Mechanisms of Bacillus subtilis biofilm formation - opportunities and limitations of AlphaFold
B.subtilis biofilms contain non-amyloid filaments composed of folded TasA protomers. Interactions of the biofilm proteins TapA and TasA supporting TasA oligomerisation and the mechanism of TapA-supported growth of TasA filaments are uncovered. In this context, the filament structure is derived by a combination of solid-state NMR and Alphafold, and it is discussed why Alphafold is not predicting the TasA monomer structure and thus the structural rearrangements correctly.
Most Microorganisms form sessile multi-cellular biofilms in which they are protected against stress by a gel-like extracellular matrix composed of proteinaceous fibrils, various extracellular polysaccharides (EPS) and often DNA. In many cases, the major biofilm-associated protein forms amyloid-like fibrils. The situation is different for Bacillus subtilis biofilms that contain filaments composed of b-sandwich domains that cannot be stained by Thioflavin T or congored. Solid-state NMR experiments revealed insertion of an initially flexible N-terminal TasA peptide segment into subsequent protomers to form a mildly helical filament, in agreement with EM and AlphaFold predictions. A combination of analytical ultra-centrifugation and NMR reveals a TapA concentration-dependent support of TasA oligomer formation. It is found that TapA serves as a template for TasA folding and oligomerization by presenting its N-terminus to unfolded TasA that appears outside the cell via the SecA pathway, yielding structures highly homologues to type I pili. We observe a donor-strand presentation to unfolded TasA that supposedly refolds around the presented strand, and becomes thus oligomerization-competent. The role of TapA is also in completion of the first TasA-TasA interface through the donation of conserved residues. Since folded monomers are present in B.subtilis biofilms, a mechanism for their inclusions into filaments is also discussed. Here, pilus-homologous structures were observed for the first time in Gram-positive bacteria.