Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties.

Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA's ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in ß-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic "raincoat" observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA's role in forming a stable film integral to B. subtilis biofilm hydrophobicity.

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA.

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibres of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.

Bifunctionality of a biofilm matrix protein controlled by redox state.

Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is critical to the success of biofilms in diverse habitats; however, many details of the composition, structure, and function remain enigmatic. Biofilms formed by the Gram-positive bacterium Bacillus subtilis depend on the production of the secreted film-forming protein BslA. Here, we show that a gradient of electron acceptor availability through the depth of the biofilm gives rise to two distinct functional roles for BslA and that these roles can be genetically separated through targeted amino acid substitutions. We establish that monomeric BslA is necessary and sufficient to give rise to complex biofilm architecture, whereas dimerization of BslA is required to render the community hydrophobic. Dimerization of BslA, mediated by disulfide bond formation, depends on two conserved cysteine residues located in the C-terminal region. Our findings demonstrate that bacteria have evolved multiple uses for limited elements in the matrix, allowing for alternative responses in a complex, changing environment.

Natural variations in the biofilm-associated protein BslA from the genus Bacillus.

BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly.

BslA-stabilized emulsion droplets with designed microstructure.

Emulsions are a central component of many modern formulations in food, pharmaceuticals, agrichemicals and personal care products. The droplets in these formulations are limited to being spherical as a consequence of the interfacial tension between the dispersed phase and continuous phase. The ability to control emulsion droplet morphology and stabilize non-spherical droplets would enable the modification of emulsion properties such as stability, substrate binding, delivery rate and rheology. One way of controlling droplet microstructure is to apply an elastic film around the droplet to prevent it from relaxing into a sphere. We have previously shown that BslA, an interfacial protein produced by the bacterial genus Bacillus, forms an elastic film when exposed to an oil- or air-water interface. Here, we highlight BslA's ability to stabilize anisotropic emulsion droplets. First, we show that BslA is capable of arresting dynamic emulsification processes leading to emulsions with variable morphologies depending on the conditions and emulsification technique applied. We then show that frozen emulsion droplets can be manipulated to induce partial coalescence. The structure of the partially coalesced droplets is retained after melting, but only when there is sufficient free BslA in the continuous phase. That the fidelity of replication can be tuned by adjusting the amount of free BslA in solution suggests that freezing BslA-stabilized droplets disrupts the BslA film. Finally, we use BslA's ability to preserve emulsion droplet structural integrity throughout the melting process to design emulsion droplets with a chosen shape and size.

A phenomenological description of BslA assemblies across multiple length scales.

Intrinsically interfacially active proteins have garnered considerable interest recently owing to their potential use in a range of materials applications. Notably, the fungal hydrophobins are known to form robust and well-organized surface layers with high mechanical strength. Recently, it was shown that the bacterial biofilm protein BslA also forms highly elastic surface layers at interfaces. Here we describe several self-assembled structures formed by BslA, both at interfaces and in bulk solution, over a range of length scales spanning from nanometres to millimetres. First, we observe transiently stable and highly elongated air bubbles formed in agitated BslA samples. We study their behaviour in a range of solution conditions and hypothesize that their dissipation is a consequence of the slow adsorption kinetics of BslA to an air-water interface. Second, we describe elongated tubules formed by BslA interfacial films when shear stresses are applied in both a Langmuir trough and a rheometer. These structures bear a striking resemblance, although much larger in scale, to the elongated air bubbles formed during agitation. Taken together, this knowledge will better inform the conditions and applications of how BslA can be used in the stabilization of multi-phase materials.This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Celebrating Soft Matter's 10th Anniversary: Simplicity in complexity--towards a soft matter physics of caramel.

Caramel is a mixture of sugars, milk proteins, fat and water cooked at high temperatures to initiate Maillard reactions. We study caramels as 'active emulsion-filled protein gels', in which fat droplets are chemically-bonded to a background gel matrix of cross-linked proteins in a concentrated aqueous sugar solution. We delimit a 'caramel region' in composition space. Oscillatory rheology within this region reveals that we can superpose the mechanical spectra of our caramels onto a single pair of G'(ω), G''(ω) master curves using time-composition superposition (tCS) over 12 decades of frequency, so that these caramels are instances of an underlying 'universal material'. This insight constrains the molecular mechanisms for structure formation, and implies that measuring a couple of parameters will suffice to predict the rheology of our caramels over 12 orders of magnitude in frequency.

Interfacial self-assembly of a bacterial hydrophobin.

