{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:32:26Z","timestamp":1787790746117,"version":"build-2784847793"},"posted":{"date-parts":[[2026,2,20]]},"group-title":"Microbiology","reference-count":72,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T00:00:00Z","timestamp":1771545600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,19]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Bacterial biofilms represent a survival strategy that enables microbial communities to withstand environmental stress.\n                  <jats:italic>Stenotrophomonas maltophilia<\/jats:italic>\n                  is an emerging, antibiotic-resistant, Gram-negative, opportunistic pathogen that frequently colonizes the lungs of individuals with cystic fibrosis. Its chaperone-usher pathway (CUP) pilus, SMF-1, is present in nearly all clinical isolates and is essential for biofilm development; however, its molecular architecture has remained unknown. Here, we present a 4.0 \u00c5 cryo-EM structure revealing that SMF-1 is an archaic, rather than a classic, CUP pilus. SMF-1 forms thin, zigzag filaments that assemble into defined antiparallel \u201cpili-couples,\u201d which further aggregate into thick bundles. These bundles act as robust intercellular tethers, facilitating the rapid cell-to-cell aggregation required for biofilm initiation. Despite high sequence and structural similarity to classic CUP systems, SMF-1 lacks the interfaces required to form a rod-like architecture, suggesting it may represent an evolutionary intermediate between the CUP classes. Finally, we demonstrate that SMF-1 producing bacteria initiate biofilm formation within 24 hours and that flagella can further accelerate this process. Together, these findings uncover a conserved bundling mechanism that promotes bacterial colonization and may contribute to pathogenicity.\n                <\/jats:p>","DOI":"10.64898\/2026.02.19.706768","type":"posted-content","created":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T16:20:17Z","timestamp":1771604417000},"source":"Crossref","is-referenced-by-count":1,"title":["The archaic CUP pilus SMF-1 utilizes a specific antiparallel bundling mechanism to initiate biofilm formation"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7221-6578","authenticated-orcid":false,"given":"Jessie Lynda","family":"Fields","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9845-3929","authenticated-orcid":false,"given":"Caitlyn C.","family":"Sebastian","sequence":"additional","affiliation":[{"name":"Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Oral Rehabilitation & Biosciences Oregon Health & Science University Portland, OR 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayisha","family":"Zia","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6300-6044","authenticated-orcid":false,"given":"Abigail N.","family":"Robertson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Oral Rehabilitation & Biosciences Oregon Health & Science University Portland, OR 97239, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megan R.","family":"Kiedrowski","sequence":"additional","affiliation":[{"name":"Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35233, USA"},{"name":"Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35233, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1008-663X","authenticated-orcid":false,"given":"Fengbin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 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