Peptidoglycan-containing Layer Research Articles

The three S‐layer‐like homology motifs of the S‐layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer

The 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(31-318). Surprisingly, rSbpA(31-202) comprising the three S-layer-like homology (SLH) motifs did not bind at all. Analysis of the SbpA sequence revealed a 58-amino-acid-long 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).

Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase.

We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.

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.

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.

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.

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.

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 functional 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 continuous 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.

Affinity cross‐flow filtration: Purification of IgG with a novel protein a affinity matrix prepared from two‐dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. 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 Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes.

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.

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.

Characterization of S-layers from mesophilic bacillaceae and studies on their protective role towards muramidases

The structure of the S-layers from eight mesophilic Bacillaceae was investigated by freeze-etching of whole cells. The S-layer lattices from Bacillus alvei CCM 2051 and B. circulans CCM 1048 exhibited oblique symmetry. Square S-layer lattices were identified for B. polymyxa CCM 1459, B. circulans CCM 2048 and B. sphaericus strains CCM 2120 and CCM 2177. Two superimposed, structurally different S-layers were detected for B. brevis strains CCM 1089 and CCM 1463. In both organisms the outer S-layer lattice showed oblique and the inner S-layer lattice hexagonal symmetry. Labelling with polycationized ferritin (a net positively charged topographical marker for electron microscopy) revealed that the S-layer surface from all strains is not net negatively charged. In thin-section preparations all strains exhibited only a 5 nm thick peptidoglycan-containing layer. Whole cells from B. circulans CCM 2048 and from both B. brevis strains were rapidly lysed by the muramidases lysozyme (MW 14,600) and mutanolysine (MW 24,000) showing that their S-layers which allowed free passage for both enzymes must possess pores larger than 3.5 nm. By contrast, whole cells from B. circulans CCM 1048, B. alvei CCM 2051, B. polymyxa CCM 1459 and B. sphaericus strains CCM 2120 and CCM 2177 were resistant to both muramidases. It could be shown that these strains possess muramidase resistant peptidoglycan-containing layers. Therefore, the latter results did not confirm the claim in the literature that muramidase resistance of S-layer carrying mesophilic Bacillaceae is caused by the S-layer itself acting as a protective coat.

Structure, surface charge, and self-assembly of the S-layer lattice from Bacillus coagulans E38-66

In freeze-etched preparations, whole cells from Bacillus coagulans E38-66 exhibited an oblique S-layer lattice (a = 9.4 nm; b = 7.4 nm; gamma = 80 degrees). The three-dimensional structure of the crystalline array was characterized by optical and computer image analysis. The lattice showed two distinctly shaped types of pores. In vitro self-assembly of isolated subunits yielded flat sheets and open-ended cylinders composed of two back-to-back monolayers. Unlike whole cells, in vitro self-assembly products were capable of binding polycationized ferritin (pI, approximately 11). This showed that only the inner S-layer face adhering to the peptidoglycan-containing layer in whole cells was net negatively charged. S-layer monomers and/or oligomers were capable of generating a closed monolayer with oblique symmetry on poly-L-lysine-coated supports. The monolayer had a typical crazy paving appearance, with numerous crystal boundaries. The handedness of the oblique lattice and ability to bind polycationized ferritin revealed that the subunits had bound with the outer, not net negatively charged face to the poly-L-lysine-coated supports. Carbodiimide-activated carboxyl groups on either cell wall fragments or self-assembly products could covalently bind high-molecular-weight nucleophiles such as ferritin. This confirmed the location of negatively charged carboxyl groups on the outermost surface of both S-layer faces. The difference in pH optimum for carbodiimide activation indicated a preponderance of alpha- and beta-carboxyl groups on the inner S-layer face and a preponderance of beta- and gamma-carboxyl groups on the outer S-layer face.

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.

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.