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.

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.

Repurposing a chemosensory macromolecular machine.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

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.

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.

Biogenic Control of Manganese Doping in Zinc Sulfide Nanomaterial Using Shewanella oneidensis MR-1.

Bacteria naturally alter the redox state of many compounds and perform atom-by-atom nanomaterial synthesis to create many inorganic materials. Recent advancements in synthetic biology have spurred interest in using biological systems to manufacture nanomaterials, implementing biological strategies to specify the nanomaterial characteristics such as size, shape, and optical properties. Here, we combine the natural synthetic capabilities of microbes with engineered genetic control circuits toward biogenically synthesized semiconductor nanomaterials. Using an engineered strain of Shewanella oneindensis with inducible expression of the cytochrome complex MtrCAB, we control the reduction of manganese (IV) oxide. Cytochrome expression levels were regulated using an inducer molecule, which enabled precise modulation of dopant incorporation into manganese doped zinc sulfide nanoparticles (Mn:ZnS). Thereby, a synthetic gene circuit controlled the optical properties of biogenic quantum dots. These biogenically assembled nanomaterials have similar physical and optoelectronic properties to chemically synthesized particles. Our results demonstrate the promise of implementing synthetic gene circuits for tunable control of nanomaterials made by biological systems.

The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type.

The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here, we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor's stator system and its structural elaboration. Motors with a single H+-driven stator have only the core periplasmic P- and L-rings; those with dual H+-driven stators have an elaborated P-ring; and motors with Na+ or Na+/H+-driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts.

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.

Tracking Electron Uptake from a Cathode into Shewanella Cells: Implications for Energy Acquisition from Solid-Substrate Electron Donors.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors, such as fumarate or oxygen, thereby providing a model system for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells than in controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool determined with a bioluminescence assay, a proton uncoupler, and a mutant of proton-pumping NADH oxidase complex I. This work suggested that the generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by complex I. A decrease in cathodic electron uptake was observed in various mutant strains, including those lacking the extracellular electron transfer components necessary for anodic-current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy acquisition, resulting in a quantifiable reduction in the cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence on cathodes, which might extend to environments where growth and division are severely limited.IMPORTANCE The majority of our knowledge of the physiology of extracellular electron transfer derives from studies of electrons moving to the exterior of the cell. The physiological mechanisms and/or consequences of the reverse processes are largely uncharacterized. This report demonstrates that when coupled to oxygen reduction, electrode oxidation can result in cellular energy acquisition. This respiratory process has potentially important implications for how microorganisms persist in energy-limited environments, such as reduced sediments under changing redox conditions. From an applied perspective, this work has important implications for microbially catalyzed processes on electrodes, particularly with regard to understanding models of cellular conversion of electrons from cathodes to microbially synthesized products.

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.

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.

Multistep hopping and extracellular charge transfer in microbial redox chains.

Dissimilatory metal-reducing bacteria are microorganisms that gain energy by transferring respiratory electrons to extracellular solid-phase electron acceptors. In addition to its importance for physiology and natural environmental processes, this form of metabolism is being investigated for energy conversion and fuel production in bioelectrochemical systems, where microbes are used as biocatalysts at electrodes. One proposed strategy to accomplish this extracellular charge transfer involves forming a conductive pathway to electrodes by incorporating redox components on outer cell membranes and along extracellular appendages known as microbial nanowires within biofilms. To describe extracellular charge transfer in microbial redox chains, we employed a model based on incoherent hopping between sites in the chain and an interfacial treatment of electrochemical interactions with the surrounding electrodes. Based on this model, we calculated the current-voltage (I-V) characteristics and found the results to be in good agreement with I-V measurements across and along individual microbial nanowires produced by the bacterium Shewanella oneidensis MR-1. Based on our analysis, we propose that multistep hopping in redox chains constitutes a viable strategy for extracellular charge transfer in microbial biofilms.