Long-duration environmental biosensing by recording analyte detection in DNA using recombinase memory.

Microbial biosensors that convert environmental information into real-time visual outputs are limited in their sensing abilities in complex environments, such as soil and wastewater, due to optical inaccessibility. Biosensors that could record transient exposure to analytes within a large time window for later retrieval represent a promising approach to solve the accessibility problem. Here, we test the performance of recombinase-memory biosensors that sense a sugar (arabinose) and a microbial communication molecule (3-oxo-C12-L-homoserine lactone) over 8 days (~70 generations) following analyte exposure. These biosensors sense the analyte and trigger the expression of a recombinase enzyme which flips a segment of DNA, creating a genetic memory, and initiates fluorescent protein expression. The initial designs failed over time due to unintended DNA flipping in the absence of the analyte and loss of the flipped state after exposure to the analyte. Biosensor performance was improved by decreasing recombinase expression, removing the fluorescent protein output, and using quantitative PCR to read out stored information. Application of memory biosensors in wastewater isolates achieved memory of analyte exposure in an uncharacterized Pseudomonas isolate. By returning these engineered isolates to their native environments, recombinase-memory systems are expected to enable longer duration and in situ investigation of microbial signaling, cross-feeding, community shifts, and gene transfer beyond the reach of traditional environmental biosensors.IMPORTANCEMicrobes mediate ecological processes over timescales that can far exceed the half-lives of transient metabolites and signals that drive their collective behaviors. We investigated strategies for engineering microbes to stably record their transient exposure to a chemical over many generations through DNA rearrangements. We identify genetic architectures that improve memory biosensor performance and characterize these in wastewater isolates. Memory biosensors are expected to be useful for monitoring cell-cell signals in biofilms, detecting transient exposure to chemical pollutants, and observing microbial cross-feeding through short-lived metabolites within cryptic methane, nitrogen, and sulfur cycling processes. They will also enable in situ studies of microbial responses to ephemeral environmental changes, or other ecological processes that are currently challenging to monitor non-destructively using real-time biosensors and analytical instruments.

The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.

Microbial sensor variation across biogeochemical conditions in the terrestrial deep subsurface.

Microbes can be found in abundance many kilometers underground. While microbial metabolic capabilities have been examined across different geochemical settings, it remains unclear how changes in subsurface niches affect microbial needs to sense and respond to their environment. To address this question, we examined how microbial extracellular sensor systems vary with environmental conditions across metagenomes at different Deep Mine Microbial Observatory (DeMMO) subsurface sites. Because two-component systems (TCSs) directly sense extracellular conditions and convert this information into intracellular biochemical responses, we expected that this sensor family would vary across isolated oligotrophic subterranean environments that differ in abiotic and biotic conditions. TCSs were found at all six subsurface sites, the service water control, and the surface site, with an average of 0.88 sensor histidine kinases (HKs) per 100 genes across all sites. Abundance was greater in subsurface fracture fluids compared with surface-derived fluids, and candidate phyla radiation (CPR) bacteria presented the lowest HK frequencies. Measures of microbial diversity, such as the Shannon diversity index, revealed that HK abundance is inversely correlated with microbial diversity (r2 = 0.81). Among the geochemical parameters measured, HK frequency correlated most strongly with variance in dissolved organic carbon (r2 = 0.82). Taken together, these results implicate the abiotic and biotic properties of an ecological niche as drivers of sensor needs, and they suggest that microbes in environments with large fluctuations in organic nutrients (e.g., lacustrine, terrestrial, and coastal ecosystems) may require greater TCS diversity than ecosystems with low nutrients (e.g., open ocean).IMPORTANCEThe ability to detect extracellular environmental conditions is a fundamental property of all life forms. Because microbial two-component sensor systems convert information about extracellular conditions into biochemical information that controls their behaviors, we evaluated how two-component sensor systems evolved within the deep Earth across multiple sites where abiotic and biotic properties vary. We show that these sensor systems remain abundant in microbial consortia at all subterranean sampling sites and observe correlations between sensor system abundances and abiotic (dissolved organic carbon variation) and biotic (consortia diversity) properties. These results suggest that multiple environmental properties may drive sensor protein evolution and highlight the need for further studies of metagenomic and geochemical data in parallel to understand the drivers of microbial sensor evolution.

The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Using Methyl Bromide for Interspecies Cell-Cell Signaling and As a Reporter in a Model Soil Consortium.

