Introduction of the electron transfer complex MtrCAB from Shewanella oneidensis MR-1 into a heterologous host provides a modular and molecularly defined route for electrons to be transferred to an extracellular inorganic solid. However, an Escherichia coli strain expressing this pathway displayed limited control of MtrCAB expression and impaired cell growth. To overcome these limitations and to improve heterologous extracellular electron transfer, we used an E. coli host with a more tunable induction system and a panel of constitutive promoters to generate a library of strains that separately transcribe the mtr and cytochrome c maturation (ccm) operons over 3 orders of magnitude. From this library, we identified strains that show 2.2 times higher levels of MtrC and MtrA and that have improved cell growth. We find that a ~300-fold decrease in the efficiency of MtrC and MtrA synthesis with increasing mtr promoter activity critically limits the maximum expression level of MtrC and MtrA. We also tested the extracellular electron transfer capabilities of a subset of the strains using a three-electrode microbial electrochemical system. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu, rather than the strain with more MtrC and MtrA. This strain also showed ~30-fold greater maximal current per cfu than its ccm-only control strain. Thus, the conditions for optimal MtrCAB expression and anode reduction are distinct, and minimal perturbations to cell morphology are correlated with improved extracellular electron transfer in E. coli.

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250 words) 1 The terrestrial deep subsurface is host to significant and diverse microbial populations. However, these 2 microbial populations remain poorly characterized, partially due to the inherent difficulty of sampling, in 3 situ studies, and isolating of the in situ microbes. Motivated by the ability of microbes to gain energy from 4 redox reactions at mineral interfaces, we here present in situ electrochemical colonization (ISEC) as a 5 method to directly study microbial electron transfer activity and to enable the capture and isolation of 6 electrochemically active microbes. We installed a potentiostatically controlled ISEC reactor containing four 7 working electrodes 1500 m below the surface at the Sanford Underground Research Facility. The working 8 electrodes were poised at different redox potentials, spanning anodic to cathodic, to mimic energy-yielding 9 mineral reducing and oxidizing reactions predicted to occur at this site. We present a 16S rRNA analysis 10 of the in situ electrode-associated microbial communities, revealing the dominance of novel bacterial 11 lineages under cathodic conditions. We also demonstrate that the in situ electrodes can be further used for 12 downstream electrochemical laboratory enrichment and isolation of novel strains. Using this workflow, we 13 isolated Bacillus, Anaerospora, Comamonas, Cupriavidus, and Azonexus strains from the electrode-14 attached biomass. Finally, the extracellular electron transfer activity of the electrode-oxidizing Comamonas 15 strain (isolated at -0.19 V vs. SHE and designated WE1-1D1) and the electrode-reducing Bacillus strain 16 (isolated at +0.53 V vs. SHE and designated WE4-1A1-BC) were confirmed in electrochemical reactors. 17 Our study highlights the utility of in situ electrodes and electrochemical enrichment workflows to shed light 18 on microbial activity in the deep terrestrial subsurface. 19