Molecular architecture of the TasA biofilm scaffold in Bacillus subtilis

Abstract

Many bacteria in nature exist in multicellular communities termed biofilms. Cells within biofilms are embedded in a primarily self-secreted extracellular polymeric matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model biofilm-forming 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. The structure of TasA fibres, however, and how fibres scaffold the biofilm at the molecular level, is not known. Here, we present electron cryomicroscopy structures of TasA fibres, which show that rather than forming amyloid fibrils, TasA monomers assemble into filaments through donor strand complementation, with each subunit donating a beta-strand to complete the fold of the next subunit along the filament. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA filaments congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. This study explains the previously observed biochemical properties of TasA and shows, for the first time, how a bacterial extracellular globular protein can assemble from monomers into beta-sheet-rich fibres, and how such fibres assemble into bundles in biofilms. We establish a hierarchical, atomic-level assembly mechanism of biofilm scaffolding that provides a structural framework for understanding bacterial biofilm formation.

Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms

“…4). Furthermore, a very recent cryo-EM study reveals globular TasA subunits arranged into a filament with a donor strand from one subunit inserted into a sheet in the next subunit (60), very similar to what we report herein for ABP. Collectively, these sequence and structural comparisons establish an evolutionary relationship between ABP and TasA.…”

Donor strand complementation, isopeptide bonds and glycosylation reinforce highly resilient archaeal thread filaments

“…Hence, it is likely that Saci_0405 forms the initial nucleating subunit for the Saci_0406 thread. Similar observations were made for the TasA filament formed by B. subtilis , where TapA a helps nucleating filament assembly of TasA, as well as the recently characterized archaeal bundle pili 46, 61 . Interestingly, we find that unlike Saci_0406, the putative cap protein Saci_0405 does not have a predicted SEC signal sequence or a transmembrane domain.…”

“…In the filament, TasA undergoes a conformational change and the binding site is free for donor strand complementation from the adjacent subunit. The filament polymerization is initialized by an accessory protein TapA which can bind to the strand accepting groove of TasA, so TasA can initiate binding to the next subunit 61 . Structural prediction of the archaeal bundle pili subunit revealed a similar mechanism 46 .…”

Electron cryo-microscopy reveals the structure of the archaeal thread filament

“…In the filament, TasA undergoes a conformational change, and the binding site is free for DSC from the adjacent subunit. The filament polymerisation is initialised by an accessory protein TapA, which can bind to the strand accepting groove of TasA, so TasA can initiate binding to the next subunit 75 . Structural prediction of the archaeal bundle pili subunit revealed a similar mechanism 62 and indeed, we also observed auto complementation in some of our Alphafold predictions for Saci_0406 (Supplementary Fig.…”