Soil microbial communities with reduced complexity are emerging as model systems for studying consortia-scale phenotypes. To establish synthetic biology tools for studying these communities in hard-to-image environmental materials, we evaluated whether a single member of a model soil consortium (MSC) can be programmed to report on gene expression without requiring matrix disruption. For these studies, we targeted a five-membered MSC that includes Dyadobacter fermentans, Ensifer adhaerens, Rhodococcus sp003130705, Streptomyces sp001905665, and Variovorax beijingensis. By coupling the expression of a methyl halide transferase to a constitutive promoter, we show that V. beijingensis can be programmed to synthesize methyl halides that accumulate in the soil headspace at levels that are ≥24-fold higher than all other MSC members across a range of environmentally relevant hydration conditions. We find that methyl halide production can report on an MSC promoter that is activated by changes in water potential, and we demonstrate that a synthetic gas signal can be read out directly using gas chromatography and indirectly using a soil-derived Methylorubrum that is programmed to produce a visual output in response to methyl halides. These tools will be useful for future studies that investigate how MSC responds to dynamic hydration conditions, such as drought and flood events induced by climate change, which can alter soil water potential and induce the release of stored carbon.

A cellular selection identifies elongated flavodoxins that support electron transfer to sulfite reductase.

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a ferredoxin, we evaluated the ability of Flds to transfer electrons from a ferredoxin-NADP reductase (FNR) to a ferredoxin-dependent SIR using growth complementation of an Escherichia coli strain with a sulfur metabolism defect. We show that Flds from cyanobacteria complement this growth defect when coexpressed with an FNR and an SIR that evolved to couple with a plant ferredoxin. When we evaluated the effect of peptide insertion on Fld-mediated electron transfer, we observed a sensitivity to insertions within regions predicted to be proximal to the cofactor and partner binding sites, while a high insertion tolerance was detected within loops distal from the cofactor and within regions of helices and sheets that are proximal to those loops. Bioinformatic analysis showed that natural Fld sequence variability predicts a large fraction of the motifs that tolerate insertion of the octapeptide SGRPGSLS. These results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of insertion tolerance is influenced by interactions with oxidoreductase partners.

Real-time bioelectronic sensing of environmental contaminants.

Real-time chemical sensing is crucial for applications in environmental and health monitoring1. Biosensors can detect a variety of molecules through genetic circuits that use these chemicals to trigger the synthesis of a coloured protein, thereby producing an optical signal2-4. However, the process of protein expression limits the speed of this sensing to approximately half an hour, and optical signals are often difficult to detect in situ5-8. Here we combine synthetic biology and materials engineering to develop biosensors that produce electrical readouts and have detection times of minutes. We programmed Escherichia coli to produce an electrical current in response to specific chemicals using a modular, eight-component, synthetic electron transport chain. As designed, this strain produced current following exposure to thiosulfate, an anion that causes microbial blooms, within 2 min. This amperometric sensor was then modified to detect an endocrine disruptor. The incorporation of a protein switch into the synthetic pathway and encapsulation of the bacteria with conductive nanomaterials enabled the detection of the endocrine disruptor in urban waterway samples within 3 min. Our results provide design rules to sense various chemicals with mass-transport-limited detection times and a new platform for miniature, low-power bioelectronic sensors that safeguard ecological and human health.

Artificial Soils Reveal Individual Factor Controls on Microbial Processes.

Soil matrix properties influence microbial behaviors that underlie nutrient cycling, greenhouse gas production, and soil formation. However, the dynamic and heterogeneous nature of soils makes it challenging to untangle the effects of different matrix properties on microbial behaviors. To address this challenge, we developed a tunable artificial soil recipe and used these materials to study the abiotic mechanisms driving soil microbial growth and communication. When we used standardized matrices with varying textures to culture gas-reporting biosensors, we found that a Gram-negative bacterium (Escherichia coli) grew best in synthetic silt soils, remaining active over a wide range of soil matric potentials, while a Gram-positive bacterium (Bacillus subtilis) preferred sandy soils, sporulating at low water potentials. Soil texture, mineralogy, and alkalinity all attenuated the bioavailability of an acyl-homoserine lactone (AHL) signaling molecule that controls community-level microbial behaviors. Texture controlled the timing of AHL sensing, while AHL bioavailability was decreased ~105-fold by mineralogy and ~103-fold by alkalinity. Finally, we built artificial soils with a range of complexities that converge on the properties of one Mollisol. As artificial soil complexity increased to more closely resemble the Mollisol, microbial behaviors approached those occurring in the natural soil, with the notable exception of organic matter. IMPORTANCE Understanding environmental controls on soil microbes is difficult because many abiotic parameters vary simultaneously and uncontrollably when different natural soils are compared, preventing mechanistic determination of any individual soil parameter's effect on microbial behaviors. We describe how soil texture, mineralogy, pH, and organic matter content can be varied individually within artificial soils to study their effects on soil microbes. Using microbial biosensors that report by producing a rare indicator gas, we identify soil properties that control microbial growth and attenuate the bioavailability of a diffusible chemical used to control community-level behaviors. We find that artificial soils differentially affect signal bioavailability and the growth of Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) microbes. These artificial soils are useful for studying the mechanisms that underlie soil controls on microbial fitness, signaling, and gene transfer.

