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U B Sleytr

Publications and source records attributed to U B Sleytr.

At least 19 recordsLinked to original sources
PubMed1992

Artificial antigens. Synthetic carbohydrate haptens immobilized on crystalline bacterial surface layer glycoproteins.

The crystalline surface-layer glycoproteins of Clostridium thermohydrosulfuricum L111-69, Bacillus stearothermophilus NRS 2004/3a and Bacillus alvei CCM 2051 were used for immobilization of spacer-linked blood group A-trisaccharide (alpha GalNAc(1----3)[alpha Fuc(1----2)]beta Gal) and of the spacer-linked, tumor-associated T-disaccharide [beta Gal(1----3)alpha GalNAc]. The immobilization involved the glycan portions of surface-layer glycoproteins. Different activation methods were used, namely, periodate oxidation, or treatment with epichlorohydrin or divinyl sulfone, followed by coupling of the hapten under appropriate conditions. The resulting conjugates are useful for assessing the application potential of haptenated surface layer preparations as carrier/adjuvants for the induction of immunity to poorly immunogenic molecules.

Animals↗
PubMed1992

Structure of the glycan chain from the surface layer glycoprotein of Clostridium thermohydrosulfuricum L77-66.

The thermophilic eubacterium Clostridium thermohydrosulfuricum L77-66 is covered by a crystalline surface layer composed of identical glycoprotein subunits which are arranged in a hexagonal lattice with centre-to-centre spacings of approx. 14.3 nm. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cell wall preparations showed the presence of several broadened, carbohydrate-containing bands in a molecular mass range of 90 to 200 kDa. A total carbohydrate content of approx. 14% was determined in the purified surface layer glycoprotein. Chemical deglycosylation of this material by trifluoromethanesulfonic acid resulted in the disappearance of the complex banding pattern. Only a single band with a molecular mass of 82 kDa remained visible upon Coomassie staining. After proteolytic digestion of the surface layer glycoprotein a single glycopeptide fraction with an apparent molecular mass of approx. 25 kDa was obtained by gel filtration. Composition analysis, methylation, periodate oxidation and a combination of homonuclear and 1H-detected heteronuclear shift-correlated nuclear magnetic resonance experiments established the following structure for the glycan chain of the surface layer glycoprotein.

Bacterial Outer Membrane Proteins↗
PubMed1992

Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein from Bacillus stearothermophilus PV72 and its application as an immobilization matrix.

The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of beta-D-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2'-dipyridyldisulphide, 550 micrograms beta-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one beta-D-galactosidase molecule [relative molecular mass (M(r)), 116,000] per two S-layer subunits (M(r), 130,000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies beta-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.

Cell Wall↗
PubMed1992

Scanning force microscopy studies of the S-layers from Bacillus coagulans E38-66, Bacillus sphaericus CCM2177 and of an antibody binding process.

In many prokaryotic cells (eubacteria and archaebacteria) the outermost cell envelope component is composed of a regularly structured protein surface layer (S-layer). The two-dimensional S-layer from Bacillus coagulans E38-66 and Bacillus sphaericus CCM2177 has been investigated by SFM at molecular resolution under physiological conditions (i.e., in buffer solution). We find the E38-66 S-layer lattice to be oblique with lattice parameters of a = 9-10 nm, b = 7-8 nm and gamma = 80 degrees -90 degrees (E38-66). The CCM2177 lattice is square with a = 12-14 nm, in good agreement with TEM data. We have used the unique possibility of the SFM to study the kinematics of biological processes and have performed experiments on the adhesion of polyclonal antibodies to the recrystallized E38-66 protein layer on a time scale of about two to ten seconds per image frame. This represents a first step in directly visualizing molecular recognition reactions.

Antibodies, Bacterial↗
PubMed1992

Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66.

We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.

Adsorption↗
PubMed1992

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.

Cell Wall↗
PubMed1992

Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70.

The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain. Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences. Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions. These are composed of the same hexasaccharide portion but are linked to oligopeptides of different length. One of them contains only a single amino acid. As concluded from chemical analyses and proton and carbon nuclear magnetic resonance spectroscopy of this preparation, the hexasaccharide moiety is linked via a novel O-glycosidic linkage. This is a beta-D-glucose residue linked to the phenolic hydroxyl group of tyrosine in intact S-layer glycoprotein.

Bacterial Outer Membrane Proteins↗
PubMed1991

Influence of an S-layer on surface properties of Bacillus stearothermophilus.

Various aspects of surface properties of the S-layer-carrying Bacillus stearothermophilus PV72 and of an S-layer-deficient mutant (strain PV72/T5) have been tested by adsorption assays on solid surfaces, electrostatic interaction chromatography and hydrophobic interaction chromatography. The adsorption assays have shown that cell adhesion of the S-layer-carrying strain was less influenced by environmental changes than it was with the S-layer-deficient mutant. Electrostatic interaction chromatography indicated that both strains have positively and negatively charged groups exposed on the cell surface but the S-layer-carrying strain reveals more positively charged groups than does the S-layer-deficient mutant. Hydrophobic interaction chromatography showed that both strains have a hydrophilic surface but that the hydrophilic properties are more pronounced with the strain lacking an S-layer.

Bacterial Adhesion↗
PubMed1991

Bacterial surface layer glycoproteins.

Crystalline cell surface layers (S-layers) are ubiquitously present in bacterial species from almost all phylogenetic branches. Recent investigations have shown that the S-layer proteins of many archaebacteria and eubacteria contain covalently linked carbohydrate chains. This evidence clearly shows that the ability for protein glycosylation is present as a common biosynthetic pathway in prokaryotic organisms.

Bacteria↗
PubMed1991

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense.

Thin sections, freeze-etched, and negatively stained preparations of Methanocorpusculum sinense cells reveal a highly lobed cell structure with a hexagonally arranged surface layer (S layer). Digital image processing of negatively stained envelope fragments show that the S layer forms a porous but strongly interconnected network. Since the S layer is the exclusive cell envelope component outside the cytoplasmic membrane it must have a cell shape determining and maintaining function. Although lattice faults such as disclinations and dislocations are a geometrical necessity on the surface of a closed protein crystal, our data indicate that they also play important roles as sites for the incorporation of new morphological units, in the formation of the lobed cell structure, and in the cell division process. In freeze-etched preparations of intact cells numerous positive and negative 60 degree wedge disclinations can be detected which form pentagons and heptagons in the hexagonal array. Complementary pairs of pentagons and heptagons are the termination points of edge dislocations. They can be expected to function both as sites for incorporation of new morphological units into the lattice and as initiation points for the cell division process. The latter is determined by the ratio between the increase of protoplast volume and the increase in actual S-layer surface area during cell growth. We postulate that this mode of cell fission represents a common feature in lobed archaebacteria which possess an S layer as the exclusive wall component.

Archaea↗
PubMed1991

Structure of the glycan chain from the surface layer glycoprotein of Bacillus alvei CCM 2051.

The cell surface of the mesophilic eubacterium Bacillus alvei CCM 2051 is covered by an oblique arranged surface layer glycoprotein. The subunits revealed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis were distinct bands of molecular masses 140,000, 128,000, and 127,000. Proteolytic degradation of the purified S-layer glycoprotein yielded a single glycopeptide fraction with an apparent molecular mass of ca. 25,000. Methylation analysis in conjunction with two-dimensional nuclear magnetic resonance experiments at 500 MHz established the branched trisaccharide (formula; see text) as the repeating unit for this glycan chain.

Bacillus↗
PubMed1978

Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures.

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.

Cell Membrane↗