Ultrastructural and atomic characterization of biofilm-associated extracellular filaments in Shewanella oneidensis MR-1.

Biofilms formed by Shewanella oneidensis MR-1 are crucial for metal reduction, underpinning bioremediation and bioenergy applications, yet detailed structural insights into biofilm components remain limited. Here we show that MR-1 biofilms contain abundant filamentous networks, membrane vesicles, and distinct square-shaped aggregates, as revealed by cryo-electron tomography and cryo-electron microscopy. We further determine near-atomic resolution structures of three filament types: bacterial flagella and two distinct pili, PilA and MshA. We present high-resolution structures of an MshA pilus and a PilA pilus from Shewanella. Structural analyses show that MshA pili exhibit a balanced surface charge distribution and extensive solvent-accessible surface area, facilitating essential interactions within the biofilm matrix. Additionally, oxygen limitation markedly increases the abundance of extracellular filaments and protein aggregates, indicating adaptive responses to environmental stress. Our findings elucidate the fundamental architecture and roles of biofilm extracellular components and provide a structural foundation for engineering enhanced Shewanella strains.