Crystalline Bacterial Cell Surface Layers Research Articles

Fabrication and characterization of zinc oxide nanoparticle coated magnetic iron oxide: Effect of S-layers adsorption on surface of oxide

Mixed zinc oxide nanoparticle coated magnetic iron oxide has been prepared by a sol–gel and co-precipitation routes. Magnetic iron oxide nanoparticles were synthesized by co-precipitation of ferric and ferrous ions with ammonia, and then zinc oxide was coated onto the surface of magnetic iron oxide by hydrolysis of zinc precursors. As a result, zinc oxide coated magnetic iron oxide nanoparticles with an average size of 68nm were obtained. The crystalline bacterial cell surface layer)S-layer (used in this study was isolated from Lactobacillus helveticus ATCC 12046. The S-layer was adsorbed onto the surface of zinc oxide nanoparticle coated magnetic iron oxide. The nanoparticles were analyzed by X-ray powder diffractometry (XRD), infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FESEM) were used to characterize the structural and the chemical features of the nanocomposites. The infrared spectra indicate that the S-layer-nanoparticle interaction occurs. This novel nanoparticle showed admirable potential in adsorption of S-layers on the surface of oxides for drug delivery.

Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

Monomolecular films of cholesterol oxidase and S-Layer proteins

Cholesterol oxidase (ChOx) is a flavoenzyme that catalyzes the oxidation of cholesterol to cholest-5-en-3-one and subsequently the isomerization to cholest-4-en-3-one. ChOx has been very commonly studied as the detection element in cholesterol biosensors. In the biosensor development field, a relatively new approach is the use of crystalline bacterial cell surface layers, known as S-Layer proteins. These proteins exhibit the ability of self-assembling at surfaces, opening a vast spectrum of applications, both in basic and applied researches. In our study, monomolecular films of ChOx and mixed films of ChOx/S-Layer proteins and DPPC/S-Layer proteins were produced using the Langmuir technique. Characterization of the films was performed by means of surface pressure–molecular area ( π– A) isotherms. Stable monolayers were obtained, which means that they can be transferred to solid substrates by Langmuir–Blodgett technique. Mixed monolayers showed an ideal like behavior.

Fluorescent S-Layer Fusionproteins; Reassembling Behaviour and Spectral Properties

S-layer proteins are crystalline bacterial cell surface layer proteins. When they are isolated or genetically modified with functional moieties they are still able to reassemble spontaneously into their distinctive monomolecular lattice symmetry as originally found on the cell surface. In solution they form differently sized self-assembly products while they build crystalline nanostructured biocoatings o nsolid supports. In our case we fused the pH-dependent green fluorescent protein (EGFP) in a 1:1 stoichiometry with a truncated form of the S-layer protein SgsE from Geobacillus stearothermophilus NRS2004/3a. We investigated the formation of the 2D nanostructured monomolecular crystalline self-assembly products with the functional moiety in defined position and orientation, representing a new patterning material for biomolecular construction kits. With transmission electron microscopy (TEM) we could demonstrate that isolated SgsE-EGFP subunits build mono- and double-layered self-assembly products, forming differently sized flat sheets and cylinders with p2 lattice symmetry (a=11.9+/-0.6nm, b=14.7+/-0.6nm, g=81.2+/-1.1nm). Atomic force microscopy (AFM) was used to resolve the crystalline domain nanostructure of the fluorescent S-layer proteins on functionalised solid supports. We demonstrated the application as building blocks by coating silica particles with the fluorescent S-layer protein, fabricating in this way a pH-dependent nanostructured surface-coating. The pKa value of the pH-dependent fluorescent S-layer coating was calculated after measuring the EGFP fluorescence emission in different pH environements using flow cytometry and fluorescence microscopy. The colloidal behaviour was investigated with Zeta-potential measurements. In conclusion, these fluorescent S-layer fusion protein assemblies can be used for investigating systematically changes in pH and surface potential at the nanoscale.

In Vivo Imaging of S-Layer Nanoarrays on Corynebacterium glutamicum

Crystalline bacterial cell surface layers (S-layers) are monomolecular arrays of (glyco)proteins that have recently produced a wealth of new opportunities in nanotechnology. Whereas the in vitro imaging of isolated S-layers is well established, their direct imaging on live cells remains very challenging. Here we use atomic force microscopy (AFM) to visualize S-layer nanoarrays on living Corynebacterium glutamicum bacteria. We demonstrate the presence of two highly ordered surface layers. The most external layer represents the hexagonal S-layer, and the inner layer displays regular patterns of nanogrooves that could act as a biomolecular template promoting the 2-D assembly of S-layer monomers. These nanoscale analyses open new avenues for understanding the structure of protein monomolecular arrays, which is a crucial challenge in current nanoscience and life science research.

