Cytochromes MtrC Research Articles

Exoelectrogenic behaviors of Shewanella oneidensis MR-1 and cytochrome knock-out mutants on smooth electrodes of different materials

The development of anodes supporting a high rate of current generation stands as a crucial and challenging aspect in microbial electrochemical systems. A well-defined electrode surface morphology is essential for comprehending the impact of material intrinsic properties on microbial current generation, offering molecular insights into the respiration of exoelectrogens with electrodes made of diverse materials. We fabricated smooth electrodes (root mean square roughness below 10 nm) using four materials, each representing a distinct class of materials commonly utilized to construct bioanodes. These electrodes were employed to investigate current generation, electrochemical characteristics, and biofilm formation by Shewanella oneidensis MR-1 and mutants lacking cytochromes MtrC (ΔmtrC), MtrA (ΔmtrA), or CymA (ΔcymA). In reactors inoculated with MR-1, the poly(3,4-ethylenedioxythiophene) polystyrene sulfonate electrodes demonstrated a current density of 12.5 ± 1.1 μA cm-2, surpassing the 3.57 ± 0.50 μA cm-2 observed with indium tin oxide electrodes, while current generation levels by gold and glassy carbon electrodes fell in between. This disparity was not attributed to distinct biofilm formations by MR-1, as it formed stable monolayer biofilms on all types of working electrodes. At least five electron transfer pathways were identified in the MR-1 Vammograms, with the activities of these pathways depending on the electrode materials. The effectiveness of both endogenous and exogenous electron mediators varied with the electrode materials involved. Additionally, a phenazine compound exhibited diverse formal potentials when interacting with the biofilm and different types of electrodes. The mutants displayed varying degrees of impaired current generation and biofilm formation capabilities, with ΔmtrA and ΔcymA being the most affected regardless of the electrode used. However, for a specific mutant, the overall current generation, relative contribution by individual pathways, and biofilm formations underwent significant changes based on the electrode materials employed.

Elongated Riboflavin-Producing Shewanella oneidensis in a Hybrid Biofilm Boosts Extracellular Electron Transfer.

Shewanella oneidensis is able to carry out extracellular electron transfer (EET), although its EET efficiency is largely limited by low flavin concentrations, poor biofilm forming-ability, and weak biofilm conductivity. After identifying an important role for riboflavin (RF) in EET via in vitro experiments, the synthesis of RF is directed to 837.74 ± 11.42µm in S. oneidensis. Molecular dynamics simulation reveals RF as a cofactor that binds strongly to the outer membrane cytochrome MtrC, which is correspondingly further overexpressed to enhance EET. Then the cell division inhibitor sulA, which dramatically enhanced the thickness and biomass of biofilm increased by 155% and 77%, respectively, is overexpressed. To reduce reaction overpotential due to biofilm thickness, a spider-web-like hybrid biofilm comprising RF, multiwalled carbon nanotubes (MWCNTs), and graphene oxide (GO) with adsorption-optimized elongated S. oneidensis, achieve a 77.83-fold increase in power (3736mW m-2 ) relative to MR-1 and dramatically reduce the charge-transfer resistance and boosted biofilm electroactivity. This work provides an elegant paradigm to boost EET based on a synthetic biology strategy and materials science strategy, opens up further opportunities for other electrogenic bacteria.

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion.

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate the binding of MtrA to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). The EET efficiency was found to be inversely correlated with the proximity of the insertion to the heme prosthetic groups. Mutants with decreased EET efficiencies also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular cytochrome MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET efficiencies. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III) respiration conditions correlated with anode reduction. The peptide insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for the regulation of EET.

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.

Proteogenomic analysis of Georgfuchsia toluolica revealed unexpected concurrent aerobic and anaerobic toluene degradation.

