We have developed a theory that lets us calculate the efficiency of microbes that absorb electricity and store CO 2 as biofuels with greater efficiency than photosynthesis. We outline 10 development scenarios including re-engineering direct electron uptake microbes with high-efficiency CO 2 fixation; scaling up H 2oxidizing microbe systems to store megawatts of electricity; engineering direct electron uptake microbes to make highly conductive artificial biofilms to enable high power density electricity storage; and engineering microbes that assimilate electrochemically reduced CO 2 with electron uptake.
A protein molecule is an intricate system whose function is highly sensitive to small external perturbations. However, no examples that correlate protein function with progressive subangstrom structural perturbations have thus far been presented. To elucidate this relationship, we have investigated a fluorescent protein, citrine, as a model system under high-pressure perturbation. The protein has been compressed to produce deformations of its chromophore by applying a high-pressure cryocooling technique. A closely spaced series of x-ray crystallographic structures reveals that the chromophore undergoes a progressive deformation of up to 0.8 Å at an applied pressure of 500 MPa. It is experimentally demonstrated that the structural motion is directly correlated with the progressive fluorescence shift of citrine from yellow to green under these conditions. This protein is therefore highly sensitive to subangstrom deformations and its function must be understood at the subangstrom level. These results have significant implications for protein function prediction and biomolecule design and engineering, because they suggest methods to tune protein function by modification of the protein scaffold.fluorescence ͉ high-pressure x-ray crystallography ͉ protein engineering ͉ protein structure-function ͉ yellow fluorescent protein
Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.
Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO2 and renewable electricity1–6. Theoretically EET could do this with an efficiency comparable to H2-oxidation7,8 but without the need for a volatile intermediate and the problems it causes for scale up9. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-110–14, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation15.
Polymorphism of water has been extensively studied, but controversy still exists over the phase transition between high-density amorphous (HDA) and low-density amorphous (LDA) ice. We report the phase behavior of HDA ice inside high-pressure cryocooled protein crystals. Using X-ray diffraction, we demonstrate that the intermediate states in the temperature range from 80 to 170 K can be reconstructed as a linear combination of HDA and LDA ice, suggesting a first-order transition. We found evidence for a liquid state of water during the ice transition based on the protein crystallographic data. These observations open the possibility that the HDA ice induced by high-pressure cryocooling is a genuine glassy form of high-density liquid.liquid-liquid hypothesis ͉ supercooled water ͉ water phases ͉ high-density liquid S upercooled water shows anomalous thermodynamic behavior (1-3). Theories that account for these anomalous properties include the stability limit (4), the singularity-free (5, 6), and the liquid-liquid (LL) critical point (7) hypotheses. The latter 2 hypotheses propose the existence of 2 distinct forms of supercooled water: high-density liquid (HDL) and low-density liquid (LDL) water (8). In the singularity-free hypothesis, HDL transforms continuously to LDL. In the LL critical point theory, HDL undergoes a first-order phase transition to LDL (8). However, experimental study of the HDL-LDL phase transition is challenging as supercooled water spontaneously converts to crystalline forms below the homogeneous nucleation temperature (Ϸ235 K at 0.1 MPa). The transition between 2 glassy forms of water, high-density amorphous (HDA) and low-density amorphous (LDA) ice, has been extensively studied (9-18), as an analogue of the HDL-LDL transition. Controversy still exists as to whether the HDA-LDA ice transition is truly a first-order phase transition (10-13) or if it occurs because of a relaxation process of an unstable amorphous structure (15-18). More importantly, the connection between the HDA-LDA ice transition and the HDL-LDL phase transition, implied by thermodynamic and structural studies on water (19-22), remains challenging to prove experimentally (8).We used X-ray diffraction to study the transition of HDA to LDA ice in protein crystals. HDA ice was induced inside protein crystals by a high-pressure cryocooling method (23) originally developed for macromolecular crystallography (23-26). The Bragg diffraction from protein crystals mainly provides information on protein structure. The simultaneously recorded water diffuse diffraction (WDD) profile reports on the phase of the water in the protein crystal, which accounts for typically 40-60% of its volume. Fig. 1 shows diffraction images and the WDD profiles from a high-pressure cryocooled crystal of the globular protein thaumatin. As the crystal temperature is increased from 80 to 170 K, the primary WDD peak, corresponding to the mean distance between neighboring water molecule oxygen atoms, shifts to lower momentum transfer (Q) region [Q ϭ 4 sin( )/ , wh...
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