The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-positive bacterium Bacillus subtilis forms biofilms that exhibit a characteristic wrinkled morphology and a highly hydrophobic surface. A critical component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. We recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also observed in the class of surface-active proteins known as hydrophobins, and are thought to mediate their interfacial activity. However, although functionally related to the hydrophobins, BslA shares no sequence nor structural similarity, and here we show that the mechanism of action is also distinct. Specifically, our results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aqueous solution by adopting a random coil conformation, enabling the protein to be soluble and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side chains into the air or oil phase and forming a three-stranded ß-sheet. This form then self-assembles into a well-ordered 2D rectangular lattice that stabilizes the interface. By replacing a hydrophobic leucine in the center of the cap with a positively charged lysine, we changed the energetics of adsorption and disrupted the formation of the 2D lattice. This limited structural metamorphosis represents a previously unidentified environmentally responsive mechanism for interfacial stabilization by proteins.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals.

Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.

In situ AFM study of amelogenin assembly and disassembly dynamics on charged surfaces provides insights on matrix protein self-assembly.

Because self-assembly of matrix proteins is a key step in hard tissue mineralization, developing an understanding of the assembly pathways and underlying mechanisms is likely to be important for successful hard tissue engineering. While many studies of matrix protein assembly have been performed on bulk solutions, in vivo these proteins are likely to be in contact with charged biological surfaces composed of lipids, proteins, or minerals. Here we report the results of an in situ atomic force microscopy (AFM) study of self-assembly by amelogenin--the principal protein of the extracellular matrix in developing enamel--in contact with two different charged substrates: hydrophilic negatively charged bare mica and positively charged 3-aminopropyl triethoxysilane (APS) silanized mica. First we demonstrate an AFM-based protocol for determining the size of both amelogenin monomers and oligomers. Using this protocol, we find that, although amelogenin exists primarily as ~26 nm in diameter nanospheres in bulk solution at a pH of 8.0 studied by dynamic light scattering, it behaves dramatically differently upon interacting with charged substrates at the same pH and exhibits complex substrate-dependent assembly pathways and dynamics. On positively charged APS-treated mica surfaces, amelogenin forms a relatively uniform population of decameric oligomers, which then transform into two main populations: higher-order assemblies of oligomers and amelogenin monomers, while on negatively charged bare mica surfaces, it forms a film of monomers that exhibits tip-induced desorption and patterning. The present study represents a successful attempt to identify the size of amelogenin oligomers and to directly monitor assembly and disassembly dynamics on surfaces. The findings have implications for amelogenin-controlled calcium phosphate mineralization in vitro and may offer new insights into in vivo self-assembly of matrix proteins as well as their control over hard tissue formation.

Dissecting amelogenin protein nanospheres: characterization of metastable oligomers.

Amelogenin self-assembles to form an extracellular protein matrix, which serves as a template for the continuously growing enamel apatite crystals. To gain further insight into the molecular mechanism of amelogenin nanosphere formation, we manipulated the interactions between amelogenin monomers by altering pH, temperature, and protein concentration to create isolated metastable amelogenin oligomers. Recombinant porcine amelogenins (rP172 and rP148) and three different mutants containing only a single tryptophan (Trp(161), Trp(45), and Trp(25)) were used. Dynamic light scattering and fluorescence studies demonstrated that oligomers were metastable and in constant equilibrium with monomers. Stable oligomers with an average hydrodynamic radius (R(H)) of 7.5 nm were observed at pH 5.5 between 4 and 10 mg · ml(-1). We did not find any evidence of a significant increase in folding upon self-association of the monomers into oligomers, indicating that they are disordered. Fluorescence experiments with single tryptophan amelogenins revealed that upon oligomerization the C terminus of amelogenin (around residue Trp(161)) is exposed at the surface of the oligomers, whereas the N-terminal region around Trp(25) and Trp(45) is involved in protein-protein interaction. The truncated rP148 formed similar but smaller oligomers, suggesting that the C terminus is not critical for amelogenin oligomerization. We propose a model for nanosphere formation via oligomers, and we predict that nanospheres will break up to form oligomers in mildly acidic environments via histidine protonation. We further suggest that oligomeric structures might be functional components during maturation of enamel apatite.

Folding, assembly, and aggregation of recombinant murine amelogenins with T21I and P41T point mutations.

Two point mutations (T21I and P40T) within amelogenin have been identified from human DNA sequences in 2 instances of amelogenesis imperfecta. We studied the folding and self-assembly of recombinant amelogenin (rM180) compared to the T21I and P40T mutants analogs. At pH 5.8 and 25°C, rM180 and the P41T mutant existed as monomers, whereas the T21I mutant formed small oligomers. At pH 8 and 25°C, all of the amelogenin samples formed nanospheres with hydrodynamic radii (R(H)) of around 15-16 nm. Upon heating to 37°C, particles of P41T increased in size (R(H) = 18 nm). During thermal denaturation at pH 5.8, both of the mutant proteins refolded more slowly than the wild-type (WT) rM180. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that the WT transformed to a partially folded conformation upon heating and remained stable. Thermal denaturation and refolding studies indicated that the mutants were less stable and exhibit a greater ability to prematurely aggregate compared to the WT. Our data suggest that in the case of P41T, alterations in the self-assembly of amelogenin are a consequence of destabilization of the secondary structure, while in the case of T21I they are a consequence of change in the overall hydrophobicity at the N-terminal region. We propose that alterations in the assembly (i.e. premature aggregation) of mutant amelogenins may have a profound effect on intra- and extracellular processes such as amelogenin secretion, proteolysis, and its interactions with nonamelogenins as well as with the forming mineral.

