scholarly journals Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

2002 ◽  
Vol 68 (7) ◽  
pp. 3251-3260 ◽  
Author(s):  
Nicola Ilk ◽  
Christine Völlenkle ◽  
Eva M. Egelseer ◽  
Andreas Breitwieser ◽  
Uwe B. Sleytr ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Fusion Protein ◽  
Secondary Cell Wall ◽  
Bacillus Sphaericus ◽  
Terminal Part ◽  
Truncated Form ◽  
Sequence Encoding ◽  
Bet V1 ◽  
Secondary Cell Wall Polymer

ABSTRACT 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 (rSbpA31-1068). 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 rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/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.

scholarly journals Structural and Functional Analyses of the Secondary Cell Wall Polymer of Bacillus sphaericus CCM 2177 That Serves as an S-Layer-Specific Anchor

1999 ◽  
Vol 181 (24) ◽  
pp. 7643-7646 ◽  
Author(s):  
Nicola Ilk ◽  
Paul Kosma ◽  
Michael Puchberger ◽  
Eva M. Egelseer ◽  
Harald F. Mayer ◽  
...  
Keyword(s):  
Cell Wall ◽  
Hydrofluoric Acid ◽  
Secondary Cell Wall ◽  
Bacillus Sphaericus ◽  
Nuclear Magnetic Resonance Analysis ◽  
Terminal Part ◽  
Resonance Analysis ◽  
Cell Wall Polymer ◽  
Functional Analyses ◽  
Secondary Cell Wall Polymer

ABSTRACT 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)]∼0.5-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→. The N-terminal part of the S-layer protein carrying S-layer homologous motifs recognizes this polymer as a binding site.

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

1998 ◽  
Vol 180 (24) ◽  
pp. 6780-6783 ◽  
Author(s):  
Margit Sára ◽  
Eva M. Egelseer ◽  
Christine Dekitsch ◽  
Uwe B. Sleytr
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Secondary Cell Wall ◽  
Bacillus Stearothermophilus ◽  
Binding Domain ◽  
Terminal Part ◽  
Binding Domains ◽  
Cell Wall Polymer ◽  
Function Relationship ◽  
Secondary Cell Wall Polymer

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

scholarly journals 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

2007 ◽  
Vol 189 (19) ◽  
pp. 7154-7158 ◽  
Author(s):  
Judith Ferner-Ortner ◽  
Christoph Mader ◽  
Nicola Ilk ◽  
Uwe B. Sleytr ◽  
Eva M. Egelseer
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Secondary Cell Wall ◽  
Geobacillus Stearothermophilus ◽  
Binding Behavior ◽  
Cell Wall Polymer ◽  
Secondary Cell Wall Polymer

ABSTRACT Surface plasmon resonance studies using C-terminal truncation forms of the S-layer protein SbsC (recombinant SbsC consisting of amino acids 31 to 270 [rSbsC31-270] and rSbsC31-443) 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.

scholarly journals Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology

2004 ◽  
Vol 186 (6) ◽  
pp. 1758-1768 ◽  
Author(s):  
Christoph Mader ◽  
Carina Huber ◽  
Dieter Moll ◽  
Uwe B. Sleytr ◽  
Margit Sára
Keyword(s):  
Cell Wall ◽  
Surface Plasmon Resonance ◽  
Real Time ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Binding Sites ◽  
Secondary Cell Wall ◽  
Sensor Chip ◽  
Geobacillus Stearothermophilus ◽  
Secondary Cell Wall Polymer

ABSTRACT The interaction between S-layer protein SbsB and the secondary cell wall polymer (SCWP) of Geobacillus stearothermophilus PV72/p2 was investigated by real-time surface plasmon resonance biosensor technology. The SCWP is an acidic polysaccharide that contains N-acetylglucosamine, N-acetylmannosamine, and pyruvic acid. For interaction studies, recombinant SbsB (rSbsB) and two truncated forms consisting of either the S-layer-like homology (SLH) domain (3SLH) or the residual part of SbsB were used. Independent of the setup, the data showed that the SLH domain was exclusively responsible for SCWP binding. The interaction was found to be highly specific, since neither the peptidoglycan nor SCWPs from other organisms nor other polysaccharides were recognized. Data analysis from that setup in which 3SLH was immobilized on a sensor chip and SCWP represented the soluble analyte was done in accordance with a model that describes binding of a bivalent analyte to a fixed ligand in terms of an overall affinity for all binding sites. The measured data revealed the presence of at least two binding sites on a single SCWP molecule with a distance of about 14 nm and an overall K d of 7.7 × 10−7 M. Analysis of data from the inverted setup in which the SCWP was immobilized on a sensor chip was done in accordance with an extension of the heterogeneous-ligand model, which indicated the existence of three binding sites with low (K d = 2.6 × 10−5 M), medium (K d = 6.1 × 10−8 M), and high (K d = 6.7 × 10−11 M) affinities. Since in this setup 3SLH was the soluble analyte and the presence of small amounts of oligomers in even monomeric protein solutions cannot be excluded, the high-affinity binding site may result from avidity effects caused by binding of at least dimeric 3SLH. Solution competition assays performed with both setups confirmed the specificity of the protein-carbohydrate interaction investigated.

scholarly journals Enterolysin A, a Cell Wall-Degrading Bacteriocin from Enterococcus faecalis LMG 2333

2003 ◽  
Vol 69 (5) ◽  
pp. 2975-2984 ◽  
Author(s):  
Trine Nilsen ◽  
Ingolf F. Nes ◽  
Helge Holo
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Amino Acid ◽  
Enterococcus Faecalis ◽  
Antimicrobial Protein ◽  
Terminal Part ◽  
Calculated Molecular Weight ◽  
Solid Media ◽  
Dependent Signal ◽  
Modular Structures

ABSTRACT A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35°C), the bacteriocin activity was increased to 5,120 bacteriocin units ml−1. Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.

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