Biology subjects

Atkinson, J. T.

Publications and source records attributed to Atkinson, J. T..

13 recordsLinked to original sources

bioRxiv2026

Living electronic transistors with tunable conductivity

Electroactive bacteria, like Shewanella oneidensis, can couple the oxidation of organic electron donors to the reduction of external conductive surfaces, such as minerals and electrodes, by utilizing multiheme cytochromes to carry charge from within the cell to external surfaces. Additionally, multiheme cytochromes facilitate gateable, long-distance (micrometer-scale) redox conduction along the outer membrane and across multiple cells bridging electrodes. While electroactive microbes are being used to develop bioelectrochemical devices, there have been limited efforts to use synthetic biology to exert additional control over microbes serving as device components. Thus, this work implements an optogenetic biofilm patterning gene circuit and a small molecule sensor in S. oneidensis to simultaneously control cell deposition and cytochrome expression. This allows for photolithographic patterning of biofilms possessing tunable electrical properties controlled with small molecules. This system demonstrates tunable electrochemical activity, redox conduction, intrinsic biofilm conductivity, and negative differential transconductance as a function of cytochrome expression. Additionally, temperature-dependent measurements of this tunable biofilm conduction reveal changes in activation energy as a function of cytochrome expression. Through this combination of synthetic biology and electrochemistry, simultaneous control over biofilm geometry and conductivity sheds light on fundamental microbial electron transport processes, and it enables the construction of living electronic devices.

synthetic biology↗

bioRxiv2026

Artificial sediments enable reproducible cultivation and recapitulate ecological interactions of cable bacteria

Cable bacteria are filamentous microbes that couple sulfide oxidation to oxygen reduction over centimeter distances via long-distance electron transport. While their activity creates characteristic biogeochemical gradients that shape sediment ecology, the study of cable bacteria has been constrained by the chemical and physical heterogeneity of the natural sediments they inhabit. To date, laboratory cultivation efforts have relied on these undefined environmental matrices. Here, we established a reproducible enrichment and cultivation platform using an artificial sediment matrix coupled with chemically defined media. This matrix successfully supported the growth of both freshwater and marine cable bacteria and enabled serial propagation over multiple transfers. Microsensor profiling confirmed that the incubations recapitulated hallmark geochemical signatures, including the sulfide, oxygen and pH gradients associated with electrogenic sulfur oxidation. Scanning electron microscopy confirmed the presence of cable bacteria, while 16S rRNA sequencing confirmed enrichment of the cable bacteria together with a stable co-enriched community that included taxa associated with sulfur and iron cycling as well as cellulose decomposition. This defined cultivation system eliminates the variability inherent to natural samples, providing a controlled platform for dissecting the physiology, genetics, and microbial interactions of cable bacteria.

microbiology↗

bioRxiv2026

Biofilm Patterning Reveals the Functional Contributions of Periplasmic Cytochromes to the Electrochemical Activity of Shewanella oneidensis

Shewanella oneidensis MR-1 is a model electroactive bacterium whose extracellular electron transfer (EET) pathway includes a sequential network of c-type cytochromes that span the inner membrane, periplasm, and outer membrane. While electrochemical studies revealed the critical role of outer-membrane cytochromes in mediating both outward EET from cells to external surfaces and lateral biofilm conduction across cells, the specific functional role of the periplasmic cytochromes in these processes is less understood. Dissecting the contributions of the periplasmic components has been challenged by the complexity of the periplasmic cytochrome network and the inherent variability of native biofilms, which confounds the electrochemical comparison of cytochrome mutants. Here, we overcome these limitations with a synthetic biology approach combining targeted deletion of genes encoding key periplasmic cytochromes with light-induced biofilm patterning to create uniform, geometrically defined biofilms on electrodes for robust electrochemical comparisons. Voltammetric measurements of patterned S. oneidensis mutant biofilms confirmed the essential role of periplasmic cytochromes in facilitating outward EET, a contribution that becomes apparent when flavins are present to accelerate interfacial electron transfer between outer-membrane cytochromes and the electrode. In contrast to this crucial role in routing outward EET across the periplasm, electrochemical gating measurements of lateral biofilm conductivity revealed that the periplasmic cytochromes do not contribute to long-distance electron transport along cellular layers bridging electrodes. These findings provide new insights into the role of periplasmic cytochromes in S. oneidensis, and distinguish their contributions to routing outward EET across the cell envelope versus biofilm conductivity. ImportanceMicrobes capable of extracellular electron transfer (EET) are central to global biogeochemical cycles and emerging bioelectrochemical technologies. In the important model EET bacterium Shewanella oneidensis MR-1, the outer-membrane components that interface with external surfaces are well-characterized. However, the functional role of the periplasmic components linking the inner and outer membranes has remained obscured by the complex network of multiple cytochromes and biofilm heterogeneity limiting precise comparisons across mutants. By combining light-induced biofilm patterning with electrochemical analysis, we successfully revealed the specific contributions of periplasmic cytochromes: these components are essential for facilitating the outward EET across the cell envelope but do not impact lateral long-distance electron transport across the biofilm. The results refine our understanding of extracellular respiration and provide design rules for engineering living electronic materials.

microbiology↗

bioRxiv2023

A red light-induced genetic system for control of extracellular electron transfer

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported into new host strains. Here, we developed and adapted a red light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. Promoter engineering and a thermodynamic model were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer (EET) within S. oneidensis. The ability to use both red and blue light-induced optogenetic circuits simultaneously was demonstrated. Our work expands the synthetic biology toolbox of Shewanella, which could facilitate future advances in applications with electrogenic bacteria.