Denitrifying Betaproteobacteria play a key role in the anaerobic degradation of monoaromatic hydrocarbons. We performed a multi‐omics study to better understand the metabolism of the representative organism Georgfuchsia toluolica strain G5G6 known as a strict anaerobe coupling toluene oxidation with dissimilatory nitrate and Fe(III) reduction. Despite the genomic potential for degradation of different carbon sources, we did not find sugar or organic acid transporters, in line with the inability of strain G5G6 to use these substrates. Using a proteomics analysis, we detected proteins of fumarate‐dependent toluene activation, membrane‐bound nitrate reductase, and key components of the metal‐reducing (Mtr) pathway under both nitrate‐ and Fe(III)‐reducing conditions. High abundance of the multiheme cytochrome MtrC implied that a porin–cytochrome complex was used for respiratory Fe(III) reduction. Remarkably, strain G5G6 contains a full set of genes for aerobic toluene degradation, and we detected enzymes of aerobic toluene degradation under both nitrate‐ and Fe(III)‐reducing conditions. We further detected an ATP‐dependent benzoyl‐CoA reductase, reactive oxygen species detoxification proteins, and cytochrome c oxidase indicating a facultative anaerobic lifestyle of strain G5G6. Correspondingly, we found diffusion through the septa a substantial source of oxygen in the cultures enabling concurrent aerobic and anaerobic toluene degradation by strain G5G6.

Metal Reduction and Protein Secretion Genes Required for Iodate Reduction by Shewanella oneidensis

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO3 -) as an anaerobic terminal electron acceptor. Microbial IO3 - electron transport pathways are postulated to terminate with nitrate (NO3 -) reductase, which reduces IO3 - as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO3 - reductase is not involved in IO3 - reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO3 - reduction by Shewanella oneidensis with lactate, formate, or H2 as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO3 - reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO3 - reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO3 - reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane β-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO3 - reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO3 - reduction activity. These findings indicate that IO3 - reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO3 - reductase to the S. oneidensis outer membrane.IMPORTANCE Microbial iodate (IO3 -) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO3 - reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO3 - reduction by S. oneidensis On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO3 - reduction by S. oneidensis These findings indicate that the IO3 - electron transport pathway terminates with an as yet unidentified IO3 - reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO3.

Shewanella putrefaciens CN32 outer membrane cytochromes MtrC and UndA reduce electron shuttles to produce electricity in microbial fuel cells

The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c-type cytochromes (OM c-Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c-Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact. Strikingly, the depression of EET by the deletion of mtrC could be partially relieved by acclimation, which might be due to an increase in the transmembrane transport of electron shuttles and/or the activation of other redox proteins. These results suggested that MtrC may be the primary reductase of flavins to ensure fast indirect EET, which plays a crucial role in MFC electricity generation.

High Performance Reduction of H2O2 with an Electron Transport Decaheme Cytochrome on a Porous ITO Electrode.

The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm–2 at 0.4 V vs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV–visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors.

The role of proteins of the outer membrane of Shewanella oneidensis MR-1 in the formation and stabilization of silver sulfide nanoparticles

Silver sulfide nanoparticles stable in aqueous solutions were obtained in presence of the cells of the bacterium Shewanella oneidensis MR-1 in aqueous solution containing an equimolar mixture of AgNO3 and Na2S2O3. Proteins absorbed on the surface of Ag2S nanoparticles were identified for the first time by MALDITOF/TOF. Among these proteins, multiheme cytochromes MtrC and OmcA, as well as the MtrB membrane porin, which forms a complex on the outer cell membrane, were detected. It was shown that an insoluble precipitate consisting of agglomerated Ag2S nanoparticles with a wide size distribution was formed in the absence of the cells. The role of the detected proteins in the mechanism of the formation and stabilization of the Ag2S nanoparticles in the studied system is discussed.

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar‐assisted chemical synthesis. In Shewanella oneidensis MR‐1 (MR‐1), trans‐outer‐membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer‐membrane‐spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR‐1. We show photoreduction of MtrC and OmcA adsorbed on RuII‐dye‐sensitized TiO2 nanoparticles and that these protein‐coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer‐membrane‐associated cytochromes of MR‐1 for solar‐driven microbial synthesis in natural and engineered bacteria.

