The CUP pilus SMF-1 utilizes a specific antiparallel bundling mechanism to initiate biofilm formation.

Bacterial biofilms enable microbial communities to withstand environmental stress and antibiotic challenge. Stenotrophomonas maltophilia is an opportunistic pathogen that frequently colonizes cystic fibrosis airways. Its chaperone-usher pathway (CUP) pilus, SMF-1, is essential for biofilm development, but its molecular architecture has remained unknown. Here, we present a 4.0 Å cryogenic electron microscopy (cryo-EM) structure demonstrating that the classic γ4 CUP pilus SMF-1 assembles into zigzag filaments that pair into antiparallel "pili couples" and higher order bundles. These bundles serve as intercellular tethers that drive rapid cell aggregation during biofilm initiation. Despite belonging to the classic CUP family, SMF-1 lacks the subunit interfaces required for canonical rod-like architectures, providing structural evidence of convergent evolution toward archaic-like biofilm bundling mechanisms. Furthermore, appendage-driven motility acts synergistically with SMF-1 pili to accelerate intercellular encounters and aggregate assembly. Together, these findings reveal a structural mechanism of pilus-mediated crosslinking promoting bacterial colonization.