24Enterococcus faecalis is an opportunistic human pathogen and the cause of biofilm-25 associated infections of the heart, catheterized urinary tract, wounds, and the dysbiotic gut 26 where it can expand to high numbers upon microbiome perturbations. The E. faecalis sortase-27 assembled endocarditis and biofilm associated pilus (Ebp) is involved in adhesion and 28 biofilm formation in vitro and in vivo. Extracellular electron transfer (EET) also promotes E. 29 faecalis biofilm formation in iron-rich environments, however neither the mechanism 30 underlying EET nor its role in virulence was previously known. Here we show that iron 31 associated with Ebp serve as a terminal electron acceptor for EET, leading to extracellular 32 iron reduction and intracellular iron accumulation. We found that a MIDAS motif within the 33 EbpA tip adhesin is required for interaction with iron, EET, and FeoB-mediated iron uptake. 34 We demonstrate that MenB and Ndh3, essential components of the aerobic respiratory chain 35 and a specialized flavin-mediated electron transport chain, respectively, are required for iron-36 mediated EET. In addition, using a mouse gastrointestinal (GI) colonization model, we show 37 that EET is essential for colonization of the GI tract, and Ebp is essential for augmented E. 38 faecalis GI colonization when dietary iron is in excess. Taken together, our findings show 39 that pilus mediated capture of iron within biofilms enables EET-mediated iron acquisition in 40 E. faecalis, and that these processes plays an important role in E. faecalis expansion in the GI 41 tract. 42 43 Significance 44 Understanding enterococcal biofilm development is the first step towards improved 45 therapeutics for the often antimicrobial resistant infections caused by these bacteria. Here we 46 report a role for Enterococcus faecalis endocarditis and biofilm associated pili (Ebp) in 47 (EET) which in turn promotes iron acquisition. Furthermore, we characterize the mechanisms 49 underlying electron transfer in the E. faecalis biofilm. Our findings support a model in which 50 E. faecalis use EET to drive the reduction of pilus-associated ferric iron, leading to iron 51 acquisition in E. faecalis biofilm, and contributing to enterococcal virulence in the GI tract.52 53 Introduction 54 Enterococcus faecalis is an important human opportunistic pathogen that causes a variety of 55 diseases including endocarditis, urinary tract infections (UTI), bacteremia, wound infection, 56 and medical device-associated infections 1 . Many of these infections are polymicrobial and 57 biofilm-associated, rendering them more tolerant to antimicrobial and immune clearance, and 58 contributing to their persistent nature 1 . Therefore, a detailed understanding of enterococcal 59 biofilm development is a critical step towards advancing new therapeutics for treatment of 60 these often antimicrobial resistant infections 1,2 . 61 62One important factor contributing to E. faecalis biofilm formation and virulence is the 63 sortase-assembled endo...