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion.

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate the binding of MtrA to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). The EET efficiency was found to be inversely correlated with the proximity of the insertion to the heme prosthetic groups. Mutants with decreased EET efficiencies also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular cytochrome MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET efficiencies. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III) respiration conditions correlated with anode reduction. The peptide insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for the regulation of EET.

Nondestructive Chemical Sensing within Bulk Soil Using 1000 Biosensors Per Gram of Matrix.

Gene expression can be monitored in hard-to-image environmental materials using gas-reporting biosensors, but these outputs have only been applied in autoclaved matrices that are hydrated with rich medium. To better understand the compatibility of indicator gas reporting with environmental samples, we evaluated how matrix hydration affects the gas signal of an engineered microbe added to a sieved soil. A gas-reporting microbe presented a gas signal in a forest soil (Alfisol) when hydrated to an environmentally relevant osmotic pressure. When the gas signal was concentrated prior to analysis, a biosensor titer of 103 cells/gram of soil produced a significant signal when soil was supplemented with halides. A signal was also observed without halide amendment, but a higher cell titer (106 cells/gram of soil) was required. A sugar-regulated gas biosensor was able to report with a similar level of sensitivity when added to an unsterilized soil matrix, illustrating how gas concentration enables biosensing within a soil containing environmental microbes. These results establish conditions where engineered microbes can report on gene expression in living environmental matrices with decreased perturbation of the soil environment compared to previously reported approaches, using biosensor titers that are orders of magnitude lower than the number of cells typically observed in a gram of soil.

Solution-Deposited and Patternable Conductive Polymer Thin-Film Electrodes for Microbial Bioelectronics.

Microbial bioelectronic devices integrate naturally occurring or synthetically engineered electroactive microbes with microelectronics. These devices have a broad range of potential applications, but engineering the biotic-abiotic interface for biocompatibility, adhesion, electron transfer, and maximum surface area remains a challenge. Prior approaches to interface modification lack simple processability, the ability to pattern the materials, and/or a significant enhancement in currents. Here, a novel conductive polymer coating that significantly enhances current densities relative to unmodified electrodes in microbial bioelectronics is reported. The coating is based on a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) crosslinked with poly(2-hydroxyethylacrylate) (PHEA) along with a thin polydopamine (PDA) layer for adhesion to an underlying indium tin oxide (ITO) electrode. When used as an interface layer with the current-producing bacterium Shewanella oneidensis MR-1, this material produces a 178-fold increase in the current density compared to unmodified electrodes, a current gain that is higher than previously reported thin-film 2D coatings and 3D conductive polymer coatings. The chemistry, morphology, and electronic properties of the coatings are characterized and the implementation of these coated electrodes for use in microbial fuel cells, multiplexed bioelectronic devices, and organic electrochemical transistor based microbial sensors are demonstrated. It is envisioned that this simple coating will advance the development of microbial bioelectronic devices.

Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential.

Recombination can be used in the laboratory to overcome component limitations in synthetic biology by creating enzymes that exhibit distinct activities and stabilities from native proteins. To investigate how recombination affects the properties of an oxidoreductase that transfers electrons in cells, we created ferredoxin (Fd) chimeras by recombining distantly related cyanobacterial and cyanomyophage Fds (53% identity) that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required for similar levels of cellular electron transfer. These results show how Fds can be diversified through recombination and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic metabolic pathway.

A Split Methyl Halide Transferase AND Gate That Reports by Synthesizing an Indicator Gas.

Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.

Prochlorococcus phage ferredoxin: structural characterization and electron transfer to cyanobacterial sulfite reductases.

Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those of photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcus marinus, revealed high levels of similarity to cyanobacterial Fds (root mean square deviations of ≤0.5 Å). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (-336 mV versus a standard hydrogen electrode), similar to other photosynthetic Fds, although it had lower thermostability (Tm = 28 °C) than did many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when coexpressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high levels of structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterially encoded oxidoreductases.

Soil organic matter attenuates the efficacy of flavonoid-based plant-microbe communication.

Plant-microbe interactions are mediated by signaling compounds that control vital plant functions, such as nodulation, defense, and allelopathy. While interruption of signaling is typically attributed to biological processes, potential abiotic controls remain less studied. Here, we show that higher organic carbon (OC) contents in soils repress flavonoid signals by up to 70%. Furthermore, the magnitude of repression is differentially dependent on the chemical structure of the signaling molecule, the availability of metal ions, and the source of the plant-derived OC. Up to 63% of the signaling repression occurs between dissolved OC and flavonoids rather than through flavonoid sorption to particulate OC. In plant experiments, OC interrupts the signaling between a legume and a nitrogen-fixing microbial symbiont, resulting in a 75% decrease in nodule formation. Our results suggest that soil OC decreases the lifetime of flavonoids underlying plant-microbe interactions.

