Biomedical subjects

U B Sleytr

Publications and source records attributed to U B Sleytr.

At least 37 records · Page 2Linked to original sources

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

Structural and functional analyses of the secondary cell wall polymer of Bacillus sphaericus CCM 2177 that serves as an S-layer-specific anchor.

Sacculi of Bacillus sphaericus CCM 2177 contain a secondary cell wall polymer which was completely extracted with 48% hydrofluoric acid. Nuclear magnetic resonance analysis showed that the polymer is composed of repeating units, as follows: -->3)-[4, 6-O-(1-carboxyethylidene)]( approximately 0. 5)-beta-D-ManpNAc-(1-->4)-beta-D-GlcpNAc-(1-->. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.

Bacillus↗

PubMed1998

Self-assembled alpha-hemolysin pores in an S-layer-supported lipid bilayer.

The effects of a supporting proteinaceous surface-layer (S-layer) from Bacillus coagulans E38-66 on a 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) bilayer were investigated. Comparative voltage clamp studies on plain and S-layer supported DPhPC bilayers revealed no significant difference in the capacitance. The conductance of the composite membrane decreased slightly upon recrystallization of the S-layer. Thus, the attached S-layer lattice did not interpenetrate or rupture the DPhPC bilayer. The self-assembly of a pore-forming protein into the S-layer supported lipid bilayer was examined. Staphylococcal alpha-hemolysin formed lytic pores when added to the lipid-exposed side. The assembly was slow compared to unsupported membranes, perhaps due to an altered fluidity of the lipid bilayer. No assembly could be detected upon adding alpha-hemolysin monomers to the S-layer-faced side of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice do not allow passage of alpha-hemolysin monomers through the S-layer pores to the lipid bilayer. In comparison to plain lipid bilayers, the S-layer supported lipid membrane had a decreased tendency to rupture in the presence of alpha-hemolysin.

Bacillus↗

PubMed1998

Voltage clamp studies on S-layer-supported tetraether lipid membranes.

Isolated subunits from the cell surface proteins (S-layer) of Bacillus coagulans E38-66 have been recrystallized on a glycerol dialkyl nonitol tetraether lipid (GDNT)-monolayer and the electrophysical features of this biomimetic membrane have been investigated in comparison to unsupported GDNT-monolayers. The GDNT-monolayer, spread on a Langmuir-Blodgett trough, was clamped with the tip of a glass patch pipette. In order to investigate the barrier function and potential to incorporate functional molecules, voltage-clamp examinations on plain and S-layer-supported GDNT-monolayers were per-formed. Our results indicate the formation of a tight GDNT-monolayer sealing the tip of the glass pipette, and a decrease in conductance of the GDNT-monolayer upon recrystallization of the S-layer protein. Thus, the S-layer protein, apparently, did not penetrate or rupture the lipid monolayer. The valinomycin-mediated increase in conductance was less pronounced for the S-layer-supported than for the plain GDNT-monolayer, indicating differences in the accessibility and/or in the fluidity of the lipid membranes. Furthermore. in contrast to plain GDNT-monolayers. S-layer supported GDNT-monolayers with high valinomycin-mediated conductance persisted over long, periods of time, indicating enhanced stability. These composite S-layer/lipid films may constitute a new tool for electrophysical and electrophysiological studies on membrane-associated and membrane-integrated biomolecules.

Anti-Bacterial Agents↗

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

A novel dipstick developed for rapid Bet v 1-specific IgE detection: recombinant allergen immobilized via a monoclonal antibody to crystalline bacterial cell-surface layers.