synthetic biology↗

bioRxiv2023

The biochemical impact of extracting an embedded adenylate kinase domain using circular permutation

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show 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 homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Laboratory evolution identifies elongated flavodoxins that support electron transfer to sulfite reductases

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. While dozens of Fld-partner oxidoreductases have been discovered, these only represent a subset of the oxidoreductases that couple with ferredoxin (Fd) protein electron carriers. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a Fd, we evaluated the ability of Flds to transfer electrons from a Fd-NADP reductase (FNR) to a Fd-dependent SIR using growth complementation of a microbe with a sulfur metabolism defect. We show that Flds from cyanobacteria complement the growth of this microbe when coexpressed with an FNR and an SIR that evolved to couple with a plant Fd. To better understand the interaction of Fld with these partner oxidoreductases, we evaluated the effect of peptide insertion on Fld-mediated electron transfer. We observe a high insertion sensitivity within regions predicted to be proximal to the cofactor and partner binding sites and a high insertion tolerance within the loop that is used to differentiate short- and long-chain flavodoxins. These results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of peptide-insertion tolerance is influenced by interactions with oxidoreductase partners in electron transfer pathways.

biochemistry↗

bioRxiv2022

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 MtrA binding 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). EET efficiency was found to be inversely correlated with insertion proximity to the heme prosthetic groups. Mutants with decreased EET 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 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. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III)-respiring 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 regulating EET.

synthetic biology↗

bioRxiv2021

Real-time environmental monitoring of contaminants using living electronic sensors

Real-time chemical sensing is needed to counter the global threats posed by pollution. We combine synthetic biology and materials engineering to develop a living bioelectronic sensor platform with minute detection times. Escherichia coli was programmed to reduce an electrode in a chemical-dependent manner using a modular, eight-component, synthetic electron transport chain. This strain produced significantly more current upon exposure to thiosulfate, an anion that causes microbial blooms. Incorporating a protein switch into the synthetic pathway and encapsulation of microbes with electrodes and conductive nanomaterials yielded a living bioelectronic sensor that could detect an endocrine disruptor within two minutes in riverine water, implicating the signal as mass transfer limited. These findings provide a new platform for miniature, low-power sensors that safeguard ecological and human health. One Sentence SummaryChemicals are detected electrically using an allosterically-regulated electron transfer pathway in designer microbes.

synthetic biology↗

bioRxiv2020

A split methyl halide transferase AND gate that reports by synthesizing an indicator gas

It is challenging to detect microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater. To develop a simple approach for monitoring 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-fold 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 can report on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this protein fragment complementation assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

ABSTRACTHomologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds 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 be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers 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 electron transfer pathway.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

Prochlorococcus phage ferredoxin: structural characterization and interactions with cyanobacterial sulfite reductases

Marine cyanobacteria are infected by phage whose genomes encode ferredoxin (Fd) electron carriers. While these Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, it is not clear how the biophysical properties and partner specificities of phage Fds relate to those in photosynthetic organisms. Bioinformatic 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 Prochlorococcus marinus, revealed high similarity to cyanobacterial Fds ([≤]0.5 [A] RMSD). Additionally, pssm2-Fd exhibits a low midpoint reduction potential (-336 mV vs. SHE) similar to other photosynthetic Fds, albeit lower thermostability (Tm = 28{degrees}C) than many Fds. When expressed in an Escherichia coli strain with a sulfite assimilation defect, pssm2-Fd complemented growth when coexpressed with a Prochlorococcus marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterial-encoded oxidoreductases.

biochemistry↗

bioRxiv2019

Family permutation profiling identifies a dynamic protein domain as functionally tolerant to increased conformational entropy

To investigate whether adenylate kinase (AK) homologs differ in their functional tolerance to mutational lesions that alter dynamics, we subjected three homologs having a range of thermostabilities to random circular permutation and evaluated where new protein termini were non-disruptive to activity using a cellular selection and deep mutational scanning. Analysis of the positional tolerance to new termini, which increase local conformational entropy by breaking peptide bonds, showed that bonds were either functionally sensitive to cleavage across all three homologs, differentially sensitive, or uniformly tolerant. The mobile AMP binding domain, which displays the highest calculated contact energies (frustration), 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. Thus, regions of high energetic frustration tolerated increases in conformational entropy in a manner that was less dependent on thermostability than regions of lower frustration. Our results suggest that family permutation profiling identifies primary structure that has been selected by evolution for high frustration that is critical to enzymatic activity. They also illustrate how deep mutational scanning can be applied to protein homologs in parallel to learn how topology and function govern mutational tolerance.

biochemistry↗

bioRxiv2019

Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer

One challenge with controlling electron flow in cells is the lack of biomolecules that directly couple the sensing of environmental conditions to electron transfer efficiency. To overcome this protein component limitation, we randomly inserted the ligand binding domain (LBD) from the human estrogen receptor (ER) into a thermostable 2Fe-2S ferredoxin (Fd) from Mastigocladus laminosus and used a bacterial selection to identify Fd-LBD fusion proteins that support electron transfer from a Fd-NADP reductase (FNR) to a Fd-dependent sulfite reductase (SIR). Mapping LBD insertion sites onto structure revealed that Fd tolerates domain insertion adjacent to or within the tetracysteine motif that coordinates the 2Fe-2S metallocluster. With both classes of the fusion proteins, cellular ET was enhanced by the ER antagonist 4-hydroxytamoxifen. In addition, one of Fds arising from ER-LBD insertion within the tetracysteine motif acquires an oxygen-tolerant 2Fe-2S cluster, suggesting that ET is regulated through post-translational ligand binding.

synthetic biology↗