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P Messner

Publications and source records attributed to P Messner.

At least 37 records · Page 2Linked to original sources
PubMed1999

Complete glycan structure of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus GS4-97.

Isolate GS4-97 was purified from an extraction juice sample of an Austrian beet sugar factory and affiliated to the newly described species Aneurinibacillus thermoaerophilus. It is closely related to the type strain of this species, A.thermoaerophilus L420-91(T), and possesses a square surface layer (S-layer) array composed of identical glycoprotein monomers as its outermost cell envelope component. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified S-layer showed an apparent molecular mass of approximately 109,000. After thorough proteolytic degradation of this material by pronase E and purification of the reaction mixture by gel permeation, chromatofocusing, and reversed-phase chromatography, a homogeneous glycopeptide fraction was obtained which was subjected to one- and two-dimensional nuclear magnetic resonance spectroscopy. The combined chemical and spectroscopic evidence, together with N-terminal sequencing, suggest the following structure of the O-glycosidically linked S-layer glycan chain of the glycopeptide: This is the first description of a beta-d-GalNAc-Thr linkage in glycoproteins.

Bacterial Proteins↗
PubMed1999

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.

Bacillus↗
PubMed1999

Thermococcus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent.

A novel barophilic, hyperthermophilic, anaerobic sulfur-metabolizing archaeon, strain MPT (T = type strain), was isolated from a hydrothermal vent site (Snakepit) on the Mid-Atlantic Ridge (depth, 3550 m). Enrichments and isolation were done under 40 MPa hydrostatic pressure at 95 degrees C. Strain MPT was barophilic at 75, 80, 85, 90, 95 and 98 degrees C, and was an obligate barophile between 95 and 100 degrees C (Tmax). For growth above 95 degrees C, a pressure of 15.0-17.5 MPa was required. The strain grew at 48-95 degrees C under atmospheric pressure. The optimal temperature for growth was 85 degrees C at both high (40 MPa) and low (0.3 MPa) pressures. The growth rate was twofold higher at 85 degrees C under in situ hydrostatic pressure compared to at low pressure. Strain MPT cells were motile, coccoid, 0.8-2.0 microns in diameter and covered by a hexagonal S-layer lattice. The optimum pH and NaCl concentration for growth at low pressure were 7.0 and 20-30 g l-1, respectively. The new isolate was an obligate heterotroph and utilized yeast extract, beef extract and peptone for growth. Growth was optimal in the presence of elemental sulfur. Rifampicin and chloramphenicol inhibited growth. The core lipids consisted of a major archaeol and a complex lipid pattern consisting of a major phospholipid. The DNA G + C content was 37.1 mol%. Sequencing of the 16S rRNA gene revealed that strain MPT belonged to the genus Thermococcus and it is proposed that this isolate should be designated as a new species, Thermococcus barophilus.

Atlantic Ocean↗
PubMed1999

Methanococcus vulcanius sp. nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp. DSM 4213T as Methanococcus fervens sp. nov.

An autotrophic, hyperthermophilic methanogen (M7T) was isolated from a deep-sea hydrothermal chimney sample collected on the East Pacific Rise at a depth of 2600 m. The coccoid-shaped cells are flagellated and exhibit a slight tumbling motility. The temperature range for growth at pH 6.5 was 49-89 degrees C, with optimum growth at 80 degrees C. The optimum pH for growth was 6.5, and the optimum NaCl concentration for growth was around 25 g l-1. The new isolate used H2 and CO2 as the only substrates for growth and methane production. Tungsten, selenium and yeast extract stimulated growth significantly. In the presence of CO2 and H2, the organism reduced elemental sulphur to hydrogen sulphide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin and streptomycin. The G + C content of the genomic DNA was 31 mol%. As determined by 16S rDNA gene sequence analysis, this organism was closely related to Methanococcus jannaschii strain JAL-1T. However, despite the high percentage of similarity between their 16S rDNA sequences (97.1%), the DNA-DNA hybridization levels between these strains were less than 5%. On the basis of these observations and physiological traits, it is proposed that this organism should be placed in a new species, Methanococcus vulcanius. The type strain is M7T (= DSM 12094T). During the course of this study, the 16S rDNA sequence analysis placed Methanococcus sp. strain AG86T (= DSM 4213T) as a close relative of M. jannaschii strain JAL-1T. However, the weak level of DNA-DNA hybridization with this strain (< 10%) allowed the proposal that strain AG86T also constitutes a new species, Methanococcus fervens.

