Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

Abstract

bBacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.

The Bacterial Extracellular Matrix Protein TapA Is a Two‐Domain Partially Disordered Protein

“…The amyloid‐like fibers in the biofilms of our model organism for biofilm formation, the soil bacterium Bacillus subtilis , are also composed of two components. The protein TasA is the major component of the fibers and the auxiliary protein, TapA, present at about 1/100 molar ratio relative to TasA . TapA has a few roles in the formation of amyloid‐like fibers: it anchors TasA fibers to the cell surface and acts as a nucleating agent in the process of TasA fiber growth; hence it has been given the name “TasA assembling/anchoring protein” .…”

“…The protein TasA is the major component of the fibers and the auxiliary protein, TapA, present at about 1/100 molar ratio relative to TasA . TapA has a few roles in the formation of amyloid‐like fibers: it anchors TasA fibers to the cell surface and acts as a nucleating agent in the process of TasA fiber growth; hence it has been given the name “TasA assembling/anchoring protein” . A previous analysis of the sequence of TapA revealed two conserved fragments (marked red in Figure A): a 37‐residue fragment, TapA 194–230, at the C terminus of the protein, which had no phenotypic effect on biofilms, and an 8‐residue fragment, TapA 50–57, at the N terminus of the protein, which affected the phenotype of the biofilm and was essential for TasA fiber assembly in vitro .…”

“…TapA has a few roles in the formation of amyloid‐like fibers: it anchors TasA fibers to the cell surface and acts as a nucleating agent in the process of TasA fiber growth; hence it has been given the name “TasA assembling/anchoring protein” . A previous analysis of the sequence of TapA revealed two conserved fragments (marked red in Figure A): a 37‐residue fragment, TapA 194–230, at the C terminus of the protein, which had no phenotypic effect on biofilms, and an 8‐residue fragment, TapA 50–57, at the N terminus of the protein, which affected the phenotype of the biofilm and was essential for TasA fiber assembly in vitro . Unlike the disordered natures of CsgA and CsgB, TasA is a structured protein, as we have previously shown, and was later corroborated by means of X‐ray diffraction .…”

Amyloid Structures as Biofilm Matrix Scaffolds

“…TasA is expressed from the tapA-sipW-tasA operon, together with two proteins, TapA and SipW, on which formation of the fibrils depends. The sipW gene encodes a type I signal peptidase that is specifically required for the processing and secretion of TasA and TapA (41), whereas TapA is an accessory protein that promotes the efficient polymerization of TasA subunits at the cell envelope (9,43,44) (Fig. 1A).…”

Composition and functions of the extracellular polymer matrix of bacterial biofilms

“…Mutants with impaired synthesis of this protein are unable to form biofilms, in spite of their retained capacity for exopolysaccharide synthesis. Existence of TasA as extracellular filaments with the properties of an amyloid protein playing an important role in the biofilm matrix was recently demonstrated [33].…”