Departamento de Microbiología, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga, (Campus Universitario de Teatinos), Málaga, Spain.
Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, Lausanne, Switzerland.
NPJ Biofilms Microbiomes. 2023 Sep 22;9(1):68. doi: 10.1038/s41522-023-00437-w.
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.
生物膜是由细胞分化过程导致部分群体分泌细胞外基质 (ECM) 而形成的细菌群落。在枯草芽孢杆菌中，ECM 的主要蛋白质成分是 TasA，它形成了纤维状支架，为 ECM 提供结构。TasA 的 N 端半胱氨酸在芽孢杆菌属物种中高度保守，包含一个蛋白质结构域，即刚性核心 (RcTasA)，对于重组蛋白的结构和功能特性至关重要。在这项研究中，我们证明了体外重组纯化的 RcTasA 保留了先前观察到的整个蛋白质的生化特性。进一步分析 RcTasA 的氨基酸序列揭示了两个易于聚集的伸展区和一个氨基酸重复不完美的区域，已知这些区域有助于功能淀粉样蛋白的组装。对 RcTasA 中发现的这些伸展区的生化特性进行分析表明，它们在体外具有淀粉样特性，这有助于 RcTasA 的淀粉样性质。此外，对不完美氨基酸重复的研究揭示了残基 D64、K68 和 D69 在 TasA 结构功能中的关键作用。携带 D64A 和 K68AD69A 取代的 TasA 版本的实验表明，该蛋白在 ECM 组装或核心和淀粉样特性稳定性方面的部分功能丧失。总之，我们的研究结果使我们能够更好地理解 TasA 在生物膜形成过程中的聚合过程，并为促进细菌中蛋白质丝分子行为的序列决定因素提供了知识。