Detection of metal binding sites on functional S-layer nanoarrays using single molecule force spectroscopy

Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.

The Structure and Binding Behavior of the Bacterial Cell Surface Layer Protein SbsC

Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC((31-844)) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.

Display of a Peptide Mimotope on a Crystalline Bacterial Cell Surface Layer (S-layer) Lattice for Diagnosis of Epstein–Barr Virus Infection

Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72/p2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein-Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 individual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.

Surfaces functionalized with self-assembling S-layer fusion proteins for nanobiotechnological applications

The fabrication of supramolecular structures and devices requires molecules that are capable of interlocking in a predictable well defined manner on surfaces required for nanobiotechnological applications. Thus, molecular self-assembly systems which exploit the molecular scale manufacturing precision of biological systems are prime candidates for supramolecular engineering. In this context, crystalline bacterial cell surface layer (S-layer) proteins of prokaryotic organisms represent a unique self-assembly system which can be exploited as patterning element for a biomolecular construction kit involving all major species of biological molecules, for example, glycans such as S-layer-specific heteropolysaccharides, lipids, and nucleic acids. One of the most fascinating properties of native or recombinant S-layer proteins is their capability to self-assemble in suspension (as flat sheets or cylinders), into monomolecular protein lattices on artificial surfaces (e.g. silicon wafers, noble metals, plastics) or on Langmuir lipid films and liposomes. Functional groups (e.g. carboxyl groups, amino or hydroxyl groups) or genetically incorporated functional domains (e.g. streptavidin) are repeated with the periodicity of the S-layer lattice at a distance resembling the lattice constants, leading to regular arrays of bound functional molecules or nanoparticles. Thus, genetically and/or chemically modified S-layer proteins can be exploited as building blocks and templates for generating functional nanostructures at meso- and macroscopic scale for both, life and non-life science applications.

High-Affinity Tags Fused to S-Layer Proteins Probed by Atomic Force Microscopy

Two-dimensional, crystalline bacterial cell surface layers, termed S-layers, are one of the most commonly observed cell surface structures of prokaryotic organisms. In the present study, genetically modified S-layer protein SbpA of Bacillus sphaericus CCM 2177 carrying the short affinity peptide Strep-tag I or Strep-tag II at the C terminus was used to generate a 2D crystalline monomolecular protein lattice on a silicon surface. Because of the genetic modification, the 2D crystals were addressable via Strep-tag through streptavidin molecules. Atomic force microscopy (AFM) was used to investigate the topography of the single-molecules array and the functionality of the fused Strep-tags. In high-resolution imaging under near-physiological conditions, structural details such as protein alignment and spacing were resolved. By applying molecular recognition force microscopy, the Strep-tag moieties were proven to be fully functional and accessible. For this purpose, streptavidin molecules were tethered to AFM tips via approximately 8-nm-long flexible polyethylene glycol (PEG) linkers. These functionalized tips showed specific interactions with 2D protein crystals containing either the Strep-tag I or Strep-tag II, with similar energetic and kinetic behavior in both cases.

S-layers as a tool kit for nanobiotechnological applications

Crystalline bacterial cell surface layers (S-layers) have been identified in a great number of different species of bacteria and represent an almost universal feature of archaea. Isolated native S-layer proteins and S-layer fusion proteins incorporating functional sequences self-assemble into monomolecular crystalline arrays in suspension, on a great variety of solid substrates and on various lipid structures including planar membranes and liposomes. S-layers have proven to be particularly suited as building blocks and patterning elements in a biomolecular construction kit involving all major classes of biological molecules (proteins, lipids, glycans, nucleic acids and combinations of them) enabling innovative approaches for the controlled 'bottom-up' assembly of functional supramolecular structures and devices. Here, we review the basic principles of S-layer proteins and the application potential of S-layers in nanobiotechnology and biomimetics including life and nonlife sciences.

Small‐Angle Scattering of S‐Layer Metallization

The formation of Pd nanoparticles on crystalline bacterial cell surface layers has been studied by means of electron microscopy and the small-angle scattering of X-rays (see figure) and neutrons. The response of the S-layer lattice to the loading with Pd complexes and metallic Pd particles as well as the effects of two different reducing agents (H2 and dimethylaminoborane) on the observed nanoparticle arrays are described.

