Single molecule tracking of bacterial cell surface cytochromes reveals dynamics that impact long-distance electron transport.

Using a series of multiheme cytochromes, the metal-reducing bacterium Shewanella oneidensis MR-1 can perform extracellular electron transfer (EET) to respire redox-active surfaces, including minerals and electrodes outside the cell. While the role of multiheme cytochromes in transporting electrons across the cell wall is well established, these cytochromes were also recently found to facilitate long-distance (micrometer-scale) redox conduction along outer membranes and across multiple cells bridging electrodes. Recent studies proposed that long-distance conduction arises from the interplay of electron hopping and cytochrome diffusion, which allows collisions and electron exchange between cytochromes along membranes. However, the diffusive dynamics of the multiheme cytochromes have never been observed or quantified in vivo, making it difficult to assess their hypothesized contribution to the collision-exchange mechanism. Here, we use quantum dot labeling, total internal reflection fluorescence microscopy, and single-particle tracking to quantify the lateral diffusive dynamics of the outer membrane-associated decaheme cytochromes MtrC and OmcA, two key components of EET in S. oneidensis. We observe confined diffusion behavior for both quantum dot-labeled MtrC and OmcA along cell surfaces (diffusion coefficients DMtrC = 0.0192 ± 0.0018 µm2/s, DOmcA = 0.0125 ± 0.0024 µm2/s) and the membrane extensions thought to function as bacterial nanowires. We find that these dynamics can trace a path for electron transport via overlap of cytochrome trajectories, consistent with the long-distance conduction mechanism. The measured dynamics inform kinetic Monte Carlo simulations that combine direct electron hopping and redox molecule diffusion, revealing significant electron transport rates along cells and membrane nanowires.

Carbonate-hosted microbial communities are prolific and pervasive methane oxidizers at geologically diverse marine methane seep sites.

At marine methane seeps, vast quantities of methane move through the shallow subseafloor, where it is largely consumed by microbial communities. This process plays an important role in global methane dynamics, but we have yet to identify all of the methane sinks in the deep sea. Here, we conducted a continental-scale survey of seven geologically diverse seafloor seeps and found that carbonate rocks from all sites host methane-oxidizing microbial communities with substantial methanotrophic potential. In laboratory-based mesocosm incubations, chimney-like carbonates from the newly described Point Dume seep off the coast of Southern California exhibited the highest rates of anaerobic methane oxidation measured to date. After a thorough analysis of physicochemical, electrical, and biological factors, we attribute this substantial metabolic activity largely to higher cell density, mineral composition, kinetic parameters including an elevated Vmax, and the presence of specific microbial lineages. Our data also suggest that other features, such as electrical conductance, rock particle size, and microbial community alpha diversity, may influence a sample's methanotrophic potential, but these factors did not demonstrate clear patterns with respect to methane oxidation rates. Based on the apparent pervasiveness within seep carbonates of microbial communities capable of performing anaerobic oxidation of methane, as well as the frequent occurrence of carbonates at seeps, we suggest that rock-hosted methanotrophy may be an important contributor to marine methane consumption.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes.

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Bacterial extracellular electron transfer components are spin selective.

Metal-reducing bacteria have adapted the ability to respire extracellular solid surfaces instead of soluble oxidants. This process requires an electron transport pathway that spans from the inner membrane, across the periplasm, through the outer membrane, and to an external surface. Multiheme cytochromes are the primary machinery for moving electrons through this pathway. Recent studies show that the chiral-induced spin selectivity (CISS) effect is observable in some of these proteins extracted from the model metal-reducing bacteria, Shewanella oneidensis MR-1. It was hypothesized that the CISS effect facilitates efficient electron transport in these proteins by coupling electron velocity to spin, thus reducing the probability of backscattering. However, these studies focused exclusively on the cell surface electron conduits, and thus, CISS has not been investigated in upstream electron transfer components such as the membrane-associated MtrA, or periplasmic proteins such as small tetraheme cytochrome (STC). By using conductive probe atomic force microscopy measurements of protein monolayers adsorbed onto ferromagnetic substrates, we show that electron transport is spin selective in both MtrA and STC. Moreover, we have determined the spin polarization of MtrA to be ∼77% and STC to be ∼35%. This disparity in spin polarizations could indicate that spin selectivity is length dependent in heme proteins, given that MtrA is approximately two times longer than STC. Most significantly, our study indicates that spin-dependent interactions affect the entire extracellular electron transport pathway.

