Saccharibacteria deploy two distinct type IV pili, driving episymbiosis, host competition, and twitching motility.

All cultivated Patescibacteria, also known as the candidate phyla radiation, are obligate episymbionts residing on other microbes. Despite being ubiquitous in many diverse environments, including mammalian microbiomes, molecular mechanisms of host identification and binding amongst ultrasmall bacterial episymbionts remain largely unknown. Type 4 pili are well conserved in this group and could potentially facilitate these symbiotic interactions. To test this hypothesis, we genetically targeted pili genes in Saccharibacteria Nanosynbacter lyticus strain TM7x to assess their essentiality and roles in symbiosis. Our results revealed that N. lyticus assembles two distinct type 4 pili: a nonessential thin pilus that has the smallest diameter of any type 4 pili and contributes to host-binding and episymbiont growth; and an essential thick pilus involved in twitching motility. To understand the role of these pili in vivo we developed Saccharibacteria competition assays and species-specific Fluorescence in situ hybridization probes. Competition between different Saccharibacteria within mock communities demonstrated consistent competitive outcomes that were not driven by priority effects but were dependent on the thin pilus. Collectively, our findings demonstrate that Saccharibacteria encode unique extracellular pili that enable their underexplored episymbiotic lifestyle and competitive fitness within a community.