S-layer-streptavidin fusion proteins and S-layer-specific heteropolysaccharides as part of a biomolecular construction kit for application in nanobiotechnology

Crystalline bacterial cell surface layer (S-layer) proteins represent the outermost envelope component of many bacteria and archaea. Isolated S-layer proteins frequently show the ability to recrystallize into monomolecular protein lattices on solid supports, Langmuir lipid films, or liposomes. Many S-layer proteins specifically recognize a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. This is the reason why such heteropolysaccharides were exploited as biomimetic linkers to solid supports, which is especially important if S-layer fusion proteins having a foreign sequence incorporated on the outermost surface are used for recrystallization to generate functional monomolecular protein lattices. The latter are currently being exploited as sensing layers for label free detection systems, as affinity matrices for binding immunoglobulins, or in the case of liposomes, as novel targeting and delivery systems.

Interaction of the crystalline bacterial cell surface layer protein SbsB and the secondary cell wall polymer of Geobacillus stearothermophilus PV72 assessed by real-time surface plasmon resonance biosensor technology.

The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall Kd of 7.7 x 10(-7) M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 10(-11) M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.

Biophysical Characterization of the Entire Bacterial Surface Layer Protein SbsB and Its Two Distinct Functional Domains

The crystalline bacterial cell surface layer (S-layer) protein SbsB of Geobacillus stearothermophilus PV72/p2 was dissected into an N-terminal part defined by the three consecutive S-layer homologous motifs and the remaining large C-terminal part. Both parts of the mature protein were produced as separate recombinant proteins (rSbsB(1-178) and rSbsB(177-889)) and compared with the full-length form rSbsB(1-889) (rSbsB). Evidence for functional and structural integrity of the two truncated forms was provided by optical spectroscopic methods and electron microscopy. In particular, binding of the secondary cell wall polymer revealed a high affinity dissociation constant of 3 nm and could be assigned solely to the soluble rSbsB(1-178), whereas rSbsB(177-889) self-assembled into the same lattice as the full-length protein. Furthermore, thermal as well as guanidinium hydrochloride induced equilibrium unfolding profiles monitored by intrinsic fluorescence, and circular dichroism spectroscopy allowed characterization of rSbsB(1-178) as an alpha-helical protein with a single cooperative unfolding transition yielding a DeltaG value of 26.5 kJ mol(-1). The C-terminal rSbsB(177-889) could be characterized as a beta-sheet protein with typical multidomain unfolding, which is partially less stable as stand-alone protein. In general, the truncated forms showed identical properties compared with the full-length rSbsB with respect to structure and function. Consequently, rSbsB is characterized by its two functionally and structurally separated parts, the specific secondary cell wall polymer binding rSbsB(1-178) and the larger rSbsB(177-889) responsible for formation of the crystalline array.

Nanotechnology and biomimetics with 2-D protein crystals.

The application potential of crystalline bacterial cell surface layers (S-layers) as nature-tailored supramolecular structures is described. The fabrication of supramolecular devices requires molecules that are capable of interlocking in a well-defined manner. Thus, molecular self-assembly systems that exploit the molecular-scale manufacturing precision of biological systems are prime candidates for supramolecular engineering. Although self-assembly of molecules is an ubiquitous strategy of morphogenesis in nature, researchers in the area of molecular nanotechnology and biomimetics are only beginning to exploit its potential for the functionalization of surfaces and interfaces as well as for the production of biomimetic membranes and encapsulation systems. This article presents an overview of a new approach in nanostructure technology and biomimetics based on crystalline bacterial cell surface layers.

Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.

S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays.

Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding d-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

ChemInform Abstract: Characterization and Use of Crystalline Bacterial Cell Surface Layers

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S-layer proteins as basic building blocks in a biomolecularconstruction kit

Crystalline bacterial cell surface layer (S-layer) proteinshave been optimized during billions of years of biological evolutionas constituent elements of one of the simplest self-assembly systems.Isolated S-layer proteins possess the intrinsic property of being ableto recrystallize into two-dimensional arrays at a broad spectrum ofsurfaces (e.g. silicon) and interfaces (e.g. air-water interface orplanar lipid films). The well-defined arrangement of functional groupson S-layer lattices allows the binding of molecules and particles indefined regular arrays. S-layers recrystallized on solid supports canbe patterned in the submicrometre range using standard opticallithography. S-layers also represent templates for the formation ofinorganic nanocrystal superlattices (e.g. CdS, Au, Ni, Pt, or Pd) asrequired for molecular electronics and nonlinear optics.