Production and cell surface anchoring of functional fusions between the SLH motifs of the Bacillus anthracis S-layer proteins and the Bacillus subtilis levansucrase

Many surface proteins of Gram-positive bacteria contain motifs, about 50 amino acids long, called S-layer homology (SLH) motifs. Bacillus anthracis, the causal agent of anthrax, synthesizes two S-layer proteins, each with three SLH motifs towards the amino-terminus. We used biochemical and genetic approaches to investigate the involvement of these motifs in cell surface anchoring. Proteinase K digestion produced polypeptides lacking these motifs, and stable three-motif polypeptides were produced in Escherichia coli that were able to bind the B. anthracis cell walls in vitro, demonstrating that the three SLH motifs were organized into a cell surface anchoring domain. We also determined the function of these SLH domains by constructing chimeric genes encoding the SLH domains fused to the normally secreted levansucrase of Bacillus subtilis. Cell fractionation and electron microscopy studies showed that each three-motif domain was sufficient for the efficient anchoring of levansucrase onto the cell surface. Proteins consisting of truncated SLH domains fused to levansucrase were unstable and associated poorly with the cell surface. Surface-exposed levansucrase retained its enzymatic and antigenic properties.     

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis

Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.     

OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively.     

Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein

Surface (S)-layers, para-crystalline arrays of protein, are deposited in the envelope of most bacterial species. These surface organelles are retained in the bacterial envelope through the non-covalent association of proteins with cell wall carbohydrates. Bacillus anthracis, a Gram-positive pathogen, produces S-layers of the protein Sap, which uses three consecutive repeats of the surface-layer homology (SLH) domain to engage secondary cell wall polysaccharides (SCWP). Using x-ray crystallography, we reveal here the structure of these SLH domains, which assume the shape of a three-prong spindle. Each SLH domain contributes to a three-helical bundle at the spindle base, whereas another α-helix and its connecting loops generate the three prongs. The inter-prong grooves contain conserved cationic and anionic residues, which are necessary for SLH domains to bind the B. anthracis SCWP. Modeling experiments suggest that the SLH domains of other S-layer proteins also fold into three-prong spindles and capture bacterial envelope carbohydrates by a similar mechanism.     

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.     

The SLH-domain protein BslO is a determinant of Bacillus anthracis chain length

The Gram-positive pathogen Bacillus anthracis grows in characteristic chains of individual, rod-shaped cells. Here, we report the cell-separating activity of BslO, a putative N-acetylglucosaminidase bearing three N-terminal S-layer homology (SLH) domains for association with the secondary cell wall polysaccharide (SCWP). Mutants with an insertional lesion in the bslO gene exhibit exaggerated chain lengths, although individual cell dimensions are unchanged. Purified BslO complements this phenotype in trans, effectively dispersing chains of bslO-deficient bacilli without lysis and localizing to the septa of vegetative cells. Compared with the extremely long chain lengths of csaB bacilli, which are incapable of binding proteins with SLH-domains to SCWP, bslO mutants demonstrate a chaining phenotype that is intermediate between wild-type and csaB. Computational simulation suggests that BslO effects a non-random distribution of B. anthracis chain lengths, implying that all septa are not equal candidates for separation.     

Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.     

In Thermoanaerobacterium thermosulfurigenes EM1 S-Layer Homology Domains Do Not Attach to Peptidoglycan

Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins). At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium. The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase. This enzyme was isolated from T. thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli. The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall. In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T. thermosulfurigenes EM1 were included in these studies. The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi. Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall. Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria.     

BslA, a pXO1-encoded adhesin of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis causes anthrax, a fulminant and lethal infection of mammals. Two large virulence plasmids, pXO1 and pXO2, harbour genes required for anthrax pathogenesis and encode secreted toxins or provide for the poly γ- d -glutamic acid capsule. In addition to capsule, B. anthracis harbours additional cell wall envelope structures, including the surface layer (S-layer), which is composed of crystalline protein arrays. We sought to identify the B. anthracis envelope factor that mediates adherence of vegetative forms to human cells and isolated BslA ( B . anthracis S - l ayer protein A ). Its structural gene, bslA , is located on the pXO1 pathogenicity island (pXO1-90) and bslA expression is both necessary and sufficient for adherence of vegetative forms to host cells. BslA assembly into S-layers and surface exposure is presumably mediated by three N-terminal SLH domains. Twenty-three B. anthracis genes, whose products harbour similar SLH domains, may provide additional surface molecules that allow bacilli to engage cells or tissues of specific hosts during anthrax pathogenesis.     

Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains

Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.     

