Physiological stratification and differential spatial distribution of amyloid curli fibres, cellulose and flagella in Escherichia coli macrocolony biofilms

SERRA, DIEGO O.; RICHTER, ANJA M.; HENGGE, REGINE

Paris

Conferencia; EMBO Conference: Microbiology after the genomics revolution (Genomes 2014); 2014

The European Molecular Biology Organization (EMBO); Pascale Cossart (Chair).

Introduction and objectives. Bacteria preferentially live in structured communities known as biofilms. Although it is widely recognized that biofilm formation depends on bacterial self-produced matrix components such as adhesins, amyloid fibres and exopolysaccharides, the precise location, organization and structural function of these components inside biofilms and the link to bacterial physiology are poorly known. Escherichia coli produces flagella during the post-exponential phase, when nutrients become limiting, but the rod-shaped cells still grow. Upon entry into stationary phase, cells stop producing flagella, become small, ovoid and multiple stress resistant and generate amyloid curli fibres and the exopolysaccharide cellulose. At the regulatory level, the synthesis of curli and cellulose depends on the biofilm regulator CsgD. Using flagella, curli fibres, cellulose, cell morphology and visualized expression of csgD as anatomical hallmarks in fluorescence and scanning electron microscopy, we seek to elucidate the spatial order of physiological stratification and matrix distribution in E. coli biofilms at high resolution. Results. Two physiologically distinct cell layers and three differential patterns of matrix distribution were distinguished at high resolution in macrocolonies of AR3110, a K-12 strain with restored capacity to produce cellulose. Macromorphologically, AR3110 macrocolonies are only about 60 micrometer thin, large and exhibit radial ridge-like structures formed by vertical buckling of the biofilm. The lower layer (about 20 um thick) features bacteria that resemble those in post-exponential phase with entangled flagella as a single and characteristic matrix component. In sharp contrast, the upper layer of flat sectors and ridges (about 40 um thick) contains starving stationary phase-like bacteria surrounded by (i) cellulose sheets/filaments in the inner zone, at the boundary with the lower layer, and (ii) a dense nanocomposite of curli and cellulose in the outermost zone. This differential pattern of matrix composition in the upper layer (cellulose only vs cellulose/curli composite) precisely correlates in space with bimodal expression of csgD. Conclusion. These results contribute to our understanding to how physiologically distinct subpopulations of bacteria arise within a community and how these subpopulation dictate a precise spatial order of matrix synthesis that ultimately defines the biofilm architecture.