Impact of surface wettability on S-layer recrystallization: a real-time characterization by QCM-D

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the assembly and recrystallization kinetics of isolated SbpA bacterial surface proteins onto silicon dioxide substrates of different surface wettability. Surface modification by UV/ozone oxidation or by vapor deposition of 1H,1H,2H,2H-perfluorododecyltrichlorosilane yielded hydrophilic or hydrophobic samples, respectively. Time evolution of frequency and dissipation factors, either individually or combined as the so-calledDfplots, showed a much faster formation of crystalline coatings for hydrophobic samples, characterized by a phase-transition peak at around the 70% of the total mass adsorbed. This behavior has been proven to mimic, both in terms of kinetics and film assembly steps, the recrystallization taking place on an underlying secondary cell-wall polymer (SCWP) as found in bacteria. Complementary atomic force microscopy (AFM) experiments corroborate these findings and reveal the impact on the final structure achieved.

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

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.

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

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.

The first biantennary bacterial secondary cell wall polymer and its influence on S-layer glycoprotein assembly

The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze—thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer—peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→4)-[α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)]-β-d-ManpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→3)-α-d-GlcpNAc-(1→3)-α-d-GlcpNAc-(1→O)-PO2--O-PO2--(O→6)-MurNAc- (where MurNAc is N-acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N-acetylmuramic acid residue, in average, of the A1γ-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S-layer glycoprotein in vitro. Initial crystallization experiments performed with the soluble S-layer glycoprotein revealed that the assembly property could be restored upon dissociation of the polymer by the addition of poly(ethylene glycols). The formed two-dimensional crystalline S-layer self-assembly products exhibited the same lattice symmetry as observed on intact bacterial cells.