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bioRxiv · 10.64898/2026.03.03.709456

A Novel Pilus System in Candidate Phyla Radiation Bacteria

Troman, L. A.·Kim, J.·Rose, J. J. A.·Johnson, M.·Banfield, J. F.·Petrovski, S.·Ghosal, D.
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Abstract

The Candidate Phyla Radiation (CPR) represents a bacterial superphylum estimated to include between 15-50% of all bacterial species, yet CPR bacteria remain challenging to culture and have been primarily identified through metagenomic approaches. Candidatus Mycosynbacter amalyticus is a parasitic CPR bacterium that specifically targets Gordonia amarae, an actinobacterium with a hydrophobic, mycolic acid-rich cell envelope. Previous cryo-electron tomography indicated that Ca. M. amalyticus assembles thin extracellular filaments that are important for host interaction, yet their molecular identity remains unknown. Here, we applied single-particle cryo-electron microscopy to determine high-resolution structures of these filaments (2.8 and 3.6 [A]), enabling the unambiguous identification of two previously uncharacterized pilins from the experimental density maps. These pilins, designated PamA and PamB, assemble into unique helical filaments distinct from all previously characterized filaments in both domain architecture and assembly mechanism. Despite low sequence identity, both PamA and PamB share conserved structural principles, including Ig-like folds and donor-strand exchange-mediated assembly. Phylogenetic analysis indicates that Pam pilins are exclusive to CPR bacteria, with homologues distributed predominantly across the classes Saccharimonadia and Microgenomatia. Analysis of the conserved pam operon identifies putative chaperones (PamC and PamD) and assembly factors structurally homologous to chaperone-usher pili components, suggesting an analogous but distinct assembly pathway. These findings expand the known diversity of bacterial pilus systems and demonstrate the power of structural approaches for characterizing uncharacterized proteins encoded within CPR genomes.

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Troman, L. A., Kim, J., Rose, J. J. A., Johnson, M., Banfield, J. F., Petrovski, S., Ghosal, D.. 2026-03-04. A Novel Pilus System in Candidate Phyla Radiation Bacteria. https://doi.org/10.64898/2026.03.03.709456

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