Serra Diego O, Hengge Regine
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Philippstr. 13 - Haus 22, 10115, Berlin, Germany.
Methods Mol Biol. 2017;1657:133-145. doi: 10.1007/978-1-4939-7240-1_11.
By adopting elaborate three-dimensional morphologies that vary according to their extracellular matrix composition, macrocolony biofilms offer a unique opportunity to interrogate about the roles of specific matrix components in shaping biofilm architecture. Here, we describe two methods optimized for Escherichia coli that profit from morphology and the high level of structural organization of macrocolonies to gain insight into the production and assembly of amyloid curli and cellulose-the two major biofilm matrix elements of E. coli-in biofilms. The first method, the macrocolony morphology assay, is based on the ability of curli and cellulose-either alone or in combination-to generate specific morphological and Congo Red-staining patterns in E. coli macrocolonies, which can then be used as a direct visual readout for the production of these matrix components. The second method involves thin sectioning of macrocolonies, which along with in situ staining of amyloid curli and cellulose and microscopic imaging allows gaining fine details of the spatial arrangement of both matrix elements inside macrocolonies. Beyond their current use with E. coli and related curli and cellulose-producing Enterobacteriaceae, both the methods offer the potential to be adapted to other bacterial species.
通过采用根据其细胞外基质组成而变化的精细三维形态，大菌落生物膜提供了一个独特的机会，来探究特定基质成分在塑造生物膜结构中的作用。在这里，我们描述了两种针对大肠杆菌优化的方法，这些方法利用大菌落的形态和高度的结构组织水平，深入了解淀粉样卷曲蛋白和纤维素（大肠杆菌生物膜的两个主要生物膜基质成分）在生物膜中的产生和组装。第一种方法，即大菌落形态分析，基于卷曲蛋白和纤维素单独或组合在大肠杆菌大菌落中产生特定形态和刚果红染色模式的能力，然后可将其用作这些基质成分产生的直接视觉读数。第二种方法涉及对大菌落进行薄切片，连同对淀粉样卷曲蛋白和纤维素的原位染色以及显微镜成像，可以获得大菌落内部两种基质成分空间排列的精细细节。除了目前用于大肠杆菌以及相关的产生卷曲蛋白和纤维素的肠杆菌科细菌外，这两种方法都有可能适用于其他细菌物种。
