Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

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 A1gamma 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.     

A synthetic medium for continuous culture of the S-layer carrying Bacillus stearothermophilus PV 72 and studies on the influence of growth conditions on cell wall properties

Bacterial cell surface layers (S-layers) which show a crystalline structure, defined pores, and a regular arrangement of functioal groups can be used for production of isoporous ultrafiltration membranes and as a matrix for immobilization of macromolecules. S-layer-carrying cell wall fragments from thermophilic Bacillaceae possess an extremely thin peptidoglycan-containing layer with pores larger than those in the S-layer lattice. Thus, they can directly be used for biotechnological applications, when an S-layer protein pool is stored in the rigid cell wall layer which is released during cell wall preparation, forming an inner S-layer. In the present study, a synthetic medium for Bacillus stearothermophilus PV 72 was developed by applying the pulse and shift technique with the aim to produce cell wall fragments with before-mentioned properties by varying the growth conditions in condtinuous culture. The organism was grown at 57 degrees C in a bioreactor with 1 L working volume equipped with exhaust gas analysis and connected to a PC-based process control system. Biomass concentration was 2.2 g/L out of 8 g/L glucose at a dilution rate of 0.3 h(-1), giving a biomass productivity of 0.66 g/L h. Although the organism was grown under different conditions, no change in peptidoglycan composition, extent of peptidoglycan crosslinking, and content of secondary cell wall polymers was observed. The amount of S-layer protein pool stored in the rigid cell wall layer and the autolytic activity depended mainly on the specific growth rate. Cell wall fragments with properties required for ultrafiltration membrane production could be produced by parameter settings in continuous culture. (c) 1995 John Wiley & Sons, Inc.     

Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980.

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.     

The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.     

Regulation of S-layer protein synthesis of Bacillus stearothermophilus PV72 through variation of continuous cultivation conditions

The crystalline cell surface layer (S-layer) of Bacillus stearothermophilus PV72 shows hexagonal lattice symmetry and is composed of a single protein species with a molecular weight of 130000. For investigating the regulation of S-layer protein synthesis, Bacillus stearothermophilus PV72 was grown in continuous culture on synthetic PVIII- medium with glucose as carbon source at constant dilution rate of 0.3 h⁻¹ at 57 ° C under different conditions and limitations. A complete outer S-layer and an S-layer protein pool sufficient for formation of about 70% inner S-layer was produced under carbon-limited growth. The inner S-layer results from an S-layer protein pool stored in the peptidoglycan-containing layer of whole cells which can emerge and assemble on the inner face of the rigid cell wall layer during the cell wall preparation procedure. Under oxygen-limited growth, only a complete outer S-layer but no S-layer protein pool was synthesized. Reduction of the methionine concentration of PVIII-medium from 100 to 10 mg l⁻¹ led to a clear decrease in S-layer protein production and to an incomplete outer S-layer. During growth in the presence of excess glucose, S-layer protein synthesis was replaced by that of an exopolysaccharide matrix. After changing to carbon limitation again, the original level of S-layer protein synthesis was achieved after only four volume exchanges. Feeding of casein hydrolysate or aromatic or basic amino acids to the continuous culture induced an irreversible loss of S-layer protein synthesis after from five to ten volume exchanges. In contrast, addition of Gly, Ala, Val, Leu, Ile, Glu, Gln, Asp, Asn, Ser and Thr in different mixtures could significantly stimulate S-layer protein production.     

Influence of an S-layer on surface properties of Bacillus stearothermophilus

Various aspects of surface properties of the S-layer-carrying Bacillus stearothermophilus PV72 and of an S-layer-deficient mutant (strain PV72/T5) have been tested by adsorption assays on solid surfaces, electrostatic interaction chromatography and hydrophobic interaction chromatography. The adsorption assays have shown that cell adhesion of the S-layer-carrying strain was less influenced by environmental changes than it was with the S-layer-deficient mutant. Electrostatic interaction chromatography indicated that both strains have positively and negatively charged groups exposed on the cell surface but the S-layer-carrying strain reveals more positively charged groups than does the S-layer-deficient mutant. Hydrophobic interaction chromatography showed that both strains have a hydrophilic surface but that the hydrophilic properties are more pronounced with the strain lacking an S-layer.     

Different Binding Specificities of S-Layer Homology Modules from Clostridium thermocellum AncA,Slp1, and Slp2

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.     

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.     

Molecular sieving through S layers of Bacillus stearothermophilus strains.

The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.     

Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition.

Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer.     

S-Layered Aneurinibacillus and Bacillus spp. Are Susceptible to the Lytic Action of Pseudomonas aeruginosa Membrane Vesicles

When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack. These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.     

