Development of a fumonisin-sensitive Saccharomyces cerevisiae indicator strain and utilization for activity testing of candidate detoxification genes.

IMPORTANCE: Fumonisins can cause diseases in animals and humans consuming Fusarium-contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.

Heterotetramers formed by an S-layer-streptavidin fusion protein and core-streptavidin as a nanoarrayed template for biochip development.

Based on the S-layer protein SbpA of Bacillus sphaericus CCM 2177, an S-layer-streptavidin fusion protein was constructed. After heterologous expression, isolation of the fusion protein, and refolding, functional heterotetramers were obtained that had retained the ability to recrystallize into the square-lattice structure on plain gold chips and on gold chips precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Monolayers generated by recrystallization of heterotetramers on plain gold chips or on gold chips precoated with thiolated SCWP were exploited for the binding of biotinylated oligonucleotides (30-mers). Hybridization experiments with complementary fluorescently labeled oligonucleotides carrying one mismatch or no mismatch (both 15-mers) were performed and evaluated with surface-plasmon-field-enhanced fluorescence spectroscopy. For surfaces generated by the recrystallization of heterotetramers on SCWP-coated gold chips, a detection limit of 1.57 pM could be determined, whereas for surfaces obtained by direct recrystallization of heterotetramers on plain gold chips, a detection limit of 8.2 pM was found. Measuring the association and dissociation processes of oligonucleotides carrying no mismatch led to a dissociation constant of K(D)=6.3 x 10(-10) m, whereas for oligonucleotides carrying one mismatch a dissociation constant of K(D)=7.9 x 10(-9) m was determined. This finding was confirmed by measuring the whole Langmuir isotherm, which resulted in a dissociation constant of K(D)=2.6 x 10(-8) m.

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.

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 Kd of 7.7 x 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 (Kd = 2.6 x 10(-5) M), medium (Kd = 6.1 x 10(-8) M), and high (Kd = 6.7 x 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.

Biophysical characterization of the entire bacterial surface layer protein SbsB and its two distinct functional domains.

The crystalline bacterial cell surface layer (S-layer) protein SbsB of Geobacillus stearothermophilus PV72/p2 was dissected into an N-terminal part defined by the three consecutive S-layer homologous motifs and the remaining large C-terminal part. Both parts of the mature protein were produced as separate recombinant proteins (rSbsB(1-178) and rSbsB(177-889)) and compared with the full-length form rSbsB(1-889) (rSbsB). Evidence for functional and structural integrity of the two truncated forms was provided by optical spectroscopic methods and electron microscopy. In particular, binding of the secondary cell wall polymer revealed a high affinity dissociation constant of 3 nm and could be assigned solely to the soluble rSbsB(1-178), whereas rSbsB(177-889) self-assembled into the same lattice as the full-length protein. Furthermore, thermal as well as guanidinium hydrochloride induced equilibrium unfolding profiles monitored by intrinsic fluorescence, and circular dichroism spectroscopy allowed characterization of rSbsB(1-178) as an alpha-helical protein with a single cooperative unfolding transition yielding a DeltaG value of 26.5 kJ mol(-1). The C-terminal rSbsB(177-889) could be characterized as a beta-sheet protein with typical multidomain unfolding, which is partially less stable as stand-alone protein. In general, the truncated forms showed identical properties compared with the full-length rSbsB with respect to structure and function. Consequently, rSbsB is characterized by its two functionally and structurally separated parts, the specific secondary cell wall polymer binding rSbsB(1-178) and the larger rSbsB(177-889) responsible for formation of the crystalline array.

S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays.

Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding d-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.

Analysis of the structure-function relationship of the S-layer protein SbsC of Bacillus stearothermophilus ATCC 12980 by producing truncated forms.

The mature surface layer (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and self-assembles into an oblique lattice type which functions as an adhesion site for a cell-associated high-molecular-mass exoamylase. To elucidate the structure-function relationship of distinct segments of SbsC, three N- and seven C-terminal truncations were produced in a heterologous expression system, isolated, purified and their properties compared with those of the recombinant mature S-layer protein rSbsC(31-1099). With the various truncated forms it could be demonstrated that the N-terminal part (aa 31-257) is responsible for anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, but this positively charged segment is not required for the self-assembly of SbsC, nor for generating the oblique lattice structure. If present, the N-terminal part leads to the formation of in vitro double-layer self-assembly products. Affinity studies further showed that the N-terminal part includes an exoamylase-binding site. Interestingly, the N-terminal part carries two sequences of 6 and 7 aa (AKAALD and KAAYEAA) that were also identified on the amylase-binding protein AbpA of Streptococcus gordonii. In contrast to the self-assembling N-terminal truncation rSbsC(258-1099), two further N-terminal truncations (rSbsC(343-1099), rSbsC(447-1099)) and three C-terminal truncations (rSbsC(31-713), rSbsC(31-844), rSbsC(31-860)) had lost the ability to self-assemble and stayed in the water-soluble state. Studies with the self-assembling C-terminal truncations rSbsC(31-880), rSbsC(31-900) and rSbsC(31-920) revealed that the C-terminal 219 aa can be deleted without interfering with the self-assembly process, while the C-terminal 179 aa are not required for the formation of the oblique lattice structure.