tubulin potentiate the electrical closure of the remaining free VDACs. The extrusion of calcium ions from the mitochondrial intermembrane space by the generated OMP, positive inside, might increase cancer cell resistance to death. Within the VGC model, the known effect of induction of ATP release from mitochondria by accumulated glucose-6-phosphate in pancreatic beta cells might result not only of the known effect of GK dissociation from the VDAC-GK complex, but also of a decrease in the free energy of glucokinase reaction, leading to the OMP decrease and VDAC opening. We suggest that the VDAC-mediated electrical control of the mitochondrial outer membrane permeability, dependent on metabolic conditions, is a fundamental physiological mechanism of global regulation of mitochondrial functions and of cell death. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatinmore » for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.« less

Actuation of flexoelectric membranes in viscoelastic fluids with applications to outer hair cells

PubMed Central

Herrera-Valencia, E. E.; Rey, Alejandro D.

2014-01-01

Liquid crystal flexoelectric actuation uses an imposed electric field to create membrane bending, and it is used by the outer hair cells (OHCs) located in the inner ear, whose role is to amplify sound through generation of mechanical power. Oscillations in the OHC membranes create periodic viscoelastic flows in the contacting fluid media. A key objective of this work on flexoelectric actuation relevant to OHCs is to find the relations and impact of the electromechanical properties of the membrane, the rheological properties of the viscoelastic media, and the frequency response of the generated mechanical power output. The model developed and used in this work is based on the integration of: (i) the flexoelectric membrane shape equation applied to a circular membrane attached to the inner surface of a circular capillary and (ii) the coupled capillary flow of contacting viscoelastic phases, such that the membrane flexoelectric oscillations drive periodic viscoelastic capillary flows, as in OHCs. By applying the Fourier transform formalism to the governing equation, analytical expressions for the transfer function associated with the curvature and electrical field and for the power dissipation of elastic storage energy were found. PMID:25332388

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.

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.

HISTOLOGY OF GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION: A Multilayer Approach.

PubMed

Li, Miaoling; Huisingh, Carrie; Messinger, Jeffrey; Dolz-Marco, Rosa; Ferrara, Daniela; Freund, K Bailey; Curcio, Christine A

2018-05-03

To systematically characterize histologic features of multiple chorioretinal layers in eyes with geographic atrophy, or complete retinal pigment epithelium (RPE) and outer retinal atrophy, secondary to age-related macular degeneration, including Henle fiber layer and outer nuclear layer; and to compare these changes to those in the underlying RPE-Bruch membrane-choriocapillaris complex and associated extracellular deposits. Geographic atrophy was delimited by the external limiting membrane (ELM) descent towards Bruch membrane. In 13 eyes, histologic phenotypes and/or thicknesses of Henle fiber layer, outer nuclear layer, underlying supporting tissues, and extracellular deposits at four defined locations on the non-atrophic and atrophic sides of the ELM descent were assessed and compared across other tissue layers, with generalized estimating equations and logit models. On the non-atrophic side of the ELM descent, distinct Henle fiber layer and outer nuclear layer became dyslaminated, cone photoreceptor inner segment myoids shortened, photoreceptor nuclei and mitochondria translocated inward, and RPE was dysmorphic. On the atrophic side of the ELM descent, all measures of photoreceptor health declined to zero. Henle fiber layer/outer nuclear layer thickness halved, and only Müller cells remained, in the absence of photoreceptors. Sub-RPE deposits remained, Bruch membrane thinned, and choriocapillaris density decreased. The ELM descent sharply delimits an area of marked gliosis and near-total photoreceptor depletion clinically defined as Geographic atrophy (or outer retinal atrophy), indicating severe and potentially irreversible tissue damage. Degeneration of supporting tissues across this boundary is gradual, consistent with steady age-related change and suggesting that RPE and Müller cells subsequently respond to a threshold of stress. Novel clinical trial endpoints should be sought at age-related macular degeneration stages before intense gliosis and thick deposits impede therapeutic intervention.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

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.

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

PubMed Central

Ruiz, Natividad; Gronenberg, Luisa S.; Kahne, Daniel; Silhavy, Thomas J.

2008-01-01

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM. PMID:18375759

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli.

PubMed

Ruiz, Natividad; Gronenberg, Luisa S; Kahne, Daniel; Silhavy, Thomas J

2008-04-08

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.

Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

PubMed Central

Geller, B. L.; Deere, J. D.; Stein, D. A.; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.

2003-01-01

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)3KC to the end of PMOs. In strains with an intact outer membrane, (KFF)3KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)3KC-PMO conjugate targeted to lacI mRNA induced β-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)3KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce β-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)3KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets. PMID:14506035

A Trojan-Horse Strategy Including a Bacterial Suicide Action for the Efficient Use of a Specific Gram-Positive Antibiotic on Gram-Negative Bacteria.

