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.

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes.

Rapid and directed electron transfer (ET) is essential for biological processes. While the rates of ET over 1-2 nm in proteins can largely be described by simplified nonadiabatic theory, it is not known how these processes scale to microscopic distances. We generated crystalline lattices of Small Tetraheme Cytochromes (STC) forming well-defined, three-dimensional networks of closely spaced redox centers that appear to be nearly ideal for multistep ET. Electrons were injected into specific locations in the STC crystals by direct photoreduction, and their redistribution was monitored by imaging. The results demonstrate ET over mesoscopic to microscopic (∼100 µm) distances through sequential hopping in a biologically based heme network. We estimate that a hypothetical "nanowire" composed of crystalline STC with a cross-section of about 100 cytochromes could support the anaerobic respiration of a Shewanella cell. The crystalline lattice insulates mobile electrons from oxidation by O2, as compared to those in cytochromes in solution, potentially allowing for efficient delivery of current without production of reactive oxygen species. The platform allows direct tests of whether the assumptions based on short-range ET hold for sequential ET over mesoscopic distances. We estimate that the interprotein ET across 6 Å between hemes in adjacent proteins was about 105 s-1, about 100-fold slower than expectations based on simplified theory. More detailed analyses implied that additional factors, possibly contributed by the crystal lattice, may strongly impact mesoscale ET mainly by increasing the reorganizational energy of interprotein ET, which suggests design strategies for engineering improved nanowires suitable for future bioelectronic materials.

Influence of the Spatial Distribution of Cationic Functional Groups at Nanoparticle Surfaces on Bacterial Viability and Membrane Interactions.

While positively charged nanomaterials induce cytotoxicity in many organisms, much less is known about how the spatial distribution and presentation of molecular surface charge impact nanoparticle-biological interactions. We systematically functionalized diamond nanoparticle surfaces with five different cationic surface molecules having different molecular structures and conformations, including four small ligands and one polymer, and we then probed the molecular-level interaction between these nanoparticles and bacterial cells. Shewanella oneidensis MR-1 was used as a model bacterial cell system to investigate how the molecular length and conformation of cationic surface charges influence their interactions with the Gram-negative bacterial membranes. Nuclear magnetic resonance (NMR) and X-ray photoelectron spectroscopy (XPS) demonstrate the covalent modification of the nanoparticle surface with the desired cationic organic monolayers. Surprisingly, bacterial growth-based viability (GBV) and membrane damage assays both show only minimal biological impact by the NPs functionalized with short cationic ligands within the concentration range tested, yet NPs covalently linked to a cationic polymer induce strong cytotoxicity, including reduced cellular viability and significant membrane damage at the same concentration of cationic groups. Transmission electron microscopy (TEM) images of these NP-exposed bacterial cells show that NPs functionalized with cationic polymers induce significant membrane distortion and the production of outer membrane vesicle-like features, while NPs bearing short cationic ligands only exhibit weak membrane association. Our results demonstrate that the spatial distribution of molecular charge plays a key role in controlling the interaction of cationic nanoparticles with bacterial cell membranes and the subsequent biological impact. Nanoparticles functionalized with ligands having different lengths and conformations can have large differences in interactions even while having nearly identical zeta potentials. While the zeta potential is a convenient and commonly used measure of nanoparticle charge, it does not capture essential differences in molecular-level nanoparticle properties that control their biological impact.

Single-Cell Mass Spectroscopic Analysis for Quantifying Active Metabolic Pathway Heterogeneity in a Bacterial Population on an Electrode.

