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.

The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated highmolecular-mass exoamylase. To elucidate the structure-function relationship of distinct segments of SbsC, three N-and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC 31-1099 . With the various truncated forms it could be demonstrated that the N-terminal part (aa 31-257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of in vitro double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of Streptococcus gordonii. In contrast to the self-assembling N-terminal truncation rSbsC 258-1099 , two further N-terminal truncations (rSbsC 343-1099 , rSbsC 447-1099 ) and three C-terminal truncations (rSbsC 31-713 , rSbsC 31-844 , rSbsC 31-860 ) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC 31-880 , rSbsC 31-900 and rSbsC 31-920 revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.

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.