The incidence of allergy to airborne proteins derived from tree and grass pollen, feces of mites, spores of molds, and pet dander has been increasing over the last decades. Since precise diagnosis is a prerequisite for successful immunotherapy, there is a rising demand for rapid, reliable, and inexpensive screening methods such as dipstick assays. With the purified recombinant major birch-pollen allergen rBet v 1a as model protein, crystalline bacterial cell-surface layers (S-layers) were tested for their applicability as an immobilization matrix for dipstick development. For this purpose, S-layers were deposited on a mechanically stable microporous support, cross-linked with glutaraldehyde, and free carboxylic acid groups of the S-layer protein were activated with carbodiimide. In the present test system, rBet v 1a was immobilized via the monoclonal mouse antibody BIP 1, which, unlike the allergen, is too large to enter the pores of the S-layer lattice, and which therefore formed a closed monolayer on the outermost surface of the crystal lattice. Moreover, BIP 1 is known to modulate IgE binding to the allergen. After incubation of the dipsticks in serum, washing of the reaction zone under tap water, and binding of an anti-IgE alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium was used as substrate, forming an IgE concentration-dependent colored precipitate on the S-layer surface. The investigation of patient sera previously tested with the CAP system confirmed the specificity of the S-layer-based dipstick assay. Since the dipstick is easy to handle and the whole test procedure takes only 90 min, this test system should be applicable for rapid determination of specific IgE and for first screening in the doctor's practice.

Allergens↗

PubMed1998

Influence of the secondary cell wall polymer on the reassembly, recrystallization, and stability properties of the S-layer protein from Bacillus stearothermophilus PV72/p2.

The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 microg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-L-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.

Bacterial Proteins↗

PubMed1998

Identification of two binding domains, one for peptidoglycan and another for a secondary cell wall polymer, on the N-terminal part of the S-layer protein SbsB from Bacillus stearothermophilus PV72/p2.

First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.

Bacterial Proteins↗

PubMed1998

The S-layer proteins of two Bacillus stearothermophilus wild-type strains are bound via their N-terminal region to a secondary cell wall polymer of identical chemical composition.

Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1gamma chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.

Amino Acid Sequence↗

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

S-layer stabilized solid support lipid bilayers.

This work describes composite structures composed of lipid bilayer or tetraetherlipid monolayer films attached to solid supports with associated crystalline bacterial cell surface layers (S-layers). The bilayer system was established by making use of the strong chemisorption of a first monolayer of thiolipids (1-octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol) on gold and attaching a second monolayer of 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine by the Langmuir Schaefer technique. The tetraetherlipid monolayer was composed of Glycerol-dialkyl-nonitol tetraetherlipid (GDNT). The monolayer of GDNT exhibits the thickness of a bilayer with hydrophilic headgroups on both sides and a hydrophobic inner part. Isolated S-layer protein from Bacillus sphaericus (CCM2177, which was injected into the subphase of an LB-trough, recrystallized into a coherent monolayer at the solid supported phospholipid bilayer and at the tetraehtherlipid monolayer. The composite lipid/S-layer structures were stable enough to allow lifting from the air-water interface, rinsing in water, and transfer into a scanning force microscope.

Bacillus↗

PubMed1997

Advances in S-layer nanotechnology and biomimetics.

Two-dimensional crystalline bacterial S-layers composed of identical protein or glycoprotein subunits turned out to be ideal materials for the development of biomimetic membranes and new approaches in molecular nanotechnology. These isoporous protein lattices have already been used as (i) structure for producing isoporous ultrafiltration membranes with very precisely defined molecular sieving properties, (ii) matrices for immobilizing monolayers of functional molecules, (iii) stabilizing structure for LB-films and liposomes, and (iv) patterning elements in molecular nanotechnology.

Archaea↗

PubMed1997

Bacterial and archaeal S-layer proteins: structure-function relationships and their biotechnological applications.

Crystalline cell surface layers (S-layers) composed of planar assemblies of protein or glycoprotein subunits are one of the most commonly observed cell envelope structures of bacteria and archaea. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at liquid-surface interfaces, including lipid films, on liposomes and on solid supports. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattices are aligned in well-defined positions and orientations. These characteristic features of S-layers have led to various applications in biotechnology, vaccine development, diagnostics, biomimetics and molecular nanotechnology.

Archaea↗

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

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Abortion, Veterinary↗

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↗

PubMed1997

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.

ATP-Binding Cassette Transporters↗