Identification, cloning, and nucleotide sequence of a silent S-layer protein gene of Lactobacillus acidophilus ATCC 4356 which has extensive similarity with the S-layer protein gene of this species.

The bacterial S-layer forms a regular structure, composed of a monolayer of one (glyco)protein, on the surfaces of many prokaryotic species. S-layers are reported to fulfil different functions, such as attachment structures for extracellular enzymes and major virulence determinants for pathogenic species. Lactobacillus acidophilus ATCC 4356, which originates from the human pharynx, possesses such an S-layer. No function has yet been assigned to the S-layer of this species. Besides the structural gene (slpA) for the S-layer protein (S-protein) which constitutes this S-layer, we have identified a silent gene (slpB), which is almost identical to slpA in two regions. From the deduced amino acid sequence, it appears that the mature SB-protein (44,884 Da) is 53% similar to the SA-protein (43,636 Da) in the N-terminal and middle parts of the proteins. The C-terminal parts of the two proteins are identical except for one amino acid residue. The physical properties of the deduced S-proteins are virtually the same. Northern (RNA) blot analysis shows that only the slpA gene is expressed in wild-type cells, in line with the results from sequencing and primer extension analyses, which reveal that only the slpA gene harbors a promoter, which is located immediately upstream of the region where the two genes are identical. The occurrence of in vivo chromosomal recombination between the two S-protein-encoding genes will be described elsewhere.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

Horizontal transference of S-layer genes within Thermus thermophilus.

The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments.     

Lactobacillus slpA promotes ESC growth through the ERK1/2 pathway

Bacterial surface layers (S-layers) are cell envelope structures ubiquitously found in gram-negative and gram-positive bacteria, including Lactobacillus. S-layers play a role in the determination and maintenance of cell shape as virulence factors, mediate cell adhesion, and regulate immature dendritic and T cells. In this study, we sought to understand the involvement of MAPK serine/threonine kinases in alterations in Endometrial epithelial cells (ESC) growth induced by Lactobacillus crispatus (L. crispatus) slpA, an S-layer protein. We applied various concentrations of L. crispatus to cultured ESCs and observed growth and changes in the phosphorylation status of ERK1/2, JNK, and p38. Similar experiments were conducted using L. crispatus lacking and overexpressing slpA. We found that ESC growth was altered by slpA primarily via ERK1/2. Our findings suggest that L. crispatus slpA promotes ESC growth mainly through an ERK1/2-dependent pathway.     

Effects of penicillin G on morphology and certain physiological parameters of Lactobacillus acidophilus ATCC 4356

Evidence shows that probiotic bacteria can undergo substantial structural and morphological changes in response to environmental stresses, including antibiotics. Therefore, this study investigated the effects of penicillin G (0.015, 0.03, and 0.06 mg/l) on the morphology and adhesion of Lactobacillus acidophilus ATCC 4356, including the colony morphotype, biofilm production, hydrophobicity, H?O? formation, S-layer structure, and slpA gene expression. Whereas only smooth colonies grew in the presence of penicillin, rough and smooth colony types were observed in the control group. L. acidophilus ATCC 4356 was found to be hydrophobic under normal conditions, yet its hydrophobicity decreased in the presence of the antibiotic. No biofilm was produced by the bacterium, despite testing a variety of different culture conditions; however, treatment with penicillin G (0.015-0.06 mg/l) significantly decreased its production of H?O? formation and altered the S-layer protein structure and slpA gene expression. The S-protein expression decreased with 0.015 mg/l penicillin G, yet increased with 0.03 and 0.06 mg/l penicillin G. In addition, the slpA gene expression decreased in the presence of 0.015 mg/l of the antibiotic. In conclusion, penicillin G was able to alter the S-layer protein production, slpA gene expression, and certain physicochemical properties of Lactobacillus acidophilus ATCC 4356.     