Microbial electrochemical catalysis based on respiratory reactions coupled with extracellular electron transport (EET), which is critical for bioenergy applications, strongly depends on the biocompatibility of the electrode material. However, the comparison of materials for such physiological responses has been difficult because of the lack of a quantitative assay for characterizing cellular metabolism at the electrode surface. Here, we developed a single-cell analysis method specific for the cells attached to the electrode to quantify active metabolic pathway heterogeneity as an index of physiological cell/electrode interaction, which generally increases with metabolic robustness in the microbial population. Nanoscale secondary ion mass spectrometry followed by microbial current production with model EET-capable bacteria, Shewanella oneidensis MR-1 and its mutant strains lacking carbon assimilation pathways, showed that different active metabolic pathways resulted in nearly identical 13C/15N assimilation ratios for individual cells in the presence of isotopically labeled nutrients, demonstrating a correlation between the 13C/15N ratio and the active metabolic pathway. Compared to the nonelectrode conditions, the heterogeneity of the assimilated 13C/15N ratio was highly enhanced on the electrode surface, suggesting that the metabolic robustness of the microbial population increased through the electrochemical interaction with the electrode. The present methodology enables us to quantitatively compare and screen electrode materials that increase the robustness of microbial electrocatalysis.

Probing Microbial Extracellular Respiration Ability Using Riboflavin.

Electrochemically active bacteria (EAB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, and thus play a great role in the fields of environment, energy, and geosciences. However, rapid and accurate quantification of the EET ability of EAB is still challenging. In this work, we develop a riboflavin-based fluorescence method for facile, accurate, and in situ measurement of the EET ability of EAB. This method is successfully used to quantify the single-cellular EET ability of Geobacter sulfurreducens DL-1 (60.29 ± 13.02 fA) and Shewanella oneidensis MR-1 (2.11 ± 0.47 fA), the two widely present EAB in the environment. It also enables quantitative identification of EET-related c-type cytochromes in the outer membrane of S. oneidensis MR-1. This method provides a useful tool to rapidly identify EAB in diverse environments and elucidate their electron transfer mechanisms.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.

Soluble electron acceptors affect bioluminescence from Shewanella woodyi.

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO3 or CoCl2 to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of <0.2 V vs Normal Hydrogen Electrode (NHE) repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicated that the luminescence intensity from S. woodyi could be systematically reduced using these two soluble electron acceptors, making S. woodyi a potential model bacterium for whole-cell luminescence bioelectrochemical sensor applications.

Pellicle development of Shewanella oneidensis is an aerotaxis-piloted and energy-dependent process.

Pellicles are biofilms found at the air-liquid interface and are widely distributed in natural environments. In this study, a simple pellicle detection method was established, and using this new method, the pellicle formation activities of Shewanella oneidensis MR-1 and its 42 cytochrome c mutants were analysed. The results showed that the pellicle was initiated at very early stages of incubation. Aerotaxis was the major external factor, while energy acquirement was the main internal factor for pellicle initiation. Among the 42 cytochrome c mutants, 17 mutants, including those deficient in aerobic respiration, sulfur or sulfite/sulfate respiration, nitrite respiration, metal respiration, DMSO respiration and fumarate respiration, exhibited delayed pellicle initiation. The results suggest that S. oneidensis utilizes the electron acceptors simultaneously under anoxic conditions and that the disruption of any of these anaerobic respiration routes would retard pellicle initiation.

The role of electron shuttle enhances Fe(III)-mediated reduction of Cr(VI) by Shewanella oneidensis MR-1.

Chromate is one of the hazardous toxic pollutants. Reduction of Cr(VI) to Cr(III) has shown to reduce the toxicity of chromate. This work examined the reduction of Cr(VI) using an anaerobic batch cultures of Shewanella oneidensis MR-1 containing Fe(III). To do so, 10 mg/L Cr(VI) was reduced to Cr(III) within 3 days along with the oxidization of Fe(II) to Fe(III). The removal rate of Cr(VI) increased with increasing the concentration of Fe(III). In the absence of Cr(VI), the Fe(II) concentration of the batch culture increased with the growth of S. oneidensis MR-1. These data showed that S. oneidensis MR-1 could reduce Fe(III) into Fe(II), resulting in reduction of Cr(VI) to Cr(III). During this process, the anthraquinone-2,6-disulfonate (AQDS) acted as an electron shuttle. Microscopic analysis showed that Cr(VI) had toxic effects on S. oneidensis MR-1 due to the appearance of Cr species on the bacterial surface. Cr2O3 or Cr(OH)3 precipitates formed during Cr(VI) reduction was identified using X-ray photoelectron spectroscopy. The AQDS as an electron shuttle enhanced the Cr(VI) reduction by S. oneidensis MR-1. Microbial reduction of Cr(VI) can be a useful technique for Cr detoxification.

