Extracellular Respiration is a Latent Energy Metabolism in Escherichia coli

Diverse microbial species utilize redox shuttles to exchange electrons with their environment through mediated extracellular electron transfer (EET). This process maintains redox homeostasis and supports anaerobic survival across diverse microbial communities. Although mediated EET has been extensively leveraged for bioelectrocatalysis and bioelectronics for decades, fundamental questions remain about how these redox shuttles are reduced within cells and their bioenergetic implications. This knowledge gap limits our understanding of the physiological roles of mediated EET in various microbes and hampers the development of efficient microbial electrochemical technologies. To address this, we developed a methodology integrating genome editing, electrochemistry, and systems biology to investigate the mechanism and bioenergetic implications of mediated EET in bacteria. Using this approach, we uncovered a mediated EET mechanism in Escherichia coli. In the absence of alternative electron sinks, the redox cycling of 2-hydroxy-1,4-naphthoquinone (HNQ) via a cytoplasmic nitroreductase enabled E. coli to respire and grow on an extracellular electrode. Genome-scale metabolic modeling suggested that HNQ- mediated EET offers a more energetically favorable route for supporting anaerobic growth than canonical fermentation. Transcriptome analysis revealed redox perturbations in response to HNQ and identified rapid metabolic adaptations that support growth. This work demonstrates that E. coli can grow independently of classical electron transport chains and fermentative pathways, unveiling a new type of anaerobic energy metabolism.