Spatiotemporal mapping of bacterial membrane potential responses to extracellular electron transfer.

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 µm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

Electrolocation? The evidence for redox-mediated taxis in Shewanella oneidensis.

Shewanella oneidensis is a dissimilatory metal reducing bacterium and model for extracellular electron transfer (EET), a respiratory mechanism in which electrons are transferred out of the cell. In the last 10 years, migration to insoluble electron acceptors for EET has been shown to be nonrandom and tactic, seemingly in the absence of molecular or energy gradients that typically allow for taxis. As the ability to sense, locate, and respire electrodes has applications in bioelectrochemical technology, a better understanding of taxis in S. oneidensis is needed. While the EET conduits of S. oneidensis have been studied extensively, its taxis pathways and their interplay with EET are not yet understood, making investigation into taxis phenomena nontrivial. Since S. oneidensis is a member of an EET-encoding clade, the genetic circuitry of taxis to insoluble acceptors may be conserved. We performed a bioinformatic analysis of Shewanella genomes to identify S. oneidensis chemotaxis orthologs conserved in the genus. In addition to the previously reported core chemotaxis gene cluster, we identify several other conserved proteins in the taxis signaling pathway. We present the current evidence for the two proposed models of EET taxis, "electrokinesis" and flavin-mediated taxis, and highlight key areas in need of further investigation.

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.

Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography.

Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that S. oneidensis OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.

Spin-Dependent Electron Transport through Bacterial Cell Surface Multiheme Electron Conduits.

Multiheme cytochromes, located on the bacterial cell surface, function as long-distance (>10 nm) electron conduits linking intracellular reactions to external surfaces. This extracellular electron transfer process, which allows microorganisms to gain energy by respiring solid redox-active minerals, also facilitates the wiring of cells to electrodes. While recent studies have suggested that a chiral induced spin selectivity effect is linked to efficient electron transmission through biomolecules, this phenomenon has not been investigated in extracellular electron conduits. Using magnetic conductive probe atomic force microscopy, Hall voltage measurements, and spin-dependent electrochemistry of the decaheme cytochromes MtrF and OmcA from the metal-reducing bacterium Shewanella oneidensis MR-1, we show that electron transport through these extracellular conduits is spin-selective. Our study has implications for understanding how spin-dependent interactions and magnetic fields may control electron transport across biotic-abiotic interfaces in both natural and biotechnological systems.

Multiheme Cytochrome Mediated Redox Conduction through Shewanella oneidensis MR-1 Cells.

Multiheme cytochromes function as extracellular electron transfer (EET) conduits that extend the metabolic reach of microorganisms to external solid surfaces. These conduits are also proposed to facilitate long-distance electron transport along cellular membranes and across multiple cells. Here we report electrochemical gating measurements of Shewanella oneidensis MR-1 cells linking interdigitated electrodes. The dependence of the source-drain current on gate potential demonstrates a redox conduction mechanism, which we link to the presence of multiheme cytochromes of the Mtr pathway. We also find that the measured thermal activation energy of 0.29 ± 0.03 eV is consistent with these obtained from electron hopping calculations through the S. oneidensis Mtr outer-membrane decaheme cytochromes. Our measurements and calculations have implications for understanding and controlling micrometer-scale electron transport in microbial systems.

Filamentous bacteria transport electrons over centimetre distances.

Oxygen consumption in marine sediments is often coupled to the oxidation of sulphide generated by degradation of organic matter in deeper, oxygen-free layers. Geochemical observations have shown that this coupling can be mediated by electric currents carried by unidentified electron transporters across centimetre-wide zones. Here we present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with electric currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, electrical cables add a new dimension to the understanding of interactions in nature and may find use in technology development.

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes.

Shewanella oneidensis MR-1 gains energy by extracellular electron transfer to solid surfaces. They employ c-type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron- and hole-transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode-bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme-to-heme interactions with the distance between the hemes.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation.

UNLABELLED: In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA The transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCE: Shewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain why S. oneidensis can be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer. Shewanella coordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Thermally activated long range electron transport in living biofilms.

