ACS Publications. Most Trusted. Most Cited. Most Read
Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Langmuir 2024, 40, 8, 4164-4173
  • Open Access
Article

Bacillus subtilis Matrix Protein TasA is Interfacially Active, but BslA Dominates Interfacial Film Properties
Click to copy article linkArticle link copied!

  • Ryan J. Morris
    Ryan J. Morris
    School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.
    National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.
    More by Ryan J. Morris
    Orcidhttps://orcid.org/0000-0003-4764-3639
  • Natalie C. Bamford
    Natalie C. Bamford
    National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.
    Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
    More by Natalie C. Bamford
    Orcidhttps://orcid.org/0000-0003-1959-856X
  • Keith M. Bromley
    Keith M. Bromley
    School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.
    More by Keith M. Bromley
  • Elliot Erskine
    Elliot Erskine
    Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
    More by Elliot Erskine
  • Nicola R. Stanley-Wall
    Nicola R. Stanley-Wall
    Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
    More by Nicola R. Stanley-Wall
  • Cait E. MacPhee*
    Cait E. MacPhee
    School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.
    National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.
    *Email: [email protected]
    More by Cait E. MacPhee
Open PDFSupporting Information (3)

Langmuir

Cite this: Langmuir 2024, 40, 8, 4164–4173
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.langmuir.3c03163
Published February 13, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

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 β-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.

This publication is licensed under

CC-BY 4.0 .
  • cc licence
  • by licence
Copyright © 2024 The Authors. Published by American Chemical Society

Subjects

what are subjects

Introduction

Click to copy section linkSection link copied!
Microbial biofilms are complex communities encapsulated in a self-produced matrix comprising proteins, carbohydrates, small metabolites, and extracellular DNA. The biofilm mode of growth confers many benefits to inhabitants such as increased resistance to environmental assault and change. (1,2) The matrix components are diverse and dependent on the microbial species as well as environmental conditions. (2,3) Some biofilm matrices have been found to include proteins with interesting properties. Bacillus subtilis is a model organism in the study of biofilm formation. The matrix produced by B. subtilis includes two key proteins: the interfacially active protein BslA, and the fiber-forming protein TasA (Figure 1A). (4−6)

Figure 1

Figure 1. TasA and BslA are B. subtilis matrix proteins. (A) Schematic representation of a B. subtilis biofilm showing that the extracellular matrix surrounding the cells confers protection from environmental pressures. The matrix proteins TasA (purple) and BslA (green) are both secreted as monomers and can take on higher-order structures. The hydrophobic BslA film coats the colony biofilm and the TasA fibers contribute to structure and biofilm formation. The representation is for illustrative purposes and not to scale. (B) Cartoon representations of the crystal structures of TasA (purple, PDB 5OF2) and BslA (green, PDB 4HBU chains J [cap-in] and H [cap-out]). The N and C-termini are labeled with the appropriate letters. (C) Diagrams of the protein domains of TasA and BslA numbered based on the amino acid sequences of each protein. The unprocessed proteins (PreTasA and PreBslA) are displayed with signal peptides (SP) in gray and the secreted domains in purple or green. The recombinant constructs (fTasA, mTasA, BslA, and BslA AxA) are also shown for clarity.

High Resolution Image
Download MS PowerPoint Slide
BslA is a secreted protein exhibiting an immunoglobulin-fold (Figure 1B,C) (3) that is required for B. subtilis sliding motility and biofilm structure. (4,7−9) At a hydrophobic/hydrophilic interface, the “cap” of BslA (constituted by the loops at one end of the β-sandwich) undergoes a structural rearrangement, exposing hydrophobic residues to the hydrophobic phase. This rearrangement causes the loops that had no secondary structure to form a new β-sheet. The increase in β-sheet structure has been seen in both the crystal structure and circular dichroism spectroscopy of BslA-stabilized refractive index matched emulsions (RIMEs). (10) This structural change has been predicted to create an energy barrier that prevents BslA from returning to the hydrophilic phase, thereby contributing to film stability. (11,12) The protein then self-assembles laterally to form a regular lattice. (10,13) Due to a cysteine motif (C178 and C180) at the C-terminus of BslA, the protein is found predominantly as a dimer in solution with the cap of one monomer adsorbing to the interface. (10) Mutation of the cysteine motif to alanine residues produces a monomeric form of BslA (BslA AxA) that produces films of equivalent stability to the wildtype protein (14) (Figure 1C). The ability of BslA to form stable elastic films at air/water and oil/water interfaces has been of interest to biotechnology, including formulation and food industries. (12,15−18)
TasA is the other main protein component of the B. subtilis matrix. Like BslA, TasA is a secreted protein with a β-sandwich fold (19) (Figure 1B). Upon secretion, the signal peptide is cleaved by SipW, and the mature protein comprising residues 28 to 261 is released. (20) The new N-terminus can participate in strand exchange with another TasA monomer, leading to polymerization of the monomers into filaments. (21) The presence of TasA filaments, or fibers, and their ability to bundle is required for biofilm structure. (6,22) These fibers have been found to polymerize spontaneously in vitro (22) Due to their mode of polymerization, the addition of a single serine at the N-terminus blocks fiber formation yielding monomeric TasA. (23) Recently, TasA was shown to associate with the lipid raft fraction of the cell membrane. (24−27) Moreover, deletion of tasA decreased sliding motility on biofilm-inducing media but not on soft LB agar. (28) In addition, TasA point mutants influence the hydrophobicity of the biofilm (27,29) suggesting to us that TasA may also have interfacial activity. We set out to determine whether TasA, like BslA, is interfacially active and whether it influences the function of BslA at an interface.

Materials and Methods

Click to copy section linkSection link copied!

Protein Production and Purification

Two versions of TasA were chosen to examine the properties of both the monomeric protein and the fibrous form (Figure 1C). The fibrous TasA construct included the mature secreted protein of residues 28–261 (fTasA), whereas the monomeric construct had an additional serine prior to residue 28 (mTasA). (23) Recombinant BslA constructs used in this study included the secreted form lacking the unstructured N-terminus (hence comprising residues 42–181), termed BslA herein (Figure 1C). In some assays, a variant of this construct, in which cysteines 178 and 180 were mutated to alanine, was used (BslA AxA). This protein is unable to make covalent linkages and remains monomeric. (14)
BslA and TasA proteins were purified as previously described. (5,14,23) In brief, for each protein, a 5 mL LB was supplemented with ampicillin (100 μg/mL) culture of Escherichia coli BL21 (DE3) pLysS cells containing the plasmid for overexpression of glutathione-S-transferase (GST)-BslA fusion, GST-BslA AxA fusion, GST-TasA fusion, or GST-mTasA fusion protein were grown overnight at 130 rpm in a 37 °C warm room (see Table S1 plasmid details). 1 L amount of autoinduction medium supplemented with ampicillin (100 μg/mL) was inoculated (1:1000 [vol/vol]) from the starter cultures and grown at 37 °C and 200 rpm until an OD600 of 0.9 was reached. Induction of protein expression was induced by reducing the temperature to 18 °C for further incubation overnight. Cells were harvested by centrifugation at 4000g for 30 min and stored at −80 °C until further use.
For purification, bacterial pellets were thawed and resuspended in a purification buffer (25 mM Tris-HCl pH 7.5, 250 mM NaCl for fTasA and mTasA, or 50 mM HEPES pH 7.5, 250 mM NaCl for BslA) supplemented with Complete EDTA-free Proteinase Inhibitors (Roche) before lysis using an Emulsiflex cell disruptor (Avestin). Cellular debris was separated by centrifugation at 27,000g for 30 min, and the resulting supernatant was incubated with Glutathione Sepharose 4B resin (GE Healthcare) agarose at a ratio of 750 μL resin per 1 L bacterial culture with gentle rotation for 2–4 h at 4 °C. The mixture of lysate plus beads was passed through a single-use 25 mL gravity flow column (Bio-Rad). The beads were washed twice with 20 mL of an appropriate purification buffer. The GST tag was removed by TEV protease treatment in 25 mL of purification buffer with 0.5 mg of TEV and 1 mM DTT. Following overnight cleavage at 4 °C, the cleaved protein was isolated using a clean gravity column. The TEV protease and any remaining GST was removed by adding 750 μL of fresh Glutathione Sepharose plus 250 μL of Ni-NTA agarose. The solution was incubated with gentle rotation overnight at 4 °C. A final gravity column step led to a flowthrough containing the clean, purified protein. The purified proteins were then concentrated and simultaneously buffer exchanged into 25 mM phosphate buffer pH 7.0 using Vivaspin 20 centrifugal concentrators. Protein purity was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The fTasA sample after purification is intrinsically heterogeneous containing a distribution of fibrils of variable lengths. (23)

Emulsions

Emulsions were created by mixing an 8 mg/mL protein solution in pH 7 phosphate buffer (mTasA or fTasA) with glyceryl trioctanoate (GTO) in an 80:20 (v/v) ratio. The oil/water solution was mixed for 1 min using a rotor stator (IKA Ultra-Turrax T10) at a shear rate of 20,000 s–1. The emulsions were allowed to cream for 20 min after which a 10 μL sample was withdrawn (from the cream fraction if separation occurred) and diluted with phosphate buffer at a ratio of 1:50. This diluted sample was then placed in a cavity slide and covered with a coverslip, which was sealed with clear nail varnish (BarryM All-in-One) to prevent evaporation. Images of the emulsion droplets were captured by using a Nikon Ti–U inverted microscope outfitted with a 10× objective. The slides were kept at room temperature for 1 week and reimaged.

Circular Dichroism Spectroscopy

Circular dichroism spectroscopy of mTasA in solution was performed by the Glasgow Structural Biology Biophysical Characterization Facility. Measurements were performed using a Jasco J-810 spectropolarimeter at an mTasA concentration of 0.5 mg/mL in a 0.02 cm quartz cuvette. Scans were performed in continuous mode between 260–200 nm at a speed of 10 nm/min. The data pitch was 0.2 nm, with a response time of 2 s. Three scans were accumulated and averaged to produce the final curve.
For fTasA, samples were measured at a protein concentration of 0.2 mg/mL (in 25 mM phosphate buffer) in a 0.1 cm quartz cuvette. A scan rate of 50 nm s–1 was used with a data pitch of 0.1 nm and a digital integration time of 1 s. Twenty scans were accumulated and averaged to produce the final curve.
RIMEs were made by first preparing an 80:20 (v/v) decane emulsion with 0.2 mg/mL of protein. The emulsion was mixed for 1 min using a rotor stator (IKA Ultra-Turrax T10) at a shear rate of 20,000 s–1. The emulsion was washed 3 times to remove any residual protein not adsorbed to the oil/water interface. Washes were performed by centrifuging at 1000 rpm for 20 s, a portion of the subphase was removed and replaced with buffer, then gently redispersed. Finally, the subphase was removed and replaced with glycerol to 59% (w/v), at which point the emulsion became transparent. The emulsion was gently remixed on a roller bank and then allowed to cream. The cream was placed in a 1 mm path length quartz cuvette for spectroscopy. To prevent creaming during the experiment, the cuvette was briefly inverted between measurements to redisperse the droplets.

Pendant Drop Tensiometry

Pendant drop experiments were performed on an EasyDrop tensiometer (Krüss, Hamburg, Germany). Protein solutions were diluted to the desired concentration with phosphate buffer made from Milli-Q water and placed in a syringe with a 1.83 mm diameter needle. For the wrinkle relaxation experiments, a 30 μL aqueous droplet of protein solution was expelled into GTO and allowed to equilibrate at room temperature for 30 min. Images were acquired at 1 fps for BslA and 2 fps for mTasA by using a digital camera. Wrinkle relaxation experiments were performed following compression of the droplet, which was achieved by retracting a volume of the protein solution (10 μL, unless otherwise stated). Compression induced the formation of wrinkles in the surface layer. The wrinkles were monitored over a 10 min period. To analyze the relaxation of the wrinkles, a line profile was drawn across the wrinkles. The line profile was plotted using the grayscale values (from 0 to 255) of each pixel along this line using ImageJ. At least 10 wrinkles were monitored over the time course of the experiment. To plot the relaxation rate, the grayscale value of the pixels was normalized, and background corrected (Supporting Figure 1).
Measurements of the dynamic interfacial tension were determined by fitting the droplet shape to the Young–Laplace equation. The dynamics of interfacial protein adsorption can often be characterized by three kinetic regimes: Regime I is a “lag time” where there is no apparent change in the interfacial tension, Regime II is when a sufficient proportion of protein adsorbs to the interface to produce a decrease in interfacial tension, and Regime III is reached when the interfacial tension plateaus to a roughly constant final value. We define the time to interface as the transition between Regime I and Regime II. This was measured by fitting linear functions to Regime I and Regime II and finding their time of intersection.

Brewster Angle Microscopy

BslA AxA (BslA 42–181 C178A C180A) was diluted to 0.005 mg/mL prior to the experiment. The solution was poured into a trough to allow the formation of a BslA single-layer film over time. The imaging area was roughly 2–3 cm away from the Wilhelmy plate. Equilibration was performed with the barriers fully withdrawn (A = 250 cm2). Sequential BAM imaging was performed using a Nanofilm E3Pse at the Diamond Light Source, U.K. In all images, black pixels represent the air/water interface, while nonblack pixels are representative of adsorbed interfacial material.

Results and Discussion

Click to copy section linkSection link copied!

TasA Stabilizes Oil-in-Water Emulsions

We first wished to determine whether TasA can stabilize emulsions, as an indication of potential interfacial activity. To test this, we produced oil-in-water (O–W) emulsions in the presence of fTasA or mTasA and found that both forms of the protein could stabilize emulsion droplets (Figure 2A,C). The emulsions stabilized by mTasA were uniformly dispersed in the continuous phase, with some droplets forming multiple emulsions (W–O–W) (Figure 2A). In contrast, when emulsions were stabilized using fTasA, we found a mixture of dispersed and clustered droplets (Figure 2C), although within the population, we also observed multiple emulsions. The clustered droplets, when viewed under the microscope, appeared bound together or bridged into units that can span hundreds of micrometers in size. We monitored the stability of the emulsions over a span of 1 week and found that the emulsions stabilized by mTasA had largely coalesced (Figure 2B). In contrast, after 7 days, the emulsions stabilized by fTasA were structurally like those observed immediately after emulsification, and both the characteristic flocculation and presence of multiple emulsions could still be clearly observed (Figure 2D). In comparison, emulsions formed under the same conditions but stabilized by BslA were stable and unchanged over the time scale of weeks. (30) These results show that both forms of TasA can stabilize emulsion droplets, indicating that TasA possesses some degree of interfacial activity.

Figure 2

Figure 2. TasA stabilizes oil–water emulsions. Microscope images of oil–water–oil droplets produced from mixing 8 mg/mL mTasA (A, B) or fTasA (C, D) in phosphate buffer with GTO (80:20 v/v). Images are from two time points: immediately after emulsification (A, C) and after 1 week of incubation at room temperature (B, D). Scale bar is 100 μm.

High Resolution Image
Download MS PowerPoint Slide

TasA Changes Structure at the Interface

Many proteins exhibit interfacial activity; however, surface interactions can often result in partial or full denaturation of the protein structure. (31−37) There are also, however, examples of natural protein biosurfactants that possess high surface activities while maintaining large-scale secondary and tertiary structures. (5,10,38,39) Using circular dichroism spectroscopy (CD), we previously showed that BslA does not denature at an interface but instead undergoes a structural transition to a more β-sheet rich structure. (10) To interrogate the structure of TasA at an oil/water interface, we compared the CD spectra of fTasA and mTasA when in an aqueous solution to the equivalent protein-stabilized emulsions. In solution, both forms of TasA possess similar CD spectra with mixed secondary structure content (Figure 3A). These results are consistent with the reported crystal and cryo-electron microscopy structures and previously published CD spectra of TasA. (19,21) When fTasA and mTasA absorb at an oil/water interface, however, both exhibit a structural shift toward greater β-sheet content (Figure 3A). This could be a transition of α-helices or unstructured regions to β-sheets. These findings are reminiscent of the structural transition observed for BslA at an interface. (10) Taken together, these results show that TasA does not denature at an interface but instead undergoes a structural transition, as in other known biosurfactants.

Figure 3

Figure 3. TasA undergoes structural changes upon adsorption to an interface. (A) CD spectroscopy of mTasA (purple) and fTasA (black) in solution (solid lines) and in RIMEs (dashed lines) shows a change in the secondary structure. (B) Pendant drop tensiometry reveals the time evolution of the interfacial tension (IFT) of a droplet of 0.1 mg/mL protein (mTasA, GST, and BslA) in GTO. The mean of three droplets is plotted for each protein with error bars representing SEM.

High Resolution Image
Download MS PowerPoint Slide

TasA Adsorbs to the Interface

Samples of fTasA are inherently heterogeneous, varying in the average length of fiber and degrees of higher-order self-assembly into bundles, with a consequent impact on sample viscosity. (23) Given that the CD spectroscopy results (Figure 3A) show that fTasA and mTasA have very similar secondary structures and undergo a similar structural change when adsorbed to an interface, we focus on the activity and properties of mTasA for the remainder of the study to remove the potential influence of sample heterogeneity. To further assess the interfacial activity of mTasA, we performed pendant drop tensiometry to monitor the kinetics of interfacial adsorption (Figure 3B). We compared mTasA to the eukaryotic detoxification enzyme Glutathione-S Transferase (GST), as a generic globular protein control with a molecular weight close to that of mTasA, and BslA as a known biosurfactant. We found that mTasA significantly lowers the interfacial tension (IFT) between the aqueous droplet and the continuous oil phase, reaching a value of ∼12.5 mN m–1 over a time scale of ∼30 min. Similarly, GST also saturates to an IFT of ∼12.5 mN m–1 (Figure 3B). In contrast, the initial decrease in IFT caused by the adsorption of BslA to the interface plateaus to a value of ∼20 mN m–1 (Figure 3B), as previously observed. (10,13) This IFT “arrest” is due to the formation of a self-assembled elastic protein layer that no longer conforms to the Young–Laplace relation. These results show that while TasA and BslA share similar secondary structural changes when interacting with an interface, the kinetic characteristics of mTasA interfacial adsorption are closer to those of a generic globular protein than to BslA.

