Crystalline Surface Layer Research Articles

Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications

Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall. These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria. In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan–peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure. In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier. An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum. These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents. All these data address new aspects regarding cell wall permeability towards macromolecules and aminoacids but also open new opportunities for biotechnology applications.

Characterisation of implanted surface layers in ion-thinned semiconductors by transmission electron microscopy

Transmission electron microscopy (TEM) rocking curves diffracted from inclined planes in argon ion-thinned Si and III–V semiconductors display a significant asymmetry around the Bragg condition. The simulation of the rocking curves shows that such an unexpected asymmetry can be attributed to the dilation of coherent crystalline surface layers implanted with argon atoms. The dark-field rocking curve asymmetry is shown to be directly related to the density of implanted argon atoms in the surface layers. Calculations also show that the rocking curve asymmetry is only observed for intermediate values of strain in the layer of a few percent, which corresponds to argon concentrations of a few at.% in the case of silicon or III–V semiconductors. This type of analysis is also relevant for the study of the strain state and chemical composition of coherently strained epitaxial layers.

Three-dimensional structuring of sapphire by sequential He + ion-beam implantation and wet chemical etching

We present a method for the selective two- and three-dimensional patterning of sapphire using light ion-beam implantation to generate severe lattice damage to depths exceeding 1 μm and subsequent selective wet chemical etching of the damaged regions by hot H3PO4. C-cut sapphire crystals were implanted through contact masks using ion fluences of 1×1016 to 5×1017 He+/cm2 and energies up to 400 keV. The etching process is characterized by a high selectivity and a rate of approximately 19 nm/min. Whereas an implantation that produces a continuously damaged pathway results in complete etching from the surface, sole in-depth implantation using only high-energy ions leads to under-etching of the crystalline surface layer. By a combination of these processes we have fabricated three-dimensional structures such as channels and bridges in sapphire.

Industrial solidification processes in polybutene‐1. Part II—influence of shear flow

AbstractFollowing some early rudimentary results on shear‐induced crystallization of polybutene‐1 (1, 2), the present paper contains more detailed results. In the course of this work the origins of the highly oriented crystalline surface layers, as well known from injection molded samples, are more closely investigated. For the purpose, a special extrusion experiment is used, in which melts of various degrees of undercooling are moved through a duct of large aspect ratio. When, after the release of the pressure at the die entry, a quench of the duct to a temperature far below the melting point is delayed, a relaxation phenomenon is observed, in accordance with the experiences with i‐PP. From these experiments one learns that the leading parameter of the process is something like the mechanical work done per unit volume, and that the relaxation time increases with decreasing temperature much faster than the viscosity of the melt. The results are qualitatively in excellent agreement with our previously obtained results for polypropylene.

Interpretation of unexpected rocking curve asymmetry in LACBED patterns of semiconductors

Transmission electron microscopy rocking curves diffracted from inclined planes in argon ion-thinned Si and III–V semiconductors display a significant asymmetry around the Bragg condition. Kinematical and dynamical calculations of the rocking curves show that such an asymmetry can be attributed to the dilation of coherent crystalline surface layers implanted with argon atoms. The surface layers are characterized with two parameters, their width h and the strain component ε z normal to the thin foil plane. The dark-field rocking curve asymmetry is shown, for sufficiently high values of h (>∼2 nm), to strongly depend on ε z which is directly related to the density of implanted argon atoms in the surface layers. Calculations also show that a significant rocking curve asymmetry is only observed for intermediate values of ε z of about a few percents.