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

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. 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.

Multiheme Cytochromes and the Bacterial Nanowires of Shewanella oneidensis MR-1: Regulation, Structure, and Extracellular Electron Transport Mechanisms

Dissimilatory metal-reducing bacteria can extract free energy from their environment by performing electron transfer to solid-phase minerals outside the cell. This extracellular electron transport (EET) has important implications in global elemental cycles as well as renewable energy technologies. Among the pathways for EET, bacterial nanowires have received significant attention in the past decade due to their unique ability to mediate long-range electron transport to electron acceptors microns away from the cell surface. Here we report a comprehensive characterization of the composition, structure, and regulatory network that underlies bacterial nanowires from the metal-reducing bacterium Shewanella oneidensis MR-1. Using fluorescent and atomic force techniques, we find that the Shewanella nanowires are extensions of the outer membrane and periplasm that contain multiple multiheme cytochromes. The localization of decaheme cytochromes MtrC and OmcA supports a multistep redox hopping mechanism, allowing long-range electron transport along a membrane network of heme cofactors that line the nanowires. The electron flux resulting from such a mechanism strongly depends on the cytochrome density and topology. Using correlated electron cryo-tomography and in vivo fluorescent microscopy, we are gaining new insight into the localization patterns of cytochromes along nanowires as well as the morphology and the formation mechanism of these structures. Finally, we report our progress on understanding the underlying regulatory network, by testing targeted mutations and analyzing the transcriptome of Shewanella chemostat cultures as they encounter electron acceptor limitation and form nanowires. The transcriptional response includes an increase in the expression of multiheme cytochromes, heme synthesis enzymes, and cytochrome maturation proteins. Our findings on the regulation, ultrastructure and electron transport mechanism help shape a biophysical understanding of these redox-functionalized membrane and vesicular extensions as a microbial strategy for electron transport and energy distribution.

A decahaem cytochrome as an electron conduit in protein-enzyme redox processes.

The decahaem cytochrome MtrC from Shewanella oneidensis MR-1 was employed as a protein electron conduit between a porous indium tin oxide electrode and redox enzymes. Using a hydrogenase and a fumarate reductase, MtrC was shown as a suitable and efficient diode to shuttle electrons to and from the electrode with the MtrC redox activity regulating the direction of the enzymatic reactions.

Flavin Binding to the Deca-heme Cytochrome MtrC: Insights from Computational Molecular Simulation

Certain dissimilatory bacteria have the remarkable ability to use extracellular metal oxide minerals instead of oxygen as terminal electron sinks, using a process known as “extracellular respiration”. Specialized multiheme cytochromes located on the outer membrane of the microbe were shown to be crucial for electron transfer from the cell surface to the mineral. This process is facilitated by soluble, biogenic flavins secreted by the organism for the purpose of acting as an electron shuttle. However, their interactions with the outer-membrane cytochromes are not established on a molecular scale. Here, we study the interaction between the outer-membrane deca-heme cytochrome MtrC from Shewanella oneidensis and flavin mononucleotide (FMN in fully oxidized quinone form) using computational docking. We find that interaction of FMN with MtrC is significantly weaker than with known FMN-binding proteins, but identify a mildly preferred interaction site close to heme 2 with a dissociation constant (Kd) = 490 μM, in good agreement with recent experimental estimates, Kd = 255 μM. The weak interaction with MtrC can be qualitatively explained by the smaller number of hydrogen bonds that the planar headgroup of FMN can form with this protein compared to FMN-binding proteins. Molecular dynamics simulation gives indications for a possible conformational switch upon cleavage of the disulphide bond of MtrC, but without concomitant increase in binding affinities according to this docking study. Overall, our results suggest that binding of FMN to MtrC is reversible and not highly specific, which may be consistent with a role as redox shuttle that facilitates extracellular respiration.