Living and Conducting: Coating Individual Bacterial Cells with In†Situ Formed Polypyrrole.

Coating individual bacterial cells with conjugated polymers to endow them with more functionalities is highly desirable. Here, we developed an in situ polymerization method to coat polypyrrole on the surface of individual Shewanella oneidensis MR-1, Escherichia coli, Ochrobacterium anthropic or Streptococcus thermophilus. All of these as-coated cells from different bacterial species displayed enhanced conductivities without affecting viability, suggesting the generality of our coating method. Because of their excellent conductivity, we employed polypyrrole-coated Shewanella oneidensis MR-1 as an anode in microbial fuel cells (MFCs) and found that not only direct contact-based extracellular electron transfer is dramatically enhanced, but also the viability of bacterial cells in MFCs is improved. Our results indicate that coating individual bacteria with conjugated polymers could be a promising strategy to enhance their performance or enrich them with more functionalities.

Wiring Bacterial Electron Flow for Sensitive Whole-Cell Amperometric Detection of Riboflavin.

A whole-cell bioelectrochemical biosensing system for amperometric detection of riboflavin was developed. A "bioelectrochemical wire" (BW) consisting of riboflavin and cytochrome C between Shewanella oneidensis MR-1 and electrode was characterized. Typically, a strong electrochemical response was observed when riboflavin (VB2) was added to reinforce this BW. Impressively, the electrochemical response of riboflavin with this BW was over 200 times higher than that without bacteria. Uniquely, this electron rewiring process enabled the development of a biosensing system for amperometric detection of riboflavin. Remarkably, this amperometric method showed high sensitivity (LOD = 2.2 nM, S/N = 3), wide linear range (5 nM ∼ 10 µM, 3 orders of magnitude), good selectivity, and high resistance to interferences. Additionally, the developed amperometric method featured good stability and reusability. It was further applied for accurate and reliable determination of riboflavin in real conditions including food, pharmaceutical, and clinical samples without pretreatment. Both the cost-effectiveness and robustness make this whole-cell amperometric system ideal for practical applications. This work demonstrated the power of bioelectrochemical signal amplification with exoelectrogen and also provided a new idea for development of versatile whole-cell amperometric biosensors.

Sulfur-Mediated Electron Shuttling Sustains Microbial Long-Distance Extracellular Electron Transfer with the Aid of Metallic Iron Sulfides.

In addition to serving as an energy source for microbial growth, iron sulfides are proposed to act as naturally occurring electrical wires that mediate long-distance extracellular electron transfer (EET) and bridge spatially discrete redox environments. These hypothetical EET reactions stand on the abilities of microbes to use the interfacial electrochemistry of metallic/semiconductive iron sulfides to maintain metabolisms; however, the mechanisms of these phenomena remain unexplored. To obtain insight into EET to iron sulfides, we monitored EET at the interface between Shewanella oneidensis MR-1 cells and biomineralized iron sulfides in an electrochemical cell. Respiratory current steeply increased with the concomitant formation of poorly crystalline mackinawite (FeS) minerals, indicating that S. oneidensis has the ability to exploit extracellularly formed metallic FeS for long-distance EET. Deletion of major proteins of the metal-reduction (Mtr) pathway (OmcA, MtrC, CymA, and PilD) caused only subtle effects on the EET efficiency, a finding that sharply contrasts the majority of studies that report that the Mtr pathway is indispensable for the reduction of metal oxides and electrodes. The gene expression analyses of polysulfide and thiosulfate reductase suggest the existence of a sulfur-mediated electron-shuttling mechanism by which HS(-) ions and water-soluble polysulfides (HS(n)(-), where n ≥ 2) generated in the periplasmic space deliver electrons from cellular metabolic processes to cell surface-associated FeS. The finding of this Mtr-independent pathway indicates that polysulfide reductases complement the function of outer-membrane cytochromes in EET reactions and, thus, significantly expand the number of microbial species potentially capable of long-distance EET in sulfur-rich anoxic environments.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes.