Microstructure and properties of crystallized melt-spun Ti50Ni25Cu25 ribbons after current-driven thermal cycling

The influence of thermal cycling driven by electrical current under a small and constant applied stress of 50 MPa on martensitic transformation, microstructure and electrical resistance of crystallized melt-spun ribbons of a Ti 50 Ni 25 Cu 25 (at.%) shape memory alloy is investigated. Cycled specimens and uncycled ones as a reference are characterized by various methods like differential scanning calorimetry, X-ray diffraction, transmission electron microscopy, and electrical resistance. After thermal cycling DSC curves during cooling exhibit two transformation peaks whereas the reverse transformation proceeds in only one step. The separation of the two peaks and also their maximum remains stable during cycling but the peak shapes become broader. The reason for the two-stage transformation is found in a crystalline surface layer already present in the as-spun state. A heat treatment between 700°C and 800°C for 600s is able to recrystallize the surface layer. Only minor changes of the microstructure due to current-driven thermal cycling could be observed. The average grain size is about 500 nm in both cycled and uncycled specimens. A few plate-like precipitates are present created during the crystallization process. Twins are frequently observed. Dislocations, however, are never observed not even in the cycled specimens.

Development of affinity microparticles for extracorporeal blood purification based on crystalline bacterial cell surface proteins.

In this article, the development of specific adsorbents for extracorporeal blood purification are described. Affinity microparticles were prepared by linking Protein A to crystalline cell surface layers (S-layers) from Thermoanaerobacter thermohydrosulfuricus 1111-69. S-layers were used in the form of cell wall fragments obtained by breaking whole cells by ultrasonification, resulting in cup-shaped structures (average size 0.5 x 1 microm) completely covered with S-layer protein. Protein A was covalently bound to carboxylic acid groups of the S-layer protein after activation with 1-ethyl-3,3'(dimethylamino)propylcarbodiimide. In batch adsorption experiments with fresh frozen human plasma, the resulting S-layer based affinity microparticles showed a high adsorption capacity for IgG (40 mg IgG were bound per g wet pellet of S-layer based affinity microparticles). Fractions eluted from the microparticles were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contained only IgG demonstrating that adsorption was specific. In biocompatibility tests, preparations of the S-layer microparticles showed no low-density lipoprotein-reactivity, no cytotoxicity, and no cytokine inducing activity.

Crystalline layer on the surface of Zr-based bulk metallic glasses

The structure, stability and microstructure of Zr 65Cu 17.5Ni 10Al 7.5 and Zr 50Cu 18Ni 17Ti 5Al 10 bulk amorphous alloys was studied by X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. A thin crystalline surface layer was observed on both bulk metallic glasses. This layer consists of zirconium carbide and some crystalline phases. The thickness of the layer varies between 5 and 10 μm, it depends on the location in the sample. The origin of the formation of the crystalline layer is discussed. The formation of the crystalline layer is connected with carbon deposition on the sample surface during the production of the metallic glass, and with select carbide formation. The resulting change of the concentration of the alloy components near the surface due to the carbide formation leads to a shift of the alloy composition from the amorphization region and results in further crystallization of near surface regions.

Electron-Beam Induced Formation of Highly Ordered Palladium and Platinum Nanoparticle Arrays on the S Layer of Bacillus sphaericus NCTC 9602

Regular arrays of metallic nanoparticles are formed by these authors using a novel technique involving electron-beam induced cluster formation in the transmission electron microscope (TEM). Nucleation is initiated in the cavities between crystalline bacterial surface layers deposited on a solid support, as schematically illustrated in the Figure.

On the origin of the two-stage behavior of the martensitic transformation of Ti 50Ni 25Cu 25 shape memory melt-spun ribbons

The observation of two successive stages in the martensitic transformation of Ti 50Ni 25Cu 25 shape memory melt-spun ribbons after long-term annealing was formerly reported (H. Rösner, A.V. Shelyakov, A.M. Glezer, K. Feit, P. Schloßmacher, Mater. Sci. Eng. A273–275 (1999) 733). Microstructural investigations revealed the formation of thin plate-like precipitates. It was concluded that the two-stage transformation behavior is governed by the precipitation of these thin plates. Recent experiments are presented which now show that a change of transformation behavior can occur in the course of isothermal heat treatments owing to a crystalline surface layer in the as-spun state of melt-spun ribbons. The previous interpretation of the two-stage transformation behavior considering the influence of plate-like precipitates is corrected in this communication.

