Bacterial macrocolony biofilms grow into intricate three-dimensional structures that depend on self-produced extracellular polymers conferring protection, cohesion and elasticity to the biofilm. InEscherichia coli, synthesis of this matrix – consisting of amyloid curli fibres and cellulose – requires CsgD, a transcription factor regulated by the stationary phase sigma factor RpoS, and occurs in the nutrient-deprived cells of the upper layer of macrocolonies. Is this asymmetric matrix distribution functionally important or is it just a fortuitous by-product of an unavoidable nutrient gradient? In order to address this question, the RpoS-dependentcsgDpromoter was replaced by a vegetative promoter. This re-wiring ofcsgDled to CsgD and matrix production in both strata of macrocolonies, with the lower layer transforming into a rigid ‘base plate’ of growing yet curli-connected cells. As a result, the two strata broke apart followed by desiccation and exfoliation of the top layer. By contrast, matrix-free cells at the bottom of wild-type macrocolonies maintain colony contact with the humid agar support by flexibly filling the space that opens up under buckling areas of the macrocolony. Precisely regulated stratification in matrix-free and matrix-producing cell layers is thus essential for the physical integrity and architecture ofE. colimacrocolony biofilms.