Donor-strand exchange drives assembly of the TasA scaffold in Bacillus subtilis biofilms

Abstract

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 chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a β-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into β-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.

TapA acts as specific chaperone in TasA filament formation by strand complementation

“…The pitch and diameter as observed by EM ( Fig. 3 A ) is reproduced by the AlphaFold model, which is in good agreement with the recently published cryo-EM structure ( 18 ). Our NMR restraints confirm these models ( SI Appendix , Figs.…”

“…A three-dimensional structure of monomeric TasA G28-239 was obtained by X-ray crystallography ( 8 ). ThT-stainable fibrils were investigated by solid-state NMR ( 10 ), and in parallel to this study nonamyloid filaments by cryo-EM ( 18 ) were studied at a resolution of 3.5 Å. Furthermore, a structural relationship between archaeal and bacterial biofilms was suggested on the basis of a 4.0 Å cryo-EM structure of Pyrobaculum calidifontis bundling pili ( 19 ).…”

Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa

“…The interaction partners of the pilus are hence unknown and warrant the focus of future imaging efforts. Since we observed isolated CupE pili forming regular mesh-like arrays in cryo-EM images (S1D Fig) , this might indicate that lateral interactions of pili may occur in the crowded conditions of the biofilm matrix, similar to other biofilm matrix fibre systems [51][52][53]. Further studies on native cellular systems-i.e., biofilms-will be required to determine the exact mode of interaction of CupE pili with other extracellular matrix components and also with cells.…”

Biophysical basis of phage liquid crystalline droplet-mediated antibiotic tolerance in pathogenic bacteria

“…However, further experiments will be needed to test this, and detailed mechanisms of how droplets mediate protection against antibiotic treatment will be an exciting future direction of experimental and theoretical research. Since bacteria often proliferate in environments rich in rod-like or filamentous molecules accompanied by biopolymers, for example in the biofilm matrix (Tarafder et al, 2020, Böhning et al, 2022, Secor et al, 2015) or on tissues covered with mucus (Wagner et al, 2018), our structural, biochemical, and imaging data suggest a paradigm where the biophysics of such associations strongly influence bacterial interactions with, and response to, external stimuli (Figure 7).…”