Envelope structure of Synechococcus sp. WH8113, a nonflagellated swimming cyanobacterium

BackgroundMany bacteria swim by rotating helical flagellar filaments [1]. Waterbury et al. [15] discovered an exception, strains of the cyanobacterium Synechococcus that swim without flagella or visible changes in shape. Other species of cyanobacteria glide on surfaces [2,7]. The hypothesis that Synechococcus might swim using traveling surface waves [6,13] prompted this investigation.ResultsUsing quick-freeze electron microscopy, we have identified a crystalline surface layer that encloses the outer membrane of the motile strain Synechococcus sp. WH8113, the components of which are arranged in a rhomboid lattice. Spicules emerge in profusion from the layer and extend up to 150 nm into the surrounding fluid. These spicules also send extensions inwards to the inner cell membrane where motility is powered by an ion-motive force [17].ConclusionThe envelope structure of Synechococcus sp. WH8113 provides new constraints on its motile mechanism. The spicules are well positioned to transduce energy at the cell membrane into mechanical work at the cell surface. One model is that an unidentified motor embedded in the cell membrane utilizes the spicules as oars to generate a traveling wave external to the surface layer in the manner of ciliated eukaryotes.

Core Losses and Soft Magnetic Properties of Nanocrystalline Fe-Zr-Nb-B Alloys with Zero-Magnetostriction

AbstractThe structure, the soft magnetic properties and the core losses for Fe-Zr-Nb-B(-Cu) nanocrystalline alloys were investigated. It has been already reported that the typical ternary nanocrystalline Fe90Zr7B3 and Fe84Nb7B9 alloys exhibit good soft magnetic properties and a small negative and a small positive magnetostriction (λs), respectively. The soft magnetic properties of the nanocrystalline Fe84–90(Zr, Nb)7B3–9 quaternary alloys with mixed compositions of Fe90Zr7B3 and Fe84Nb7B9 cannot be improved whereas very small magnetostrictions are obtained. The quaternary alloys with 7 at% (Zr + Nb) have structures of an amorphous with α-Fe phases on the free and roll-contacted surfaces at an as-quenched state. The crystalline surface layers deteriorate the soft magnetic properties at a crystallized state. The high saturation magnetic induction of 1.64 T, the high permeability of 60,000 at 1 kHz and the very low core loss of 0.9 W/kg at 1.4 T and 50 Hz are obtained for the Fe85.5(Zr1/3Nb2/3)6B8.5 alloy containing 6 at% of (Zr + Nb) content with nearly zero-λs produced by crystallizing the single amorphous phase without the crystalline surface layers. The thermal stability of the core loss of the quaternary alloy is significantly higher than that of the Fe78Si9B13 amorphous alloy. The crystalline surface layers of the Fe84–90(Zr, Nb)7B3–9 quaternary alloys disappear by 1 at% Cu addition, which results in significant improvement of the soft magnetic properties at a crystallized state.

Fabrication of highly oriented nanocluster arrays by biomolecular templating

The formation of highly oriented cluster arrays by chemical deposition of platinum onto a regular 2D protein template is studied. The crystalline bacterial surface layer (S layer) of Sporosarcina ureae exhibiting a p4 symmetry with a lattice spacing of 13.2 nm is used as the protein template. Cluster deposition onto the S layer is achieved by chemical reduction of a platinum salt. The growth kinetics of the clusters has been examined by UV–VIS spectrometry. Transmission electron microscopy reveals the formation of well separated metal clusters with an average diameter of 1.9±0.6 nm. The lateral cluster arrangement is defined by the underlying protein crystal. We observe 7 cluster sites per unit cell corresponding to a density of 4×1012 cm-2. They are located in the nano-sized pores and gaps of the crystalline S layer.

