Vanessa Saldivar

Microbial respiration via extracellular electron transfer (EET) drives various globally-important environmental processes and applications in bioenergy, bioremediation, and bioelectronics. However, despite ~40 years of research, the EET components and pathways have remained unclear due to their high redundancy. e.g., depending on nutrient availability, Geobacter sulfurreducens selects distinct sets of membrane-embedded porin-cytochrome complexes (PCCs) that are long-believed to transfer electrons to surface-displayed polymerized cytochrome “nanowires”. In contrast, here we show that PCC Om(abc)B and OmcS nanowires form sequential, independent EET pathways to environmentally-abundant Fe(III). Genetic studies show that PCCs enable switching from intracellular to extracellular ET and provide energy to synthesize nanowires that then perform long-range (>1 μm) EET to access ~50-times the cell’s biovolume of Fe(III) essential for replication. Surprisingly, wired cells completely shut-off PCC production without altering their expression. Our biophysical, biochemical, and coevolution analyses, using native-lipid nanodiscs, spectroelectrochemistry, UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies, reveal that Om(ab)B and OmcB protein and electronic structures (Em, -182, -167 mV, respectively) are evolutionarily-optimized for efficient EET, enabling OmcB to transfer electrons ~5 times faster than nanowires. This microbial strategy to switch from wireless to wired EET network could enable engineering and controlling EET in diverse environmentally-important species and environments.

Read counts mapped to mitochondrial genome

RIP-seq Histogram of H1944, H23 and PC9 with and without IMT-1 Treatment

Representative peak analysis for heteroplasmy

IF of cell lines with IMT-1 Treatment

Enrichment of mtdsRNAs is captured through dsRNA-seq in selected NSCLC cell lines. A, Workflow of RIP-seq. B, Quantification of unique reads detected by RIP-seq. Reads from the heavy (red) and light (blue) strands were mapped to the mitochondrial genome ChrMT. See Supplementary Fig. S1 for an enlarged view of the sequencing data. C and D, Statistical analysis of successfully aligned reads per genomic feature (gene, CDS, rRNA, exon, and D-loop, with either tRNA included or excluded). Bar represents average. Error bars represent ± SEM. P values are represented as is. One-way ANOVA was completed. Each dot represents an independent experiment (n = 3). E, Mitochondrial genome mapped with primers used for long-read sequencing. Primers can be found in Supplementary Table S1. Green: ND5 Fwd → RNR2 Rev. Blue: RNR2 Fwd → Cox3 Rev. Orange: Cox3 Fwd → ND5 Rev. F, Gel showing the bands generated by PCR across three different cell lines used for ADAR sequencing. Representative of three independent experiments (n = 3).

IF of cell lines with IMT-1 Treatment

Enrichment of mtdsRNAs is captured through dsRNA-seq in selected NSCLC cell lines. A, Workflow of RIP-seq. B, Quantification of unique reads detected by RIP-seq. Reads from the heavy (red) and light (blue) strands were mapped to the mitochondrial genome ChrMT. See Supplementary Fig. S1 for an enlarged view of the sequencing data. C and D, Statistical analysis of successfully aligned reads per genomic feature (gene, CDS, rRNA, exon, and D-loop, with either tRNA included or excluded). Bar represents average. Error bars represent ± SEM. P values are represented as is. One-way ANOVA was completed. Each dot represents an independent experiment (n = 3). E, Mitochondrial genome mapped with primers used for long-read sequencing. Primers can be found in Supplementary Table S1. Green: ND5 Fwd → RNR2 Rev. Blue: RNR2 Fwd → Cox3 Rev. Orange: Cox3 Fwd → ND5 Rev. F, Gel showing the bands generated by PCR across three different cell lines used for ADAR sequencing. Representative of three independent experiments (n = 3).