Binding to pyruvylated compounds as an ancestral mechanism to anchor the outer envelope in primitive bacteria

Electron microscopy of isolated cell walls of the ancient bacterium Thermus thermophilus revealed that most of the peptidoglycan (PG) surface, apart from the septal region, was shielded against specific alphaPG antibodies. On the other hand, an antiserum raised against S-layer-attached cell wall fragments (alphaSAC) bound to most of the surface except for the septal regions. Treatments with alpha-amylase and pronase E made the entire cell wall surface uniformly accessible to alphaPG and severely decreased the binding of alphaSAC. We concluded that a layer of strongly bound secondary cell wall polymers (SCWPs) covers most of the cell wall surface in this ancient bacterium. A preliminary analysis revealed that such SCWPs constitute 14% of the cell wall and are essentially composed of sugars. Enzyme treatments of the cell walls revealed that SCWP was required in vitro for the binding of the S-layer protein through the S-layer homology (SLH) motif. The csaB gene was necessary for the attachment of the S-layer-outer membrane (OM) complex to the cell wall in growing cells of T. thermophilus. In vitro experiments confirmed that cell walls from a csaB mutant bound to the S-layer with a much lower affinity ( approximately 1/10) than that of the wild type. CsaB was found to be required for pyruvylation of components of the SCWP and for immunodetection with alpha-SAC antiserum. Therefore, the S-layer-OM complex of T. thermophilus binds to the cell wall through the SLH motif of the S-layer protein via a strong interaction with a highly immunogenic pyruvylated component of the SCWP. Immuno-cross-reactive compounds were detected with alphaSAC on cell walls of other Thermus spp. and in the phylogenetically related microorganism Deinococcus radiodurans. These results imply that the interaction between the SLH motif and pyruvylated components of the cell wall arose early during bacterial evolution as an ancestral mechanism for anchoring proteins and outer membranes to the cell walls of primitive bacteria.     

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.     

Surface-layer (S-layer) proteins sap and EA1 govern the binding of the S-layer-associated protein BslO at the cell septa of Bacillus anthracis

The Gram-positive pathogen Bacillus anthracis contains 24 genes whose products harbor the structurally conserved surface-layer (S-layer) homology (SLH) domain. Proteins endowed with the SLH domain associate with the secondary cell wall polysaccharide (SCWP) following secretion. Two such proteins, Sap and EA1, have the unique ability to self-assemble into a paracrystalline layer on the surface of bacilli and form S layers. Other SLH domain proteins can also be found within the S layer and have been designated Bacillus S-layer-associated protein (BSLs). While both S-layer proteins and BSLs bind the same SCWP, their deposition on the cell surface is not random. For example, BslO is targeted to septal peptidoglycan zones, where it catalyzes the separation of daughter cells. Here we show that an insertional lesion in the sap structural gene results in elongated chains of bacilli, as observed with a bslO mutant. The chain length of the sap mutant can be reduced by the addition of purified BslO in the culture medium. This complementation in trans can be explained by an increased deposition of BslO onto the surface of sap mutant bacilli that extends beyond chain septa. Using fluorescence microscopy, we observed that the Sap S layer does not overlap the EA1 S layer and slowly yields to the EA1 S layer in a growth-phase-dependent manner. Although present all over bacilli, Sap S-layer patches are not observed at septa. Thus, we propose that the dynamic Sap/EA1 S-layer coverage of the envelope restricts the deposition of BslO to the SCWP at septal rings.     

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     

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).     

S-layer homology domain proteins Csac_0678 and Csac_2722 are implicated in plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus

The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.     

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.     

Species-Specific Cell Wall Binding Affinity of the S-Layer Proteins of Mosquitocidal Bacterium Bacillus sphaericus C3-41