Different binding specificities of S-layer homology modules from Clostridium thermocellum AncA, Slp1, and Slp2

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.     

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits.

Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.     

Influence of the Secondary Cell Wall Polymer on the Reassembly,Recrystallization, and Stability Properties of the S-Layer Protein from Bacillus stearothermophilus PV72/p2

The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 μg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-l-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.Many bacteria and archaea possess crystalline bacterial cell surface layers (S-layers) as their outermost cell envelope component (3, 36, 38). S-layers are composed of identical protein or glycoprotein subunits which assemble into two-dimensional crystalline arrays showing oblique, square, or hexagonal lattice symmetry. S-layer subunits from bacteria are linked to each other and to the underlying cell envelope layer by noncovalent interactions and may therefore be isolated from whole cells or cell wall fragments by different procedures involving chaotropic agents, detergents, chelating agents, or high salt concentrations or by alkaline or acidic pH conditions. During removal of the disrupting agents, e.g., by dialysis, the S-layer subunits frequently reassemble into flat sheets or open-ended cylinders (in vitro self-assembly in suspension; for reviews, see references 37 and 38).Studies regarding the binding mechanism between the S-layer protein and the underlying cell envelope layer have shown that in gram-negative bacteria, the N-terminal region of the S-layer subunits recognizes specific lipopolysaccharides in the outer membrane (9, 29, 41). For Aeromonas hydrophila it was found, however, that the C-terminal part of the S-layer protein is essential for interaction with the outer membrane (40). A similar observation was reported for the S-layer protein from the gram-positive Corynebacterium glutamicum. A hydrophobic stretch of 21 amino acids located at the C-terminal end of the S-layer protein was found to interact with a hydrophobic layer in the cell wall proper that most probably consisted of mycolic acid (8). In earlier studies it was suggested that secondary cell wall polymers could represent the binding sites for the S-layer proteins from Bacillus sphaericus (15, 16) and Lactobacillus buchneri (24).Recently, a high-molecular-weight secondary cell wall polymer (SCWP) containing glucose and N-acetylglucosamine (GlcNAc) was extracted from peptidoglycan-containing sacculi of two Bacillus stearothermophilus wild-type strains (PV72/p6 and ATCC 12980 [10]). An SCWP of different chemical composition could be isolated from peptidoglycan-containing sacculi of an oxygen-induced variant strain from B. stearothermophilus PV72/p6 (35). The SCWP produced by this variant strain (B. stearothermophilus PV72/p2) is mainly composed of GlcNAc and N-acetylmannosamine (ManNAc) and shows a molecular weight of about 24,000 (33). Binding studies with proteolytic cleavage fragments and native peptidoglycan-containing sacculi revealed that the N-terminal region is involved in anchoring the S-layer subunits to the rigid cell wall layer (10, 11, 33). Several observations have supported the notion that a specific recognition and binding mechanism exists between the SCWP and the N-terminal region of the S-layer proteins from B. stearothermophilus strains. (i) Despite the overall heterogeneity, S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region and are capable of binding to an SCWP of identical chemical composition. (ii) B. stearothermophilus PV72/p6 and the oxygen-induced p2 variant produce an SCWP of different chemical composition and structure. (iii) The S-layer protein from B. stearothermophilus PV72/p2 did not recognize native peptidoglycan-containing sacculi from B. stearothermophilus wild-type strains as binding sites (35). (iv) The S-layer protein from B. stearothermophilus PV72/p6 (SbsA) and the oxygen-induced p2 variant (SbsB) are encoded by different genes which show little overall identity (19, 20), and only SbsB possesses a typical S-layer homologous (SLH) domain (23) at the N-terminal part.By sequence comparison, SLH domains (23) were identified on the N-terminal part of several S-layer proteins (6, 13, 23, 27, 30) or at the very C-terminal end of cell-associated exoenzymes and exoproteins (21, 22, 25, 26). SLH domains were suggested to anchor these proteins permanently or transiently to the cell surface. So far, evidence for a binding function of an SLH domain was provided for the S-layer protein of Thermus thermophilus (30) and for the outer-layer proteins of the cellulosome complex from Clostridium thermocellum (21, 22).In the present study, the influence of the SCWP on the formation of self-assembly products in suspension and on the recrystallization properties of the S-layer protein from B. stearothermophilus PV72/p2 on solid supports such as poly-l-lysine-coated electron microscopy (EM) grids was investigated. Moreover, studies on the stability of the S-layer protein against endoproteinase Glu-C attack in the presence and the absence of the SCWP were carried out.     

Specific interaction of the tetragonally arrayed protein layer of Bacillus sphaericus with its peptidoglycan sacculus.

