Bacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σ(S) (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as "anatomical" hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in "curli baskets." OBJECTIVE Heterogeneity or cellular differentiation in biofilms is a commonly accepted concept, but direct evidence at the microscale has been difficult to obtain. Our study reveals the microanatomy and microphysiology of an Escherichia coli macrocolony biofilm at an unprecedented cellular resolution, with physiologically different zones and strata forming as a function of known global regulatory networks that respond to biofilm-intrinsic gradients of nutrient supply. In addition, this study identifies zones of heterogeneous and potentially bistable CsgD and curli expression, shows bacterial curli networks to strikingly resemble Alzheimer plaques, and suggests a new role of flagella as an architectural element in biofilms.