TasA Affects BslA Film Formation via Competition

In view of the previous results, we wished to understand whether there is competition or interaction between BslA and TasA at an interface since they originate from the same biological system and are both produced during biofilm formation. First, we studied the change in the IFT as a function of time using pendant drop tensiometry when both BslA and TasA, or BslA and GST, were copresent in the aqueous phase. For both mixed samples, we did not observe an arrest at high IFT values as seen with BslA alone (c.f. Figure 3B). Since this arrest is due to the formation of an elastic film, this result implies that there is no continuous elastic surface layer formed when there is a mixture of both BslA and other interfacially active proteins at the interface.
To explore this hypothesis further, we investigated the robustness of the BslA elastic film formed in the presence or absence of mTasA. An aqueous droplet containing protein was expelled into the oil phase on the end of a needle, and the protein adsorption was allowed to reach equilibrium (30 min). The droplet was then compressed by the retraction of a known volume of fluid back into the needle. Previous work has shown that BslA forms highly elastic films that wrinkle on droplet compression and that these wrinkles do not relax over time (5) (Supporting Information (SI), Movie 1). In contrast, mTasA alone does not produce a wrinkled elastic layer upon compression (SI, Movie 2). To see how the BslA elastic layer is influenced by the presence of mTasA, we added increasing amounts of mTasA mixed with a constant concentration of BslA (0.2 mg/mL or 6.6 mM of BslA dimer) and performed the wrinkle relaxation experiments. We find that at high concentrations of mTasA (0.1 mg/mL or 3.8 mM) at a molar ratio of TasA to dimeric BslA of ∼1:1.7, an elastic layer is formed but relaxes very quickly (∼10 s) (Figure 4B). This implies that a BslA film can form in the presence of mTasA since mTasA on its own does not display the formation of wrinkles under compression. However, the presence of mTasA significantly weakens the robustness of the BslA film at these concentrations. A 10-fold decrease in the mTasA concentration (0.38 mM, ratio of ∼1:17) resulted in a film relaxation time that was nearly 30 times longer (Figure 4B). Another 10-fold decrease in mTasA concentration (0.038 mM, ratio of ∼1:171) produced relaxation behavior that was longer than the experimental time window and resembled the relaxation behavior of BslA alone (Figure 4B). These findings reveal a dose-dependency in which the BslA film has decreased stability with an increase in the presence of another interfacially active protein, mTasA. It is possible that the presence of mTasA at the interface prevents BslA from forming an interconnected network that would otherwise stabilize the surface of the droplet. Alternatively, mTasA may interact with BslA at the interface, modulating the film-forming ability of BslA.

Figure 4

Figure 4. TasA affects the film formation of BslA. (A) Pendant drop tensiometry of BslA (0.2 mg/mL, 6.6 mM) with mTasA (0.1 mg/mL, 3.8 mM) or GST (0.1 mg/mL, 3.8 mM) at an oil/water interface shows a drop in the IFT over time. (B) Effect of mTasA on BslA film formation is dose-dependent as measured by wrinkle relaxation assays. Retraction of 10 μL from an equilibrium state 40 μL droplet in GTO led to visible wrinkles. The relaxation of wrinkles was plotted as a function of time for three different ratios of TasA to the BslA dimer. The concentration of BslA was the same as that in panel (A) at 0.2 mg/mL. (C) Wrinkle relaxation of mTasA/BslA mixture (1:1.7 molar ratio) plotted as a function of time for varied retraction volumes. (D) Time to interface calculated from pendant drop tensiometry for 0.03 mg/mL GST, mTasA, and BslA at an air–water interface for three independent experiments. All plots show the mean of three droplets with error bars representing SEM.

High Resolution Image
Download MS PowerPoint Slide
To further analyze TasA’s effects, we performed experiments where we studied the relaxation of mixed mTasA/BslA droplets with increasing retraction volumes (i.e., increasing compressions) at a constant molar ratio of 1:1.7. We find that by increasing the retraction volume, we observe films that have longer relaxation times (Figure 4C). At high droplet compressions (smaller droplet surface areas), BslA forms more robust films that maintain wrinkle formation. These results suggest that under compression, mTasA is desorbed from the interface while BslA remains. We propose that as the surface area is reduced, mTasA leaves the interface, allowing BslA to make more interactions with neighboring BslA molecules, thereby permitting the establishment of a space-spanning network that is more robust to mechanical perturbations. Since the concentration of mTasA in solution does not change, this result also suggests that mTasA does not modulate BslA self-assembly and film formation via protein–protein interactions.
Finally, we wished to understand how mTasA and BslA potentially compete for space at an interface. We measured the time to the interface for the two proteins using pendant drop tensiometry, this time at an air–water interface. Here, we define the time to interface as the transition time between Regime I and Regime II in the kinetics of interfacial adsorption. We find that BslA is approximately an order of magnitude slower adsorbing to the interface as compared to mTasA and also GST as a control protein (Figure 4D). This very rapid adsorption by mTasA and GST explains why the IFT decreases significantly in the mixed samples containing BslA (Figure 4A): mTasA and GST get to the interface first and dominate the kinetics. This finding also provides insight into why mTasA at high concentrations impacts the film stability, as seen in the mixed ratio pendant drop wrinkle relaxation (Figure 4B). In the wrinkle relaxation assay, pressure is applied after the proteins at the interface have reached an equilibrium state. The kinetics occurring at the beginning lead to a certain ratio of BslA to mTasA at the interface, with mTasA reaching the interface more quickly. Based on the emulsions which are stable for long periods, we suggest that TasA stably associates until the retraction of the droplet and the compressive force induces desorption.

Evolution of BslA Film Formation as Revealed by Brewster Angle Microscopy

To provide additional insight into the results above, we investigated the structure and evolution over time of BslA film formation at an interface using Brewster Angle Microscopy (BAM). To ensure a monolayer of protein at the interface, we employed BslA AxA, which is monomeric in solution. (14) In this experiment, sequential BAM images were taken, and the resultant morphology of the surface layer was imaged (Figure 5A). Small anisotropic clusters of micrometer-scale domains were prevalent at the early stages of film development. From ∼350 s onward and a surface coverage of ∼51%, we observed continuously connected regions of BslA (Figure 5B). This was followed by the formation of large, smooth rafts or islands. It was also seen that movement of surface material slowed down until it essentially stopped by ∼500 s. Finally, we observe the formation of a continuous BslA layer at ∼1000 s within the field of view. We measured surface pressures during equilibration and during compression experiments, which indicated that a continuous layer across the entire trough was not completely achieved after this time frame (Supporting Figure 3). However, after only an area reduction of 15 cm2, we observed a sharp rise in surface pressure, which indicates a large surface layer was present after the initial equilibration. Taken together, these results show that BslA forms distinct, interconnected regions at early times, which grow to form larger rafts that ultimately results in a continuous protein layer at the interface. We additionally performed experiments using the pendant drop to test the film robustness as a function of droplet equilibration time. After a set equilibration time, the pendant drop volume was retracted to provide a compressive force, and we observed whether interfacial layer wrinkling occurred and, if so, how long those wrinkles persisted (Supporting Figure 1). We found that no wrinkling occurred for equilibration times up to 60 s. After 180 s equilibration times, wrinkles do form but relax within ∼40 s. We found longer persisting wrinkles that relax more slowly after 300 s equilibration. Finally, we found that after 600 s of equilibration, wrinkles persisted for the entirety of the observation window (10 min). Although the experimental conditions between trough and pendant drop experiments are different (e.g., protein concentration/surface area and hydrophobic phase), the time scales for film formation and film robustness as probed by pendant drop compression are broadly coincident.

Figure 5

Figure 5. BslA film formation viewed by Brewster angle microscopy. (A) Images of a single region of the buffer/air interface over time labeled in seconds (s). Black pixels represent solution, and brighter pixels are interfacial material (0.005 mg/mL BslA protein). The first image at 247 s shows the microdomains forming. Then clear islands become visible that migrate across the field of view 448 and 509 s. The last 3 time points show the filling of the film into a monolayer. (B) Network of BslA film domains t = 372 s with each large continuous region given a unique color (e.g., red, cyan, yellow, and green) to highlight the extent of interconnectivity. The image was binarized after the threshold greyscale value of 12 was set. All scale bars are 50 μm.

High Resolution Image
Download MS PowerPoint Slide
Biofilms are biological communities of microorganisms formed by the secretion of materials that bind the collective together. In B. subtilis biofilms, TasA plays multiple roles, including structuring and maturation of the biofilm. (6) TasA was also found to affect cellular physiology upon biofilm induction, (25) and part of this was linked to its association with the detergent resistance fraction of the bacterial membrane. (27) The multifunctionality, its location in the extracellular milieux, and its association with a membrane fraction led us to investigate whether TasA had an interfacial activity. Here, we have shown that the B. subtilis biofilm matrix proteins BslA and TasA both have interfacial activity but have very different biophysical properties.
Our results demonstrate that both monomeric and fiber forms of TasA can stabilize oil-in-water emulsion droplets. Interestingly, fTasA stabilized emulsions for significantly longer than the monomeric form. There are a few possible reasons for this. The fibrous form of TasA is more resistant to proteolysis and degradation than mTasA, (23) and hence, intrinsic properties of the proteins could cause the variation in emulsion lifetime. Another possibility is that the multimeric structure affects the stability and/or kinetics of interfacial binding. The fibrous form is likely to create multivalent interactions with the interface. Thus, dissociation of one interfacially active unit (monomer) from the interface could occur, but dissociation of the whole fiber is unlikely. Dissociated units would also be held close to the interface and not diffuse away, leading to increased reassociation events. Additionally, there is evidence that TasA filaments bundle, and this side-to-side interaction may further stabilize the protein at the interface. Another interesting difference between fTasA and nonfibrous mTasA is that fTasA emulsions are nonuniform with persistent clusters of droplets evident. This could be due to filament-filament bundling or bridging between droplets. Another possibility is that one filament participates in the stabilization of more than one droplet. Previous work studying protein fibril stabilization of emulsions found that fibrils above a critical concentration can act as a depletant, inducing flocculation. (40,41) Other work studying Pickering emulsion stabilized by cellulose fibers found that three-dimensional (3D) fiber-flocculated droplet networks could be created. Network formation was dependent on fibril morphology, where highly bundled fibers induced flocculation via depletion, whereas well-separated fibrils created networks of emulsion droplets via interfacial adsorption and bridging. (42) These networks were highly stable and are reminiscent of those observed for fTasA (Figure 1C). Indeed, the size and morphology of the cellulose fibrils are similar to fTasA. (23) It is, therefore, plausible that such a mechanism is at play in the formation of the fTasa-stabilized emulsion networks.
Both multimerization states of the protein undergo a structural change at the interface with an increased β-sheet content. The recent cryo-EM structure of the TasA fiber and the crystal structure of the TasA monomer show very little structural difference between the two, which was also observed by CD previously (23) and herein. This raises the question of which region of the protein undergoes rearrangement at the interface. From the structures, we found a cluster of three helices on the side of the β-sandwich that is not involved in filament formation. The helicity of this region is not predicted by secondary structure servers (JPred and Phyre2 (43,44)), and the region has high B-factors in the crystal structure (indicative of variation between monomers in the crystal or motion of the region); thus, it may represent a region capable of structural rearrangement. This would explain the primary β-sheet minima found in the CD-RIMEs spectra for the interfacially associated protein.
Protein-interfacial interactions can often result in denaturation of the protein structure. However, this work has shown that TasA undergoes a similar restructuring as BslA when adsorbed to an interface. (10) There are other instances of proteins that interact with interfaces that demonstrate this type of reorganization while retaining their overall secondary structure. Ranaspumin-2 (Rsn-2), a biosurfactant associated with the foam nests of the Túngara frog, was shown to undergo a dramatic “clam-shell-like” reorganization of its tertiary structure when adsorbed to an interface while retaining its secondary structure motifs. (38) Moreover, it was found that this process was reversible: the protein could be removed from the interface through compressive forces and then return via diffusion to readsorb to the interface. A similar reversible restructuring has also been reported for the apolipoprotein apolipophorin-III produced by the insect Galleria mellonella. (45) This structural plasticity and reversibility of adsorption would be advantageous to the organism, as it permits the versatile use of protein resources. For instance, we have shown that BslA has a multipurpose function beyond simply forming a hydrophobic layer at the exterior of the biofilm. (14) Similarly, the reversibility of apolipophorin-III allows the organism to reuse this resource for lipoprotein transport, lipopolysaccharide detoxification, and pattern recognition during an innate immune response. (46) We suggest that TasA may also fall within this class of reusable or multipurpose biosurfactants.
The relatively longer TTI of BslA has been attributed to the necessity for the protein to restructure at or prior to reaching the interface. Indeed, point mutants to the BslA “cap” caused changes to the TTI supporting dependency on the cap restructuring. (13) The TTI of mTasA is much shorter than that of BslA, suggesting a lower barrier to adsorption despite also going through restructuring (Figure 4D). It is likely that many factors affect adsorption dynamics, causing the differences between BslA and TasA TTIs, including surface hydrophobicity, the area that restructures, and the energetic barrier to restructuring in solution.
When BslA is adsorbed to the interface, it forms a robust elastic layer that dissipates the energy of compression through deformation or wrinkling. These deformations last indefinitely, contrasting sharply with the TasA films, which form transient wrinkles that very rapidly dissipate. This behavior is like that observed for the biosurfactant Rsn-2 and implies that TasA is removable from an interface when compressive forces are applied. (38) Further work is required to investigate the reversibility of not only monomeric but also fibrous TasA adsorption to an interface, given that fibrous TasA appears to make multivalent interactions.
We have shown that the coadsorption of BslA with other proteins can have a marked effect on both the interfacial tension as well as the ability to form a strong elastic layer. Moreover, our results show that BslA is outcompeted with the interface: BslA is significantly slower to adsorb to the interface compared to TasA and our control protein GST, which prevents BslA from making a strong film. This observation suggests that BslA lateral interactions are integral to the stability of the film. In concert with the BAM results, we posit that the adsorption of other proteins is likely to impede or disrupt the time evolution of BslA network formation. The data in Figure 4B can then be understood within this context: more TasA will take up more space at the interface, interfering with the ability of BslA to form a space-spanning film. Therefore, at those higher TasA concentrations, we observe modified film relaxation dynamics. Moreover, the data from Figure 4C can be rationalized within this model. Under compression, mTasA returns to the aqueous phase, while BslA remains. At greater volume reductions, more mTasA is eliminated from the interface, allowing more contact between BslA molecules and resulting in greater film stability. Similar behavior was observed for BslA when mixed emulsions were made with casein, wherein BslA maintained stable emulsions despite cooling, Tween-80 addition, and melting. (30)
In view of these results, a question arises: how does BslA form a hydrophobic layer at the surface of a biofilm in vivo when there are a multitude of other molecular components possibly interacting with and competing at the air/water interface? Our pendant drop results (Figure 4) show that some film rigidity is still possible when BslA is coadsorbed to an interface. Importantly, compressive forces enhance BslA film formation since BslA remains so tightly bound to the interface relative to other proteins. We speculate that compressive forces within the biofilm (driven by e.g., evaporation and cellular growth) favor the retention of BslA at the interface over other surface-active molecules. Indeed, the highly wrinkled phenotype of B. subtilis biofilms shows that many compressive forces are at work within a growing biofilm, which could aid in the establishment of robust BslA surface layers.

Conclusions

Click to copy section linkSection link copied!
In this work, we explored the interfacial behavior of two primary protein matrix components of B. subtilis biofilms. We demonstrated that the protein TasA stabilizes oil-in water emulsions and that the fibrillar form of the protein exhibits long-term emulsion stability versus the monomer. We showed that the adsorption of TasA to an interface does not result in the denaturation of the protein but rather induces a partial conformational change with an enhancement of β-sheet content. We further investigated the dynamics of TasA interfacial adsorption using pendant drop tensiometry and found that TasA rapidly adsorbs to an interface but does not form an elastic film. This behavior contrasts with the other important biofilm matrix component, BslA, which adsorbs to the interface an order magnitude slower than TasA but forms a robust elastic layer. The stability and mechanical properties of the BslA film could be modulated through the presence and coadsorption of TasA. We performed Brewster Angle Microscopy and imaged the dynamical development and morphological characteristics of the BslA layer, which showed that the protein forms an interconnected gel-like network before forming a continuous layer at an air–water interface. These observations pose new questions about how BslA and TasA may function in an in vivo setting as well as opening new approaches to control the structure and mechanical properties of interfacial protein films.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.3c03163.

  • Plasmids used in this study; diagram of wrinkle analysis; air/water interface kinetics; BAM surface pressure isotherms, and film relaxation dynamics as a function of equilibration time (PDF)

  • BslA forms a robust interfacial film. A 40 L droplet of 0.1 mg/mL BslA is expelled into GTO after 30 min of equilibration time. 10 L volume is withdrawn and subsequently, long-lived wrinkles form within the elastic BslA interfacial layer. The frame rate is 0.1 frame/s (AVI)

  • mTasA does not form a robust interfacial film. A 40 L droplet of 0.1 mg/mL mTasA is expelled into GTO after 30 min of equilibration time. 10 L volume is withdrawn, and it is observed that there is no wrinkling. Additional volumes are withdrawn until the droplet is very small, at which time a very transiently wrinkled film is observed. The frame rate is 1 frames/s (AVI)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
    • Cait E. MacPhee - School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.;  National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.;  Email: [email protected]
  • Authors
    • Ryan J. Morris - School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.;  National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.;  Orcidhttps://orcid.org/0000-0003-4764-3639
    • Natalie C. Bamford - National Biofilms Innovation Centre, Southampton SO17 1GB, U.K.;  Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.;  Orcidhttps://orcid.org/0000-0003-1959-856X
    • Keith M. Bromley - School of Physics & Astronomy, University of Edinburgh, Peter Guthrie Tait Road, Edinburgh EH9 3FD, U.K.;  Present Address: Plantible Foods, 1340 Specialty Drive Ste H, Vista, California 92081, United States
    • Elliot Erskine - Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.;  Present Address: IGS, James Hutton Institute, Invergowrie, Dundee DD2 5DA, U.K
    • Nicola R. Stanley-Wall - Division of Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
  • Author Contributions

    R.J.M., N.C.B., K.M.B., and E.E. designed and performed experiments, R.J.M., N.C.B., K.M.B., and E.E. performed data analysis. N.R.S.W. and C.E. obtained funding. R.J.M. and N.C.B. wrote the manuscript. R.J.M.B., N.C.B., N.R.S.W., and C.E. edited the manuscript.