Microbial biofilms grown utilizing electrodes as metabolic electron acceptors or donors are a new class of biomaterials with distinct electronic properties. Here we report that electron transport through living electrode-grown Geobacter sulfurreducens biofilms is a thermally activated process with incoherent redox conductivity. The temperature dependency of this process is consistent with electron-transfer reactions involving hemes of c-type cytochromes known to play important roles in G. sulfurreducens extracellular electron transport. While incoherent redox conductivity is ubiquitous in biological systems at molecular-length scales, it is unprecedented over distances it appears to occur through living G. sulfurreducens biofilms, which can exceed 100 microns in thickness.

Shewanella oneidensis MR-1 bacterial nanowires exhibit p-type, tunable electronic behavior.

The study of electrical transport in biomolecular materials is critical to our fundamental understanding of physiology and to the development of practical bioelectronics applications. In this study, we investigated the electronic transport characteristics of Shewanella oneidensis MR-1 nanowires by conducting-probe atomic force microscopy (CP-AFM) and by constructing field-effect transistors (FETs) based on individual S. oneidensis nanowires. Here we show that S. oneidensis nanowires exhibit p-type, tunable electronic behavior with a field-effect mobility on the order of 10(-1) cm(2)/(V s), comparable to devices based on synthetic organic semiconductors. This study opens up opportunities to use such bacterial nanowires as a new semiconducting biomaterial for making bioelectronics and to enhance the power output of microbial fuel cells through engineering the interfaces between metallic electrodes and bacterial nanowires.

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 to 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. A thermodynamic model and promoter engineering 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 within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1.

Bacterial nanowires are extracellular appendages that have been suggested as pathways for electron transport in phylogenetically diverse microorganisms, including dissimilatory metal-reducing bacteria and photosynthetic cyanobacteria. However, there has been no evidence presented to demonstrate electron transport along the length of bacterial nanowires. Here we report electron transport measurements along individually addressed bacterial nanowires derived from electron-acceptor-limited cultures of the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. Transport along the bacterial nanowires was independently evaluated by two techniques: (i) nanofabricated electrodes patterned on top of individual nanowires, and (ii) conducting probe atomic force microscopy at various points along a single nanowire bridging a metallic electrode and the conductive atomic force microscopy tip. The S. oneidensis MR-1 nanowires were found to be electrically conductive along micrometer-length scales with electron transport rates up to 10(9)/s at 100 mV of applied bias and a measured resistivity on the order of 1 Ω·cm. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA produce appendages that are morphologically consistent with bacterial nanowires, but were found to be nonconductive. The measurements reported here allow for bacterial nanowires to serve as a viable microbial strategy for extracellular electron transport.

Soluble Iron Enhances Extracellular Electron Uptake by Shewanella oneidensis MR-1.

Extracellular electron transfer (EET) is a process that microorganisms use to reduce or oxidize external insoluble electron acceptors or donors. Much of our mechanistic understanding of this process is derived from studies of transmembrane cytochrome complexes and extracellular redox shuttles that mediate outward EET to anodes and external electron acceptors. In contrast, there are knowledge gaps concerning the reverse process of inward EET from external electron donors to cells. Here, we describe a role for soluble iron (exogenous FeCl2) in enhancing EET from cathodes to the model EET bacterium Shewanella oneidensis MR-1, with fumarate serving as the intracellular electron acceptor. This iron concentration-dependent electron uptake was eradicated upon addition of an iron chelator and occurred only in the presence of fumarate reductase, confirming an electron pathway from cathodes to this periplasmic enzyme. Moreover, S. oneidensis mutants lacking specific outer membrane and periplasmic cytochromes exhibited significantly decreased current levels relative to wild-type. These results indicate that soluble iron can function as an electron carrier to the EET machinery of S. oneidensis.

Living electronics: A catalogue of engineered living electronic components.

Biology leverages a range of electrical phenomena to extract and store energy, control molecular reactions and enable multicellular communication. Microbes, in particular, have evolved genetically encoded machinery enabling them to utilize the abundant redox-active molecules and minerals available on Earth, which in turn drive global-scale biogeochemical cycles. Recently, the microbial machinery enabling these redox reactions have been leveraged for interfacing cells and biomolecules with electrical circuits for biotechnological applications. Synthetic biology is allowing for the use of these machinery as components of engineered living materials with tuneable electrical properties. Herein, we review the state of such living electronic components including wires, capacitors, transistors, diodes, optoelectronic components, spin filters, sensors, logic processors, bioactuators, information storage media and methods for assembling these components into living electronic circuits.