Tetragonal layer protein (T-layer) isolated from Bacillus sphaericus NTCC 9602 (wild type) or 9602 Lmw (variant) bonded specifically to the sacculi (peptidoglycan) of either cell type. Only uncleaved T-layer subunits were capable of specific recognition of the B. sphaericus sacculi; other Bacillus strains and gram-positive bacterial sacculi would not adsorb B. sphaericus strain 9602 T-layer. The peptidogylcan did not function as a template since isolated T-layer subunits self-assembled into characteristic pattern. Upon reassociation with sacculi, T-layer assemblies were randomly oriented patches compared with more continuous strictly oriented pattern on cells or fresh cell walls. T-layer associated with the sacculus was less susceptible to conditions that dissociated in vitro-assembled T-layer. Mild proteolysis of both wild-type and variant T-layer subunits by a variety of enzymes reduced the molecular weight by 18,000 in all cases, indicating that one region of the molecule was particularly susceptible to cleavage. Subunits from which the minor fragment had been cleaved upon aging retained the capacity to assemble in vitro, but would no longer adsorb to sacculi. Thus, the ability of T-layer to form networks was separate from its ability to bind cell walls, and the 18,000-dalton piece of the T-layer polypeptide was necessary for attachment to the cell wall.     

Identification of Two Binding Domains, One for Peptidoglycan and Another for a Secondary Cell Wall Polymer, on the N-Terminal Part of the S-Layer Protein SbsB from Bacillus stearothermophilus PV72/p2

First studies on the structure-function relationship of the S-layer protein from B. stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP). The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain. The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.     

Binding of S-layer homology modules from Clostridium thermocellum SdbA to peptidoglycans

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.     

Novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe a novel type of affinity matrix which was prepared by covalently binding Protein A to crystalline cell surface layers (S-layers) from the gram-positive Clostridium thermohydrosulfuricum L111-69. S-layers were used in the form of cell wall fragments, which were obtained by breaking whole cells by ultrasonification and removing the cell content and the plasma membrane. In these thimble shaped structures, revealing a size of 1 to 2 mum, the peptidoglycan-containing layer was covered on both faces with a hexagonally ordered S-layer lattice composed of identical glycoprotein subunits. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein A. Quantitative determination confirmed that up to two Protein A molecules were bound per S-layer subunit leading to a dense monomolecular coverage of the immobilization matrix with the ligand.Affinity microparticles were capable of adsorbing lgG from solutions of purified preparations, from artificial lgG-albumin mixtures, and from serum. The amount of lgG bound to affinity microparticles corresponded to the theoretical saturation capacity. Under appropriate conditions, up to 95% of the adsorbed lgG could be eluted again. Affinity microparticles were found to have an extremely low Protein A leakage and a high stability toward mechanical forces. Because pores in the S-layer lattice revealed a size of 4 to 5 nm, immobilization of Protein A and adsorption of lgG was restricted to the outermost surface area. This excludes mass transfer problems usually encountered with affinity matrices prepared from amorphous polymers where more than 90% of the ligands are immobilized in the interior. (c) 1994 John Wiley & Sons, Inc.     

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1. Received: 23 October 1998 / Accepted: 21 December 1998     

Freeze-Etching of the Cell Envelope of an Acinetobacter Species Which Carries a Regular Array of Surface Subunits

Freeze-etching was applied to preparations, with and without glycerol, of Acinetobacter sp. strain MJT/F5/199A, consisting of intact cells after normal growth or after incubation with chloramphenicol, spheroplasts, and isolated cell walls and outer membranes. Etched preparations show that a regular array of subunits forms the surface of normal cells. Near the zones of constriction in dividing cells, blebs and irregularities are seen, and some blebs, consisting of both surface subunits and outer membrane, are released from the cells. The cross-fractured cell envelope shows four layers which are related to the structures seen in section as follows: cw1, which is not visible in section, contains the surface subunits; cw2 consists of all or part of the outer membrane; cw3 includes the intermediate and dense, peptidoglycan-containing layers; within these cell wall layers is the plasma membrane. Internal fracture of the plasma membrane occurs under all conditions tested, but the fracture plane in the cell wall is only revealed in chloramphenicol-treated cells or normal cells freeze-fractured with glycerol present; the characteristic fracture faces are not seen in spheroplasts or isolated outer membranes. The concave fracture face cw2 consists of densely packed granules, while the convex face cw3 is fibrillar. The probable location of this fracture plane is discussed. After incubation with chloramphenicol, the outer surface of the cells is obscured by extracellular material, the dense peptidoglycan-containing layer is increased in thickness, and the cytoplasm contains rounded bodies bounded by one or more unit membranes.