The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1␥ chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.

The S-layer lattice from Bacillus stearothermophilus DSM 2358 completely covers the cell surface and exhibits oblique symmetry. During growth of B. stearothermophilus DSM 2358 on starch medium, three amylases with molecular weights of 58,000, 98,000, and 184,000 were secreted into the culture fluid, but only the highmolecular-weight enzyme was found to be cell associated. Studies of interactions between cell wall components and amylases revealed no affinity of the high-molecular-weight amylase to isolated peptidoglycan. On the other hand, this enzyme was always found to be associated with S-layer self-assembly products or S-layer fragments released during preparation of spheroplasts by treatment of whole cells with lysozyme. The molar ratio of S-layer subunits to the bound amylase was approximately 8:1, which corresponded to one enzyme molecule per four morphological subunits. Immunoblotting experiments with polyclonal antisera against the high-molecular-weight amylase revealed a strong immunological signal in response to the enzyme but no cross-reaction with the S-layer protein or the smaller amylases. Immunogold labeling of whole cells with anti-amylase antiserum showed that the high-molecular-weight amylase is located on the outer face of the S-layer lattice. Because extraction of the amylase was possible without disintegration of the S-layer lattice into its constituent subunits, it can be excluded that the enzyme is incorporated into the crystal lattice and participates in the self-assembly process. Affinity experiments strongly suggest the presence of a specific recognition mechanism between the amylase molecules and S-layer protein domains either exposed on the outermost surface or inside the pores. In summary, results obtained in this study confirmed that the S-layer protein from B. stearothermophilus DSM 2358 functions as an adhesion site for a high-molecular-weight amylase.

During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid. Only the high-molecular-weight amylase (hmwA) was also identified as cell associated. Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface. For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells. The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth. The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels. This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions. During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites. Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.