  • Funding

    The work was funded by the Biotechnology and Biological Science Research Council (BBSRC) [BB/R012415/1] [BB/P001335/1] [BB/X002950/1] and the Wellcome Trust [200208/Z/15/Z]. Dr. Natalie Bamford was supported by an EMBO long-term fellowship (ALTF 471–2020), Dr. Elliot Erskine by the Wellcome Institutional Strategic Support Fund (Award no. 097818/Z/11).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

The authors acknowledge the MVLS Structural Biology and Biophysical Characterization Facility, University of Glasgow, for the CD spectroscopy of mTasA in solution. We thank Tetyana Sukhodub for her help with protein purification, Zoe O.G. Schyns, Marieke Schor, and Lucia Baldauf for helpful discussions during the course of this work.

Abbreviations

Click to copy section linkSection link copied!
CD

circular dichroism

IFT

interfacial tension

BAM

Brewster angle microscopy

References

Click to copy section linkSection link copied!

This article references 46 other publications.

  1. 1
    Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2 (2), 95– 108,  DOI: 10.1038/nrmicro821
    Google Scholar
    1
    Bacterial biofilms: From the natural environment to infectious diseases
    Hall-Stoodley, Luanne; Costerton, J. William; Stoodley, Paul
    Nature Reviews Microbiology (2004), 2 (2), 95-108CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. Biofilms, matrix-enclosed microbial accretions that adhere to biol. or non-biol. surfaces, represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (∼3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.
  2. 2
    Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol. 2010, 8 (9), 623– 633,  DOI: 10.1038/nrmicro2415
    Google Scholar
    2
    The biofilm matrix
    Flemming, Hans-Curt; Wingender, Jost
    Nature Reviews Microbiology (2010), 8 (9), 623-633CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
    A review. The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mech. stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addn., the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
  3. 3
    Hobley, L.; Harkins, C.; MacPhee, C. E.; Stanley-Wall, N. R. Giving Structure to the Biofilm Matrix: An Overview of Individual Strategies and Emerging Common Themes. FEMS Microbiol. Rev. 2015, 39 (5), 649– 669,  DOI: 10.1093/femsre/fuv015
    Google Scholar
    3
    Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes
    Hobley, Laura; Harkins, Catriona; MacPhee, Cait E.; Stanley-Wall, Nicola R.
    FEMS Microbiology Reviews (2015), 39 (5), 649-669CODEN: FMREE4; ISSN:1574-6976. (Oxford University Press)
    Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Crit. to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnol. uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the mols. and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, exptl. techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars.
  4. 4
    Kobayashi, K.; Iwano, M. BslA (YuaB) Forms a Hydrophobic Layer on the Surface of Bacillus Subtilis Biofilms. Mol. Microbiol. 2012, 85 (1), 51– 66,  DOI: 10.1111/j.1365-2958.2012.08094.x
    Google Scholar
    4
    BslA (YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms
    Kobayashi, Kazuo; Iwano, Megumi
    Molecular Microbiology (2012), 85 (1), 51-66CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
    Biofilms are surface-assocd. bacterial aggregates, in which bacteria are enveloped by polymeric substances known as the biofilm matrix. Bacillus subtilis biofilms display persistent resistance to liq. wetting and gas penetration, which probably explains the broad-spectrum resistance of the bacteria in these biofilms to antimicrobial agents. In this study, BslA (formerly YuaB) was identified as a major contributor to the surface repellency of B. subtilis biofilms. Disruption of bslA resulted in the loss of surface repellency and altered the biofilm surface microstructure. BslA localized to the biofilm matrix in an exopolysaccharide-dependent manner. Purified BslA exhibited amphiphilic properties and formed polymers in response to increases in the area of the air-water interface in vitro. Genetic and biochem. analyses showed that the self-polymn. activity of BslA was essential for its ability to localize to the biofilm matrix. Confocal laser scanning microscopy showed that BslA formed a layer on the biofilm surface. Taken together, the authors propose that BslA, standing for biofilm-surface layer protein, is responsible for the hydrophobic layer on the surface of biofilms.
  5. 5
    Hobley, L.; Ostrowski, A.; Rao, F. V.; Bromley, K. M.; Porter, M.; Prescott, A. R.; MacPhee, C. E.; Van Aalten, D. M. F.; Stanley-Wall, N. R. BslA Is a Self-Assembling Bacterial Hydrophobin That Coats the Bacillus Subtilis Biofilm. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (33), 13600– 13605,  DOI: 10.1073/pnas.1306390110
    Google Scholar
    5
    BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm
    Hobley, Laura; Ostrowski, Adam; Rao, Francesco V.; Bromley, Keith M.; Porter, Michael; Prescott, Alan R.; MacPhee, Cait E.; van Aalten, Daan M. F.; Stanley-Wall, Nicola R.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (33), 13600-13605,S13600/1-S13600/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-pos. 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, the authors show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophys. anal. demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Mol. function is revealed from anal. of the crystal structure of BslA, which consists of an Ig-type fold with the addn. of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophys. anal. 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 physiochem. properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. The authors suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.
  6. 6
    Branda, S. S.; Chu, F.; Kearns, D. B.; Losick, R.; Kolter, R. A Major Protein Component of the Bacillus Subtilis Biofilm Matrix. Mol. Microbiol. 2006, 59 (4), 1229– 1238,  DOI: 10.1111/j.1365-2958.2005.05020.x
    Google Scholar
    6
    A major protein component of the Bacillus subtilis biofilm matrix
    Branda, Steven S.; Chu, Frances; Kearns, Daniel B.; Losick, Richard; Kolter, Roberto
    Molecular Microbiology (2006), 59 (4), 1229-1238CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
    Microbes construct structurally complex multi-cellular communities (biofilms) through prodn. of an extracellular matrix. Here we present evidence from SEM showing that a wild strain of the Gram pos. bacterium Bacillus subtilis builds such a matrix. Genetic, biochem. and cytol. evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multi-cellular communities. Extracellular complementation expts. revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.
  7. 7
    Arnaouteli, S.; MacPhee, C. E.; Stanley-Wall, N. R. Just in Case It Rains: Building a Hydrophobic Biofilm the Bacillus Subtilis Way. Curr. Opin. Microbiol. 2016, 34, 7– 12,  DOI: 10.1016/j.mib.2016.07.012
    Google Scholar
    7
    Just in case it rains: building a hydrophobic biofilm the Bacillus subtilis way
    Arnaouteli, Sofia; MacPhee, Cait E.; Stanley-Wall, Nicola R.
    Current Opinion in Microbiology (2016), 34 (), 7-12CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
    A review. Over the millennia, diverse species of bacteria have evolved multiple independent mechanisms to structure sessile biofilm communities that confer protection and stability to the inhabitants. The Gram-pos. soil bacterium Bacillus subtilis biofilm presents as an architecturally complex, highly hydrophobic community that resists wetting by water, solvents, and biocides. This remarkable property is conferred by a small secreted protein called BslA, which self-assembles into an organized lattice at an interface. In the biofilm, prodn. of BslA is tightly regulated and the resultant protein is secreted into the extracellular environment where it forms a very effective communal barrier allowing the resident B. subtilis cells to shelter under the protection of a protein raincoat.
  8. 8
    Hölscher, T.; Kovács, Á. T. Sliding on the Surface: Bacterial Spreading without an Active Motor. Environ. Microbiol. 2017, 19 (7), 2537– 2545,  DOI: 10.1111/1462-2920.13741
    Google Scholar
    8
    Sliding on the surface: bacterial spreading without an active motor
    Holscher Theresa; Kovacs Akos T
    Environmental microbiology (2017), 19 (7), 2537-2545 ISSN:.
    Bacteria are able to translocate over surfaces using different types of active and passive motility mechanisms. Sliding is one of the passive types of movement since it is powered by the pushing force of dividing cells and additional factors facilitating the expansion over surfaces. In this review, we describe the sliding proficient bacteria that were previously investigated in details highlighting the sliding facilitating compounds and the regulation of sliding motility. Besides surfactants that reduce the friction between cells and substratum, other compounds including exopolysaccharides, hydrophobic proteins, or glycopeptidolipids where discovered to promote sliding. Therefore, we present the sliding bacteria in three groups depending on the additional compound required for sliding. Despite recent accomplishments in sliding research there are still many open questions about the mechanisms underlying sliding motility and its regulation in diverse bacterial species.
  9. 9
    Grau, R. R.; de Oña, P.; Kunert, M.; Leñini, C.; Gallegos-Monterrosa, R.; Mhatre, E.; Vileta, D.; Donato, V.; Hölscher, T.; Boland, W. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus Subtilis. mBio 2015, 6 (4), 10– 1128,  DOI: 10.1128/mBio.00581-15
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Bromley, K. M.; Morris, R. J.; Hobley, L.; Brandani, G.; Gillespie, R. M. C.; McCluskey, M.; Zachariae, U.; Marenduzzo, D.; Stanley-Wall, N. R.; MacPhee, C. E. Interfacial Self-Assembly of a Bacterial Hydrophobin. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (17), 5419– 5424,  DOI: 10.1073/pnas.1419016112
    Google Scholar
    10
    Interfacial self-assembly of a bacterial hydrophobin
    Bromley, Keith M.; Morris, Ryan J.; Hobley, Laura; Brandani, Giovanni; Gillespie, Rachel M. C.; McCluskey, Matthew; Zachariae, Ulrich; Marenduzzo, Davide; Stanley-Wall, Nicola R.; MacPhee, Cait. E.
    Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (17), 5419-5424CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-pos. bacterium, Bacillus subtilis, forms biofilms that exhibit a characteristic wrinkled morphol. and a highly hydrophobic surface. A crit. component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. The authors recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also obsd. 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 the authors show that the mechanism of action is also distinct. Specifically, the results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aq. soln. by adopting a random coil conformation, enabling the protein to be sol. and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side-chains into the air or oil phase and forming a 3-stranded β-sheet. This form then self-assembles into a well-ordered 2-dimensional (2D) rectangular lattice that stabilizes the interface. By replacing a hydrophobic Leu residue in the center of the cap with a pos. charged Lys residue, the authors 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.
  11. 11
    Brandani, G. B.; Schor, M.; Morris, R.; Stanley-Wall, N.; MacPhee, C. E.; Marenduzzo, D.; Zachariae, U. The Bacterial Hydrophobin BslA Is a Switchable Ellipsoidal Janus Nanocolloid. Langmuir 2015, 31 (42), 11558– 11563,  DOI: 10.1021/acs.langmuir.5b02347
    Google Scholar
    11
    The Bacterial Hydrophobin BslA is a Switchable Ellipsoidal Janus Nanocolloid
    Brandani, Giovanni B.; Schor, Marieke; Morris, Ryan; Stanley-Wall, Nicola; MacPhee, Cait E.; Marenduzzo, Davide; Zachariae, Ulrich
    Langmuir (2015), 31 (42), 11558-11563CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    BslA is an amphiphilic protein that forms a highly hydrophobic coat around Bacillus subtilis biofilms, shielding the bacterial community from external aq. soln. It has a unique structure featuring a distinct partition between hydrophilic and hydrophobic surfaces. This surface property is reminiscent of synthesized Janus colloids. By investigating the behavior of BslA variants at water-cyclohexane interfaces through a set of multiscale simulations informed by exptl. data, we show that BslA indeed represents a biol. example of an ellipsoidal Janus nanoparticle, whose surface interactions are, moreover, readily switchable. BslA contains a local conformational toggle, which controls its global affinity for, and orientation at, water-oil interfaces. This adaptability, together with single-point mutations, enables the fine-tuning of its solvent and interfacial interactions, and suggests that BslA could be a basis for biotechnol. applications.
  12. 12
    Morris, R. J.; Bromley, K. M.; Stanley-Wall, N.; MacPhee, C. E. A Phenomenological Description of BslA Assemblies across Multiple Length Scales. Philos. Trans. R. Soc., A 2016, 374 (2072), 20150131  DOI: 10.1098/rsta.2015.0131
    Google Scholar
    12
    A phenomenological description of BslA assemblies across multiple length scales
    Morris, Ryan J.; Bromley, Keith M.; Stanley-Wall, Nicola; MacPhee, Cait E.
    Philosophical Transactions of the Royal Society, A: Mathematical, Physical & Engineering Sciences (2016), 374 (2072), 20150131/1-20150131/14CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)
    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 mech. 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 soln., over a range of length scales spanning from nanometers to millimetres. First, we observe transiently stable and highly elongated air bubbles formed in agitated BslA samples. We study their behavior in a range of soln. 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.
  13. 13
    Morris, R. J.; Schor, M.; Gillespie, R. M. C.; Ferreira, A. S.; Baldauf, L.; Earl, C.; Ostrowski, A.; Hobley, L.; Bromley, K. M.; Sukhodub, T. Natural Variations in the Biofilm-Associated Protein BslA from the Genus Bacillus. Sci. Rep. 2017, 7 (1), 6730  DOI: 10.1038/s41598-017-06786-9
    Google Scholar
    13
    Natural variations in the biofilm-associated protein BslA from the genus Bacillus
    Morris Ryan J; Schor Marieke; Baldauf Lucia; Earl Chris; Bromley Keith M; MacPhee Cait E; Gillespie Rachel M C; Ferreira Ana Sofia; Ostrowski Adam; Sukhodub Tetyana; Arnaouteli Sofia; Stanley-Wall Nicola R; Hobley Laura
    Scientific reports (2017), 7 (1), 6730 ISSN:.
    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.
  14. 14
    Arnaouteli, S.; Ferreira, A. S.; Schor, M.; Morris, R. J.; Bromley, K. M.; Jo, J.; Cortez, K. L.; Sukhodub, T.; Prescott, A. R.; Dietrich, L. E. P. Bifunctionality of a Biofilm Matrix Protein Controlled by Redox State. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (30), E6184– E6191,  DOI: 10.1073/pnas.1707687114
    Google Scholar
    14
    Bifunctionality of a biofilm matrix protein controlled by redox state
    Arnaouteli, Sofia; Ferreira, Ana Sofia; Schor, Marieke; Morris, Ryan J.; Bromley, Keith M.; Jo, Jeanyoung; Cortez, Krista L.; Sukhodub, Tetyana; Prescott, Alan R.; Dietrich, Lars E. P.; MacPhee, Cait E.; Stanley-Wall, Nicola R.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (30), E6184-E6191CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is crit. to the success of biofilms in diverse habitats; however, many details of the compn., structure, and function remain enigmatic. Biofilms formed by the Gram-pos. bacterium Bacillus subtilis depend on the prodn. 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 2 distinct functional roles for BslA and that these roles can be genetically sepd. 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 2 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.
  15. 15
    Stanley-Wall, N. R.; MacPhee, C. E. Connecting the Dots between Bacterial Biofilms and Ice Cream. Phys. Biol. 2015, 12 (6), 063001  DOI: 10.1088/1478-3975/12/6/063001
    Google Scholar
    15
    Connecting the dots between bacterial biofilms and ice cream
    Stanley-Wall Nicola R; MacPhee Cait E
    Physical biology (2015), 12 (6), 063001 ISSN:.
    Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.
  16. 16
    Kaufman, G.; Liu, W.; Williams, D. M.; Choo, Y.; Gopinadhan, M.; Samudrala, N.; Sarfati, R.; Yan, E. C. Y.; Regan, L.; Osuji, C. O. Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA. Langmuir 2017, 33 (47), 13590– 13597,  DOI: 10.1021/acs.langmuir.7b03226
    Google Scholar
    16
    Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA
    Kaufman, Gilad; Liu, Wei; Williams, Danielle M.; Choo, Youngwoo; Gopinadhan, Manesh; Samudrala, Niveditha; Sarfati, Raphael; Yan, Elsa C. Y.; Regan, Lynne; Osuji, Chinedum O.
    Langmuir (2017), 33 (47), 13590-13597CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Protein adsorption and assembly at interfaces provides a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil-water interfaces leading to the prodn. of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates in a low areal d. of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small mol. surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic-lipophilic balance (HLB no.) calcd. based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries reveal intriguing reversible and non-reversible modes of mech. deformation, resp.
  17. 17
    Bromley, K. M.; MacPhee, C. E. BslA-Stabilized Emulsion Droplets with Designed Microstructure. Interface Focus 2017, 7 (4), 20160124  DOI: 10.1098/rsfs.2016.0124
    Google Scholar
    17
    BslA-stabilized emulsion droplets with designed microstructure
    Bromley Keith M; MacPhee Cait E
    Interface focus (2017), 7 (4), 20160124 ISSN:2042-8898.
    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.
  18. 18
    Schloss, A. C.; Liu, W.; Williams, D. M.; Kaufman, G.; Hendrickson, H. P.; Rudshteyn, B.; Fu, L.; Wang, H.; Batista, V. S.; Osuji, C. Fabrication of Modularly Functionalizable Microcapsules Using Protein-Based Technologies. ACS Biomater. Sci. Eng. 2016, 2 (11), 1856– 1861,  DOI: 10.1021/acsbiomaterials.6b00447
    Google Scholar
    18
    Fabrication of Modularly Functionalizable Microcapsules Using Protein-Based Technologies
    Schloss, Ashley C.; Liu, Wei; Williams, Danielle M.; Kaufman, Gilad; Hendrickson, Heidi P.; Rudshteyn, Benjamin; Fu, Li; Wang, Hongfei; Batista, Victor S.; Osuji, Chinedum; Yan, Elsa C. Y.; Regan, Lynne
    ACS Biomaterials Science & Engineering (2016), 2 (11), 1856-1861CODEN: ABSEBA; ISSN:2373-9878. (American Chemical Society)
    Proteins are desirable building blocks to create self-assembled, spatially defined structures and interfaces on length-scales that are inaccessible by traditional methods. Here, we describe a novel approach to create functionalized monolayers using the proteins BslA and SpyCatcher/SpyTag. BslA is a bacterial hydrophobin whose amphiphilic character underlies its ability to assemble into a monolayer at both air/water and oil/water interfaces. We demonstrate that having the SpyTag peptide fused at the N- or C-terminus does not affect the formation of such monolayers. We establish the creation of stable oil-in-water microcapsules using BslA, and also show the fabrication of capsules outwardly displaying the reactive SpyTag peptide by fusing it to the C-terminus of BslA. Such capsules can be covalently labeled by reacting the surface-displayed SpyTag with SpyCatcher fused to any desired protein. We demonstrate this principle by labeling microcapsules using green fluorescent protein (GFP). All components are genetically encodable, the reagents can be readily prepd. in large quantities, and all reactions occur at ambient temp. in aq. soln. Thus, this straightforward, modular, scalable strategy has myriad potential applications in the creation of novel, functional materials and interfaces.
  19. 19
    Diehl, A.; Roske, Y.; Ball, L.; Chowdhury, A.; Hiller, M.; Molière, N.; Kramer, R.; Stöppler, D.; Worth, C. L.; Schlegel, B. Structural Changes of TasA in Biofilm Formation of Bacillus Subtilis. Proc. Natl. Acad. Sci. U.S.A. 2018, 115 (13), 3237– 3242,  DOI: 10.1073/pnas.1718102115
    Google Scholar
    19
    Structural changes of TasA in biofilm formation of Bacillus subtilis
    Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Moliere, Noel; Kramer, Regina; Stoeppler, Daniel; Worth, Catherine L.; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; Rossum, Barth-Jan van; Schmieder, Peter; Heinemann, Udo; Turgay, Kuersad; Akbey, Umit; Oschkinat, Hartmut
    Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (13), 3237-3242CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Microorganisms form surface-attached communities, termed bio- films, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addn. to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a sol., stably folded monomer, whose structure we have detd. by X-ray crystallog. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and anal. ultracentrifugation (AUC) expts. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet-rich fibrils, was obsd. in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the mol. level.
  20. 20
    Stöver, A. G.; Driks, A. Secretion, Localization, and Antibacterial Activity of TasA, a Bacillus Subtilis Spore-Associated Protein. J. Bacteriol. 1999, 181 (5), 1664– 1672,  DOI: 10.1128/JB.181.5.1664-1672.1999
    Google Scholar
    20
    Secretion, localization, and antibacterial activity of TasA, a Bacillus subtilis spore-associated protein
    Stover, Axel G.; Driks, Adam
    Journal of Bacteriology (1999), 181 (5), 1664-1672CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    The synthesis and subcellular localization of the proteins that comprise the Bacillus subtilis spore are under a variety of complex controls. To better understand these controls, the authors have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore. TasA synthesis begins approx. 30 min after the onset of sporulation and requires the sporulation transcription factor genes spoOH and spoOA. The first 81 nucleotides of tasA encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from Tas isolated from a sporulating cell lysate. In B. subtilis cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores. Cells unable to produce SipW produce a 34-kDa from of TasA, consistent with a failure to remove the N-terminal 27 amino acids. In cells engineered to express sipW and tasA during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium. These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores. Although TasA is dispensable for sporulation under lab. conditions, we find that TasA has a broad spectrum antibacterial activity. The authors discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus confering a competitive advantage to the spore.
  21. 21
    Böhning, J.; Ghrayeb, M.; Pedebos, C.; Abbas, D. K.; Khalid, S.; Chai, L.; Bharat, T. A. M. Donor-Strand Exchange Drives Assembly of the TasA Scaffold in Bacillus Subtilis Biofilms. Nat. Commun. 2022, 13 (1), 7082  DOI: 10.1038/s41467-022-34700-z
    Google Scholar
    21
    Donor-strand exchange drives assembly of the TasA scaffold in Bacillus subtilis biofilms
    Bohning, Jan; Ghrayeb, Mnar; Pedebos, Conrado; Abbas, Daniel K.; Khalid, Syma; Chai, Liraz; Bharat, Tanmay A. M.
    Nature Communications (2022), 13 (1), 7082CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chem. and mech. stresses. In the Gram-pos. model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibers contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibers, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibers through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fiber. Combining electron cryotomog., at. force microscopy, and mutational studies, we show how TasA fibers congregate in three dimensions to form abundant fiber bundles that are essential for B. subtilis biofilm formation. Our study explains the previously obsd. biochem. properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibers, and how such fibers assemble into bundles in biofilms.
  22. 22
    Romero, D.; Aguilar, C.; Losick, R.; Kolter, R. Amyloid Fibers Provide Structural Integrity to Bacillus Subtilis Biofilms. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (5), 2230– 2234,  DOI: 10.1073/pnas.0910560107
    Google Scholar
    22
    Amyloid fibers provide structural integrity to Bacillus subtilis biofilms
    Romero, Diego; Aguilar, Claudio; Losick, Richard; Kolter, Roberto
    Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (5), 2230-2234, S2230/1-S2230/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, the authors characterized the physicochem. properties of this protein. They report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochem. analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymd. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphol., indicating that the purified protein retained biol. activity. It is proposed that TasA forms amyloid fibers that bind cells together in the biofilm.
  23. 23
    Erskine, E.; Morris, R. J.; Schor, M.; Earl, C.; Gillespie, R. M. C.; Bromley, K. M.; Sukhodub, T.; Clark, L.; Fyfe, P. K.; Serpell, L. C. Formation of Functional, Non-amyloidogenic Fibres by Recombinant Bacillus Subtilis TasA. Mol. Microbiol. 2018, 110 (6), 897– 913,  DOI: 10.1111/mmi.13985
    Google Scholar
    23
    Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA
    Erskine, Elliot; Morris, Ryan J.; Schor, Marieke; Earl, Chris; Gillespie, Rachel M. C.; Bromley, Keith M.; Sukhodub, Tetyana; Clark, Lauren; Fyfe, Paul K.; Serpell, Louise C.; Stanley-Wall, Nicola R.; MacPhee, Cait E.
    Molecular Microbiology (2018), 110 (6), 897-913CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
    Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibers of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fiber forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibers form spontaneously, and share the biol. activity of TasA fibers extd. from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophys. properties of both native and recombinant TasA fibers indicate that they are not functional amyloid-like fibers. A gel formed by TasA fibers can recover after phys. 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 fibers formed by TasA orthologues we uncover species variability in the ability of heterologous fibers to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophys. requirements of the TasA fibers across different species and/or reflect the precise mol. interactions needed for biofilm matrix assembly.
  24. 24
    Malishev, R.; Abbasi, R.; Jelinek, R.; Chai, L. Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein. Biochemistry 2018, 57 (35), 5230– 5238,  DOI: 10.1021/acs.biochem.8b00002
    Google Scholar
    24
    Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein
    Malishev, Ravit; Abbasi, Razan; Jelinek, Raz; Chai, Liraz
    Biochemistry (2018), 57 (35), 5230-5238CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Biofilms are aggregates of cells that form surface-assocd. communities. The cells in a biofilm are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins and sometimes nucleic acids. Some extracellular matrix proteins form fibers, often termed functional amyloids or amyloid-like fibrils, to differentiate their constructive function from disease-related amyloid fibers. Recent studies of functional amyloid assembly have neglected their interaction with membranes, despite their native assembly in a cellular environment. Here, we use the protein TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein, and ask whether a bacterial functional amyloid interacts with membranes. Using biochem., spectroscopic and microscopic tools, we show that TasA interacts distinctively with model bacterial membranes and that this interaction mutually influences the protein and the membranes' morphol. and structure. At the protein's level, TasA fibers of similar structure and morphol. are formed in the absence of membranes and in the presence of the eukaryotic model membranes. However, in the presence of the bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane's level, fluorescence microscopy and fluorescence anisotropy measurements indicate that the bacterial membranes deform more considerably than the eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial model membranes more than eukaryotic membranes and that this, in turn, disrupts the membranes and alters the fiber formation pathway of TasA. Considering the important role of TasA in providing integrity to biofilms, our study of the TasA-membrane interactions may direct the design of anti-biofilm drugs to the protein-membrane interface.