The binding affinities and specificities of six truncated S-layer homology domain (SLH) polypeptides of mosquitocidal Bacillus sphaericus strain C3-41 with the purified cell wall sacculi have been assayed. The results indicated that the SLH polypeptide comprised of amino acids 31 to 210 was responsible for anchoring the S-layer subunits to the rigid cell wall layer via a distinct type of secondary cell wall polymer and that a motif of the recombinant SLH polypeptide comprising amino acids 152 to 210 (rSLH_152-210) was essential for the stable binding of the S-layer with the bacterial cell walls. The quantitative assays revealed that the K_D (equilibrium dissociation constant) values of rSLH_152-210 and rSLH_31-210 with purified cell wall sacculi were 1.11 × 10⁻⁶ M and 1.40 × 10⁻⁶ M, respectively. The qualitative assays demonstrated that the SLH domain of strain C3-41 could bind only to the cell walls or the cells treated with 5 M guanidinium hydrochloride of both toxic and nontoxic B. sphaericus strains but not to those from other bacteria, indicating the species-specific binding of the SLH polypeptide of B. sphaericus with bacterial cell walls.Crystalline bacterial cell surface layers (S-layers) cover the cell surfaces of many bacteria and archaea during all stages of growth and division. S-layers are composed of identical protein or glycoprotein subunits, which can assemble into two-dimensional crystalline arrays and exhibit oblique, square, or hexagonal symmetry (27, 28, 30). S-layers play key roles in the interaction between bacterial cells and environment as protective coats, molecular sieves, ion traps, cell adhesion mediators, and attachment structures (4, 21, 26, 29). Many S-layer proteins possess an N-terminal region with highly conserved amino acid sequences, which is called an S-layer homology (SLH) domain. An SLH domain contains one, two, or three repeating SLH motifs (6, 16). Each SLH motif is composed of about 55 amino acids containing 10 to 15 conserved residues (6, 17). It is suggested that the SLH domain of S-layer proteins is responsible for the binding of the S-layer subunits to the rigid cell wall layer (6, 15, 17, 19, 25), while the middle and C-terminal parts include the domains which are involved in the self-assembly process (27). In the case of Bacillaceae, secondary cell wall polymers (SCWP) are responsible for binding with SLH domains (13, 18, 19), but the SLH domains of some other bacteria have an affinity for peptidoglycan (33).Bacillus sphaericus is a gram-positive soil bacterium that represents a strictly aerobic group of mesophilic endospore-forming bacteria. Due to its specific toxicity to target mosquito larvae and the limited environment impact, some strains of this bacterium have been successfully used worldwide in integrated mosquito control programs. Previous studies revealed that some nontoxic strains of B. sphaericus contained S-layer proteins, and the S-layer proteins of B. sphaericus NCTC 9602, JG-A12, P1, and CCM 2177 have been studied in detail elsewhere (3, 7-9, 12, 22).B. sphaericus C3-41, a highly active strain isolated from a mosquito-breeding site in China in 1987, has different levels of toxicity against Culex spp., Anopheles spp., and Aedes spp. This strain belongs to the flagella serotype H5a5b, like strains 2362 and 1593 (32), and it has been developed as a commercial larvicide (JianBao) for mosquito larva control in China during the last decade (31). The genomic analysis of strain C3-41 revealed that an S-layer protein gene (slpC) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF535606","term_id":"145975949","term_text":"EF535606"}}EF535606) exists on the chromosomal genome and its sequence is identical to the S-layer protein of B. sphaericus 2362 (1, 10), composed of 3,531 bp encoding a protein of 1,176 amino acids with a molecular size of 125 kDa. Although the binding function of S-layers has been identified in some nontoxic B. sphaericus strains (6, 11), it is not well documented in mosquitocidal B. sphaericus strains, and there are few reports on the binding function of each SLH motif and the binding specificity.In this study, the binding affinities and specificities of each SLH motif of S-layer protein from mosquitocidal B. sphaericus C3-41 alone and in combination with the different cell wall preparations have been investigated, and the species-specific binding of SLH polypeptide with bacterial cell walls has been demonstrated.     

Glucosamine substitution and muramidase susceptibility in Bacillus anthracis

Cell walls of Bacillus anthracis were found to be resistant to lysozyme, and partially resistant to mutanolysin, a muramidase from Streptomyces globisporus. Following treatment with acetic anhydride, it was observed that the walls were highly susceptible to hydrolysis by lysozyme or mutanolysin. Analyses of cell walls, prior to and following derivatization with fluorodinitrobenzene, revealed that approximately 88% of the glucosamine residues and 34% of the muramic acid residues of the peptidoglycan contained unsubstituted amino groups, thereby providing an explanation for the resistance of the walls to lysozyme. The walls of B. anthracis were approximately 19% cross-linked, based on the findings that 81% of the diaminopimelic acid residues could be modified by fluorodinitrobenzene. Walls of B. thuringiensis 4040 and B. cereus ATCC 19637 also contained high percentages of unsubstituted amino sugars, and unless acetylated, were also relatively resistant to lysozyme and mutanolysin. When B. anthracis, B. cereus, or B. thuringiensis were grown in the presence of 100 micrograms/mL lysozyme, there was a decrease in the average number of cells per chain, but there was no decrease in growth rates, suggesting that the enzyme was acting at septa. It is unlikely that lysozyme and autolysins act synergistically in Bacillus, because azide anion, which activates autolysins, did not enhance the lytic action of lysozyme in B. anthracis, B. cereus, or B. thuringiensis.