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Nanoparticle facilitated extracellular electron transfer in microbial fuel cells.

Microbial fuel cells (MFCs) have been the focus of substantial research interest due to their potential for long-term, renewable electrical power generation via the metabolism of a broad spectrum of organic substrates, although the low power densities have limited their applications to date. Here, we demonstrate the potential to improve the power extraction by exploiting biogenic inorganic nanoparticles to facilitate extracellular electron transfer in MFCs. Simultaneous short-circuit current recording and optical imaging on a nanotechnology-enabled platform showed substantial current increase from Shewanella PV-4 after the formation of cell/iron sulfide nanoparticle aggregates. Detailed characterization of the structure and composition of the cell/nanoparticle interface revealed crystalline iron sulfide nanoparticles in intimate contact with and uniformly coating the cell membrane. In addition, studies designed to address the fundamental mechanisms of charge transport in this hybrid system showed that charge transport only occurred in the presence of live Shewanella, and moreover demonstrated that the enhanced current output can be attributed to improved electron transfer at cell/electrode interface and through the cellular-networks. Our approach of interconnecting and electrically contacting bacterial cells through biogenic nanoparticles represents a unique and promising direction in MFC research and has the potential to not only advance our fundamental knowledge about electron transfer processes in these biological systems but also overcome a key limitation in MFCs by constructing an electrically connected, three-dimensional cell network from the bottom-up.

Uranium reduction by Shewanella oneidensis MR-1 as a function of NaHCO3 concentration: surface complexation control of reduction kinetics.

It is crucial to determine the controls on the kinetics of U(VI) bioreduction in order to understand and model the fate and mobility of U in groundwater systems and also to enhance the effectiveness of U bioremediation strategies. In this study, we measured the rate of U(VI) reduction by Shewanella oneidensis strain MR-1 as function of NaHCO3 concentration. The experiments demonstrate that increasing concentrations of NaHCO3 in the system lead to slower U(VI) reduction kinetics. The NaHCO3 concentration also strongly affects the speciation of U(VI) on the bacterial cell envelope. We used a thermodynamic surface complexation modeling approach to determine the speciation and concentration of U(VI) adsorbed onto the bacteria as a function of the NaHCO3 concentration in the experimental systems. We observed a strong positive correlation between the measured U(VI) reduction rates and the calculated total concentration of U(VI) surface complexes formed on the bacterial cell envelope. This positive correlation indicates that the speciation and concentration of U(VI) adsorbed on the bacterial cell envelope control the kinetics of U(VI) bioreduction under the experimental conditions. The results of this study serve as a basis for developing speciation-based kinetic rate laws for enzymatic reduction of U(VI) by bacteria.

Occurrence of a bacterial membrane microdomain at the cell division site enriched in phospholipids with polyunsaturated hydrocarbon chains.