  25. 25
    Mielich-Süss, B.; Schneider, J.; Lopez, D. Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus Subtilis. mBio 2013, 4 (6), e00719-13  DOI: 10.1128/mBio.00719-13
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    López, D.; Kolter, R. Functional Microdomains in Bacterial Membranes. Genes Dev. 2010, 24 (17), 1893– 1902,  DOI: 10.1101/gad.1945010
    Google Scholar
    26
    Functional microdomains in bacterial membranes
    Lopez, Daniel; Kolter, Roberto
    Genes & Development (2010), 24 (17), 1893-1902CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
    The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid-a known inhibitor of squalene synthases-impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiol. processes in microdomains may be a more widespread feature of membranes than previously appreciated.
  27. 27
    Cámara-Almirón, J.; Navarro, Y.; Díaz-Martínez, L.; Magno-Pérez-Bryan, M. C.; Molina-Santiago, C.; Pearson, J. R.; de Vicente, A.; Pérez-García, A.; Romero, D. Dual Functionality of the Amyloid Protein TasA in Bacillus Physiology and Fitness on the Phylloplane. Nat. Commun. 2020, 11 (1), 1859  DOI: 10.1038/s41467-020-15758-z
    Google Scholar
    27
    Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane
    Camara-Almiron, Jesus; Navarro, Yurena; Diaz-Martinez, Luis; Magno-Perez-Bryan, Maria Concepcion; Molina-Santiago, Carlos; Pearson, John R.; de Vicente, Antonio; Perez-Garcia, Alejandro; Romero, Diego
    Nature Communications (2020), 11 (1), 1859CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiol. and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytol. symptoms indicative of excessive cellular stress leading to increased cell death. TasA assocs. to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addn. to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.
  28. 28
    Van Gestel, J.; Vlamakis, H.; Kolter, R. From Cell Differentiation to Cell Collectives: Bacillus Subtilis Uses Division of Labor to Migrate. PLoS Biol. 2015, 13 (4), e1002141  DOI: 10.1371/journal.pbio.1002141
    Google Scholar
    28
    From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate
    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto
    PLoS Biology (2015), 13 (4), e1002141/1-e1002141/29CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)
    We show how flagellum-independent migration is driven by the division of labor of 2 cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called van Gogh bundles) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles det. the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecol. advantage. This is a key factor underlying the diverse origins of multicellularity.
  29. 29
    Dragoš, A.; Martin, M.; Falcón García, C.; Kricks, L.; Pausch, P.; Heimerl, T.; Bálint, B.; Maróti, G.; Bange, G.; López, D. Collapse of Genetic Division of Labour and Evolution of Autonomy in Pellicle Biofilms. Nat. Microbiol. 2018, 3 (12), 1451– 1460,  DOI: 10.1038/s41564-018-0263-y
    Google Scholar
    29
    Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms
    Dragos, Anna; Martin, Marivic; Garcia, Carolina Falcon; Kricks, Lara; Pausch, Patrick; Heimerl, Thomas; Balint, Balazs; Maroti, Gergely; Bange, Gert; Lopez, Daniel; Lieleg, Oliver; Kovacs, Akos T.
    Nature Microbiology (2018), 3 (12), 1451-1460CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
    Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain debatable. Here, we track the evolution of pellicle biofilms formed through genetic division of labor and ask whether partially deficient partners can evolve autonomy. Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exopolysaccharide (EPS) and the fiber protein TasA. In monocultures, Δeps and ΔtasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale and ΔtasA gradually outcompetes its partner Δeps. Pellicle formation can evolve independently from division of labour in Δeps and ΔtasA monocultures, by selection acting on the residual matrix component, TasA or EPS, resp. Using a set of interdisciplinary tools, we unravel that the TasA producer (Δeps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochem. properties of the protein. Conversely, the EPS producer (ΔtasA) undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomech. properties. Finally, we revisit the collapse of division of labour between Δeps and ΔtasA in light of a strong frequency vs. exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an addnl. barrier to evolution of division of labour between genetically distinct microorganisms.
  30. 30
    MacPhee, C.; Stanley-wall, N.; Bromley, K.; Morris, R.; Hobley, L. Synthetic Multiphase Systems, Google Patents, 2020.
    Google Scholar
    There is no corresponding record for this reference.
  31. 31
    Clarkson, J. R.; Cui, Z. F.; Darton, R. C. Protein Denaturation in Foam: I. Mechanism Study. J. Colloid Interface Sci. 1999, 215 (2), 323– 332,  DOI: 10.1006/jcis.1999.6255
    Google Scholar
    31
    Protein denaturation in foam I. Mechanism study
    Clarkson, J. R.; Cui, Z. F.; Darton, R. C.
    Journal of Colloid and Interface Science (1999), 215 (2), 323-332CODEN: JCISA5; ISSN:0021-9797. (Academic Press)
    The aim of this study was to elucidate the mechanism by which protein mols. become denatured in foam. Damage to the protein is mainly due to surface denaturation at the gas-liq. interface. A fraction of the mols. adsorbed do not refold to their native state when they desorb. The degree of denaturation was found to correlate directly with the interfacial exposure, which, for mobile or partially mobile interfaces, is increased by drainage. Expts. with two different proteins showed that, under the conditions of the tests, around 10% of BSA mols. which had adsorbed at the surface remained denatured when they desorbed. For pepsin the figure was around 75%. Oxidn., which was previously thought to be a major cause of protein damage in foam, was found to be minimal. Neither do the high shear stresses in the liq. bulk encountered during bubble bursting cause denaturation, because energy is dissipated at a much greater length scale than that of the protein mol. (c) 1999 Academic Press.
  32. 32
    Graham, D. E.; Phillips, M. C. Proteins at Liquid Interfaces: I. Kinetics of Adsorption and Surface Denaturation. J. Colloid Interface Sci. 1979, 70 (3), 403– 414,  DOI: 10.1016/0021-9797(79)90048-1
    Google Scholar
    32
    Proteins at liquid interfaces. I. Kinetics of adsorption and surface denaturation
    Graham, D. E.; Phillips, M. C.
    Journal of Colloid and Interface Science (1979), 70 (3), 403-14CODEN: JCISA5; ISSN:0021-9797.
    The rates of change of film pressure (π) and surface concn. (Γ) of protein during the adsorption of β-casein, bovine serum albumin (BSA), and lysozyme at the air-water interface were monitored by the Wilhelmy plate and surface radioactivity methods, resp. The increases in π and Γ for the relatively flexible β-casein mol. occur simultaneously with both parameters attaining their steady-state values at about the same time. In contrast, π and Γ follow different time courses for the globular lysozyme mol.; Γ can reach a steady state value while π is still increasing significantly. The kinetics indicate that initially adsorption is diffusion-controlled, but at higher surface coverages there is an energy barrier to adsorption. Under these conditions, the ability of the protein mols. to create space in the existing film and penetrate and rearrange in the surface is rate-detg. Two regions exist: the relaxation time τ1 (typically ∼2 h when Γ ∼2 mg m-2) describes the adsorption when both π and Γ are increasing, whereas τ2 (in the range 1-8 h for all 3 proteins) relates to the situation when π is increasing at const. Γ because the protein mols. are changing conformation in the surface.
  33. 33
    Jungbauer, A.; Machold, C.; Hahn, R. Hydrophobic Interaction Chromatography of Proteins: III. Unfolding of Proteins upon Adsorption. J. Chromatogr. A 2005, 1079 (1–2), 221– 228,  DOI: 10.1016/j.chroma.2005.04.002
    Google Scholar
    33
    Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption
    Jungbauer, Alois; Machold, Christine; Hahn, Rainer
    Journal of Chromatography A (2005), 1079 (1-2), 221-228CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
    Hydrophobic interaction chromatog. (HIC) exploits the hydrophobic properties of protein surfaces for sepn. and purifn. by performing interactions with chromatog. sorbents of hydrophobic nature. In contrast to reversed-phase chromatog., this methodol. is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in the presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concns. under isocratic conditions only, a fraction of the applied amt. is eluted. The higher the salt concn., the lower is the amt. of eluted protein. The rest can be desorbed with a buffer of low salt concn. or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochem. measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, the authors hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.
  34. 34
    Tronin, A.; Dubrovsky, T.; Dubrovskaya, S.; Radicchi, G.; Nicolini, C. Role of Protein Unfolding in Monolayer Formation on Air– Water Interface. Langmuir 1996, 12 (13), 3272– 3275,  DOI: 10.1021/la950879+
    Google Scholar
    34
    Role of Protein Unfolding in Monolayer Formation on Air-Water Interface
    Tronin, Andrey; Dubrovsky, Timothy; Dubrovskaya, Svetlana; Radicchi, Giuliano; Nicolini, Claudio
    Langmuir (1996), 12 (13), 3272-3275CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Mol. exchange kinetics between a monolayer of antibody mols. formed on the air-water interface and the protein soln. was studied by means of fluorescent labeling. It was shown that there is no inclusion of dissolved mols. in the previously formed monolayer during even 6 h of exposure regardless of monolayer surface d. The surface activity of IgG and horseradish peroxidase mols. was studied by means of surface compression isotherms, and the specific biol. activity of the monolayers formed from these proteins was measured by enzyme and immunoassay techniques. It was shown that the surface activity of the proteins increases while specific biol. activity decreases with exposure of the mols. on the water surface. Since the same effects were caused by denaturing agents, we propose that the surface activity of the proteins and the absence of surface-vol. exchange are due to partial unfolding of the mols. which takes place on the water surface. Two models of the partial unfolding are discussed: complete denaturation of some part of the mols. and partial unfolding of each mol. The process of surface denaturation was shown to be slow and controllable. One can achieve a pronounced increase of protein surface activity with low degrdn. of the specific biol. activity of the monolayer; thus, it can be used in the practice of protein Langmuir film deposition.
  35. 35
    Yano, Y. F. Kinetics of Protein Unfolding at Interfaces. J. Phys.: Condens. Matter 2012, 24 (50), 503101  DOI: 10.1088/0953-8984/24/50/503101
    Google Scholar
    35
    Kinetics of protein unfolding at interfaces
    Yano, Yohko
    Journal of Physics: Condensed Matter (2012), 24 (50), 503101/1-503101/16CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
    A review. The conformation of protein mols. is detd. by a balance of various forces, including van der Waals attraction, electrostatic interaction, H-bonding, and conformational entropy. When protein mols. encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein mol. strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that the protein conformation changes during the adsorption process due to protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. Here, the author considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.
  36. 36
    Dickinson, E.; Matsumura, Y. Proteins at Liquid Interfaces: Role of the Molten Globule State. Colloids Surf., B 1994, 3 (1–2), 1– 17,  DOI: 10.1016/0927-7765(93)01116-9
    Google Scholar
    36
    Proteins at liquid interfaces: role of the molten globule state
    Dickinson, Eric; Matsumura, Yasuki
    Colloids and Surfaces, B: Biointerfaces (1994), 3 (1/2), 1-17CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier)
    A review, with 108 refs. Recent advances in understanding the structure and dynamics of proteins at liq. interfaces are reviewed with particular ref. to adsorbed layers at the oil-water interface in protein-stabilized emulsions. The authors discuss the importance of mol. flexibility in detg. the properties of adsorbed layers and the ease of exchange of protein mols. between bulk and surface in mixed systems. While a statistical model of a nearly random copolymer can be used to describe the adsorption of a disordered protein such as β-casein, such a representation is unrealistic for an adsorbed globular protein, which is typically a compact deformable macromol. particle having a structure lying somewhere between the native state and the completely unfolded form. It is proposed that such a state of an adsorbed globular protein at a liq. interface is close to what is now called the "molten globule" state. This is the partially denatured state of a globular protein which retains the ordered secondary structure but not the tertiary structure of the native protein. The authors describe the various ways of producing the molten globule state, and the authors review the exptl. evidence for the molten globule state of α-lactalbumin in some detail. In the final part of the paper, the authors discuss some new results on the surface activity of α-lactalbumin and the competitive adsorption of α-lactalbumin and β-lactoglobulin in emulsions at acidic pH or in the presence of EDTA. This discussion shows how the concept of the molten globule state provides new insight into the relation between protein structure and the properties of adsorbed layers at liq. interfaces.
  37. 37
    Husband, F. A.; Garrood, M. J.; Mackie, A. R.; Burnett, G. R.; Wilde, P. J. Adsorbed Protein Secondary and Tertiary Structures by Circular Dichroism and Infrared Spectroscopy with Refractive Index Matched Emulsions. J. Agric. Food Chem. 2001, 49 (2), 859– 866,  DOI: 10.1021/jf000688z
    Google Scholar
    37
    Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions
    Husband, Fiona A.; Garrood, Martin J.; Mackie, Alan R.; Burnett, Gary R.; Wilde, Peter J.
    Journal of Agricultural and Food Chemistry (2001), 49 (2), 859-866CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    The secondary structure of protein adsorbed at the emulsion interface has been studied in refractive index matched emulsions using the techniques of CD (CD) and Fourier transform IR spectroscopy. Bovine serum albumin (BSA) and bovine β-lactoglobulin (βlg) stabilized emulsions were studied, and the refractive index was altered by the addn. of glycerol or polyethylene glycol. The effect of additive on the soln. and adsorbed protein structure in addn. to the effect of adsorption time was considered. Both adsorption and glycerol addn. alter protein secondary structure; however, the majority of secondary structure remains. Small changes are obsd. in the secondary structure of adsorbed protein with time. Near-UV CD studies showed the effect of glycerol and adsorption on the arom. groups. BSA showed small changes both upon the addn. of glycerol to protein in soln. and upon adsorption. βlg showed slightly larger changes upon the addn. of glycerol to protein in soln. and a larger change upon adsorption.
  38. 38
    Morris, R. J.; Brandani, G. B.; Desai, V.; Smith, B. O.; Schor, M.; MacPhee, C. E. The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway. Biophys. J. 2016, 111 (4), 732– 742,  DOI: 10.1016/j.bpj.2016.06.006
    Google Scholar
    38
    The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway
    Morris, Ryan J.; Brandani, Giovanni B.; Desai, Vibhuti; Smith, Brian O.; Schor, Marieke; MacPhee, Cait E.
    Biophysical Journal (2016), 111 (4), 732-742CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Ranaspumin-2 (Rsn-2) is a surfactant protein found in the foam nests of the t´ungara frog. Previous exptl. work has led to a proposed model of adsorption that involves an unusual clam-shell-like unhinging of the protein at an interface. Interestingly, there is no concomitant denaturation of the secondary structural elements of Rsn-2 with the large-scale transformation of its tertiary structure. In this work we use both expt. and simulation to better understand the driving forces underpinning this unusual process. We develop a modified G‾o-model approach where we have included explicit representation of the side chains to realistically model the interaction between the secondary structure elements of the protein and the interface. Doing so allows for the study of the underlying energy landscape that governs the mechanism of Rsn-2 interfacial adsorption. Exptl., we study targeted mutants of Rsn-2, using the Langmuir trough, pendant drop tensiometry, and CD, to demonstrate that the clam-shell model is correct. We find that Rsn-2 adsorption is in fact a two-step process: the hydrophobic N-terminal tail recruits the protein to the interface after which Rsn-2 undergoes an unfolding transition that maintains its secondary structure. Intriguingly, our simulations show that the conformation Rsn-2 adopts at an interface is an arrested state along the denaturation pathway. More generally, our computational model should prove a useful, and computationally efficient, tool in studying the dynamics and energetics of protein-interface interactions.
  39. 39
    Wösten, H. A. B. Hydrophobins: Multipurpose Proteins. Annu. Rev. Microbiol. 2001, 55 (1), 625– 646,  DOI: 10.1146/annurev.micro.55.1.625
    Google Scholar
    39
    Hydrophobins: Multipurpose proteins
    Wosten, Han A. B.
    Annual Review of Microbiology (2001), 55 (), 625-646CODEN: ARMIAZ; ISSN:0066-4227. (Annual Reviews Inc.)
    A review. Class I and class II hydrophobins are small secreted fungal proteins that play a role in a broad range of processes in the growth and development of filamentous fungi. For instance, they are involved in the formation of aerial structures and in the attachment of hyphae to hydrophobic surfaces. The mechanisms by which hydrophobins fulfill these functions are based on their property to self-assemble at hydrophilic-hydrophobic interfaces into a 10 nm-thin highly amphipathic film. Complementation studies have shown that class I hydrophobins belong to a closely related group of morphogenetic proteins, but that they have evolved to function at specific interfaces. Recent evidence indicates that hydrophobins do not only function by self-assembly. Monomeric hydrophobin has been implicated in cell-wall assembly, but the underlying mechanism is not yet clear. In addn., hydrophobin monomers could act as toxins and elicitors.
  40. 40
    Blijdenstein, T. B. J.; Veerman, C.; van der Linden, E. Depletion– Flocculation in Oil-in-Water Emulsions Using Fibrillar Protein Assemblies. Langmuir 2004, 20 (12), 4881– 4884,  DOI: 10.1021/la0497447
    Google Scholar
    40
    Depletion-Flocculation in Oil-in-Water Emulsions Using Fibrillar Protein Assemblies
    Blijdenstein, Theo B. J.; Veerman, Cecile; Van der Linden, Erik
    Langmuir (2004), 20 (12), 4881-4884CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    This paper shows that low concns. of β-lactoglobulin fibrils can induce depletion-flocculation in β-lactoglobulin-stabilized oil-in-H2O emulsions. The min. required fibril concn. for flocculation was detd. exptl. for fibril lengths of ∼3 and 0.1 μm. The min. fibril concn. for flocculation is 2 orders of magnitude higher for the short fibrils than for the long ones. These exptl. results correspond well with a theor. prediction based on a model of spinodal decompn. Rheol. measurements were performed, verifying that flocculation was induced by a depletion mechanism. The results of this study show that the min. concn. required for depletion-flocculation can be tuned by varying the length of the fibrils.
  41. 41
    Peng, J.; Simon, J. R.; Venema, P.; Van Der Linden, E. Protein Fibrils Induce Emulsion Stabilization. Langmuir 2016, 32 (9), 2164– 2174,  DOI: 10.1021/acs.langmuir.5b04341
    Google Scholar
    41
    Protein Fibrils Induce Emulsion Stabilization
    Peng, Jinfeng; Simon, Joana Ralfas; Venema, Paul; van der Linden, Erik
    Langmuir (2016), 32 (9), 2164-2174CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The behavior of an oil-in-water emulsion was studied in the presence of protein fibrils for a wide range of fibril concns. by using rheol., diffusing wave spectroscopy, and confocal laser scanning microscopy. Results showed that above a min. fibril concn. depletion flocculation occurred, leading to oil droplet aggregation and enhanced creaming of the emulsion. Upon further increasing the concn. of the protein fibrils, the emulsions were stabilized. In this stable regime both aggregates of droplets and single droplets are present, and these aggregates are smaller than the aggregates in the flocculated emulsion samples at the lower fibril concns. The size of the droplet aggregates in the stabilized emulsions is independent of fibril concn. In addn., the droplet aggregation was reversible upon diln. both by a pH 2 HCl soln. and by a fibril soln. at the same concn. The viscosity of the emulsions contg. fibrils was comparable to that of the pure fibril soln. Neither fibril networks nor droplet gel networks were obsd. in our study. The stabilization mechanism of emulsions contg. long protein fibrils at high protein fibril concns. points toward the mechanism of a kinetic stabilization.
  42. 42
    Yuan, T.; Zeng, J.; Wang, B.; Cheng, Z.; Chen, K. Pickering Emulsion Stabilized by Cellulosic Fibers: Morphological Properties-Interfacial Stabilization-Rheological Behavior Relationships. Carbohydr. Polym. 2021, 269, 118339  DOI: 10.1016/j.carbpol.2021.118339
    Google Scholar
    42
    Pickering emulsion stabilized by cellulosic fibers: Morphological properties-interfacial stabilization-rheological behavior relationships
    Yuan, Tianzhong; Zeng, Jinsong; Wang, Bin; Cheng, Zheng; Chen, Kefu
    Carbohydrate Polymers (2021), 269 (), 118339CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
    This work aimed to study the stabilization mechanism induced by different morphologies of cellulosic fiber in O/W emulsion. Three types of cellulosic fibers were named squashed cellulose, incompletely nanofibrillated cellulose, and completely nanofibrillated cellulose, resp. Squashed cellulose acted as barriers between the droplets to stabilize emulsion via depletion flocculation, whereas incompletely nanofibrillated and completely nanofibrillated cellulose formed covering layer via interfacial adsorption and connected adjacent droplets to create the droplet-fiber network structure via bridging flocculation. Differently, completely nanofibrillated cellulose formed the denser covering layer leading to a more stability of droplet. Importantly, it had the higher capacity of bridging flocculation, which can tightly connect the adjacent droplets to form a stronger droplet-fiber 3D network structure. Consequently, in rheol. anal. including creep compliance, and dynamic modulus, the corresponding emulsions showed excellent anti-deformation ability and dynamic stability. This study provides practical guidance on the productions of foodstuff and cosmetic.
  43. 43
    Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, M. N.; Sternberg, M. J. E. The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis. Nat. Protoc. 2015, 10 (6), 845– 858,  DOI: 10.1038/nprot.2015.053
    Google Scholar
    43
    The Phyre2 web portal for protein modeling, prediction and analysis
    Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M.; Wass, Mark N.; Sternberg, Michael J. E.
    Nature Protocols (2015), 10 (6), 845-858CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which the authors previously published a paper in Nature Protocols. In this updated protocol, the authors describe Phyre2, which uses advanced remote homol. detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they det. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain compn. and model quality. A range of addnl. available tools is described to find a protein structure in a genome, to submit large no. of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.
  44. 44
    Cole, C.; Barber, J. D.; Barton, G. J. The Jpred 3 Secondary Structure Prediction Server. Nucleic Acids Res. 2008, 36, W197– W201,  DOI: 10.1093/nar/gkn238
    Google Scholar
    44
    The Jpred 3 secondary structure prediction server
    Cole, Christian; Barber, Jonathan D.; Barton, Geoffrey J.
    Nucleic Acids Research (2008), 36 (Web Server), W197-W201CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Jpred (http://www.compbio.dundee.ac.uk/jpred) is a secondary structure prediction server powered by the Jnet algorithm. Jpred performs over 1000 predictions per wk for users in more than 50 countries. The recently updated Jnet algorithm provides a three-state (α-helix, β-strand and coil) prediction of secondary structure at an accuracy of 81.5%. Given either a single protein sequence or a multiple sequence alignment, Jpred derives alignment profiles from which predictions of secondary structure and solvent accessibility are made. The predictions are presented as colored HTML, plain text, PostScript, PDF and via the Jalview alignment editor to allow flexibility in viewing and applying the data. The new Jpred 3 server includes significant usability improvements that include clearer feedback of the progress or failure of submitted requests. Functional improvements include batch submission of sequences, summary results via email and updates to the search databases. A new software pipeline will enable Jnet/Jpred to continue to be updated in sync with major updates to SCOP and UniProt and so ensures that Jpred 3 will maintain high-accuracy predictions.
  45. 45
    Leon, L. J.; Idangodage, H.; Wan, C.-P. L.; Weers, P. M. M. Apolipophorin III: Lipopolysaccharide Binding Requires Helix Bundle Opening. Biochem. Biophys. Res. Commun. 2006, 348 (4), 1328– 1333,  DOI: 10.1016/j.bbrc.2006.07.199
    Google Scholar
    45
    Apolipophorin III: Lipopolysaccharide binding requires helix bundle opening
    Leon, Leonardo J.; Idangodage, Hasitha; Wan, Chung-Ping L.; Weers, Paul M. M.
    Biochemical and Biophysical Research Communications (2006), 348 (4), 1328-1333CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
    Apolipophorin III (apoLp-III) is a prototypical apolipoprotein used for structure-function studies. Besides its crucial role in lipid transport, apoLp-III is able to assoc. with fungal and bacterial membranes and stimulate cellular immune responses. We recently demonstrated a binding interaction of apoLp-III from the greater wax moth, Galleria mellonella, with lipopolysaccharides (LPS). In the present study, the requirement of helix bundle opening for LPS binding interaction was investigated. Using site-directed mutagenesis, two cysteine residues were introduced in close spatial proximity (P5C/A135C). When the helix bundle was locked by disulfide bond formation, the tethered helix bundle failed to assoc. with LPS. In contrast, the mutant protein regained its ability to bind upon redn. with dithiothreitol. Thus, helix bundle opening is a crit. event in apoLp-III binding interaction with LPS. This mechanism implies that the hydrophobic interior of the protein interacts directly with LPS, analogous to that obsd. for lipid interaction.
  46. 46
    Whitten, M. M. A.; Tew, I. F.; Lee, B. L.; Ratcliffe, N. A. A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1, 3-Glucan Pattern Recognition and Cellular Encapsulation Reactions. J. Immunol. 2004, 172 (4), 2177– 2185,  DOI: 10.4049/jimmunol.172.4.2177
    Google Scholar
    46
    A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions
    Whitten, Miranda M. A.; Tew, Ian F.; Lee, Bok L.; Ratcliffe, Norman A.
    Journal of Immunology (2004), 172 (4), 2177-2185CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
    Lipoproteins and mols. for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study the authors describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide prodn. in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study the authors show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and β-1,3-glucan and therefore may act as a pattern recognition mol. for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiol. levels, and, with the notable exception of β-glucan binding, are only obsd. with the lipid-assocd. form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition mol. and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunol., particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