The cooperation of enamelin and amelogenin in controlling octacalcium phosphate crystal morphology.

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation.

Structural analysis of a repetitive protein sequence motif in strepsirrhine primate amelogenin.

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates.

Probing the self-association, intermolecular contacts, and folding propensity of amelogenin.

Amelogenins are an intrinsically disordered protein family that plays a major role in the development of tooth enamel, one of the most highly mineralized materials in nature. Monomeric porcine amelogenin possesses random coil and residual secondary structures, but it is not known which sequence regions would be conformationally attractive to potential enamel matrix targets such as other amelogenins (self-assembly), other matrix proteins, cell surfaces, or biominerals. To address this further, we investigated recombinant porcine amelogenin (rP172) using "solvent engineering" techniques to simultaneously promote native-like structure and induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules. We discovered that in the presence of 2,2,2-trifluoroethanol (TFE) significant folding transitions and stabilization occurred primarily within the N- and C-termini, while the polyproline Type II central domain was largely resistant to conformational transitions. Seven Pro residues (P2, P127, P130, P139, P154, P157, P162) exhibited conformational response to TFE, and this indicates these Pro residues act as folding enhancers in rP172. The remaining Pro residues resisted TFE perturbations and thus act as conformational stabilizers. We also noted that TFE induced rP172 self-association via the formation of intermolecular contacts involving P4-H6, V19-P33, and E40-T58 regions of the N-terminus. Collectively, these results confirm that the N- and C-termini of amelogenin are conformationally responsive and represent potential interactive sites for amelogenin-target interactions during enamel matrix mineralization. Conversely, the Pro, Gln central domain is resistant to folding and this may have important functional significance for amelogenin.

Perturbed amelogenin secondary structure leads to uncontrolled aggregation in amelogenesis imperfecta mutant proteins.

Mutations in amelogenin sequence result in defective enamel, and the diverse group of genetically altered conditions is collectively known as amelogenesis imperfecta (AI). Despite numerous studies, the detailed molecular mechanism of defective enamel formation is still unknown. In this study, we have examined the biophysical properties of a recombinant murine amelogenin (rM180) and two point mutations identified from human DNA sequences in two cases of AI (T21I and P41T). At pH 5.8 and 25 °C, wild type (WT) rM180 and mutant P41T existed as monomers, and mutant T21I formed lower order oligomers. CD, dynamic light scattering, and fluorescence studies indicated that rM180 and P41T can be classified as a premolten globule-like subclass protein at 25 °C. Thermal denaturation and refolding monitored by CD ellipticity at 224 nm indicated the presence of a strong hysteresis in mutants compared with WT. Variable temperature tryptophan fluorescence and dynamic light scattering studies showed that WT transformed to a partially folded conformation upon heating and remained stable. The partially folded conformation formed by P41T, however, readily converted into a heterogeneous population of aggregates. T21I existed in an oligomeric state at room temperature and, upon heating, rapidly formed large aggregates over a very narrow temperature range. Thermal denaturation and refolding studies indicated that the mutants are less stable and exhibit poor refolding ability compared with WT rM180. Our results suggest that alterations in self-assembly of amelogenin are a consequence of destabilization of the intrinsic disorder. Therefore, we propose that, like a number of other human diseases, AI appears to be due to the destabilization of the secondary structure as a result of amelogenin mutations.

Tooth enamel proteins enamelin and amelogenin cooperate to regulate the growth morphology of octacalcium phosphate crystals.

To examine the hypothetical cooperative role of enamelin and amelogenin in controlling the growth morphology of enamel crystals in the post-secretory stage, we applied a cation selective membrane system for the growth of octacalcium phosphate (OCP) in the truncated recombinant porcine amelogenin (rP148) with and without the 32kDa enamelin fragment. Enamelin alone inhibited the growth in the c-axis direction more than rP148, yielding OCP crystals with the smallest aspect ratio of all conditions tested. When enamelin was added to the amelogenin "gel-like matrix", the inhibitory action of the protein mixture on the growth of OCP in the c-axis direction was diminished, while that in the b-axis direction was increased. As a result, the length to width ratio (aspect ratio) of OCP crystal was markedly increased. Addition of enamelin to amelogenin enhanced the potential of amelogenin to stabilize the amorphous calcium phosphate (ACP) transient phase. The ratio of enamelin and amelogenin was crucial for stabilization of ACP and the growth of OCP crystals with larger aspect ratio. The cooperative regulatory action of enamelin and amelogenin was attained, presumably, through co-assembling of enamelin and amelogenin. These results have important implications in understanding the growth mechanism of enamel crystals with large aspect ratio.