共查询到20条相似文献,搜索用时 10 毫秒
1.
Structural and functional analyses of the secondary cell wall polymer of Bacillus sphaericus CCM 2177 that serves as an S-layer-specific anchor 下载免费PDF全文
Ilk N Kosma P Puchberger M Egelseer EM Mayer HF Sleytr UB Sára M 《Journal of bacteriology》1999,181(24):7643-7646
Sacculi of Bacillus sphaericus CCM 2177 contain a secondary cell wall polymer which was completely extracted with 48% hydrofluoric acid. Nuclear magnetic resonance analysis showed that the polymer is composed of repeating units, as follows: -->3)-[4, 6-O-(1-carboxyethylidene)]( approximately 0. 5)-beta-D-ManpNAc-(1-->4)-beta-D-GlcpNAc-(1-->. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site. 相似文献
2.
Ilk N Völlenkle C Egelseer EM Breitwieser A Sleytr UB Sára M 《Applied and environmental microbiology》2002,68(7):3251-3260
The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices. 相似文献
3.
Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer. 总被引:1,自引:0,他引:1 下载免费PDF全文
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. 相似文献
4.
5.
Stability and self-assembly of the S-layer protein of the cell wall of Bacillus stearothermophilus 总被引:6,自引:0,他引:6
The surface layer of the cell envelope of Bacillus stearothermophilus consists of a regular array of protein subunits. As shown by dodecyl sulfate polyacrylamide gel-electrophoresis and ultracentrifugation, the fully solubilized S-layer protein represents a homogeneous entity with a subunit molecular mass of 115 +/- 5 kDa. Solubilization of the protein may be accomplished at acid pH, or using high concentrations of urea or guanidine X HCl. It is accompanied by (partial) denaturation, thus interfering with the characterization of the protein in its unperturbed native state. Removal of the solubilizing agent by dialysis or dilution allows the S-layer to be reassembled into two-dimensional crystalline lattices identical to those observed in intact cells. To determine the kinetics of association, optimum conditions are found to be rapid mixing with 0.1 M sodium phosphate pH 7.0, 20 degrees C, final protein concentration greater than 10 micrograms/ml. If the time course of the self-assembly is monitored by light scattering, as well as by chemical cross-linking with glutardialdehyde, multiphasic kinetics with a rapid initial phase and slow consecutive processes of higher than second-order are observed. The rapid phase may be attributed to the formation of oligomeric precursors (Mr greater than 10(6) ). Concentration-dependent light scattering measurements give evidence for a "critical concentration" of association, suggesting that patches of 12-16 protein subunits fuse and recrystallize into the final (native) S-layer structure. Recrystallization tends to be complete. 相似文献
6.
Max S.G. Legg Fiona F. Hager-Mair Simon Krauter Susannah M.L. Gagnon Arturo Lpez-Guzmn Charlie Lim Markus Blaukopf Paul Kosma Christina Schffer Stephen V. Evans 《The Journal of biological chemistry》2022,298(4)
Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division. 相似文献
7.
Christina Scha¨ffer Norbert Mu¨ller Pravat K. Mandal Rudolf Christian Sonja Zayni Paul Messner 《Glycoconjugate journal》2000,17(10):681-690
The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei
CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed:[(Pyr4,6)--D-Manp
NAc-(14)--D-Glcp
NAc-(13)]ñ11-(Pyr4,6)--D-Manp
NAc-(14)--D-Glcp
NAc-(1O)-PO2-O-PO2-(O6)-MurNAc-Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. 31P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex.In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed. 相似文献
8.
Expansion of the tetragonally arrayed cell wall protein layer during growth of Bacillus sphaericus. 总被引:1,自引:5,他引:1 下载免费PDF全文
The outermost layer of the cell wall of Bacillus sphaericus strain P-1 is a tetragonally arrayed structure (T-layer) which is assembled from a single polypeptide. No turnover of T-layer was detected during growth of cultures. In contrast, the turnover of peptidoglycan was between 20 and 25% per generation. The sites of deposition of new T-layer on the cell surface were identified by the indirect fluorescent antibody technique, which labeled old T-layer, and by the reverse technique, which labeled new T-layer. These experiments demonstrated that the major area of T-layer deposition was a band at the site of an incipient cell division. This band subsequently split and covered the new pole of each progeny cell. Little or no T-layer was inserted into existing poles. In addition, multiple bands of new T-layer, which probably accommodate cell elongation, were inserted along the lateral surface of the cell. 相似文献
9.
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. 相似文献
10.
High-affinity interaction between the S-layer protein SbsC and the secondary cell wall polymer of Geobacillus stearothermophilus ATCC 12980 determined by surface plasmon resonance technology 下载免费PDF全文
Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC(31-270)] and rSbsC(31-443)) and the secondary cell wall polymer (SCWP) isolated from Geobacillus stearothermophilus ATCC 12980 confirmed the exclusive responsibility of the N-terminal region comprising amino acids 31 to 270 for SCWP binding. Quantitative analyses indicated binding behavior demonstrating low, medium, and high affinities. 相似文献
11.
