Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community.

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding, Escherichia coli assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1-R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as "gatekeepers" to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.A myloids are ordered proteinaceous fibers commonly associated with mammalian neurodegenerative diseases and prion-based encephalopathies (1). Amyloid fibers have distinct biochemical and biophysical properties, such as remarkable resistance to chemical and thermal denaturation, and specific tinctorial properties when bound to Congo red and thioflavin T (ThT) (1). The molecular basis of neurodegenerative disease development induced by amyloid propagation remains elusive, partially because of the seemingly erratic and uncontrolled nature of amyloidogenesis. An emerging focus of amyloid biosynthesis has shown that amyloids can also be an integral part of physiology found in different organisms including bacteria, fungi, and mammals (2, 3). How nature coordinates functional amyloid propagation and reduces the associated cytotoxicity is poorly understood.Curli, a bacterially produced functional amyloid, is an important component of the extracellular matrix and is involved in bacterial community behaviors (4). Because of the amyloid properties of curli fibers (5, 6), the colonies of curli-producing Escherichia coli stain red when grown on Congo red indicator plates, which provides a convenient assay to monitor curli assembly in vivo (7). In E. coli, at least six proteins are dedicated to directing efficient curli formation. Curli fibers are composed of a major subunit CsgA and a minor subunit CsgB. CsgA remains unpolymerized until it encounters the surface-tethered nucleator CsgB, which initiates CsgA polymerization (8). CsgD is a transcriptional activator for the csgBA operon (4). CsgG, CsgE, and CsgF are nonfiber structural accessory proteins involved in secretion and stabilization of the fiber subunits and modulation of fiber assembly (6). CsgG is prop...

SummaryBacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.

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Background: Seeding of eukaryotic amyloids is highly specific. Results: Curli subunits from different bacteria can cross-seed and such interspecies interactions restore surface attachment and biofilm formation. Conclusion: Curli cross-seeding is relaxed, which promotes interspecies biofilms. Significance: This is the first study on cross-seeding of bacterial amyloids and will help define the roles of bacterial amyloids in multispecies biofilms.