Camara-Almiron, J. | bioRxiv Science

bioRxiv2023

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.

microbiology↗

bioRxiv2019

Dual functionality of the TasA amyloid protein in Bacillus physiology and fitness on the phylloplane

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). Previous studies have demonstrated that genetic pathways involved in biofilm formation are activated under a variety of environmental conditions to enhance bacterial fitness; however, the functions of the individual ECM components are still poorly understood. In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. In this study, we demonstrate that beyond their well-known defect in biofilm formation,{Delta} tasA cells also exhibit a range of cytological symptoms indicative of excessive cellular stress, including DNA damage accumulation, changes in membrane potential, higher susceptibility to oxidative stress, and alterations in membrane dynamics. Collectively, these events can lead to increased programmed cell death in the colony. We show that these major physiological changes in{Delta} tasA cells are likely independent of the structural role of TasA during amyloid fiber formation in the ECM. The presence of TasA in cellular membranes, which would place it in proximity to functional membrane microdomains, and mislocalization of the flotillin-like protein FloT in{Delta} tasA cells, led us to propose a role for TasA in the stabilization of membrane dynamics as cells enter stationary phase. We found that these alterations caused by the absence of TasA impair the survival, colonization and competition of Bacillus cells on the phylloplane. Taken together, our results allow the separation of two complementary roles of this functional amyloid protein: i) structural functions during ECM assembly and interactions with plants, and ii) a physiological function in which TasA, via its localization to the cell membrane, stabilizes membrane dynamics and supports more effective cellular adaptation to environmental cues.

microbiology↗