Zhilan Sun Jian Kong Shumin Hu Wentao Kong Wenwei Lu Wei Liu 《Applied microbiology and biotechnology》2013,97(5):1941-1952
It was previously shown that the surface (S)-layer proteins covering the cell surface of Lactobacillus crispatus K313 were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-layer protein genes (slpA, slpB, and slpC) of L. crispatus K313 were amplified by PCR, sequenced, and characterized in detail. Quantitative real-time PCR analysis reveals that slpA was silent under the tested conditions; whereas slpB and slpC, the putative amino acid sequences which exhibited minor similarities to the previously reported S-layer proteins in L. crispatus, were actively expressed. slpB, which was predominantly expressed in L. crispatus K313, was further investigated for its functional domains. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-layer protein expression. By producing a set of N- and C-terminally truncated recombinant SlpB proteins in Escherichia coli, the cell wall-binding region was mapped to the C terminus, where rSlpB380–501 was sufficient for binding to isolated cell wall fragments. Moreover, the binding ability of the C terminus was variable among the Lactobacillus species (S-layer- and non-S-layer-producing strains), and teichoic acid may be acting as the receptor of SlpB. To determine the adhesion region of SlpB to extracellular matrix proteins, ELISA was performed. Binding to immobilized types I and IV collagen was observed with the His-SlpB1–379 peptides, suggesting that the extracellular matrix protein-binding domain was located in the N terminus. 相似文献
12.
Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII 下载免费PDF全文
Stephanie E. Willing Thomas Candela Helen Alexandra Shaw Zoe Seager Stéphane Mesnage Robert P. Fagan Neil F. Fairweather 《Molecular microbiology》2015,96(3):596-608
Gram‐positive surface proteins can be covalently or non‐covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non‐identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock‐down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram‐positive bacteria. 相似文献
13.
The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain 总被引:2,自引:1,他引:1
Mohamed Chami Nicolas Bayan Jean Louis Peyret Thaddée Gulik-Krzywicki Gérard Leblon & Emanuel Shechter 《Molecular microbiology》1997,23(3):483-492
PS2 is the S-layer protein of Corynebacterium glutamicum . The S-layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70°C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S-layer, which remains organized. Because we show that trypsin cleaves the C-terminal part of the protein, we conclude that this domain is involved in the association of the S-layer to the cell but is not essential in the interaction between individual PS2 proteins within the S-layer. A modified form of PS2, deleted of its C-terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S-layer and is mainly released into the medium. We suggest that PS2 is anchored via its C-terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane. 相似文献
14.
One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein 下载免费PDF全文
The N-terminal repeat (SAC1) of the S-protein of Lactobacillus acidophilus bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not. Treatment of CWFs with hydrofluoric acid, but not phenol, prevented binding. Apparently, SAC1 is necessary and sufficient for cell wall binding. Our data suggest that SAC anchors the S-protein to a cell wall teichoic acid. 相似文献
15.
Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei. 相似文献
16.
17.
Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli. 总被引:1,自引:0,他引:1 下载免费PDF全文
M J Loessner S K Maier H Daubek-Puza G Wendlinger S Scherer 《Journal of bacteriology》1997,179(9):2845-2851
The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts. 相似文献
18.
Forsberg LS Abshire TG Friedlander A Quinn CP Kannenberg EL Carlson RW 《Glycobiology》2012,22(8):1103-1117
Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1?→?4)-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1?→?4)-[3-O-acetyl]-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNH(2)-(1→. 相似文献
19.
A polypeptide bacteriophage receptor: modified cell wall protein subunits in bacteriophage-resistant mutants of Bacillus sphaericus strain P-1 总被引:3,自引:17,他引:3 下载免费PDF全文
Bacillus sphaericus strain P-1 has previously been shown to have a tetragonally arrayed (T layer) protein which forms the outer layer of the cell wall. The T layer was quantitatively extracted from whole cells by 6 M urea, and the T layer subunits were purified by electrophoresis of the extracts on acrylamide gels containing 0.1% sodium dodecyl sulfate or 6 M urea. Using ethylene diacrylate cross-linked gels, the T layer was found to make up 16% of the total cellular protein. A virulent bacteriophage which is inactivated by purified T layer was isolated from soil. Twenty-four phage-resistant mutants were isolated, of which 17 had T layer subunits of increased mobility on sodium dodecyl sulfate acrylamide gels. No mutants devoid of T layer were found. Mutants were grouped into six classes according to the molecular weight of their T layer subunits. These ranged from that of the wild type, 150,000 down to 86,000. Two mutants from different classes were examined in detail. Cells of the mutant strains did not adsorb phage nor did cell walls isolated from these mutants inactivate phage. The amino acid composition of the T layers from mutants differed little from that of the wild-type T layer. 相似文献