Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers.

Long-range (>10 µm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within ∼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.

Decoding the metabolic response of Escherichia coli for sensing trace heavy metals in water.

Heavy metal contamination due to industrial and agricultural waste represents a growing threat to water supplies. Frequent and widespread monitoring for toxic metals in drinking and agricultural water sources is necessary to prevent their accumulation in humans, plants, and animals, which results in disease and environmental damage. Here, the metabolic stress response of bacteria is used to report the presence of heavy metal ions in water by transducing ions into chemical signals that can be fingerprinted using machine learning analysis of vibrational spectra. Surface-enhanced Raman scattering surfaces amplify chemical signals from bacterial lysate and rapidly generate large, reproducible datasets needed for machine learning algorithms to decode the complex spectral data. Classification and regression algorithms achieve limits of detection of 0.5 pM for As3+ and 6.8 pM for Cr6+, 100,000 times lower than the World Health Organization recommended limits, and accurately quantify concentrations of analytes across six orders of magnitude, enabling early warning of rising contaminant levels. Trained algorithms are generalizable across water samples with different impurities; water quality of tap water and wastewater was evaluated with 92% accuracy.

Electrically Fueled Active Supramolecular Materials.

Fuel-driven dissipative self-assemblies play essential roles in living systems, contributing both to their complex, dynamic structures and emergent functions. Several dissipative supramolecular materials have been created using chemicals or light as fuel. However, electrical energy, one of the most common energy sources, has remained unexplored for such purposes. Here, we demonstrate a new platform for creating active supramolecular materials using electrically fueled dissipative self-assembly. Through an electrochemical redox reaction network, a transient and highly active supramolecular assembly is achieved with rapid kinetics, directionality, and precise spatiotemporal control. As electronic signals are the default information carriers in modern technology, the described approach offers a potential opportunity to integrate active materials into electronic devices for bioelectronic applications.

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.

Conformations of peptoids in nanosheets result from the interplay of backbone energetics and intermolecular interactions.

The conformations adopted by the molecular constituents of a supramolecular assembly influence its large-scale order. At the same time, the interactions made in assemblies by molecules can influence their conformations. Here we study this interplay in extended flat nanosheets made from nonnatural sequence-specific peptoid polymers. Nanosheets exist because individual polymers can be linear and untwisted, by virtue of polymer backbone elements adopting alternating rotational states whose twists oppose and cancel. Using molecular dynamics and quantum mechanical simulations, together with experimental data, we explore the design space of flat nanostructures built from peptoids. We show that several sets of peptoid backbone conformations are consistent with their being linear, but the specific combination observed in experiment is determined by a combination of backbone energetics and the interactions made within the nanosheet. Our results provide a molecular model of the peptoid nanosheet consistent with all available experimental data and show that its structure results from a combination of intra- and intermolecular interactions.

Correction: Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution.

Correction for 'Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution' by Nicole L. Ing et al., Phys. Chem. Chem. Phys., 2017, 19, 21791-21799.

The Phe-Ile Zipper: A Specific Interaction Motif Drives Antiparallel Coiled-Coil Hexamer Formation.

Coiled coils are a robust motif for exploring amino acid interactions, generating unique supramolecular structures, and expanding the functional properties of biological materials. A recently discovered antiparallel coiled-coil hexamer (ACC-Hex, peptide 1) exhibits a unique interaction in which Phe and Ile residues from adjacent α-helices interact to form a Phe-Ile zipper within the hydrophobic core. Analysis of the X-ray crystallographic structure of ACC-Hex suggests that the stability of the six-helix bundle relies on specific interactions between the Phe and Ile residues. The Phe-Ile zipper is unprecedented and represents a powerful tool for utilizing the Phe-Ile interactions to direct supramolecular assembly. To further probe and understand the limits of the Phe-Ile zipper, we designed peptide sequences with natural and unnatural amino acids placed at the Phe and Ile residue positions. Using size exclusion chromatography and small-angle X-ray scattering, we found that the proper assembly of ACC-Hex from monomers is sensitive to subtle changes in side chain steric bulk and hydrophobicity introduced by mutations at the Phe and Ile residue positions. Of the sequence variants that formed ACC-Hex, mutations in the hydrophobic core significantly affected the stability of the hexamer, from a ΔGuw of 2-8 kcal mol-1. Additional sequences were designed to further probe and enhance the stability of the ACC-Hex system by maximizing salt bridging between the solvent-exposed residues. Finally, we expanded on the generality of the Phe-Ile zipper, creating a unique sequence that forms an antiparallel hexamer that is topologically similar to ACC-Hex but atomistically unique.

Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution.

The bacterium Geobacter sulfurreducens is a model biological catalyst in microbial electrochemical devices. G. sulfurreducens forms electrically conductive, electrode-associated biofilms, but the biological structures mediating electrical conduction from cells to the electrodes are a matter of debate. Bacteria in these communities produce a network of fiber-like Type IV pili, which have been proposed to act either as inherent, protein-based electronic conductors, or as electronically inert scaffolds for cytochromes mediating long-range charge transport. Previous studies have examined pilus conduction mechanisms under vacuum and in dry conditions, but their conduction mechanism under physiologically relevant conditions has yet to be characterized. In this work, we isolate G. sulfurreducens pili, and compare the electronic conduction mechanism of both live biofilms and purified pili networks under dry and aqueous conditions. Solid-state I-V characteristics indicate that electronic transport in films of purified pili is representative of conduction in a fiber percolation network. Electrochemical gating measurements in a bipotentiostat device configuration confirm previous results suggesting redox currents dominate live biofilm conduction. Purified pili films, however, exhibit non-redox electronic conduction under aqueous, buffered conditions, and their conductivity increases with decreasing temperature. These findings show that isolated pili possess inherent, non-redox-mediated conductivity consistent with a metallic-like model of charge carrier transport. The results demonstrate an experimental platform for studying electronic transport in biomaterials and suggest that pili serve as an exemplary model for designing bioelectronic interfaces.

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides.

The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.

Enhanced thermoelectric performance of rough silicon nanowires.

Approximately 90 per cent of the world's power is generated by heat engines that use fossil fuel combustion as a heat source and typically operate at 30-40 per cent efficiency, such that roughly 15 terawatts of heat is lost to the environment. Thermoelectric modules could potentially convert part of this low-grade waste heat to electricity. Their efficiency depends on the thermoelectric figure of merit ZT of their material components, which is a function of the Seebeck coefficient, electrical resistivity, thermal conductivity and absolute temperature. Over the past five decades it has been challenging to increase ZT > 1, since the parameters of ZT are generally interdependent. While nanostructured thermoelectric materials can increase ZT > 1 (refs 2-4), the materials (Bi, Te, Pb, Sb, and Ag) and processes used are not often easy to scale to practically useful dimensions. Here we report the electrochemical synthesis of large-area, wafer-scale arrays of rough Si nanowires that are 20-300 nm in diameter. These nanowires have Seebeck coefficient and electrical resistivity values that are the same as doped bulk Si, but those with diameters of about 50 nm exhibit 100-fold reduction in thermal conductivity, yielding ZT = 0.6 at room temperature. For such nanowires, the lattice contribution to thermal conductivity approaches the amorphous limit for Si, which cannot be explained by current theories. Although bulk Si is a poor thermoelectric material, by greatly reducing thermal conductivity without much affecting the Seebeck coefficient and electrical resistivity, Si nanowire arrays show promise as high-performance, scalable thermoelectric materials.

Correlating electrochemical stimulus to structural change in liquid electron microscopy videos using the structural dissimilarity metric.

