Light-directed biofilm formation reveals the functional contributions of periplasmic cytochromes to the electrochemical activity of Shewanella oneidensis

ABSTRACT Shewanella oneidensis MR-1 is a model electroactive bacterium whose extracellular electron transfer (EET) pathway includes a sequential network of c -type cytochromes that span the inner membrane, periplasm, and outer membrane. While electrochemical studies have revealed the critical role of outer-membrane cytochromes in mediating both outward EET from cells to external surfaces and lateral biofilm conduction across cells, the specific functional role of periplasmic cytochromes in these processes remains less understood. Dissecting the contributions of periplasmic components has been challenged by the complexity of the periplasmic cytochrome network and the variability of native biofilms, which confound electrochemical comparisons of cytochrome mutants. Here, we overcome these limitations with a synthetic biology approach combining targeted deletion of genes encoding key periplasmic cytochromes with light-induced biofilm patterning to create uniform, geometrically defined biofilms on electrodes for robust electrochemical comparisons. Voltammetric measurements of patterned S. oneidensis mutant biofilms confirmed the essential role of periplasmic cytochromes in facilitating outward EET, a contribution that becomes apparent when flavins are present, accelerating interfacial electron transfer between outer-membrane cytochromes and the electrode. In contrast to this role in routing outward EET across the periplasm, electrochemical gating measurements of lateral biofilm conductivity revealed that the periplasmic cytochromes do not contribute to long-distance electron transport along cellular layers bridging electrodes. These findings provide new insights into the role of periplasmic cytochromes in S. oneidensis , highlighting a robust functional redundancy within the periplasmic network, and distinguish their contributions to routing outward EET across the cell envelope versus biofilm conductivity. IMPORTANCE Microbes capable of extracellular electron transfer (EET) are central to global biogeochemical cycles and emerging bioelectrochemical technologies. In the important model EET bacterium Shewanella oneidensis MR-1, the outer-membrane components that interface with external surfaces are well characterized. However, the functional role of the periplasmic components linking the inner and outer membranes has remained obscured by the complex network of multiple cytochromes and biofilm heterogeneity, limiting precise comparisons across mutants. By combining light-induced biofilm patterning with electrochemical analysis, we successfully revealed the specific contributions of periplasmic cytochromes: these components are essential for facilitating outward EET across the cell envelope but do not impact lateral long-distance electron transport across the biofilm. The results refine our understanding of extracellular respiration and provide design rules for engineering living electronic materials.