Fibrillation of the Pseudomonas Extracellular Functional Amyloid FapC is Controlled by Oligomeric State, Hierarchical Architecture and the C-Terminus

Functional amyloids enable bacteria to harness the exceptional stability of the amyloid fold while preventing uncontrolled aggregation. However, the molecular mechanisms that encode this balance between robustness and regulation remain incompletely understood. Here, we define how the Pseudomonas functional amyloid FapC, a secreted extracellular functional amyloid in bacterial biofilm, achieves controlled fibril assembly through a modular architecture and a short C-terminal regulatory element. Using controlled purification, modular truncation constructs, and quantitative kinetic assays, we reveal that full-length FapC fibrillation, comprising three irregular layers, is highly sensitive to its initial oligomeric state. Non-monomeric species exert opposing effects: high-molecular-weight assemblies accelerate aggregation via heterogeneous nucleation, while low-molecular-weight species potently inhibit productive assembly. In contrast, isolated single Layer 3, a structurally defined {beta}-solenoid segment, fibrillates rapidly with minimal lag and is largely insensitive to oligomeric heterogeneity, identifying it as a dominant amyloidogenic unit. Yet, despite its minimal architecture, Layer 3 still benefits from the presence of the C-terminal region, which enhances fibrillation rate and yield. Removal of this C-terminal segment delays nucleation, slows elongation, and reduces final fibril formation in both full-length and Layer 3 contexts, highlighting its role as a non-structural regulator of productive folding. Comparative studies show that hierarchical layering imposes kinetic checkpoints and expands regulatory potential, tuning the timing and efficiency of assembly. Together, our results establish that FapC encodes a fibrillation mechanism built on hierarchical architectural design and localized sequence-specific regulation. This suggests that precise control, rather than mere aggregation propensity, maybe the defining hallmark of functional amyloids, distinguishing them from their pathological counterparts.