Prophage-dependent membrane vesicles in Geobacter soli: Implications for biofilm formation and extracellular electron...

Geobacter species are among the most efficient biocatalysts in bioelectrochemical systems (BES), with their performance dependent on effective extracellular electron transfer (EET) between electroactive bacteria and electrodes. Although membrane vesicles (MVs) are known to participate in EET, the formation of prophage-dependent MVs (PMVs) in Geobacter species and their specific contribution to EET have remained unclear. This study demonstrated that Geobacter soli produced PMVs via prophage-mediated explosive cell lysis, as evidenced by their characteristic morphological and compositional features. Compared with MVs from prophage-deficient Δpro2 mutant, wild-type MVs (WT-MVs) exhibited a higher proportion of bilayer-membrane vesicles (10.1% vs. 4.2%), 2.6-fold increase in DNA content, and significant enrichment in cytoplasmic and inner membrane proteins. Compared with the wild-type-inoculated BES, the Δpro2-inoculated BES showed a 24.4-29.5% reduction in current generation over three operational cycles, and a decrease in biofilm thickness from 40 μm to 35 μm. Notably, supplementation with WT-MVs restored the electrochemical performance of Δpro2-biofilm, increasing current output by 50.9-86.3%. Comparative proteomic and biochemical analyses revealed that WT-MVs were enriched in c-type cytochromes as well as biofilm-promoting proteins, including adhesins, polysaccharide-associated proteins and stress response factors, relative to Δpro2-MVs, indicating that PMVs actively facilitated both biofilm formation and EET. This work provided the first direct evidence of PMV production in Geobacter and established their association with enhanced EET and biofilm formation. These findings advance our understanding of vesicle-mediated electron transfer mechanism in electroactive bacteria and highlight prophage elements as promising targets for engineering high-performance BES for energy recovery and environmental applications.