This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the “Candidatus Brocadia sinica”-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel β-strands, and circular dichroism conducted on the isolated surface protein indicated that β-strands are the dominant higher-order structure. The isolated protein was stained positively by the β-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation.

中文翻译：

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Anammox细胞外基质中大量蛋白质的相变介导细胞间聚集和生物膜形成。

这项研究描述了来自关键环境和生物技术生物膜的高度丰富的细胞外蛋白的第一个直接功能分配，该膜执行厌氧铵氧化（anammox）过程。Brosi_A1236的表达水平，属于一类蛋白质的先前建议被细胞表面相关联的，是在最上面的一个百分位的所有基因中的“暂定富含Brocadia sinica”的生物膜。通过计算预测，Brosi_A1236结构由免疫球蛋白样的反平行β链组成，并且对分离的表面蛋白进行的圆二色性表明β链是主要的高阶结构。分离的蛋白质被β-折叠特异性染色剂thioflavin T以及生物膜的细胞表面和基质相关区域阳性染色。表面蛋白具有较大的非结构化含量，包括在其C末端的两个高度无序的结构域。无序域与基质结合，从而促进了带负电荷的乳胶微球的粘附，该微球被用作细胞的代理。无序域和分离的整个表面蛋白也经历了液-液相分离以形成悬浮液中的液滴。无序蛋白的液滴润湿了微球和细菌细胞的表面，并促进了它们的结合。此外，表面层蛋白形成凝胶以及有序的晶体结构。这些观察表明，通过相变的生物物理重塑促进聚集和生物膜形成。