In this study, we found that phospholipids containing an eicosapentaenyl group form a novel membrane microdomain at the cell division site of a Gram-negative bacterium, Shewanella livingstonensis Ac10, using chemically synthesized fluorescent probes. The occurrence of membrane microdomains in eukaryotes and prokaryotes has been demonstrated with various imaging tools for phospholipids with different polar headgroups. However, few studies have focused on the hydrocarbon chain-dependent localization of membrane-resident phospholipids in vivo. We previously found that lack of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid found at the sn-2 position of glycerophospholipids, causes a defect in cell division after DNA replication of S. livingstonensis Ac10. Here, we synthesized phospholipid probes labeled with a fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) group to study the localization of EPA-containing phospholipids by fluorescence microscopy. A fluorescent probe in which EPA was bound to the glycerol backbone via an ester bond was found to be unsuitable for imaging because EPA was released from the probe by in vivo hydrolysis. To overcome this problem, we synthesized hydrolysis-resistant ether-type phospholipid probes. Using these probes, we found that the fluorescence localized between two nucleoids at the cell center during cell division when the cells were grown in the presence of the eicosapentaenyl group-containing probe (N-NBD-1-oleoyl-2-eicosapentaenyl-sn-glycero-3-phosphoethanolamine), whereas this localization was not observed with the oleyl group-containing control probe (N-NBD-1-oleoyl-2-oleyl-sn-glycero-3-phosphoethanolamine). Thus, phospholipids containing an eicosapentaenyl group are specifically enriched at the cell division site. Formation of a membrane microdomain enriched in EPA-containing phospholipids at the nucleoid occlusion site probably facilitates cell division.

Dynamic modeling of aerobic growth of Shewanella oneidensis. Predicting triauxic growth, flux distributions, and energy requirement for growth.

A model-based analysis is conducted to investigate metabolism of Shewanella oneidensis MR-1 strain in aerobic batch culture, which exhibits an intriguing growth pattern by sequentially consuming substrate (i.e., lactate) and by-products (i.e., pyruvate and acetate). A general protocol is presented for developing a detailed network-based dynamic model for S. oneidensis based on the Lumped Hybrid Cybernetic Model (L-HCM) framework. The L-HCM, although developed from only limited data, is shown to accurately reproduce exacting dynamic metabolic shifts, and provide reasonable estimates of energy requirement for growth. Flux distributions in S. oneidensis predicted by the L-HCM compare very favorably with (13)C-metabolic flux analysis results reported in the literature. Predictive accuracy is enhanced by incorporating measurements of only a few intracellular fluxes, in addition to extracellular metabolites. The L-HCM developed here for S. oneidensis is consequently a promising tool for the analysis of intracellular flux distribution and metabolic engineering.

Regulation of cytochrome c- and quinol oxidases, and piezotolerance of their activities in the deep-sea piezophile Shewanella violacea DSS12 in response to growth conditions.

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.

Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system.

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

Soluble electron shuttles can mediate energy taxis toward insoluble electron acceptors.

Shewanella species grow in widely disparate environments and play key roles in elemental cycling, especially in environments with varied redox conditions. To obtain a system-level understanding of Shewanella's robustness and versatility, the complex interplay of cellular growth, metabolism, and transport under conditions of limiting carbon sources, energy sources, and electron acceptors must be elucidated. In this paper, population-level taxis of Shewanella oneidensis MR-1 cells in the presence of a rate-limiting, insoluble electron acceptor was investigated. A novel mechanism, mediated energy taxis, is proposed by which Shewanella use riboflavin as both an electron shuttle and an attractant to direct cell movement toward local sources of insoluble electron acceptors. The cells secrete reduced riboflavin, which diffuses to a nearby particle containing an insoluble electron acceptor and is oxidized. The oxidized riboflavin then diffuses away from the particle, establishing a spatial gradient that draws cells toward the particle. Experimental and modeling results are presented to support this mechanism. S. oneidensis MR-1 cells inoculated into a uniform dispersion of MnO(2) particles in dilute agar exhibited taxis outward, creating a clear zone within which riboflavin was detected by mass spectrometry. Cells inoculated into dilute agar containing oxidized riboflavin similarly exhibited taxis, rapidly forming an expanding zone of reduced riboflavin. A mathematical model based on the proposed mechanism was able to predict experimental trends, including how concentrations of riboflavin and insoluble electron acceptors (e.g., MnO(2)) affected tactic cell migration.