S‐Layers

Abstract

Microbial surface layer (S‐layer) proteins assemble into two‐dimensional (2D) crystalline lattices on the cell surface of many species of Archaea and Bacteria and represent the outermost cell wall component, except for the existence of a carbohydrate capsule. Despite their widespread occurrence, the function of S‐layers remained obscure. Analysing the S‐layer structure on the one hand and the interaction of the 2D crystal with the underlying cell membrane on the other, reveals the S‐layers' cell wall function and the mechanism of cell stabilisation in Archaea. This basic function is not obvious in Bacteria. Here, experimental data suggest a major role in mediating and controlling interactions of S‐layers with the microbes' environment. The hybrid functional role of S‐layers will be understood more clearly if structural research is combined with the investigation of S‐layer interactions with the adjacent cell envelope components and the complex environmental factors.

Key Concepts

  • Microbial S‐layers have a hybrid functional role, that is, mediating cell stability and controlling cell surface properties.
  • The architecture of S‐layers and their close interactions with the cell membrane determine and explain the S‐layers' cell wall function in Archaea.
  • Attracting and repelling surface characteristics of S‐layers in Bacteria mediate the interactions with the environment and contribute to the proliferation and survival of cells.
  • The investigation of S‐layer functions requires the combined analysis of the S‐layer structure and its impact on the cell envelope components as well as of interactions with environmental factors.

Keywords: surface layer; S‐layer; microbial cell wall; microbial cell envelope; S‐layer function; S‐layer structure; two‐dimensional protein lattice; archaeal cell wall; S‐layer interactions; microbial adhesion

