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

Abstract

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.

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.

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.

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.

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.

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.