Lactobacillus surface layers and their applications

Surface (S-) layers are crystalline arrays of proteinaceous subunits present as the outermost component of cell wall in several species of the genus Lactobacillus, as well as in many other bacteria and Archaea. Despite the high similarity of the amino acid composition of all known S-layer proteins, the overall sequence similarity is, however, surprisingly small even between the Lactobacillus S-layer proteins. In addition, the typical characteristics of Lactobacillus S-layer proteins, distinguishing them from other S-layer proteins, are small size and high-predicted pI value. Several lactobacilli possess multiple S-layer protein genes, which can be differentially or simultaneously expressed. To date, the characterized functions of Lactobacillus S-layers are involved in mediating adhesion to different host tissues. A few applications for the S-layer proteins of lactobacilli already exist, including their use as antigen delivery vehicles.     

Bacterial and Archaeal S-Layers as a Subject of Nanobiotechnology

Many bacteria and archaea have a crystalline surface layer (S-layer), which overlies the cell envelope. S-layers each consist of one protein or glycoprotein species. Protein subunits of the S-layer noncovalently interact with each other and with the underlying cell-envelope component. On average, the S-layer lattice has pores of 2–6 nm and is 5–10 nm high. Isolated S-layer proteins recrystallize to form two-dimensional crystalline structures in solution, on a solid support, and on planar lipid membranes. Owing to this unique property, S-layers have a broad range of applications. This review focuses on the structural features and applications of S-layers and their proteins, with special emphasis on their use in nanobiotechnology.     

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.     

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.     

Structural and chemical characterization of S-layers of selected strains ofBacillus stearothermophilus andDesulfotomaculum nigrificans

The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each ofBacillus stearothermophilus andDesulfotomaculum nigrificans were compared. Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent. The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices. In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products. The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic. With the exception of the S-layers of two strains ofB. stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated. Among these strains significant differences in the amount and composition of the glycan portions were found. Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.     

S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene.

The cell surfaces of several Lactobacillus species are covered by a regular layer composed of a single species of protein, the S-protein. The 43-kDa S-protein of the neotype strain Lactobacillus acidophilus ATCC 4356, which originated from the pharynx of a human, was purified. Antibodies generated against purified S-protein were used to screen a lambda library containing chromosomal L. acidophilus ATCC 4356 DNA. Several phages showing expression of this S-protein in Escherichia coli were isolated. A 4.0-kb DNA fragment of one of those phages hybridized to a probe derived from an internal tryptic fragment of the S-protein. The slpA gene, coding for the surface layer protein, was located entirely on the 4.0-kb fragment as shown by deletion analysis. The nucleotide sequence of the slpA gene was determined and appeared to encode a protein of 444 amino acids. The first 24 amino acids resembled a putative secretion signal, giving rise to a mature S-protein of 420 amino acids (44.2 kDa). The predicted isoelectric point of 9.4 is remarkably high for an S-protein but is in agreement with the data obtained during purification. The expression of the entire S-protein or of large, C-terminally truncated S-proteins is unstable in E. coli.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Interchange of the active and silent S-layer protein genes of Lactobacillus acidophilus by inversion of the chromosomal slp segment

The most-dominant surface-exposed protein in many bacterial species is the S-protein. This protein crystallises into a regular monolayer on the outside surface of the bacteria: the S-layer. Lactobacillus acidophilus harbours two S-protein-encoding genes, slpA and slpB , only one of which ( slpA  ) is expressed. In this study, we show by polymerase chain reaction (PCR) analysis that slpA and slpB are located on a 6 kb chromosomal segment, in opposite orientations. In a small fraction of the bacterial population, this segment is inverted. The inversion leads to interchanging of the expressed and silent S-protein-encoding genes, and places the formerly silent gene behind the S-promoter which is located outside the inverted segment. A 26 bp sequence showing a high degree of similarity with the consensus sequence recognized by the Din family of invertases is present in the region where recombination occurs. Expression of the slpA gene seems to be favoured under laboratory growth conditions because 99.7% of the chromosomes of an L. acidophilus ATCC 4356 broth culture had the slpA gene present at the slp expression site.     

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051^T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051^T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.