Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.
The low pressure at the surface of Mars (average: 6 mbar) is one potentially biocidal factor that any extant life on the planet would need to endure. Near subsurface life, while shielded from ultraviolet radiation, would also be exposed to this low pressure environment, as the atmospheric gas-phase pressure increases very gradually with depth. Few studies have focused on low pressure as inhibitory to the growth or survival of organisms. However, recent work has uncovered a potential constraint to bacterial growth below 25 mbar. The study reported here tested the survivability of four methanogen species (Methanothermobacter wolfeii, Methanosarcina barkeri, Methanobacterium formicicum, Methanococcus maripaludis) under low pressure conditions approaching average martian surface pressure (6 mbar - 143 mbar) in an aqueous environment. Each of the four species survived exposure of varying length (3 days - 21 days) at pressures down to 6 mbar. This research is an important stepping-stone to determining if methanogens can actively metabolize/grow under these low pressures. Additionally, the recently discovered recurring slope lineae suggest that liquid water columns may connect the surface to deeper levels in the subsurface. If that is the case, any organism being transported in the water column would encounter the changing pressures during the transport.
Electroactive bacteria are living catalysts, mediating energy-generating reactions at anodes or energy storage reactions at cathodes via extracellular electron transfer (EET). The Cathode-ANode (CANode) biofilm community was recently shown to facilitate both reactions, however, the identity of the primary constituents and underlying molecular mechanisms remain unknown. Here, we used metagenomics and metatranscriptomics to characterize the CANode biofilm. We show that a previously uncharacterized member of the family Desulfobulbaceae, Desulfobulbaceae -2, which had <1% relative abundance, had the highest relative gene expression and accounted for over 60% of all differentially expressed genes. At the anode potential, differential expression of genes for a conserved flavin oxidoreductase (Flx) and heterodisulfide reductase (Hdr) known to be involved in ethanol oxidation suggests a source of electrons for the energy-generating reaction. Genes for sulfate and carbon dioxide reduction pathways were expressed by Desulfobulbaceae -2 at both potentials and are the proposed energy storage reactions. Reduction reactions may be mediated by direct electron uptake from the electrode, or from hydrogen generated at the cathode potential. The Desulfobulbaceae -2 genome is predicted to encode at least 85 multi-heme (≥3 hemes) c -type cytochromes, some with as many as 26 heme-binding domains, that could facilitate reversible electron transfer with the electrode. Gene expression in other CANode biofilm species was also affected by the electrode potential, although to a lesser extent, and we cannot rule out their contribution to observed current. Results provide evidence of gene expression linked to energy storage and energy-generating reactions and will enable development of the CANode biofilm as a microbially-driven rechargeable battery. IMPORTANCE Microbial electrochemical technologies (METs) rely on electroactive bacteria to catalyze energy-generating and energy storage reactions at electrodes. Known electroactive bacteria are not equally capable of both reactions and METs are typically configured to be unidirectional. Here we report on genomic and transcriptomic characterization of a recently described microbial electrode community called the Cathode-ANode (CANode). The CANode community is able to generate or store electrical current based on the electrode potential. During periods where energy is not needed, electrons generated from a renewable source, such as solar power, could be converted into energy storage compounds to later be reversibly oxidized by the same microbial catalyst. Thus, the CANode system can be thought of as a living “rechargeable battery”. Results show that a single organism may be responsible for both reactions demonstrating a new paradigm for electroactive bacteria.
Life on Earth is found in a wide range of environments as long as the basic requirements of a liquid solvent, a nutrient source, and free energy are met. Previous hypotheses have speculated how extraterrestrial microbial life may function, among them that particle radiation might power living cells indirectly through radiolytic products. On Earth, so-called electrophilic organisms can harness electron flow from an extracellular cathode to build biomolecules. Here, we describe two hypothetical mechanisms, termed "direct electrophy" and "indirect electrophy" or "fluorosynthesis," by which organisms could harness extracellular free electrons to synthesize organic matter, thus expanding the ensemble of potential habitats in which extraterrestrial organisms might be found in the Solar System and beyond. The first mechanism involves the direct flow of secondary electrons from particle radiation to a microbial cell to power the organism. The second involves the indirect utilization of impinging secondary electrons and a fluorescing molecule, either biotic or abiotic in origin, to drive photosynthesis. Both mechanisms involve the attenuation of an incoming particle's energy to create low-energy secondary electrons. The validity of the hypotheses is assessed through simple calculations showing the biomass density attainable from the energy supplied. Also discussed are potential survival strategies that could be used by organisms living in possible habitats with a plentiful supply of secondary electrons, such as near the surface of an icy moon. While we acknowledge that the only definitive test for the hypothesis is to collect specimens, we also describe experiments or terrestrial observations that could support or nullify the hypotheses. Key Words: Radiation-Electrophiles-Subsurface life. Astrobiology 18, 73-85.
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