PubMed

Schalk, Isabelle J

2018-05-10

In the alarming context of rising bacterial antibiotic resistance, there is an urgent need to discover new antibiotics or increase and/or enlarge the activity of those currently in use. The need for new antibiotics is even more urgent in the case of Gram-negative bacteria, such as Acinetobacter, Pseudomonas, and Enterobacteria, which have become resistant to many antibiotics and have an outer membrane with very low permeability to drugs. Vectorization of antibiotics using siderophores may be a solution to bypass such a bacterial wall: the drugs use the iron transporters of the outer membrane as gates to enter bacteria in a Trojan-horse strategy. Designing siderophore-antibiotics that can cross outer membranes has become almost routine, but their transport across the inner membrane is still a limiting step, as well as a strategy that allows dissociation of the antibiotic from the siderophore once inside the bacteria. Liu et al. ( J. Med. Chem. 2018 , DOI: 10.1021/acs.jmedchem.8b00218 ) report the synthesis of a siderophore-cephalosporin compound and demonstrate that β-lactams, such as cephalosporins, can serve as β-lactamase-triggered releasable linkers to allow intracellular delivery of Gram-positive antibiotics to Gram-negative bacteria.

Modeling 3-D deformation of outer hair cells and their production of the active force in the cochlea.

PubMed

Spector, A A; Ameen, M; Schmiedt, R A

2002-10-01

We analyze the deformation of the outer hair cell and its production of active force under physiological conditions. The active force has two components. One results from the strain caused by loading in the organ of Corti in the cochlea and depends on the level of the acoustic signal; the other is related to the intrinsic active properties of the cell membrane. We demonstrate our approach by considering, as a basic model of an outer hair cell in the organ of Corti, a cylindrical shell that is filled with an incompressible fluid and located between two planes that move relative to each other. These planes represent the basilar membrane and tectorial membrane complexes. We show that the deformed state of the cell has a 3-D nature, including bending and twisting components. This is different from the experimental conditions in which the active force is usually measured. We estimate the active force as a function of the relative position of the planes, angle of the cell's inclination, and the cell length.

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.

Sequence of the fhuE outer-membrane receptor gene of Escherichia coli K12 and properties of mutants.

PubMed

Sauer, M; Hantke, K; Braun, V

1990-03-01

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.

Bioinformatic analysis of meningococcal Msf and Opc to inform vaccine antigen design

PubMed Central

Andreae, Clio A.; Sessions, Richard B.; Virji, Mumtaz

2018-01-01

Neisseria meningitidis is an antigenically and genetically variable Gram-negative bacterium and a causative agent of meningococcal meningitis and septicaemia. Meningococci encode many outer membrane proteins, including Opa, Opc, Msf, fHbp and NadA, identified as being involved in colonisation of the host and evasion of the immune response. Although vaccines are available for the prevention of some types of meningococcal disease, none currently offer universal protection. We have used sequences within the Neisseria PubMLST database to determine the variability of msf and opc in 6,500 isolates. In-silico analysis revealed that although opc is highly conserved, it is not present in all isolates, with most isolates in clonal complex ST-11 lacking a functional opc. In comparison, msf is found in all meningococcal isolates, and displays diversity in the N-terminal domain. We identified 20 distinct Msf sequence variants (Msf SV), associated with differences in number of residues within the putative Vn binding motifs. Moreover, we showed distinct correlations with certain Msf SVs and isolates associated with either hyperinvasive lineages or those clonal complexes associated with a carriage state. We have demonstrated differences in Vn binding between three Msf SVs and generated a cross reactive Msf polyclonal antibody. Our study has highlighted the importance of using large datasets to inform vaccine development and provide further information on the antigenic diversity exhibited by N. meningitidis. PMID:29547646

Bioinformatic analysis of meningococcal Msf and Opc to inform vaccine antigen design.

PubMed

Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz; Hill, Darryl J

2018-01-01

Neisseria meningitidis is an antigenically and genetically variable Gram-negative bacterium and a causative agent of meningococcal meningitis and septicaemia. Meningococci encode many outer membrane proteins, including Opa, Opc, Msf, fHbp and NadA, identified as being involved in colonisation of the host and evasion of the immune response. Although vaccines are available for the prevention of some types of meningococcal disease, none currently offer universal protection. We have used sequences within the Neisseria PubMLST database to determine the variability of msf and opc in 6,500 isolates. In-silico analysis revealed that although opc is highly conserved, it is not present in all isolates, with most isolates in clonal complex ST-11 lacking a functional opc. In comparison, msf is found in all meningococcal isolates, and displays diversity in the N-terminal domain. We identified 20 distinct Msf sequence variants (Msf SV), associated with differences in number of residues within the putative Vn binding motifs. Moreover, we showed distinct correlations with certain Msf SVs and isolates associated with either hyperinvasive lineages or those clonal complexes associated with a carriage state. We have demonstrated differences in Vn binding between three Msf SVs and generated a cross reactive Msf polyclonal antibody. Our study has highlighted the importance of using large datasets to inform vaccine development and provide further information on the antigenic diversity exhibited by N. meningitidis.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans

PubMed Central

Holzweber, Markus; Lippitz, Andreas; Krueger, Katharina; Jankowski, Joachim; Unger, Wolfgang E. S.

2015-01-01

The surfaces of polymeric dialyzer membranes consisting of polysulfone and poly-vinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied. PMID:25711334