Figure 1. Space groups of two‐dimensional (2D; planar) crystals formed by a single type of (macro)molecules (symbolised as B). Filled symbols indicate molecules that are rotated by 180° around an axis in the crystal plane; they occur mirrored in projection. The rectangles and parallelograms delineate the crystallographic unit cells, that is, the repetitive structural elements of the 2D lattices. The notation codes identify the unit cell type (p: primitive; c: centred), the rotational symmetry (1; 2; 3; 4; 6) and the existence of main mirror (m) and glide mirror (g) axes within the unit cell or none (1). Natural 2D crystals of S‐layer proteins never show mirror symmetries in projection.
Figure 2. Structure of selected S‐layers as obtained from 3D electron microscopy. (a) S‐layer from the Gram‐positive bacterium Sporosarcina ureae; image of the negatively stained layer and 3D models of four unit cells, each with views of the outer and inner surface (size of the crystallographic unit cell 12.9 by 12.9 nm). (b) 3D models illustrating the individual complexity of selected unit cell structures. From left to right: S‐layers with p4 symmetry from the archaeon Desulfurococcus mobils (lattice constant 18 nm), from the bacteria Clostridium aceticum (12 nm) and Sp. ureae (12.9 nm), and with p6 symmetry from the archaeon Pyrobaculum islandicum (29.9 nm). The images are to scale (image size 45 nm). The models are based on electron microscopical 3D reconstructions of negatively stained S‐layers (Baumeister et al., ; Woodcock et al., ; Engelhardt et al., ; Phipps et al., ). (c) Schematic representation of proteins in unit cells of the p4 symmetric S‐layers shown in (b) in the same order, illustrating the patterns of protein connections with increasing complexity. The different connections are colour‐coded in blue, red and green. The connection types are classified as C42, C44′, C424′; the single digits denote the respective symmetry axes in the unit cell (identified in the scheme on the right) and indicate where protein connections are located. Symmetry axes: filled square (4‐fold axis), open square (4′‐fold axis), biangle (2‐fold axis).
Figure 3. Representative structures of the most common protein domains connecting S‐layers to the underlying component of the cell envelope. (a) Fragment of the stalk of the archaeal S‐layer from Staphylothermus marinus forming a tetragonal coiled‐coil structure. The C‐terminal end of the stalk (oriented to the bottom, not shown) is anchored in the cell membrane (model displayed with Chimera Software Version 1.6.2 and coordinates from the Protein Data Bank (PDB) accession number 1FE6). (b) S‐layer homology (SLH) domain from the Bacillus anthracis S‐layer protein with three homologous but not identical repeats. The binding site for the pyruvylated SCWP is located in the clefts between two adjacent repeats (PDB accession number 3PYW).
Figure 4. Examples for archaeal S‐layers with stalks being anchored in the cell membrane. (a) Staphylothermus marinus tetragonal S‐layer unit Tetrabrachion with associated protease dimer in the middle of the stalk (electron micrograph of isolated, negatively stained S‐layer units). (b) Pyrodictium abyssi S‐layer (side view from 3D reconstruction) illustrating the periplasmic space with enclosed protein particles (top), 3D model of the S‐layer (bottom). (Images courtesy of S. Nickell, Martinsried.)
Figure 5. Schemes illustrating the mechanism of cell stabilisation by S‐layers in Archaea. (a) Forces generated by the intracellular turgor and transmitted to the membrane‐anchored S‐layer. FP denotes the pressure force acting on the S‐layer stalk of height s, FA indicates the resulting tensile force acting on the S‐layer arms and the S‐layer protein connections (only one force vector is shown). SL: S‐layer, CM: cell membrane, R: cell radius without S‐layer (cell radius including S‐layer: RC = R + s), d′: maximum distance of membrane anchors in the S‐layer unit cell with cell constant d, φ: half angle defined by the respective stalks and the middle of the cell. (b,c) Model of bulge formation in S‐layer‐membrane associations. Scheme of tetragonal and hexagonal insertion of S‐layer stalks in membranes and formation of membrane bulges between the locally fixed insertion points upon a difference in osmotic pressure inside and outside of the membrane. The bulges are characterised by the bending radius r (r1 and r2 for different bulges), that is, the radius of a hypothetical sphere that fits the shape of the bulge, and by the corresponding bulge height h. A and B indicate insertion points of S‐layer stalks. (Reproduced and adapted from Engelhardt, 2007b © Elsevier.) (d) Theoretical relation between osmotic pressure differences on S‐layer‐membrane systems and the formation of membrane bulges between membrane anchors of S‐layers unit cells. Curves for a stiff membrane and tetragonal and hexagonal S‐layer unit cells with lattice constants of d = 15 nm. The total pressure for bulge formation is the sum of the pressures for membrane bending and expansion. The critical total pressure is assessed by the stability limit of lipid vesicles of the same (bending) radius. The critical lower bending radius is given by a maximum membrane expansion of 2%. The minimal bending radius is derived from the maximum distance between S‐layer anchors in unit cells (shown for the more pessimistic value of the tetragonal unit cell). (e) Maximum pressure allowed for membrane bulges at the critical lower bending radius as a function of the S‐layer lattice constant. Parameters for flexible (stiff) membranes: Bending stiffnes 10−19 Nm (2.5 × 10−19 Nm), elasticity modulus 0.2 N m−1 (0.7 N m−1), linear tension for pore formation 2 × 10−11 N (5 × 10−11 N). (Calculations based on the approach given in Engelhardt, ; here with distinct values for bulges over unit cells of p4 and p6 symmetry).
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Further Reading

Albers SV and Mey BH (2011) The archaeal cell envelope. Nature Reviews 9: 414–426.

Engelhardt H and Peters J (1998) Structural research on surface layers – a focus on stability, surface layer homology domains, surface layer‐cell wall interactions. Journal of Structural Biology 124: 276–302.

Engelhardt H (2007) Are S‐layers exoskeletons? The basic function of surface protein layers revisited. Journal of Structural Biology 160: 115–124.

Pavkov‐Keller T, Howorka S and Keller W (2011) The structure of bacterial S‐layer proteins. In: Howorka S (ed) Progress in Molecular Biology and Translational Science, Molecular Assembly in Natural and Engineered Systems, vol. 103, pp. 73–130.

Sleytr UB, Schuster B, Egelseer E‐M and Pum D (2014) S‐layers: principles and applications. FEMS Microbiology Reviews 38: 823–864.

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Engelhardt, Harald(Feb 2016) S‐Layers. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021936]