Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component of many bacteria and archaea (35,37,38). S-layers are composed of identical protein or glycoprotein subunits, and they completely cover the cell surface during all stages of bacterial growth and division. The S-layer subunits assemble into either oblique, square, or hexagonal lattices. In the case of Bacillaceae, the N-terminal part is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer (SCWP) to the rigid cell wall layer (2,5,21,25,34,35).S-layers are unique biomaterials with properties most relevant for applications in molecular nanotechnology, nanobiotechnology, and biomimetics (38). Many S-layer proteins recrystallize into regularly structured monolayers on solid supports, such as silicon wafers, gold chips, and silanized glass or plastic materials, as well as on Langmuir lipid films, on liposomes (20,23), and at the air-water interface. Pores passing through S-layer lattices are of identical size and morphology, and functional groups show a regular distribution and high density. For production of S-layer-based biosensors (38), affinity microparticles (45), and solid-phase immunoassays (3,4,39), functional groups in the S-layer lattice were exploited as covalent binding sites for biologically active macromolecules, such as enzymes, antibodies, or ligands. As alternatives to the existing technology, namely, immobilization by chemical methods, genetic approaches are particularly attractive for incorporation of functional peptide sequences into S-layer proteins, which must be done at positions that do not interfere with their self-assembly properties and their interaction with SCWP.

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.

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.

Highlights► Bacterial S-layer proteins have a great potential as nanoscale building blocks. ► By genetic engineering, functional domains were incorporated into S-layers. ► Recombinant fusion proteins were functional and maintained self-assembly capability. ► Nanobiotechnological applications are sensor technology, diagnostics, vaccine development, and biocatalysts.

SummaryThe S-layer protein SbpA of Bacillus sphaericus CCM 2177 recognizes a pyruvylated secondary cell wall polymer (SCWP) as anchoring structure to the peptidoglycan-containing layer. Data analysis from surface plasmon resonance (SPR) spectroscopy revealed the existence of three different binding sites with high, medium and low affinity for rSbpA on SCWP immobilized to the sensor chip. The shortest C-terminal truncation with specific affinity to SCWP was rSbpA . Surprisingly, rSbpA 31-202 comprising the three S-layerlike homology (SLH) motifs did not bind at all. Analysis of the SbpA sequence revealed a 58-amino-acidlong SLH-like motif starting 11 amino acids after the third SLH motif. The importance of this motif for reconstituting the functional SCWP-binding domain was further demonstrated by construction of a chimaeric protein consisting of the SLH domain of SbsB, the S-layer protein of Geobacillus stearothermophilus PV72/p2 and the C-terminal part of SbpA. In contrast to SbsB or its SLH domain which did not recognize SCWP of B. sphaericus CCM 2177 as binding site, the chimaeric protein showed specific affinity. Deletion of 213 C-terminal amino acids of SbpA had no impact on the square (p4) lattice structure, whereas deletion of 350 amino acids was linked to a change in lattice type from square to oblique (p1).

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)]∼0.5-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA 31-1068 /ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-m-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm 2 , whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm 2 was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.

The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.