Molecular characterization of the N-terminal half of TasA during functional amyloid assembly and its contribution to Bacillus subtilis biofilm formation
Biofilms are bacterial communities that result from a cell differentiation process that leads to the secretion of an extracellular matrix (ECM) by part of the bacterial population. In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA, which forms a fiber-based scaffold that confers structure to the ECM. The N-terminal half of TasA is strongly conserved among Bacillus species and contains a protein domain, the amyloid core (AcTasA), which is critical for the formation of the amyloid architecture. In this study, we demonstrate that recombinantly purified AcTasA in vitro retains biochemical properties previously observed for the entire protein. Further analysis of the AcTasA amino acid sequence revealed two amyloidogenic stretches and a region of imperfect amino acid repeats, which are known to contribute to functional amyloid assembly. Biochemical characterization of these amyloidogenic stretches found in AcTasA revealed their amyloid capacity in vitro, contributing to the amyloid nature of AcTasA. Moreover, the study of the imperfect amino acid repeats revealed the critical role of residues D64, K68 and D69 in the structural function of TasA. In vivo and in vitro experiments with versions of TasA carrying the substitutions D64A, K68A, and D69A demonstrated a partial loss of function of the protein either in the assembly of the ECM or in the stability of the core and amyloid polymerization. Taken together, our findings allow us to better understand the polymerization process of TasA during biofilm formation and provide knowledge into the sequence determinants that promote the molecular behavior of functional amyloids.