Interfacial self-assembly of a bacterial hydrophobin

Abstract

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

Keywords: Bacillus subtilis; BslA; bacterial hydrophobin; biofilm; interfacial self-assembly.

Fig. 1.

A plot of regime I times versus concentration of WT-BslA (closed circles) and BslA-L77K (open circles). The dashed line represents the predicted time to reach a surface coverage of 1.57 mg⋅m⁻² using Eq. 1.

Fig. 2.

(A) CD spectra of WT-BslA (black line) and BslA-L77K (gray line) in 25 mM phosphate buffer (pH 7). (B) CD spectra of refractive index matched emulsions stabilized by WT-BslA (black line) and BslA-L77K (gray line). Dotted lines: raw data; solid lines: smoothed data (SI Appendix).

Fig. 3.

TEM images of (A) WT-BslA and (B) BslA-L77K stained with uranyl acetate. Scale bar = 50 nm. Insets: FFTs of the entire TEM image. The numbers in A correspond to the Miller indices of the 2D lattice structure.

Fig. 4.

(A) The secondary structures of chain C and chain I of BslA, derived from the crystal structure PDB: 4BHU (10). Color code: β-strand (blue), α-helix (magenta), residue Leu-77 (green), cap strands (yellow highlight). Amino acids 43–46, 155–159, and 171–172 of chain I are not defined in the crystal structure, suggesting structural heterogeneity. (B) A depiction of chain C (Left) and chain I (Right), highlighting the different orientations of the hydrophobic residues in the cap (black). Images generated using Visual Molecular Dynamics (33).

Fig. 5.

(A) Interfacial tension profiles of a droplet of unfractionated WT-BslA (0.02 mg⋅mL⁻¹) in air (black line) and in glyceryl trioctanoate (gray line). (B) A 50-µL droplet of WT-BslA (0.03 mg⋅mL⁻¹) on highly ordered pyrolytic graphite after 0 (Left) and 30 (Right) min. (C) A 25-µL droplet of WT-BslA (0.02 mg⋅mL⁻¹) in air before and after compression. (D) A 40-µL droplet of WT-BslA (0.2 mg⋅mL⁻¹) in glyceryl trioctanoate before and after compression.

Fig. 6.

Schematic of BslA adsorption. When unbound, the conformation of the hydrophobic cap of WT-BslA (A) orients the hydrophobic residues away from the aqueous medium, slowing the rate of adsorption (indicated by a small arrow). The L77K mutation (B) removes the adsorption barrier by exposing some or all of the hydrophobic residues within the hydrophobic cap, increasing the rate of adsorption (indicated by a bold arrow). Once adsorbed onto the interface, the surface-bound WT-BslA refolds to a conformation rich in β-sheet and is able to form strong lateral interactions with adjacent molecules, forming an organized lattice that under normal circumstances will not be removed from the interface (indicated by the crossed arrow). Surface-bound BslA-L77K forms a less well-organized lattice and can be removed from the interface with only minimal energy, such as droplet compression.