Cited By

Click to copy section linkSection link copied!
Citation Statements
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 4 publications.

  1. Hu Wang, Chunzhong Li, Yanmin Wang, Huanming Zhang. Bacterial Species in Engineered Living Materials: Strategies and Future Directions. Microbial Biotechnology 2025, 18 (5) https://doi.org/10.1111/1751-7915.70164
  2. Emmanuelle Baudu, Eric Raspaud, Catherine Fontagné-Faucher, Yassine Nait Chabane, Claire-Emmanuelle Marcato-Romain. Morphogenesis and mechanical properties of Bacillus amyloliquefaciens biofilms: a comparative study of rough and smooth morphotypes. Current Research in Microbial Sciences 2025, 8 , 100403. https://doi.org/10.1016/j.crmicr.2025.100403
  3. Natalie C. Bamford, Ryan J. Morris, Alan Prescott, Paul Murphy, Elliot Erskine, Cait E. MacPhee, Nicola R. Stanley‐Wall. TasA Fibre Interactions Are Necessary for Bacillus subtilis Biofilm Structure. Molecular Microbiology 2024, 122 (4) , 598-609. https://doi.org/10.1111/mmi.15315
  4. Antoine Danchin. Exploring overlooked growth-promoting mechanisms by plant-associated bacteria. Sustainable Microbiology 2024, 1 (1) https://doi.org/10.1093/sumbio/qvae011
Download PDF
Go to Langmuir
Get e-Alerts
Get e-Alerts

Langmuir

Cite this: Langmuir 2024, 40, 8, 4164–4173
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.langmuir.3c03163
Published February 13, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Article Views

1151

Altmetric

-

Citations

4
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. TasA and BslA are B. subtilis matrix proteins. (A) Schematic representation of a B. subtilis biofilm showing that the extracellular matrix surrounding the cells confers protection from environmental pressures. The matrix proteins TasA (purple) and BslA (green) are both secreted as monomers and can take on higher-order structures. The hydrophobic BslA film coats the colony biofilm and the TasA fibers contribute to structure and biofilm formation. The representation is for illustrative purposes and not to scale. (B) Cartoon representations of the crystal structures of TasA (purple, PDB 5OF2) and BslA (green, PDB 4HBU chains J [cap-in] and H [cap-out]). The N and C-termini are labeled with the appropriate letters. (C) Diagrams of the protein domains of TasA and BslA numbered based on the amino acid sequences of each protein. The unprocessed proteins (PreTasA and PreBslA) are displayed with signal peptides (SP) in gray and the secreted domains in purple or green. The recombinant constructs (fTasA, mTasA, BslA, and BslA AxA) are also shown for clarity.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. TasA stabilizes oil–water emulsions. Microscope images of oil–water–oil droplets produced from mixing 8 mg/mL mTasA (A, B) or fTasA (C, D) in phosphate buffer with GTO (80:20 v/v). Images are from two time points: immediately after emulsification (A, C) and after 1 week of incubation at room temperature (B, D). Scale bar is 100 μm.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. TasA undergoes structural changes upon adsorption to an interface. (A) CD spectroscopy of mTasA (purple) and fTasA (black) in solution (solid lines) and in RIMEs (dashed lines) shows a change in the secondary structure. (B) Pendant drop tensiometry reveals the time evolution of the interfacial tension (IFT) of a droplet of 0.1 mg/mL protein (mTasA, GST, and BslA) in GTO. The mean of three droplets is plotted for each protein with error bars representing SEM.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. TasA affects the film formation of BslA. (A) Pendant drop tensiometry of BslA (0.2 mg/mL, 6.6 mM) with mTasA (0.1 mg/mL, 3.8 mM) or GST (0.1 mg/mL, 3.8 mM) at an oil/water interface shows a drop in the IFT over time. (B) Effect of mTasA on BslA film formation is dose-dependent as measured by wrinkle relaxation assays. Retraction of 10 μL from an equilibrium state 40 μL droplet in GTO led to visible wrinkles. The relaxation of wrinkles was plotted as a function of time for three different ratios of TasA to the BslA dimer. The concentration of BslA was the same as that in panel (A) at 0.2 mg/mL. (C) Wrinkle relaxation of mTasA/BslA mixture (1:1.7 molar ratio) plotted as a function of time for varied retraction volumes. (D) Time to interface calculated from pendant drop tensiometry for 0.03 mg/mL GST, mTasA, and BslA at an air–water interface for three independent experiments. All plots show the mean of three droplets with error bars representing SEM.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. BslA film formation viewed by Brewster angle microscopy. (A) Images of a single region of the buffer/air interface over time labeled in seconds (s). Black pixels represent solution, and brighter pixels are interfacial material (0.005 mg/mL BslA protein). The first image at 247 s shows the microdomains forming. Then clear islands become visible that migrate across the field of view 448 and 509 s. The last 3 time points show the filling of the film into a monolayer. (B) Network of BslA film domains t = 372 s with each large continuous region given a unique color (e.g., red, cyan, yellow, and green) to highlight the extent of interconnectivity. The image was binarized after the threshold greyscale value of 12 was set. All scale bars are 50 μm.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 46 other publications.