In-situ liquid cell transmission electron microscopy (LCTEM) with electrical biasing capabilities has emerged as an invaluable tool for directly imaging electrode processes with high temporal and spatial resolution. However, accurately quantifying structural changes that occur on the electrode and subsequently correlating them to the applied stimulus remains challenging. Here, we present structural dissimilarity (DSSIM) analysis as segmentation-free video processing algorithm for locally detecting and quantifying structural change occurring in LCTEM videos. In this study, DSSIM analysis is applied to two in-situ LCTEM videos to demonstrate how to implement this algorithm and interpret the results. We show DSSIM analysis can be used as a visualization tool for qualitative data analysis by highlighting structural changes which are easily missed when viewing the raw data. Furthermore, we demonstrate how DSSIM analysis can serve as a quantitative metric and efficiently convert 3-dimensional microscopy videos to 1-dimenional plots which makes it easy to interpret and compare events occurring at different timepoints in a video. In the analyses presented here, DSSIM is used to directly correlate the magnitude and temporal scale of structural change to the features of the applied electrical bias. ImageJ, Python, and MATLAB programs, including a user-friendly interface and accompanying documentation, are published alongside this manuscript to make DSSIM analysis easily accessible to the scientific community.

Observing the Dynamics of an Electrochemically Driven Active Material with Liquid Electron Microscopy.

Electrochemical liquid electron microscopy has revolutionized our understanding of nanomaterial dynamics by allowing for direct observation of their electrochemical production. This technique, primarily applied to inorganic materials, is now being used to explore the self-assembly dynamics of active molecular materials. Our study examines these dynamics across various scales, from the nanoscale behavior of individual fibers to the micrometer-scale hierarchical evolution of fiber clusters. To isolate the influences of the electron beam and electrical potential on material behavior, we conducted thorough beam-sample interaction analyses. Our findings reveal that the dynamics of these active materials at the nanoscale are shaped by their proximity to the electrode and the applied electrical current. By integrating electron microscopy observations with reaction-diffusion simulations, we uncover that local structures and their formation history play a crucial role in determining assembly rates. This suggests that the emergence of nonequilibrium structures can locally accelerate further structural development, offering insights into the behavior of active materials under electrochemical conditions.

Structure of Geobacter OmcZ filaments suggests extracellular cytochrome polymers evolved independently multiple times.

While early genetic and low-resolution structural observations suggested that extracellular conductive filaments on metal-reducing organisms such as Geobacter were composed of type IV pili, it has now been established that bacterial c-type cytochromes can polymerize to form extracellular filaments capable of long-range electron transport. Atomic structures exist for two such cytochrome filaments, formed from the hexaheme cytochrome OmcS and the tetraheme cytochrome OmcE. Due to the highly conserved heme packing within the central OmcS and OmcE cores, and shared pattern of heme coordination between subunits, it has been suggested that these polymers have a common origin. We have now used cryo-electron microscopy (cryo-EM) to determine the structure of a third extracellular filament, formed from the Geobacter sulfurreducens octaheme cytochrome, OmcZ. In contrast to the linear heme chains in OmcS and OmcE from the same organism, the packing of hemes, heme:heme angles, and between-subunit heme coordination is quite different in OmcZ. A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments. This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins.

Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens were first observed two decades ago, with genetic and biochemical data suggesting that conductive fibres were type IV pili. Recently, an extracellular conductive filament of G. sulfurreducens was found to contain polymerized c-type cytochrome OmcS subunits, not pilin subunits. Here we report that G. sulfurreducens also produces a second, thinner appendage comprised of cytochrome OmcE subunits and solve its structure using cryo-electron microscopy at ~4.3 Å resolution. Although OmcE and OmcS subunits have no overall sequence or structural similarities, upon polymerization both form filaments that share a conserved haem packing arrangement in which haems are coordinated by histidines in adjacent subunits. Unlike OmcS filaments, OmcE filaments are highly glycosylated. In extracellular fractions from G. sulfurreducens, we detected type IV pili comprising PilA-N and -C chains, along with abundant B-DNA. OmcE is the second cytochrome filament to be characterized using structural and biophysical methods. We propose that there is a broad class of conductive bacterial appendages with conserved haem packing (rather than sequence homology) that enable long-distance electron transport to chemicals or other microbial cells.

Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development.

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.

Bacteria pattern spontaneously on periodic nanostructure arrays.

Surface-associated bacteria typically form self-organizing communities called biofilms. Spatial segregation is important for various bacterial processes associated with cellular and community development. Here, we demonstrate bacterial ordering and oriented attachment on the single-cell level induced by nanometer-scale periodic surface features. These surfaces cause spontaneous and distinct patterning phases, depending on their periodicity, which is observed for several strains, both gram positive and negative. This patterning is a general phenomenon that can control natural biofilm organization on the cellular level.

Single crystalline mesoporous silicon nanowires.

Herein we demonstrate a novel electroless etching synthesis of monolithic, single-crystalline, mesoporous silicon nanowire arrays with a high surface area and luminescent properties consistent with conventional porous silicon materials. These porous nanowires also retain the crystallographic orientation of the wafer from which they are etched. Electron microscopy and diffraction confirm their single-crystallinity and reveal the silicon surrounding the pores is as thin as several nanometers. Confocal fluorescence microscopy showed that the photoluminescence (PL) of these arrays emanate from the nanowires themselves, and their PL spectrum suggests that these arrays may be useful as photocatalytic substrates or active components of nanoscale optoelectronic devices.

Field-effect modulation of Seebeck coefficient in single PbSe nanowires.

In this Letter, we present a novel strategy to control the thermoelectric properties of individual PbSe nanowires. Using a field-effect gated device, we were able to tune the Seebeck coefficient of single PbSe nanowires from 64 to 193 microV x K(-1). This direct electrical field control of sigma and S suggests a powerful strategy for optimizing ZT in thermoelectric devices. These results represent the first demonstration of field-effect modulation of the thermoelectric figure of merit in a single semiconductor nanowire. This novel strategy for thermoelectric property modulation could prove especially important in optimizing the thermoelectric properties of semiconductors where reproducible doping is difficult to achieve.

Deep Learning Analysis of Vibrational Spectra of Bacterial Lysate for Rapid Antimicrobial Susceptibility Testing.

Rapid antimicrobial susceptibility testing (AST) is an integral tool to mitigate the unnecessary use of powerful and broad-spectrum antibiotics that leads to the proliferation of multi-drug-resistant bacteria. Using a sensor platform composed of surface-enhanced Raman scattering (SERS) sensors with control of nanogap chemistry and machine learning algorithms for analysis of complex spectral data, bacteria metabolic profiles post antibiotic exposure are correlated with susceptibility. Deep neural network models are able to discriminate the responses of Escherichia coli and Pseudomonas aeruginosa to antibiotics from untreated cells in SERS data in 10 min after antibiotic exposure with greater than 99% accuracy. Deep learning analysis is also able to differentiate responses from untreated cells with antibiotic dosages up to 10-fold lower than the minimum inhibitory concentration observed in conventional growth assays. In addition, analysis of SERS data using a generative model, a variational autoencoder, identifies spectral features in the P. aeruginosa lysate data associated with antibiotic efficacy. From this insight, a combinatorial dataset of metabolites is selected to extend the latent space of the variational autoencoder. This culture-free dataset dramatically improves classification accuracy to select effective antibiotic treatment in 30 min. Unsupervised Bayesian Gaussian mixture analysis achieves 99.3% accuracy in discriminating between susceptible versus resistant to antibiotic cultures in SERS using the extended latent space. Discriminative and generative models rapidly provide high classification accuracy with small sets of labeled data, which enormously reduces the amount of time needed to validate phenotypic AST with conventional growth assays. Thus, this work outlines a promising approach toward practical rapid AST.

Structural Determination of a Filamentous Chaperone to Fabricate Electronically Conductive Metalloprotein Nanowires.

The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.