Woodard, T. | bioRxiv Science

bioRxiv2022

Microbial nanowires with genetically modified peptide ligands to sustainably fabricate electronic sensing devices

Nanowires have substantial potential as the sensor component in electronic sensing devices. However, surface functionalization of traditional nanowire and nanotube materials with short peptides that increase sensor selectivity and sensitivity requires complex chemistries with toxic reagents. In contrast, microorganisms can assemble pilin monomers into protein nanowires with intrinsic conductivity from renewable feedstocks, yielding an electronic material that is robust and stable in applications, but also biodegradable. Here we report that the sensitivity and selectivity of protein nanowire-based sensors can be modified with a simple plug and play genetic approach in which a short peptide sequence, designed to bind the analyte of interest, is incorporated into the pilin protein that is microbially assembled into nanowires. We employed a scalable Escherichia coli chassis to fabricate protein nanowires that displayed either a peptide previously demonstrated to effectively bind ammonia, or a peptide known to bind acetic acid. Sensors comprised of thin films of the nanowires amended with the ammonia-specific peptide had a ca. 100-fold greater response to ammonia than sensors made with unmodified protein nanowires. Protein nanowires with the peptide that binds acetic acid yielded a 4-fold higher response than nanowires without the peptide. The results demonstrate that protein nanowires with enhanced sensor response for analytes of interest can be fabricated with a flexible genetic strategy that sustainably eliminates the energy, environmental, and health concerns associated with other common nanomaterials.

bioengineering↗

bioRxiv2021

Mechanisms for Electron Uptake by Methanosarcina acetivorans During Direct Interspecies Electron Transfer

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in co-culture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in co-culture versus pure culture growth on acetate revealed that transcripts for the outer-surface, multi-heme, c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based co-culture as well as the archaella-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based co-cultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. ImportanceThe conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connects on the outer surface of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina that play an important role in methane production in soils and sediments participate in DIET.

microbiology↗