    1. 1
      Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2 (2), 95– 108,  DOI: 10.1038/nrmicro821
      1
      Bacterial biofilms: From the natural environment to infectious diseases
      Hall-Stoodley, Luanne; Costerton, J. William; Stoodley, Paul
      Nature Reviews Microbiology (2004), 2 (2), 95-108CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. Biofilms, matrix-enclosed microbial accretions that adhere to biol. or non-biol. surfaces, represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (∼3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.
    2. 2
      Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol. 2010, 8 (9), 623– 633,  DOI: 10.1038/nrmicro2415
      2
      The biofilm matrix
      Flemming, Hans-Curt; Wingender, Jost
      Nature Reviews Microbiology (2010), 8 (9), 623-633CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
      A review. The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mech. stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addn., the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
    3. 3
      Hobley, L.; Harkins, C.; MacPhee, C. E.; Stanley-Wall, N. R. Giving Structure to the Biofilm Matrix: An Overview of Individual Strategies and Emerging Common Themes. FEMS Microbiol. Rev. 2015, 39 (5), 649– 669,  DOI: 10.1093/femsre/fuv015
      3
      Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes
      Hobley, Laura; Harkins, Catriona; MacPhee, Cait E.; Stanley-Wall, Nicola R.
      FEMS Microbiology Reviews (2015), 39 (5), 649-669CODEN: FMREE4; ISSN:1574-6976. (Oxford University Press)
      Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Crit. to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnol. uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the mols. and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, exptl. techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars.
    4. 4
      Kobayashi, K.; Iwano, M. BslA (YuaB) Forms a Hydrophobic Layer on the Surface of Bacillus Subtilis Biofilms. Mol. Microbiol. 2012, 85 (1), 51– 66,  DOI: 10.1111/j.1365-2958.2012.08094.x
      4
      BslA (YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms
      Kobayashi, Kazuo; Iwano, Megumi
      Molecular Microbiology (2012), 85 (1), 51-66CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
      Biofilms are surface-assocd. bacterial aggregates, in which bacteria are enveloped by polymeric substances known as the biofilm matrix. Bacillus subtilis biofilms display persistent resistance to liq. wetting and gas penetration, which probably explains the broad-spectrum resistance of the bacteria in these biofilms to antimicrobial agents. In this study, BslA (formerly YuaB) was identified as a major contributor to the surface repellency of B. subtilis biofilms. Disruption of bslA resulted in the loss of surface repellency and altered the biofilm surface microstructure. BslA localized to the biofilm matrix in an exopolysaccharide-dependent manner. Purified BslA exhibited amphiphilic properties and formed polymers in response to increases in the area of the air-water interface in vitro. Genetic and biochem. analyses showed that the self-polymn. activity of BslA was essential for its ability to localize to the biofilm matrix. Confocal laser scanning microscopy showed that BslA formed a layer on the biofilm surface. Taken together, the authors propose that BslA, standing for biofilm-surface layer protein, is responsible for the hydrophobic layer on the surface of biofilms.
    5. 5
      Hobley, L.; Ostrowski, A.; Rao, F. V.; Bromley, K. M.; Porter, M.; Prescott, A. R.; MacPhee, C. E.; Van Aalten, D. M. F.; Stanley-Wall, N. R. BslA Is a Self-Assembling Bacterial Hydrophobin That Coats the Bacillus Subtilis Biofilm. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (33), 13600– 13605,  DOI: 10.1073/pnas.1306390110
      5
      BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm
      Hobley, Laura; Ostrowski, Adam; Rao, Francesco V.; Bromley, Keith M.; Porter, Michael; Prescott, Alan R.; MacPhee, Cait E.; van Aalten, Daan M. F.; Stanley-Wall, Nicola R.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (33), 13600-13605,S13600/1-S13600/14CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-pos. 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, the authors show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophys. anal. demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Mol. function is revealed from anal. of the crystal structure of BslA, which consists of an Ig-type fold with the addn. of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophys. anal. 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 physiochem. properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. The authors suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.
    6. 6
      Branda, S. S.; Chu, F.; Kearns, D. B.; Losick, R.; Kolter, R. A Major Protein Component of the Bacillus Subtilis Biofilm Matrix. Mol. Microbiol. 2006, 59 (4), 1229– 1238,  DOI: 10.1111/j.1365-2958.2005.05020.x
      6
      A major protein component of the Bacillus subtilis biofilm matrix
      Branda, Steven S.; Chu, Frances; Kearns, Daniel B.; Losick, Richard; Kolter, Roberto
      Molecular Microbiology (2006), 59 (4), 1229-1238CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)
      Microbes construct structurally complex multi-cellular communities (biofilms) through prodn. of an extracellular matrix. Here we present evidence from SEM showing that a wild strain of the Gram pos. bacterium Bacillus subtilis builds such a matrix. Genetic, biochem. and cytol. evidence indicates that the matrix is composed predominantly of a protein component, TasA, and an exopolysaccharide component. The absence of TasA or the exopolysaccharide resulted in a residual matrix, while the absence of both components led to complete failure to form complex multi-cellular communities. Extracellular complementation expts. revealed that a functional matrix can be assembled even when TasA and the exopolysaccharide are produced by different cells, reinforcing the view that the components contribute to matrix formation in an extracellular manner. Having defined the major components of the biofilm matrix and the control of their synthesis by the global regulator SinR, we present a working model for how B. subtilis switches between nomadic and sedentary lifestyles.
    7. 7
      Arnaouteli, S.; MacPhee, C. E.; Stanley-Wall, N. R. Just in Case It Rains: Building a Hydrophobic Biofilm the Bacillus Subtilis Way. Curr. Opin. Microbiol. 2016, 34, 7– 12,  DOI: 10.1016/j.mib.2016.07.012
      7
      Just in case it rains: building a hydrophobic biofilm the Bacillus subtilis way
      Arnaouteli, Sofia; MacPhee, Cait E.; Stanley-Wall, Nicola R.
      Current Opinion in Microbiology (2016), 34 (), 7-12CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)
      A review. Over the millennia, diverse species of bacteria have evolved multiple independent mechanisms to structure sessile biofilm communities that confer protection and stability to the inhabitants. The Gram-pos. soil bacterium Bacillus subtilis biofilm presents as an architecturally complex, highly hydrophobic community that resists wetting by water, solvents, and biocides. This remarkable property is conferred by a small secreted protein called BslA, which self-assembles into an organized lattice at an interface. In the biofilm, prodn. of BslA is tightly regulated and the resultant protein is secreted into the extracellular environment where it forms a very effective communal barrier allowing the resident B. subtilis cells to shelter under the protection of a protein raincoat.
    8. 8
      Hölscher, T.; Kovács, Á. T. Sliding on the Surface: Bacterial Spreading without an Active Motor. Environ. Microbiol. 2017, 19 (7), 2537– 2545,  DOI: 10.1111/1462-2920.13741
      8
      Sliding on the surface: bacterial spreading without an active motor
      Holscher Theresa; Kovacs Akos T
      Environmental microbiology (2017), 19 (7), 2537-2545 ISSN:.
      Bacteria are able to translocate over surfaces using different types of active and passive motility mechanisms. Sliding is one of the passive types of movement since it is powered by the pushing force of dividing cells and additional factors facilitating the expansion over surfaces. In this review, we describe the sliding proficient bacteria that were previously investigated in details highlighting the sliding facilitating compounds and the regulation of sliding motility. Besides surfactants that reduce the friction between cells and substratum, other compounds including exopolysaccharides, hydrophobic proteins, or glycopeptidolipids where discovered to promote sliding. Therefore, we present the sliding bacteria in three groups depending on the additional compound required for sliding. Despite recent accomplishments in sliding research there are still many open questions about the mechanisms underlying sliding motility and its regulation in diverse bacterial species.
    9. 9
      Grau, R. R.; de Oña, P.; Kunert, M.; Leñini, C.; Gallegos-Monterrosa, R.; Mhatre, E.; Vileta, D.; Donato, V.; Hölscher, T.; Boland, W. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus Subtilis. mBio 2015, 6 (4), 10– 1128,  DOI: 10.1128/mBio.00581-15
      There is no corresponding record for this reference.
    10. 10
      Bromley, K. M.; Morris, R. J.; Hobley, L.; Brandani, G.; Gillespie, R. M. C.; McCluskey, M.; Zachariae, U.; Marenduzzo, D.; Stanley-Wall, N. R.; MacPhee, C. E. Interfacial Self-Assembly of a Bacterial Hydrophobin. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (17), 5419– 5424,  DOI: 10.1073/pnas.1419016112
      10
      Interfacial self-assembly of a bacterial hydrophobin
      Bromley, Keith M.; Morris, Ryan J.; Hobley, Laura; Brandani, Giovanni; Gillespie, Rachel M. C.; McCluskey, Matthew; Zachariae, Ulrich; Marenduzzo, Davide; Stanley-Wall, Nicola R.; MacPhee, Cait. E.
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (17), 5419-5424CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The majority of bacteria in the natural environment live within the confines of a biofilm. The Gram-pos. bacterium, Bacillus subtilis, forms biofilms that exhibit a characteristic wrinkled morphol. and a highly hydrophobic surface. A crit. component in generating these properties is the protein BslA, which forms a coat across the surface of the sessile community. The authors recently reported the structure of BslA, and noted the presence of a large surface-exposed hydrophobic patch. Such surface patches are also obsd. 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 the authors show that the mechanism of action is also distinct. Specifically, the results suggest that the amino acids making up the large, surface-exposed hydrophobic cap in the crystal structure are shielded in aq. soln. by adopting a random coil conformation, enabling the protein to be sol. and monomeric. At an interface, these cap residues refold, inserting the hydrophobic side-chains into the air or oil phase and forming a 3-stranded β-sheet. This form then self-assembles into a well-ordered 2-dimensional (2D) rectangular lattice that stabilizes the interface. By replacing a hydrophobic Leu residue in the center of the cap with a pos. charged Lys residue, the authors 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.
    11. 11
      Brandani, G. B.; Schor, M.; Morris, R.; Stanley-Wall, N.; MacPhee, C. E.; Marenduzzo, D.; Zachariae, U. The Bacterial Hydrophobin BslA Is a Switchable Ellipsoidal Janus Nanocolloid. Langmuir 2015, 31 (42), 11558– 11563,  DOI: 10.1021/acs.langmuir.5b02347
      11
      The Bacterial Hydrophobin BslA is a Switchable Ellipsoidal Janus Nanocolloid
      Brandani, Giovanni B.; Schor, Marieke; Morris, Ryan; Stanley-Wall, Nicola; MacPhee, Cait E.; Marenduzzo, Davide; Zachariae, Ulrich
      Langmuir (2015), 31 (42), 11558-11563CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      BslA is an amphiphilic protein that forms a highly hydrophobic coat around Bacillus subtilis biofilms, shielding the bacterial community from external aq. soln. It has a unique structure featuring a distinct partition between hydrophilic and hydrophobic surfaces. This surface property is reminiscent of synthesized Janus colloids. By investigating the behavior of BslA variants at water-cyclohexane interfaces through a set of multiscale simulations informed by exptl. data, we show that BslA indeed represents a biol. example of an ellipsoidal Janus nanoparticle, whose surface interactions are, moreover, readily switchable. BslA contains a local conformational toggle, which controls its global affinity for, and orientation at, water-oil interfaces. This adaptability, together with single-point mutations, enables the fine-tuning of its solvent and interfacial interactions, and suggests that BslA could be a basis for biotechnol. applications.
    12. 12
      Morris, R. J.; Bromley, K. M.; Stanley-Wall, N.; MacPhee, C. E. A Phenomenological Description of BslA Assemblies across Multiple Length Scales. Philos. Trans. R. Soc., A 2016, 374 (2072), 20150131  DOI: 10.1098/rsta.2015.0131
      12
      A phenomenological description of BslA assemblies across multiple length scales
      Morris, Ryan J.; Bromley, Keith M.; Stanley-Wall, Nicola; MacPhee, Cait E.
      Philosophical Transactions of the Royal Society, A: Mathematical, Physical & Engineering Sciences (2016), 374 (2072), 20150131/1-20150131/14CODEN: PTRMAD; ISSN:1364-503X. (Royal Society)
      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 mech. 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 soln., over a range of length scales spanning from nanometers to millimetres. First, we observe transiently stable and highly elongated air bubbles formed in agitated BslA samples. We study their behavior in a range of soln. 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.
    13. 13
      Morris, R. J.; Schor, M.; Gillespie, R. M. C.; Ferreira, A. S.; Baldauf, L.; Earl, C.; Ostrowski, A.; Hobley, L.; Bromley, K. M.; Sukhodub, T. Natural Variations in the Biofilm-Associated Protein BslA from the Genus Bacillus. Sci. Rep. 2017, 7 (1), 6730  DOI: 10.1038/s41598-017-06786-9
      13
      Natural variations in the biofilm-associated protein BslA from the genus Bacillus
      Morris Ryan J; Schor Marieke; Baldauf Lucia; Earl Chris; Bromley Keith M; MacPhee Cait E; Gillespie Rachel M C; Ferreira Ana Sofia; Ostrowski Adam; Sukhodub Tetyana; Arnaouteli Sofia; Stanley-Wall Nicola R; Hobley Laura
      Scientific reports (2017), 7 (1), 6730 ISSN:.
      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.
    14. 14
      Arnaouteli, S.; Ferreira, A. S.; Schor, M.; Morris, R. J.; Bromley, K. M.; Jo, J.; Cortez, K. L.; Sukhodub, T.; Prescott, A. R.; Dietrich, L. E. P. Bifunctionality of a Biofilm Matrix Protein Controlled by Redox State. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (30), E6184– E6191,  DOI: 10.1073/pnas.1707687114
      14
      Bifunctionality of a biofilm matrix protein controlled by redox state
      Arnaouteli, Sofia; Ferreira, Ana Sofia; Schor, Marieke; Morris, Ryan J.; Bromley, Keith M.; Jo, Jeanyoung; Cortez, Krista L.; Sukhodub, Tetyana; Prescott, Alan R.; Dietrich, Lars E. P.; MacPhee, Cait E.; Stanley-Wall, Nicola R.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (30), E6184-E6191CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Biofilms are communities of microbial cells that are encapsulated within a self-produced polymeric matrix. The matrix is crit. to the success of biofilms in diverse habitats; however, many details of the compn., structure, and function remain enigmatic. Biofilms formed by the Gram-pos. bacterium Bacillus subtilis depend on the prodn. 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 2 distinct functional roles for BslA and that these roles can be genetically sepd. 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 2 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.
    15. 15
      Stanley-Wall, N. R.; MacPhee, C. E. Connecting the Dots between Bacterial Biofilms and Ice Cream. Phys. Biol. 2015, 12 (6), 063001  DOI: 10.1088/1478-3975/12/6/063001
      15
      Connecting the dots between bacterial biofilms and ice cream
      Stanley-Wall Nicola R; MacPhee Cait E
      Physical biology (2015), 12 (6), 063001 ISSN:.
      Emerging research is revealing a diverse array of interfacially-active proteins that are involved in varied biological process from foaming horse sweat to bacterial raincoat formation. We describe an interdisciplinary approach to study the molecular and biophysical mechanisms controlling the activity of an unusual bacterial protein called BslA. This protein is needed for biofilm formation and forms a protective layer or raincoat over the bacterial community, but also has a multitude of potential applications in multiphase formulations. Here we document our journey from fundamental research to an examination of the applications for this surface-active protein in ice cream.
    16. 16
      Kaufman, G.; Liu, W.; Williams, D. M.; Choo, Y.; Gopinadhan, M.; Samudrala, N.; Sarfati, R.; Yan, E. C. Y.; Regan, L.; Osuji, C. O. Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA. Langmuir 2017, 33 (47), 13590– 13597,  DOI: 10.1021/acs.langmuir.7b03226
      16
      Flat Drops, Elastic Sheets, and Microcapsules by Interfacial Assembly of a Bacterial Biofilm Protein, BslA
      Kaufman, Gilad; Liu, Wei; Williams, Danielle M.; Choo, Youngwoo; Gopinadhan, Manesh; Samudrala, Niveditha; Sarfati, Raphael; Yan, Elsa C. Y.; Regan, Lynne; Osuji, Chinedum O.
      Langmuir (2017), 33 (47), 13590-13597CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Protein adsorption and assembly at interfaces provides a potentially versatile route to create useful constructs for fluid compartmentalization. In this context, we consider the interfacial assembly of a bacterial biofilm protein, BslA, at air-water and oil-water interfaces. Densely packed, high modulus monolayers form at air-water interfaces leading to the formation of flattened sessile water drops. BslA forms elastic sheets at oil-water interfaces leading to the prodn. of stable monodisperse oil-in-water microcapsules. By contrast, water-in-oil microcapsules are unstable but display arrested rather than full coalescence on contact. The disparity in stability likely originates in a low areal d. of BslA hydrophobic caps on the exterior surface of water-in-oil microcapsules, relative to the inverse case. In direct analogy with small mol. surfactants, the lack of stability of individual water-in-oil microcapsules is consistent with the large value of the hydrophilic-lipophilic balance (HLB no.) calcd. based on the BslA crystal structure. The occurrence of arrested coalescence indicates that the surface activity of BslA is similar to that of colloidal particles that produce Pickering emulsions, with the stability of partially coalesced structures ensured by interfacial jamming. Micropipette aspiration and flow in tapered capillaries reveal intriguing reversible and non-reversible modes of mech. deformation, resp.
    17. 17
      Bromley, K. M.; MacPhee, C. E. BslA-Stabilized Emulsion Droplets with Designed Microstructure. Interface Focus 2017, 7 (4), 20160124  DOI: 10.1098/rsfs.2016.0124
      17
      BslA-stabilized emulsion droplets with designed microstructure
      Bromley Keith M; MacPhee Cait E
      Interface focus (2017), 7 (4), 20160124 ISSN:2042-8898.
      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.
    18. 18
      Schloss, A. C.; Liu, W.; Williams, D. M.; Kaufman, G.; Hendrickson, H. P.; Rudshteyn, B.; Fu, L.; Wang, H.; Batista, V. S.; Osuji, C. Fabrication of Modularly Functionalizable Microcapsules Using Protein-Based Technologies. ACS Biomater. Sci. Eng. 2016, 2 (11), 1856– 1861,  DOI: 10.1021/acsbiomaterials.6b00447
      18
      Fabrication of Modularly Functionalizable Microcapsules Using Protein-Based Technologies
      Schloss, Ashley C.; Liu, Wei; Williams, Danielle M.; Kaufman, Gilad; Hendrickson, Heidi P.; Rudshteyn, Benjamin; Fu, Li; Wang, Hongfei; Batista, Victor S.; Osuji, Chinedum; Yan, Elsa C. Y.; Regan, Lynne
      ACS Biomaterials Science & Engineering (2016), 2 (11), 1856-1861CODEN: ABSEBA; ISSN:2373-9878. (American Chemical Society)
