Comparative transcriptomics and scarless genome editing uncover a periplasmic <i>c</i> -type cytochrome important for extracellular electron uptake in <i>Thioclava electrotropha</i> ElOx9 ^T

ABSTRACT Extracellular electron uptake (EEU) is a form of extracellular electron transfer that enables microorganisms to use solid-phase electron donors for respiration and biosynthesis, with important implications for biogeochemical cycling and biotechnology. Thioclava electrotropha is a metabolically versatile marine Alphaproteobacterium capable of autotrophic sulfur oxidation, heterotrophic growth, in addition to cathodic electron uptake despite lacking homologs to known extracellular electron transfer proteins from mineral-reducing or mineral-oxidizing microbes. Here, we explore the genetic basis for EEU in T. electrotropha through integrated transcriptomic, genetic, and electrochemical approaches. RNA sequencing revealed distinct transcriptional profiles for cathode oxidation versus sulfur oxidation, with 584 genes uniquely upregulated during EEU. Among the most highly upregulated genes were putative c -type cytochromes predicted to localize to the periplasm. A putative monoheme c -type cytochrome gene (AKL02_08760) designated pmcA showed &gt;100-fold upregulation during cathode oxidation. We developed a genetic system enabling scarless deletions in T. electrotropha and demonstrated that deletion of pmcA significantly decreased EEU capacity, while complementation restored and enhanced electron uptake beyond wild-type levels. Phylogenetic analysis revealed PmcA homologs in 2,587 bacterial species across multiple phyla, with distribution patterns indicative of horizontal gene transfer. The pmcA gene belongs to a putative four-gene cytochrome c -multicopper oxidase operon, of which all members were upregulated during EEU, with the operon displaying atypical codon usage and elevated GC content consistent with horizontal acquisition. These findings establish a novel and potentially horizontally transferred mechanism for bacterial EEU. Further elucidation of this pathway will provide new targets for engineering enhanced bioelectrochemical systems with EEU capabilities. IMPORTANCE Though extracellular electron transfer (EET) has been shown to drive critical biogeochemical processes in a range of environments, there are only a limited number of biomarkers that help us assign the genetic potential for EET to other microbes. This is especially true for organisms that use EET to acquire electrons from external electron donors. This work identifies a novel gene involved in the extracellular electron uptake mechanism employed by the marine sediment chemolithoautotroph and sulfur-oxidizing bacterium, Thioclava electrotropha . Homologs of this novel gene are found in over 2,500 species spanning multiple phyla. Though biochemical characterization is necessary to fully understand the role of this protein in EET, this work supports the potential for a widely distributed and previously uncharacterized mechanism of extracellular electron uptake. As EET has enabled the potential for multiple biotechnological applications, including microbial fuel cells and microbial electrosynthesis, further characterization of this system has implications for improved engineering of bioelectrochemical systems or use of novel electrotrophic microorganisms.