Base Composition↗
PubMed1999

Purification, crystallization and preliminary structural studies of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC), the third enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 A. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 A and alpha = beta = 90, gamma = 120 degrees.

Carbohydrate Epimerases↗
PubMed1999

Overexpression, purification, crystallization and preliminary structural study of dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD), the fourth enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from -Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two--step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.

Carbohydrate Dehydrogenases↗
PubMed1999

[Mid-term prognostic value of plasma heavy-chain myosin in thrombolysed myocardial infarction].

Plasma myosin heavy chain assay, which can be easily performed during the acute phase of myocardial infarction, is a recent method allowing quantitative assessment of the extent of infarction. However, to our knowledge, its prognostic value has not been studied in contrast with serum myosin light chain assay. We monitored the state of health of 40 patients (including 37 men with a mean age of 56 years) for two years after a first myocardial infarction, thrombolized during the acute phase. Their survival (mortality) and the development of "cardiac events" (MI, angina, sudden death, etc.) were evaluated at 2 years. The results observed at 2 years were correlated with the initial plasma myosin assay results and other direct and indirect methods of assessment of the extent of infarction, performed during the acute phase of myocardial infarction (cardiac enzymes, contrast angiography). The main result of this study is the demonstration that an unusual plasma myosin release kinetic (complex appearance) is predictive for the medium-term development of heart failure (p = 0.04) and/or destabilization of coronary insufficiency (p = 0.02). These results need to be emphasized, as with only 5 serum myosin assays performed over a 10-day period, it seems possible to identify a group of patients at high risk of medium-term complications, who possess a complex release kinetic during the acute phase of myocardial infarction and a value for area under the curve greater than 10.470 microliters U/L (cut-off value, p = 0.043).

Acute Disease↗
PubMed1998

Desulfurobacterium thermolithotrophum gen. nov., sp. nov., a novel autotrophic, sulphur-reducing bacterium isolated from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, strictly autotrophic, sulphur-reducing bacterium, designated BSAT (T = type strain), was isolated from a deep-sea hydrothermal chimney sample collected at the mid-Atlantic ridge. Gram-negative cells occurred singly or in pairs as small highly motile rods. Spores were not observed. The temperature range for growth was 40 to 75 degrees C, with an optimum at 70 degrees C. The pH range for growth at 70 degrees C was from 4.4 to 7.5, with an optimum around 6.0. The sea salt concentration range for growth was 15-70 gI(-1) with an optimum at 35 gI(-1). Elemental sulphur, thiosulphate and sulphite were reduced to hydrogen sulphide. Sulphate and cystine were not reduced. The G+C content of the genomic DNA was 35 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the strain was a member of the domain Bacteria and formed a branch that was almost equidistant from members of the orders Aquificales and Thermotogales. The new organism possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Desulfurobacterium gen. nov., of which Desulfurobacterium thermolithotrophum sp. nov. is the type species. The type strain is BSAT (= DMS 11699T).

Base Sequence↗
PubMed1998

Methanococcus infernus sp. nov., a novel hyperthermophilic lithotrophic methanogen isolated from a deep-sea hydrothermal vent.

An autotrophic, extremely thermophilic methanogen (ME(T)) was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge at a depth of 3000 m. The heavily flagellated cells are motile and coccoid shaped. The new strain growths between 55 and 91 degrees C, with an optimum growth temperature at 85 degree C. The optimum pH for growth is 6.5, and the optimum sea salt concentration for growth is around 25 g l-1. The organism uses H2 and CO2 as the only substrate for growth and methane production. Tungsten, selenium and yeast extract stimulate growth significantly. In the presence of CO2 and H2, the organism reduces elemental sulphur to hydrogen sulphide. The G+C content of the genomic DNA is 33 mol%. As determined by 16S gene sequence analysis, this organism is closely related to Methanococcus jannaschii strain JAL-1T. However, no significant homology was observed between them with DNA-DNA hybridization. It is proposed that this organism should be placed in a new species, Methanococcus infernus. The type strain is ME(T) (= DSM 11812T).