Stabilizing effect of an S-layer on liposomes towards thermal or mechanical stress

Isolated subunits of the crystalline cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were recrystallized on positively charged unilamellar liposomes. Liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine (HDA) in a molar ratio of 10:5:4 and they were prepared by the dehydration–rehydration method followed by an extrusion procedure. The S-layer protein to DPPC ratio was 5.7 nmol/μmol which approximately corresponds to the theoretical value estimated by using the areas occupied by the S-layer lattice and the lipid membrane. Coating of the positively charged liposomes with S-layer protein resulted in inversion of the ζ-potential from +29.1 mV to −27.1 mV. Covalent crosslinking of the recrystallized S-layer protein was achieved with glutaraldehyde. Chemical analysis revealed that almost all amino groups (>95%) from HDA in the liposomal membrane were involved in the reaction. To study the influence of an S-layer lattice on the stability of the liposomes, the hydrophilic marker carboxyfluoresceine (CF) was encapsulated and its release was determined for plain and S-layer-coated liposomes in the course of mechanical and thermal challenges. In comparison to plain liposomes, S-layer-coated liposomes released only half the amount of enclosed CF upon exposure to shear forces or ultrasonication as mechanical stress factors. Furthermore, temperature shifts from 25°C to 55°C and vice versa induced considerably less CF release from S-layer-coated than from plain liposomes. A similar stabilizing effect of the S-layer lattice was observed after glutaraldehyde treatment of plain and S-layer-coated liposomes.

Interaction of crystalline bacterial cell surface proteins with lipid bilayers in liposomes. A sound velocity study

Sound velocity and density measurements have been used to study physical properties of plain unilamellar liposomes and covered by a crystalline cell surface layer (S-layer) of Bacillus coagulans, strain E38-66. The liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine. The measured parameters — velocity number [ ul and density ρ — decrease with increasing temperature for both suspensions of liposomes with S-layers (S-DPPC) and without S-layers. The values of [ u] at T<29°C are smaller, and at T>29°C larger for S-DPPC than those of plain liposomes. The density of the S-DPPC system was slightly greater than those of plain bilayer suspensions. The specific adiabatic compressibility, ϕ k/ β 0, calculated from the values of [ u] and of the specific volume, ϕ k, increases with temperature for both liposome systems. The comparison of the parameter ϕ k/ β 0 for S-DPPC and DPPC indicates that the adiabatic compressibility of S-DPPC at T<20°C was higher and at T>20°C was lower in comparison with that of plain DPPC. This is evidence of an interesting phenomenon of softening and condensing effects of S-proteins on the lipid bilayer depending on the temperature.

The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus, designated slp, was sequenced and the recombinant gene product was expressed in Escherichia coli. The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus. However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.

The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein.

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus, designated slp, was sequenced and the recombinant gene product was expressed in Escherichia coli. The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus. However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.

Radiation effects of poly(vinylidene fluoride) II

Seven kinds of poly(vinylidene fluoride) (PVDF) specimens with different crystallinity are irradiated with electron beam. The radiation effects on the amorphous and crystal zones and the relationship between the structures of the materials and the radiation effects are discussed. Differential scanning calorimeter (DSC), electron spin resonance (ESR) and X ray diffraction are used to study the radiation damage of PVDF crystal. The zero entropy production phase transition temperature, the irradiation damage yield of crystal zone and the destiny of the residual free radical of macromolecules produced by irradiation are all discussed. It is shown that appreciable structural defects and distortions start from both of the deformed points of regularly folded crystalline stems and the irregular loop zone of the crystalline surface layers.

Radiation effects of poly(vinylidene fluoride) (PVDF) (I)

Seven kinds of poly (vinylidene fluoride) (PMF) specimens with different crystallinity are irradiated with electron beam. The radiation effects on the amorphous and crystal zones and the relationship between the structures of the materials and the radiation effects are discussed. Differential scanning calorimeter (DSC), electron spin resonance (ESR) and X-ray diffraction are used to study the radiation damage of PMF crystal. The zero-entropy-production phase transition temperature, the irradiation damage yield of crystal zone and the destiny of the residual free radical of macromolecules produced by irradiation are all discussed. It is shown that appreciable structural defects and distortions start from both of the deformed points of regularly folded crystalline stems and the irregular loop zone of the crystalline surface layers.

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.