      Proteins are desirable building blocks to create self-assembled, spatially defined structures and interfaces on length-scales that are inaccessible by traditional methods. Here, we describe a novel approach to create functionalized monolayers using the proteins BslA and SpyCatcher/SpyTag. BslA is a bacterial hydrophobin whose amphiphilic character underlies its ability to assemble into a monolayer at both air/water and oil/water interfaces. We demonstrate that having the SpyTag peptide fused at the N- or C-terminus does not affect the formation of such monolayers. We establish the creation of stable oil-in-water microcapsules using BslA, and also show the fabrication of capsules outwardly displaying the reactive SpyTag peptide by fusing it to the C-terminus of BslA. Such capsules can be covalently labeled by reacting the surface-displayed SpyTag with SpyCatcher fused to any desired protein. We demonstrate this principle by labeling microcapsules using green fluorescent protein (GFP). All components are genetically encodable, the reagents can be readily prepd. in large quantities, and all reactions occur at ambient temp. in aq. soln. Thus, this straightforward, modular, scalable strategy has myriad potential applications in the creation of novel, functional materials and interfaces.
    19. 19
      Diehl, A.; Roske, Y.; Ball, L.; Chowdhury, A.; Hiller, M.; Molière, N.; Kramer, R.; Stöppler, D.; Worth, C. L.; Schlegel, B. Structural Changes of TasA in Biofilm Formation of Bacillus Subtilis. Proc. Natl. Acad. Sci. U.S.A. 2018, 115 (13), 3237– 3242,  DOI: 10.1073/pnas.1718102115
      19
      Structural changes of TasA in biofilm formation of Bacillus subtilis
      Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Moliere, Noel; Kramer, Regina; Stoeppler, Daniel; Worth, Catherine L.; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; Rossum, Barth-Jan van; Schmieder, Peter; Heinemann, Udo; Turgay, Kuersad; Akbey, Umit; Oschkinat, Hartmut
      Proceedings of the National Academy of Sciences of the United States of America (2018), 115 (13), 3237-3242CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Microorganisms form surface-attached communities, termed bio- films, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addn. to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a sol., stably folded monomer, whose structure we have detd. by X-ray crystallog. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and anal. ultracentrifugation (AUC) expts. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet-rich fibrils, was obsd. in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the mol. level.
    20. 20
      Stöver, A. G.; Driks, A. Secretion, Localization, and Antibacterial Activity of TasA, a Bacillus Subtilis Spore-Associated Protein. J. Bacteriol. 1999, 181 (5), 1664– 1672,  DOI: 10.1128/JB.181.5.1664-1672.1999
      20
      Secretion, localization, and antibacterial activity of TasA, a Bacillus subtilis spore-associated protein
      Stover, Axel G.; Driks, Adam
      Journal of Bacteriology (1999), 181 (5), 1664-1672CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      The synthesis and subcellular localization of the proteins that comprise the Bacillus subtilis spore are under a variety of complex controls. To better understand these controls, the authors have identified and characterized a 31-kDa sporulation protein, called TasA, which is secreted into the culture medium early in sporulation and is also incorporated into the spore. TasA synthesis begins approx. 30 min after the onset of sporulation and requires the sporulation transcription factor genes spoOH and spoOA. The first 81 nucleotides of tasA encode a 27-amino-acid sequence that resembles a signal peptide and which is missing from Tas isolated from a sporulating cell lysate. In B. subtilis cells unable to synthesize the signal peptidase SipW, TasA is not secreted, nor is it incorporated into spores. Cells unable to produce SipW produce a 34-kDa from of TasA, consistent with a failure to remove the N-terminal 27 amino acids. In cells engineered to express sipW and tasA during exponential growth, TasA migrates as a 31-kDa species and is secreted into the culture medium. These results indicate that SipW plays a crucial role in the export of TasA out of the cell and its incorporation into spores. Although TasA is dispensable for sporulation under lab. conditions, we find that TasA has a broad spectrum antibacterial activity. The authors discuss the possibility that during the beginning of sporulation as well as later, during germination, TasA inhibits other organisms in the environment, thus confering a competitive advantage to the spore.
    21. 21
      Böhning, J.; Ghrayeb, M.; Pedebos, C.; Abbas, D. K.; Khalid, S.; Chai, L.; Bharat, T. A. M. Donor-Strand Exchange Drives Assembly of the TasA Scaffold in Bacillus Subtilis Biofilms. Nat. Commun. 2022, 13 (1), 7082  DOI: 10.1038/s41467-022-34700-z
      21
      Donor-strand exchange drives assembly of the TasA scaffold in Bacillus subtilis biofilms
      Bohning, Jan; Ghrayeb, Mnar; Pedebos, Conrado; Abbas, Daniel K.; Khalid, Syma; Chai, Liraz; Bharat, Tanmay A. M.
      Nature Communications (2022), 13 (1), 7082CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chem. and mech. stresses. In the Gram-pos. model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibers contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibers, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibers through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fiber. Combining electron cryotomog., at. force microscopy, and mutational studies, we show how TasA fibers congregate in three dimensions to form abundant fiber bundles that are essential for B. subtilis biofilm formation. Our study explains the previously obsd. biochem. properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibers, and how such fibers assemble into bundles in biofilms.
    22. 22
      Romero, D.; Aguilar, C.; Losick, R.; Kolter, R. Amyloid Fibers Provide Structural Integrity to Bacillus Subtilis Biofilms. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (5), 2230– 2234,  DOI: 10.1073/pnas.0910560107
      22
      Amyloid fibers provide structural integrity to Bacillus subtilis biofilms
      Romero, Diego; Aguilar, Claudio; Losick, Richard; Kolter, Roberto
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (5), 2230-2234, S2230/1-S2230/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, the authors characterized the physicochem. properties of this protein. They report that purified TasA forms fibers of variable length and 10-15 nm in width. Biochem. analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymd. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphol., indicating that the purified protein retained biol. activity. It is proposed that TasA forms amyloid fibers that bind cells together in the biofilm.
    23. 23
      Erskine, E.; Morris, R. J.; Schor, M.; Earl, C.; Gillespie, R. M. C.; Bromley, K. M.; Sukhodub, T.; Clark, L.; Fyfe, P. K.; Serpell, L. C. Formation of Functional, Non-amyloidogenic Fibres by Recombinant Bacillus Subtilis TasA. Mol. Microbiol. 2018, 110 (6), 897– 913,  DOI: 10.1111/mmi.13985
      23
      Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA
      Erskine, Elliot; Morris, Ryan J.; Schor, Marieke; Earl, Chris; Gillespie, Rachel M. C.; Bromley, Keith M.; Sukhodub, Tetyana; Clark, Lauren; Fyfe, Paul K.; Serpell, Louise C.; Stanley-Wall, Nicola R.; MacPhee, Cait E.
      Molecular Microbiology (2018), 110 (6), 897-913CODEN: MOMIEE; ISSN:0950-382X. (Wiley-Blackwell)
      Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibers of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fiber forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibers form spontaneously, and share the biol. activity of TasA fibers extd. from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophys. properties of both native and recombinant TasA fibers indicate that they are not functional amyloid-like fibers. A gel formed by TasA fibers can recover after phys. 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 fibers formed by TasA orthologues we uncover species variability in the ability of heterologous fibers to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophys. requirements of the TasA fibers across different species and/or reflect the precise mol. interactions needed for biofilm matrix assembly.
    24. 24
      Malishev, R.; Abbasi, R.; Jelinek, R.; Chai, L. Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein. Biochemistry 2018, 57 (35), 5230– 5238,  DOI: 10.1021/acs.biochem.8b00002
      24
      Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein
      Malishev, Ravit; Abbasi, Razan; Jelinek, Raz; Chai, Liraz
      Biochemistry (2018), 57 (35), 5230-5238CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Biofilms are aggregates of cells that form surface-assocd. communities. The cells in a biofilm are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins and sometimes nucleic acids. Some extracellular matrix proteins form fibers, often termed functional amyloids or amyloid-like fibrils, to differentiate their constructive function from disease-related amyloid fibers. Recent studies of functional amyloid assembly have neglected their interaction with membranes, despite their native assembly in a cellular environment. Here, we use the protein TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein, and ask whether a bacterial functional amyloid interacts with membranes. Using biochem., spectroscopic and microscopic tools, we show that TasA interacts distinctively with model bacterial membranes and that this interaction mutually influences the protein and the membranes' morphol. and structure. At the protein's level, TasA fibers of similar structure and morphol. are formed in the absence of membranes and in the presence of the eukaryotic model membranes. However, in the presence of the bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane's level, fluorescence microscopy and fluorescence anisotropy measurements indicate that the bacterial membranes deform more considerably than the eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial model membranes more than eukaryotic membranes and that this, in turn, disrupts the membranes and alters the fiber formation pathway of TasA. Considering the important role of TasA in providing integrity to biofilms, our study of the TasA-membrane interactions may direct the design of anti-biofilm drugs to the protein-membrane interface.
    25. 25
      Mielich-Süss, B.; Schneider, J.; Lopez, D. Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus Subtilis. mBio 2013, 4 (6), e00719-13  DOI: 10.1128/mBio.00719-13
      There is no corresponding record for this reference.
    26. 26
      López, D.; Kolter, R. Functional Microdomains in Bacterial Membranes. Genes Dev. 2010, 24 (17), 1893– 1902,  DOI: 10.1101/gad.1945010
      26
      Functional microdomains in bacterial membranes
      Lopez, Daniel; Kolter, Roberto
      Genes & Development (2010), 24 (17), 1893-1902CODEN: GEDEEP; ISSN:0890-9369. (Cold Spring Harbor Laboratory Press)
      The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid-a known inhibitor of squalene synthases-impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiol. processes in microdomains may be a more widespread feature of membranes than previously appreciated.
    27. 27
      Cámara-Almirón, J.; Navarro, Y.; Díaz-Martínez, L.; Magno-Pérez-Bryan, M. C.; Molina-Santiago, C.; Pearson, J. R.; de Vicente, A.; Pérez-García, A.; Romero, D. Dual Functionality of the Amyloid Protein TasA in Bacillus Physiology and Fitness on the Phylloplane. Nat. Commun. 2020, 11 (1), 1859  DOI: 10.1038/s41467-020-15758-z
      27
      Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane
      Camara-Almiron, Jesus; Navarro, Yurena; Diaz-Martinez, Luis; Magno-Perez-Bryan, Maria Concepcion; Molina-Santiago, Carlos; Pearson, John R.; de Vicente, Antonio; Perez-Garcia, Alejandro; Romero, Diego
      Nature Communications (2020), 11 (1), 1859CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiol. and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytol. symptoms indicative of excessive cellular stress leading to increased cell death. TasA assocs. to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addn. to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.
    28. 28
      Van Gestel, J.; Vlamakis, H.; Kolter, R. From Cell Differentiation to Cell Collectives: Bacillus Subtilis Uses Division of Labor to Migrate. PLoS Biol. 2015, 13 (4), e1002141  DOI: 10.1371/journal.pbio.1002141
      28
      From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate
      van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto
      PLoS Biology (2015), 13 (4), e1002141/1-e1002141/29CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)
      We show how flagellum-independent migration is driven by the division of labor of 2 cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called van Gogh bundles) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles det. the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecol. advantage. This is a key factor underlying the diverse origins of multicellularity.
    29. 29
      Dragoš, A.; Martin, M.; Falcón García, C.; Kricks, L.; Pausch, P.; Heimerl, T.; Bálint, B.; Maróti, G.; Bange, G.; López, D. Collapse of Genetic Division of Labour and Evolution of Autonomy in Pellicle Biofilms. Nat. Microbiol. 2018, 3 (12), 1451– 1460,  DOI: 10.1038/s41564-018-0263-y
      29
      Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms
      Dragos, Anna; Martin, Marivic; Garcia, Carolina Falcon; Kricks, Lara; Pausch, Patrick; Heimerl, Thomas; Balint, Balazs; Maroti, Gergely; Bange, Gert; Lopez, Daniel; Lieleg, Oliver; Kovacs, Akos T.
      Nature Microbiology (2018), 3 (12), 1451-1460CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
      Closely related microorganisms often cooperate, but the prevalence and stability of cooperation between different genotypes remain debatable. Here, we track the evolution of pellicle biofilms formed through genetic division of labor and ask whether partially deficient partners can evolve autonomy. Pellicles of Bacillus subtilis rely on an extracellular matrix composed of exopolysaccharide (EPS) and the fiber protein TasA. In monocultures, Δeps and ΔtasA mutants fail to form pellicles, but, facilitated by cooperation, they succeed in co-culture. Interestingly, cooperation collapses on an evolutionary timescale and ΔtasA gradually outcompetes its partner Δeps. Pellicle formation can evolve independently from division of labour in Δeps and ΔtasA monocultures, by selection acting on the residual matrix component, TasA or EPS, resp. Using a set of interdisciplinary tools, we unravel that the TasA producer (Δeps) evolves via an unconventional but reproducible substitution in TasA that modulates the biochem. properties of the protein. Conversely, the EPS producer (ΔtasA) undergoes genetically variable adaptations, all leading to enhanced EPS secretion and biofilms with different biomech. properties. Finally, we revisit the collapse of division of labour between Δeps and ΔtasA in light of a strong frequency vs. exploitability trade-off that manifested in the solitarily evolving partners. We propose that such trade-off differences may represent an addnl. barrier to evolution of division of labour between genetically distinct microorganisms.
    30. 30
      MacPhee, C.; Stanley-wall, N.; Bromley, K.; Morris, R.; Hobley, L. Synthetic Multiphase Systems, Google Patents, 2020.
      There is no corresponding record for this reference.
    31. 31
      Clarkson, J. R.; Cui, Z. F.; Darton, R. C. Protein Denaturation in Foam: I. Mechanism Study. J. Colloid Interface Sci. 1999, 215 (2), 323– 332,  DOI: 10.1006/jcis.1999.6255
      31
      Protein denaturation in foam I. Mechanism study
      Clarkson, J. R.; Cui, Z. F.; Darton, R. C.
      Journal of Colloid and Interface Science (1999), 215 (2), 323-332CODEN: JCISA5; ISSN:0021-9797. (Academic Press)
      The aim of this study was to elucidate the mechanism by which protein mols. become denatured in foam. Damage to the protein is mainly due to surface denaturation at the gas-liq. interface. A fraction of the mols. adsorbed do not refold to their native state when they desorb. The degree of denaturation was found to correlate directly with the interfacial exposure, which, for mobile or partially mobile interfaces, is increased by drainage. Expts. with two different proteins showed that, under the conditions of the tests, around 10% of BSA mols. which had adsorbed at the surface remained denatured when they desorbed. For pepsin the figure was around 75%. Oxidn., which was previously thought to be a major cause of protein damage in foam, was found to be minimal. Neither do the high shear stresses in the liq. bulk encountered during bubble bursting cause denaturation, because energy is dissipated at a much greater length scale than that of the protein mol. (c) 1999 Academic Press.
    32. 32
      Graham, D. E.; Phillips, M. C. Proteins at Liquid Interfaces: I. Kinetics of Adsorption and Surface Denaturation. J. Colloid Interface Sci. 1979, 70 (3), 403– 414,  DOI: 10.1016/0021-9797(79)90048-1
      32
      Proteins at liquid interfaces. I. Kinetics of adsorption and surface denaturation
      Graham, D. E.; Phillips, M. C.
      Journal of Colloid and Interface Science (1979), 70 (3), 403-14CODEN: JCISA5; ISSN:0021-9797.
      The rates of change of film pressure (π) and surface concn. (Γ) of protein during the adsorption of β-casein, bovine serum albumin (BSA), and lysozyme at the air-water interface were monitored by the Wilhelmy plate and surface radioactivity methods, resp. The increases in π and Γ for the relatively flexible β-casein mol. occur simultaneously with both parameters attaining their steady-state values at about the same time. In contrast, π and Γ follow different time courses for the globular lysozyme mol.; Γ can reach a steady state value while π is still increasing significantly. The kinetics indicate that initially adsorption is diffusion-controlled, but at higher surface coverages there is an energy barrier to adsorption. Under these conditions, the ability of the protein mols. to create space in the existing film and penetrate and rearrange in the surface is rate-detg. Two regions exist: the relaxation time τ1 (typically ∼2 h when Γ ∼2 mg m-2) describes the adsorption when both π and Γ are increasing, whereas τ2 (in the range 1-8 h for all 3 proteins) relates to the situation when π is increasing at const. Γ because the protein mols. are changing conformation in the surface.
    33. 33
      Jungbauer, A.; Machold, C.; Hahn, R. Hydrophobic Interaction Chromatography of Proteins: III. Unfolding of Proteins upon Adsorption. J. Chromatogr. A 2005, 1079 (1–2), 221– 228,  DOI: 10.1016/j.chroma.2005.04.002
      33
      Hydrophobic interaction chromatography of proteins. III. Unfolding of proteins upon adsorption
      Jungbauer, Alois; Machold, Christine; Hahn, Rainer
      Journal of Chromatography A (2005), 1079 (1-2), 221-228CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      Hydrophobic interaction chromatog. (HIC) exploits the hydrophobic properties of protein surfaces for sepn. and purifn. by performing interactions with chromatog. sorbents of hydrophobic nature. In contrast to reversed-phase chromatog., this methodol. is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. Hydrophobic interactions are promoted by salt and thus proteins are retained in the presence of a cosmotropic salt. When proteins are injected on HIC columns with increasing salt concns. under isocratic conditions only, a fraction of the applied amt. is eluted. The higher the salt concn., the lower is the amt. of eluted protein. The rest can be desorbed with a buffer of low salt concn. or water. It has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. This has been also corroborated by physicochem. measurements. The retention data of 5 different model proteins and 10 different stationary phases were evaluated. Partial unfolding of proteins upon adsorption on surfaces of HIC media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. Furthermore, the authors hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain.
    34. 34
      Tronin, A.; Dubrovsky, T.; Dubrovskaya, S.; Radicchi, G.; Nicolini, C. Role of Protein Unfolding in Monolayer Formation on Air– Water Interface. Langmuir 1996, 12 (13), 3272– 3275,  DOI: 10.1021/la950879+