Anti-Bacterial Agents↗
PubMed1998

Methanoculleus palmolei sp. nov., an irregularly coccoid methanogen from an anaerobic digester treating wastewater of a palm oil plant in north-Sumatra, Indonesia.

Strain INSLUZT (= DSM 4273T) was isolated from a biogas-producing bioreactor treating wastewater of a palm oil mill on North-Sumatra (Indonesia). Cells of strain INSLUZT were highly irregularly coccoid, 1.25-2.0 microns in diameter, had a cell envelope consisting of the cytoplasmic membrane and an S-layer of hexagonally arranged glycoprotein subunits with an M(r) of 120,000, and were flagellated (motility was not observed). Cells were mesophilic and grew most rapidly at 40 degrees C on H2/CO2' formate, 2-propanol/CO2 2-butanol/CO2 and cyclopentanol/CO2 to give methane. Tungstate promoted growth on H2/CO2 with acetate as the solely required organic medium supplement. The G + C content of DNA was 59 mol% (Tm method) and 59.5 mol% (HPLC method). 16S rDNA analysis revealed a phylogenetic relationship to Methanoculleus species; the name Methanoculleus palmolei sp. nov. is therefore proposed for strain INSLUZT (= DSM 4273T).

Anaerobiosis↗
PubMed1998

S-layered Aneurinibacillus and Bacillus spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles.

When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.

Bacillus↗
PubMed1997

Bacterial glycoproteins.

Glycoproteins are a diverse group of complex macromolecules that are present in virtually all forms of life. Their presence in prokaryotes, however, has been demonstrated, and accepted, only recently. Bacterial glycoproteins have been identified in many archaeobacteria and in eubacteria. They comprise a wide range of different cell envelope components such as membrane-associated glycoproteins, surface-associated glycoproteins and crystalline surface layers (S-layers), as well as secreted glycoproteins and exoenzymes. Even their occurrence in the cytoplasm cannot yet be ruled out. This minireview tries to cover the whole subject as completely as possible and refers to available information on presence, structure, biosynthesis, and molecular biology of bacterial glycoproteins.

Bacterial Outer Membrane Proteins↗
PubMed1997

Bet v 1, the major birch pollen allergen, conjugated to crystalline bacterial cell surface proteins, expands allergen-specific T cells of the Th1/Th0 phenotype in vitro by induction of IL-12.

Modulation of allergic immune responses by using adequate adjuvants is a promising concept for future immunotherapy of type I hypersensitivity. In the present study, recombinant Bet v 1 (rBet v 1, the major birch pollen allergen) was conjugated to cross-linked crystalline surface layer proteins (SL) derived from Gram-positive eubacteria. T cell lines (TCL) and clones (TCC) were established from peripheral blood of birch pollen-allergic patients. TCL and TCC were induced either using rBet v 1 alone or rBet v 1/SL conjugates (rBet v 1/SL) as initial antigen stimulus. Cytokine production after re-stimulation with rBet v 1 was investigated. TCL initiated with rBet v 1/SL showed significantly increased IFN-gamma production as compared to rBet v 1 -selected TCL. TCC were established from TCL of five patients. As expected, the majority of CD4+ TCC induced by rBet v 1 (55%) displayed a Th2-like pattern of cytokine production. However, only 21% of Bet v 1-specific TCC isolated from TCL established with the Bet v 1/SL revealed this phenotype. The majority of SL-specific TCC (80%) belonged to the Th1 phenotype. In cultures of peripheral blood mononuclear cells, both, SL and Bet v 1/SL (but not rBet v 1) stimulated the production of high levels of IL-12, a pivotal mediator of Th1 responses. Moreover, stimulation of rBet v 1-induced TCC with rBet v 1/SL led to an increased IFN-gamma production. This effect could be reversed by neutralizing anti-IL-12 mAb. Together these results indicate an adjuvant effect of SL mediated by IL-12. Our results indicate that bacterial components, such as SL, displaying adjuvant effects may be suitable for immunotherapeutical vaccines for type I allergy.

Adjuvants, Immunologic↗
PubMed1997

Biochemistry of S-layers.

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.

Archaea↗
PubMed1997

Applications of S-layers.

