Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts 13C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.

Bacterial biofilms are complex, multicellular communities made up of bacteria enmeshed in a self-produced extracellular matrix (ECM) that protects against environmental stress. The ECM often comprises insoluble components, which complicates the study of biofilm composition, structure, and function. Wrinkled, agar-grown Escherichia coli biofilms require 2 insoluble macromolecules: curli amyloid fibers and cellulosic polymers. We quantified these components with solid-state nuclear magnetic resonance (NMR) and determined that curli contributed 85% of the isolated uropathogenic E coli ECM dry mass. The remaining 15% was cellulosic, but, surprisingly, was not ordinary cellulose. We tracked the identity of the unanticipated peak in the 13C NMR spectrum of the cellulosic component and discovered that E coli secrete phosphoethanolamine (pEtN)-modified cellulose. Cellulose is the most abundant biopolymer on the planet, and this marked the first identification of a naturally, chemically modified cellulose. To investigate potential roles of pEtN cellulose, we customized a newly designed live-cell monolayer rheometer and demonstrated that pEtN cellulose facilitated E coli attachment to bladder epithelial cells and acted as a glue, keeping curli cell associated. The discovery of pEtN cellulose opens questions regarding its biological function(s) and provides opportunities in materials science to explore this newly discovered biopolymer.