100th Anniversary of Macromolecular Science Viewpoint: Soft Materials for Microbial Bioelectronics.

Bioelectronics brings together the fields of biology and microelectronics to create multifunctional devices with the potential to address longstanding technological challenges and change our way of life. Microbial electrochemical devices are a growing subset of bioelectronic devices that incorporate naturally occurring or synthetically engineered microbes into electronic devices and have broad applications including energy harvesting, chemical production, water remediation, and environmental and health monitoring. The goal of this Viewpoint is to highlight recent advances and ongoing challenges in the rapidly developing field of microbial bioelectronic devices, with an emphasis on materials challenges. We provide an overview of microbial bioelectronic devices, discuss the biotic-abiotic interface in these devices, and then present recent advances and ongoing challenges in materials related to electron transfer across the abiotic-biotic interface, microbial adhesion, redox signaling, electronic amplification, and device miniaturization. We conclude with a summary and perspective of the field of microbial bioelectronics.

Translating New Synthetic Biology Advances for Biosensing Into the Earth and Environmental Sciences.

The rapid diversification of synthetic biology tools holds promise in making some classically hard-to-solve environmental problems tractable. Here we review longstanding problems in the Earth and environmental sciences that could be addressed using engineered microbes as micron-scale sensors (biosensors). Biosensors can offer new perspectives on open questions, including understanding microbial behaviors in heterogeneous matrices like soils, sediments, and wastewater systems, tracking cryptic element cycling in the Earth system, and establishing the dynamics of microbe-microbe, microbe-plant, and microbe-material interactions. Before these new tools can reach their potential, however, a suite of biological parts and microbial chassis appropriate for environmental conditions must be developed by the synthetic biology community. This includes diversifying sensing modules to obtain information relevant to environmental questions, creating output signals that allow dynamic reporting from hard-to-image environmental materials, and tuning these sensors so that they reliably function long enough to be useful for environmental studies. Finally, ethical questions related to the use of synthetic biosensors in environmental applications are discussed.

De novo design of symmetric ferredoxins that shuttle electrons in vivo.

A symmetric origin for bacterial ferredoxins was first proposed over 50 y ago, yet, to date, no functional symmetric molecule has been constructed. It is hypothesized that extant proteins have drifted from their symmetric roots via gene duplication followed by mutations. Phylogenetic analyses of extant ferredoxins support the independent evolution of N- and C-terminal sequences, thereby allowing consensus-based design of symmetric 4Fe-4S molecules. All designs bind two [4Fe-4S] clusters and exhibit strongly reducing midpoint potentials ranging from -405 to -515 mV. One of these constructs efficiently shuttles electrons through a designed metabolic pathway in Escherichia coli These finding establish that ferredoxins consisting of a symmetric core can be used as a platform to design novel electron transfer carriers for in vivo applications. Outer-shell asymmetry increases sequence space without compromising electron transfer functionality.

Overcoming component limitations in synthetic biology through transposon-mediated protein engineering.

Protein fission and fusion can be used to create biomolecules with new structures and functions, including circularly permuted proteins that require post-translational modifications for activity, split protein AND gates that require multiple inputs for activity, and fused domains that function as chemical-dependent protein switches. Herein we describe how transposon mutagenesis can be used for protein design to create libraries of permuted, split, or domain-inserted proteins. When coupled with a functional screen or selection, these approaches can rapidly diversify the topologies and functions of natural proteins and create useful protein components for synthetic biology.

Protein tolerance to random circular permutation correlates with thermostability and local energetics of residue-residue contacts.

Adenylate kinase (AK) orthologs with a range of thermostabilities were subjected to random circular permutation, and deep mutational scanning was used to evaluate where new protein termini were nondisruptive to activity. The fraction of circularly permuted variants that retained function in each library correlated with AK thermostability. In addition, analysis of the positional tolerance to new termini, which increase local conformational flexibility, showed that bonds were either functionally sensitive to cleavage across all homologs, differentially sensitive, or uniformly tolerant. The mobile AMP-binding domain, which displays the highest calculated contact energies, presented the greatest tolerance to new termini across all AKs. In contrast, retention of function in the lid and core domains was more dependent upon AK melting temperature. These results show that family permutation profiling identifies primary structure that has been selected by evolution for dynamics that are critical to activity within an enzyme family. These findings also illustrate how deep mutational scanning can be applied to protein homologs in parallel to differentiate how topology, stability, and local energetics govern mutational tolerance.