The wealth of information existing on the general principle of S-layers has revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from many organisms are capable of recrystallizing as closed monolayers onto solid supports at the air-water interface, on lipid monolayers or onto the surface of liposomes. Particularly their repetitive physicochemical properties down to the subnanometer scale make S-layers unique structures for functionalization of surfaces and interfaces down to the ultimate resolution limit. The following review focuses on selected applications in biotechnology, diagnostics, vaccine development, biomimetic membranes, supramolecular engineering and nanotechnology. Despite progress in the characterization of S-layers and the exploitation of S-layers for the applications described in this chapter, it is clear that the field lags behind others (e.g. enzyme engineering) in applying recent advances in protein engineering. Genetic modification and targeted chemical modification would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein. The application of protein engineering to S-layers will require the development of straightforward expression systems, the development of simple assays for assembly and function that are suitable for the rapid screening of numerous mutants and the acquisition of structural information at atomic resolution. Attention should be given to these areas in the coming years.

Bacteria↗
PubMed1996

Toward selective elicitation of TH1-controlled vaccination responses: vaccine applications of bacterial surface layer proteins.

Bacterial surface layer proteins have been utilized as combined vaccine carrier/adjuvants and offer a number of advantages in these applications. The crystalline protein arrays contain functional groups in precisely defined orientations for coupling of haptens. Conventional applications of S-layer vaccines do not cause observable trauma or side effects. Depending on the nature of the S-layer preparations, antigenic conjugates will induce immune responses of a predominantly cellular or predominantly humoral nature. Immune responses to S-layer-hapten conjugates are also observed following oral/nasal application. In the present contribution, the status of investigations with S-layer conjugates in three main immunological projects is reviewed. In a project aimed at immunotherapy of cancer, conjugates of S-layer with small, tumor-associated oligosaccharides have been found to elicit hapten-specific DTH responses. An enlarged program of chemical synthesis has now been initiated to prepare a complete set of mucin-derived, tumor-associated oligosaccharides and their chemically modified analogues for elicitation of cell-mediated immune responses to certain tumors in humans. In another application, oligosaccharides derived from capsules of Streptococcus pneumoniae type 8 have been linked to S-layer proteins and have been found to elicit protective antibody responses in animals. Most recently, allergen S-layer conjugates have been prepared with the intention to suppress the TH2-directed, IgE-mediated allergic responses to Bet nu 1, the major allergen of birch pollen. In the former two applications, the S-layer vaccine technology appears to offer the versatility needed to direct vaccination responses toward predominant control by TH1 or TH2 lymphocytes to meet the different therapeutic or prophylactic requirements in each case. In the third application, work has progressed to a preliminary stage only.

Adjuvants, Immunologic↗
PubMed1996

Immunoreactivity of allergen (Bet v 1) conjugated to crystalline bacterial cell surface layers (S-layers).

BACKGROUND: Crystalline cell surface layers (S-layers) from Gram-positive eubacteria had been demonstrated as carrier/adjuvants for chemically synthesized tumor-associated oligosaccharide haptens and capsular polysaccharide antigens of Streptococcus pneumoniae strains. OBJECTIVES: The applicability of S-layers as vaccine carrier for treatment of Type I allergy was investigated. STUDY DESIGN: Native or cross-linked S-layer self-assembly products and cell wall preparations from Bacillus sphaericus CCM 2177 and Thermoanaerobacter thermohydrosulfuricus L111-69 and L110-69 were used for immobilization of recombinant major birch pollen allergen Bet v 1. RESULTS AND CONCLUSIONS: Depending on the carrier used, amounts of approximately 20-40 micrograms allergen per mg conjugate could be immobilized. By application of L-glutamic acid dimethyl ester as a spacer, this value could be increased approximately 10-fold. The functionality of the rBet v 1-conjugates was assessed in immunological systems. (i) The presence of intact B-cell epitopes was demonstrated in inhibition experiments using human Bet v 1-specific IgE. (ii) The rBet v 1-S-layer conjugates were immunogenic in mice. (iii) The proliferation of rBet v 1-specific T-cell clones suggested that the peptides created by processing of immobilized Bet v 1 were similar to those derived from natural allergen. (iv) Stimulation of human allergen-specific TH2 lymphocytes with S-layer-conjugated Bet v 1 led to a modulation of the cytokine production pattern from TH2 to TH0/TH1. This study indicates that S-layers may be suitable carriers for few immunotherapeutical vaccines for Type 1 hypersensitivity.

Adjuvants, Immunologic↗