      34
      Role of Protein Unfolding in Monolayer Formation on Air-Water Interface
      Tronin, Andrey; Dubrovsky, Timothy; Dubrovskaya, Svetlana; Radicchi, Giuliano; Nicolini, Claudio
      Langmuir (1996), 12 (13), 3272-3275CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Mol. exchange kinetics between a monolayer of antibody mols. formed on the air-water interface and the protein soln. was studied by means of fluorescent labeling. It was shown that there is no inclusion of dissolved mols. in the previously formed monolayer during even 6 h of exposure regardless of monolayer surface d. The surface activity of IgG and horseradish peroxidase mols. was studied by means of surface compression isotherms, and the specific biol. activity of the monolayers formed from these proteins was measured by enzyme and immunoassay techniques. It was shown that the surface activity of the proteins increases while specific biol. activity decreases with exposure of the mols. on the water surface. Since the same effects were caused by denaturing agents, we propose that the surface activity of the proteins and the absence of surface-vol. exchange are due to partial unfolding of the mols. which takes place on the water surface. Two models of the partial unfolding are discussed: complete denaturation of some part of the mols. and partial unfolding of each mol. The process of surface denaturation was shown to be slow and controllable. One can achieve a pronounced increase of protein surface activity with low degrdn. of the specific biol. activity of the monolayer; thus, it can be used in the practice of protein Langmuir film deposition.
    35. 35
      Yano, Y. F. Kinetics of Protein Unfolding at Interfaces. J. Phys.: Condens. Matter 2012, 24 (50), 503101  DOI: 10.1088/0953-8984/24/50/503101
      35
      Kinetics of protein unfolding at interfaces
      Yano, Yohko
      Journal of Physics: Condensed Matter (2012), 24 (50), 503101/1-503101/16CODEN: JCOMEL; ISSN:0953-8984. (IOP Publishing Ltd.)
      A review. The conformation of protein mols. is detd. by a balance of various forces, including van der Waals attraction, electrostatic interaction, H-bonding, and conformational entropy. When protein mols. encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein mol. strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that the protein conformation changes during the adsorption process due to protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. Here, the author considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.
    36. 36
      Dickinson, E.; Matsumura, Y. Proteins at Liquid Interfaces: Role of the Molten Globule State. Colloids Surf., B 1994, 3 (1–2), 1– 17,  DOI: 10.1016/0927-7765(93)01116-9
      36
      Proteins at liquid interfaces: role of the molten globule state
      Dickinson, Eric; Matsumura, Yasuki
      Colloids and Surfaces, B: Biointerfaces (1994), 3 (1/2), 1-17CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier)
      A review, with 108 refs. Recent advances in understanding the structure and dynamics of proteins at liq. interfaces are reviewed with particular ref. to adsorbed layers at the oil-water interface in protein-stabilized emulsions. The authors discuss the importance of mol. flexibility in detg. the properties of adsorbed layers and the ease of exchange of protein mols. between bulk and surface in mixed systems. While a statistical model of a nearly random copolymer can be used to describe the adsorption of a disordered protein such as β-casein, such a representation is unrealistic for an adsorbed globular protein, which is typically a compact deformable macromol. particle having a structure lying somewhere between the native state and the completely unfolded form. It is proposed that such a state of an adsorbed globular protein at a liq. interface is close to what is now called the "molten globule" state. This is the partially denatured state of a globular protein which retains the ordered secondary structure but not the tertiary structure of the native protein. The authors describe the various ways of producing the molten globule state, and the authors review the exptl. evidence for the molten globule state of α-lactalbumin in some detail. In the final part of the paper, the authors discuss some new results on the surface activity of α-lactalbumin and the competitive adsorption of α-lactalbumin and β-lactoglobulin in emulsions at acidic pH or in the presence of EDTA. This discussion shows how the concept of the molten globule state provides new insight into the relation between protein structure and the properties of adsorbed layers at liq. interfaces.
    37. 37
      Husband, F. A.; Garrood, M. J.; Mackie, A. R.; Burnett, G. R.; Wilde, P. J. Adsorbed Protein Secondary and Tertiary Structures by Circular Dichroism and Infrared Spectroscopy with Refractive Index Matched Emulsions. J. Agric. Food Chem. 2001, 49 (2), 859– 866,  DOI: 10.1021/jf000688z
      37
      Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions
      Husband, Fiona A.; Garrood, Martin J.; Mackie, Alan R.; Burnett, Gary R.; Wilde, Peter J.
      Journal of Agricultural and Food Chemistry (2001), 49 (2), 859-866CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      The secondary structure of protein adsorbed at the emulsion interface has been studied in refractive index matched emulsions using the techniques of CD (CD) and Fourier transform IR spectroscopy. Bovine serum albumin (BSA) and bovine β-lactoglobulin (βlg) stabilized emulsions were studied, and the refractive index was altered by the addn. of glycerol or polyethylene glycol. The effect of additive on the soln. and adsorbed protein structure in addn. to the effect of adsorption time was considered. Both adsorption and glycerol addn. alter protein secondary structure; however, the majority of secondary structure remains. Small changes are obsd. in the secondary structure of adsorbed protein with time. Near-UV CD studies showed the effect of glycerol and adsorption on the arom. groups. BSA showed small changes both upon the addn. of glycerol to protein in soln. and upon adsorption. βlg showed slightly larger changes upon the addn. of glycerol to protein in soln. and a larger change upon adsorption.
    38. 38
      Morris, R. J.; Brandani, G. B.; Desai, V.; Smith, B. O.; Schor, M.; MacPhee, C. E. The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway. Biophys. J. 2016, 111 (4), 732– 742,  DOI: 10.1016/j.bpj.2016.06.006
      38
      The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway
      Morris, Ryan J.; Brandani, Giovanni B.; Desai, Vibhuti; Smith, Brian O.; Schor, Marieke; MacPhee, Cait E.
      Biophysical Journal (2016), 111 (4), 732-742CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Ranaspumin-2 (Rsn-2) is a surfactant protein found in the foam nests of the t´ungara frog. Previous exptl. work has led to a proposed model of adsorption that involves an unusual clam-shell-like unhinging of the protein at an interface. Interestingly, there is no concomitant denaturation of the secondary structural elements of Rsn-2 with the large-scale transformation of its tertiary structure. In this work we use both expt. and simulation to better understand the driving forces underpinning this unusual process. We develop a modified G‾o-model approach where we have included explicit representation of the side chains to realistically model the interaction between the secondary structure elements of the protein and the interface. Doing so allows for the study of the underlying energy landscape that governs the mechanism of Rsn-2 interfacial adsorption. Exptl., we study targeted mutants of Rsn-2, using the Langmuir trough, pendant drop tensiometry, and CD, to demonstrate that the clam-shell model is correct. We find that Rsn-2 adsorption is in fact a two-step process: the hydrophobic N-terminal tail recruits the protein to the interface after which Rsn-2 undergoes an unfolding transition that maintains its secondary structure. Intriguingly, our simulations show that the conformation Rsn-2 adopts at an interface is an arrested state along the denaturation pathway. More generally, our computational model should prove a useful, and computationally efficient, tool in studying the dynamics and energetics of protein-interface interactions.
    39. 39
      Wösten, H. A. B. Hydrophobins: Multipurpose Proteins. Annu. Rev. Microbiol. 2001, 55 (1), 625– 646,  DOI: 10.1146/annurev.micro.55.1.625
      39
      Hydrophobins: Multipurpose proteins
      Wosten, Han A. B.
      Annual Review of Microbiology (2001), 55 (), 625-646CODEN: ARMIAZ; ISSN:0066-4227. (Annual Reviews Inc.)
      A review. Class I and class II hydrophobins are small secreted fungal proteins that play a role in a broad range of processes in the growth and development of filamentous fungi. For instance, they are involved in the formation of aerial structures and in the attachment of hyphae to hydrophobic surfaces. The mechanisms by which hydrophobins fulfill these functions are based on their property to self-assemble at hydrophilic-hydrophobic interfaces into a 10 nm-thin highly amphipathic film. Complementation studies have shown that class I hydrophobins belong to a closely related group of morphogenetic proteins, but that they have evolved to function at specific interfaces. Recent evidence indicates that hydrophobins do not only function by self-assembly. Monomeric hydrophobin has been implicated in cell-wall assembly, but the underlying mechanism is not yet clear. In addn., hydrophobin monomers could act as toxins and elicitors.
    40. 40
      Blijdenstein, T. B. J.; Veerman, C.; van der Linden, E. Depletion– Flocculation in Oil-in-Water Emulsions Using Fibrillar Protein Assemblies. Langmuir 2004, 20 (12), 4881– 4884,  DOI: 10.1021/la0497447
      40
      Depletion-Flocculation in Oil-in-Water Emulsions Using Fibrillar Protein Assemblies
      Blijdenstein, Theo B. J.; Veerman, Cecile; Van der Linden, Erik
      Langmuir (2004), 20 (12), 4881-4884CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      This paper shows that low concns. of β-lactoglobulin fibrils can induce depletion-flocculation in β-lactoglobulin-stabilized oil-in-H2O emulsions. The min. required fibril concn. for flocculation was detd. exptl. for fibril lengths of ∼3 and 0.1 μm. The min. fibril concn. for flocculation is 2 orders of magnitude higher for the short fibrils than for the long ones. These exptl. results correspond well with a theor. prediction based on a model of spinodal decompn. Rheol. measurements were performed, verifying that flocculation was induced by a depletion mechanism. The results of this study show that the min. concn. required for depletion-flocculation can be tuned by varying the length of the fibrils.
    41. 41
      Peng, J.; Simon, J. R.; Venema, P.; Van Der Linden, E. Protein Fibrils Induce Emulsion Stabilization. Langmuir 2016, 32 (9), 2164– 2174,  DOI: 10.1021/acs.langmuir.5b04341
      41
      Protein Fibrils Induce Emulsion Stabilization
      Peng, Jinfeng; Simon, Joana Ralfas; Venema, Paul; van der Linden, Erik
      Langmuir (2016), 32 (9), 2164-2174CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The behavior of an oil-in-water emulsion was studied in the presence of protein fibrils for a wide range of fibril concns. by using rheol., diffusing wave spectroscopy, and confocal laser scanning microscopy. Results showed that above a min. fibril concn. depletion flocculation occurred, leading to oil droplet aggregation and enhanced creaming of the emulsion. Upon further increasing the concn. of the protein fibrils, the emulsions were stabilized. In this stable regime both aggregates of droplets and single droplets are present, and these aggregates are smaller than the aggregates in the flocculated emulsion samples at the lower fibril concns. The size of the droplet aggregates in the stabilized emulsions is independent of fibril concn. In addn., the droplet aggregation was reversible upon diln. both by a pH 2 HCl soln. and by a fibril soln. at the same concn. The viscosity of the emulsions contg. fibrils was comparable to that of the pure fibril soln. Neither fibril networks nor droplet gel networks were obsd. in our study. The stabilization mechanism of emulsions contg. long protein fibrils at high protein fibril concns. points toward the mechanism of a kinetic stabilization.
    42. 42
      Yuan, T.; Zeng, J.; Wang, B.; Cheng, Z.; Chen, K. Pickering Emulsion Stabilized by Cellulosic Fibers: Morphological Properties-Interfacial Stabilization-Rheological Behavior Relationships. Carbohydr. Polym. 2021, 269, 118339  DOI: 10.1016/j.carbpol.2021.118339
      42
      Pickering emulsion stabilized by cellulosic fibers: Morphological properties-interfacial stabilization-rheological behavior relationships
      Yuan, Tianzhong; Zeng, Jinsong; Wang, Bin; Cheng, Zheng; Chen, Kefu
      Carbohydrate Polymers (2021), 269 (), 118339CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)
      This work aimed to study the stabilization mechanism induced by different morphologies of cellulosic fiber in O/W emulsion. Three types of cellulosic fibers were named squashed cellulose, incompletely nanofibrillated cellulose, and completely nanofibrillated cellulose, resp. Squashed cellulose acted as barriers between the droplets to stabilize emulsion via depletion flocculation, whereas incompletely nanofibrillated and completely nanofibrillated cellulose formed covering layer via interfacial adsorption and connected adjacent droplets to create the droplet-fiber network structure via bridging flocculation. Differently, completely nanofibrillated cellulose formed the denser covering layer leading to a more stability of droplet. Importantly, it had the higher capacity of bridging flocculation, which can tightly connect the adjacent droplets to form a stronger droplet-fiber 3D network structure. Consequently, in rheol. anal. including creep compliance, and dynamic modulus, the corresponding emulsions showed excellent anti-deformation ability and dynamic stability. This study provides practical guidance on the productions of foodstuff and cosmetic.
    43. 43
      Kelley, L. A.; Mezulis, S.; Yates, C. M.; Wass, M. N.; Sternberg, M. J. E. The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis. Nat. Protoc. 2015, 10 (6), 845– 858,  DOI: 10.1038/nprot.2015.053
      43
      The Phyre2 web portal for protein modeling, prediction and analysis
      Kelley, Lawrence A.; Mezulis, Stefans; Yates, Christopher M.; Wass, Mark N.; Sternberg, Michael J. E.
      Nature Protocols (2015), 10 (6), 845-858CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which the authors previously published a paper in Nature Protocols. In this updated protocol, the authors describe Phyre2, which uses advanced remote homol. detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they det. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain compn. and model quality. A range of addnl. available tools is described to find a protein structure in a genome, to submit large no. of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.
    44. 44
      Cole, C.; Barber, J. D.; Barton, G. J. The Jpred 3 Secondary Structure Prediction Server. Nucleic Acids Res. 2008, 36, W197– W201,  DOI: 10.1093/nar/gkn238
      44
      The Jpred 3 secondary structure prediction server
      Cole, Christian; Barber, Jonathan D.; Barton, Geoffrey J.
      Nucleic Acids Research (2008), 36 (Web Server), W197-W201CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Jpred (http://www.compbio.dundee.ac.uk/jpred) is a secondary structure prediction server powered by the Jnet algorithm. Jpred performs over 1000 predictions per wk for users in more than 50 countries. The recently updated Jnet algorithm provides a three-state (α-helix, β-strand and coil) prediction of secondary structure at an accuracy of 81.5%. Given either a single protein sequence or a multiple sequence alignment, Jpred derives alignment profiles from which predictions of secondary structure and solvent accessibility are made. The predictions are presented as colored HTML, plain text, PostScript, PDF and via the Jalview alignment editor to allow flexibility in viewing and applying the data. The new Jpred 3 server includes significant usability improvements that include clearer feedback of the progress or failure of submitted requests. Functional improvements include batch submission of sequences, summary results via email and updates to the search databases. A new software pipeline will enable Jnet/Jpred to continue to be updated in sync with major updates to SCOP and UniProt and so ensures that Jpred 3 will maintain high-accuracy predictions.
    45. 45
      Leon, L. J.; Idangodage, H.; Wan, C.-P. L.; Weers, P. M. M. Apolipophorin III: Lipopolysaccharide Binding Requires Helix Bundle Opening. Biochem. Biophys. Res. Commun. 2006, 348 (4), 1328– 1333,  DOI: 10.1016/j.bbrc.2006.07.199
      45
      Apolipophorin III: Lipopolysaccharide binding requires helix bundle opening
      Leon, Leonardo J.; Idangodage, Hasitha; Wan, Chung-Ping L.; Weers, Paul M. M.
      Biochemical and Biophysical Research Communications (2006), 348 (4), 1328-1333CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
      Apolipophorin III (apoLp-III) is a prototypical apolipoprotein used for structure-function studies. Besides its crucial role in lipid transport, apoLp-III is able to assoc. with fungal and bacterial membranes and stimulate cellular immune responses. We recently demonstrated a binding interaction of apoLp-III from the greater wax moth, Galleria mellonella, with lipopolysaccharides (LPS). In the present study, the requirement of helix bundle opening for LPS binding interaction was investigated. Using site-directed mutagenesis, two cysteine residues were introduced in close spatial proximity (P5C/A135C). When the helix bundle was locked by disulfide bond formation, the tethered helix bundle failed to assoc. with LPS. In contrast, the mutant protein regained its ability to bind upon redn. with dithiothreitol. Thus, helix bundle opening is a crit. event in apoLp-III binding interaction with LPS. This mechanism implies that the hydrophobic interior of the protein interacts directly with LPS, analogous to that obsd. for lipid interaction.
    46. 46
      Whitten, M. M. A.; Tew, I. F.; Lee, B. L.; Ratcliffe, N. A. A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1, 3-Glucan Pattern Recognition and Cellular Encapsulation Reactions. J. Immunol. 2004, 172 (4), 2177– 2185,  DOI: 10.4049/jimmunol.172.4.2177
      46
      A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions
      Whitten, Miranda M. A.; Tew, Ian F.; Lee, Bok L.; Ratcliffe, Norman A.
      Journal of Immunology (2004), 172 (4), 2177-2185CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
      Lipoproteins and mols. for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study the authors describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide prodn. in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study the authors show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and β-1,3-glucan and therefore may act as a pattern recognition mol. for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiol. levels, and, with the notable exception of β-glucan binding, are only obsd. with the lipid-assocd. form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition mol. and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunol., particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.3c03163.

    • Plasmids used in this study; diagram of wrinkle analysis; air/water interface kinetics; BAM surface pressure isotherms, and film relaxation dynamics as a function of equilibration time (PDF)

    • BslA forms a robust interfacial film. A 40 L droplet of 0.1 mg/mL BslA is expelled into GTO after 30 min of equilibration time. 10 L volume is withdrawn and subsequently, long-lived wrinkles form within the elastic BslA interfacial layer. The frame rate is 0.1 frame/s (AVI)

    • mTasA does not form a robust interfacial film. A 40 L droplet of 0.1 mg/mL mTasA is expelled into GTO after 30 min of equilibration time. 10 L volume is withdrawn, and it is observed that there is no wrinkling. Additional volumes are withdrawn until the droplet is very small, at which time a very transiently wrinkled film is observed. The frame rate is 1 frames/s (AVI)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.