Molecular characterization of the N-terminal half of TasA during amyloid-like assembly and its contribution to Bacillus subtilis biofilm formation

Abstract

Biofilms are bacterial communities that result from a cell differentiation process leading to the secretion of an extracellular matrix (ECM) by part of the population. In Bacillus subtilis, the main protein component of the ECM is 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 rigid core (RcTasA), which is critical for the structural and functional properties of the recombinant protein. In this study, we demonstrate that recombinantly purified RcTasA in vitro retains biochemical properties previously observed for the entire protein. Further analysis of the RcTasA amino acid sequence revealed two aggregation-prone stretches and a region of imperfect amino acid repeats, which are known to contribute to functional amyloid assembly. Biochemical characterization of these stretches found in RcTasA revealed their amyloid-like capacity in vitro, contributing to the amyloid nature of RcTasA. 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. Experiments with versions of TasA carrying the substitutions D64A and K68AD69A 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-like properties. 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 protein filaments in bacteria.

Uncovering the Molecular Composition and Architecture of the Bacillus subtilis Biofilm via Solid-State NMR Spectroscopy

“…The protein signals are primarily from the structural proteins TasA and potential BslA, as evidenced by severe loss of protein signals in the Δmatrix biofilm (Figure c). Moreover, the chemical shifts of Cα for the resolved protein residues, which are sensitive to φ and ψ torsion angles in proteins, indicate a predominant β-strand secondary structure (Figure S10d), consistent with the molecular conformation of TasA ,,− and BslA. , Meanwhile, a previous report also proposed TasA and BslA as the source of protein signals in the 1D CP spectrum of the B. subtilis 3610 biofilm .…”

“…87,88 Meanwhile, a previous report also proposed TasA and BslA as the source of protein signals in the 1D CP spectrum of the B. subtilis 3610 biofilm. 37 2D spectra of TasA filaments, derived from previous studies, 84,86 exhibit a significant overall alignment with our data (Figure S5). Nonetheless, nuanced spectral deviations are observed, which may stem from variations in absolute quantities, subtle alterations in structural conformations, the presence of additional protein components, or differences in the measurement conditions.…”

Involvement of the Bacillus SecYEG Pathway in Biosurfactant Production and Biofilm Formation

“…Te products of the secD and secF genes form another heterotrimeric membrane complex, SecDF-yajC, which interacts with the SecYEG complex [7][8][9][10]. Bacterial secretory proteins are known to perform several essential "remote control" functions, such as food supply, cell-to-cell communication, and bioflm formation [3,[11][12][13]. It is therefore important to note that with the evolution and selection of bacterial species, many features remain unknown in terms of the SecYEG export component.…”