A ROLE OF PROTEINS OF THE OUTER MEMBRANE OF Shewanella oneidensis MR-1 BACTERIA IN FORMATION AND STABILIZATION OF SILVER SULFIDE NANOPARTICLES

Preparations of Ag 2 S nanoparticles obtained in equimolar AgNO3 and Na2S2O3 aqueous solutions in the presence of Shewanella oneidensis MR-1 bacteria cells have been shown to be stable in aqueous solutions. Proteins absorbed on the surface of these nanoparticles were identified for the first time by the MALDI TOF/TOF method. Among them, multi-heme cytochromes MtrC and OmcA, as well as a MtrB membrane protein porin that forms a complex with these cytochromes on the outer cell membrane were detected. It was shown that in the absence of the cells, an insoluble precipitate consisting of agglomerated Ag 2 S nanoparticles with broad size distribution was formed. The role of the identified proteins in the mechanism of the formation and stabilization of the Ag 2 S nanoparticles in this system is discussed.

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.

Single-cell imaging and spectroscopic analyses of Cr(VI) reduction on the surface of bacterial cells.

We investigate the single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic, highly soluble Cr(VI) can be efficiently reduced to less toxic, nonsoluble Cr(2)O(3) nanoparticles by MR-1. Cr(2)O(3) is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr(2)O(3) and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1 surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of MR-1 is wild type > single mutant ΔmtrC or mutant ΔomcA > double mutant (ΔomcA-ΔmtrC). Moreover, our results also suggest that direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may coexist in the reduction processes.

Effects of the Anaerobic Respiration of <i>Shewanella oneidensis</i> MR-1 on the Stability of Extracellular U(VI) Nanofibers

Uranium (VI) is considered to be one of the most widely dispersed and problematic environmental contaminants, due in large part to its high solubility and great mobility in natural aquatic systems. We previously reported that under anaerobic conditions, Shewanella oneidensis MR-1 grown in medium containing uranyl acetate rapidly accumulated long, extracellular, ultrafine U(VI) nanofibers composed of polycrystalline chains of discrete meta-schoepite (UO3·2H2O) nanocrystallites. Wild-type MR-1 finally transformed the uranium (VI) nanofibers to uranium (IV) nanoparticles via further reduction. In order to investigate the influence of the respiratory chain in the uranium transformation process, a series of mutant strains lacking a periplasmic cytochrome MtrA, outer membrane (OM) cytochrome MtrC and OmcA, a tetraheme cytochrome CymA anchored to the cytoplasmic membrane, and a trans-OM protein MtrB, were tested in this study. Although all the mutants produced U(VI) nanofibers like the wild type, the transformation rates from U(VI) nanofibers to U(IV) nanoparticles varied; in particular, the mutant with deletion in tetraheme cytochrome CymA stably maintained the uranium (VI) nanofibers, suggesting that the respiratory chain of S. oneidensis MR-1 is probably involved in the stability of extracellular U(VI) nanofibers, which might be easily treated via the physical processes of filtration or flocculation for the remediation of uranium contamination in sediments and aquifers, as well as the recovery of uranium in manufacturing processes.

Genomic Plasticity Enables a Secondary Electron Transport Pathway in Shewanella oneidensis

Microbial dissimilatory iron reduction is an important biogeochemical process. It is physiologically challenging because iron occurs in soils and sediments in the form of insoluble minerals such as hematite or ferrihydrite. Shewanella oneidensis MR-1 evolved an extended respiratory chain to the cell surface to reduce iron minerals. Interestingly, the organism evolved a similar strategy for reduction of dimethyl sulfoxide (DMSO), which is reduced at the cell surface as well. It has already been established that electron transfer through the outer membrane is accomplished via a complex in which β-barrel proteins enable interprotein electron transfer between periplasmic oxidoreductases and cell surface-localized terminal reductases. MtrB is the β-barrel protein that is necessary for dissimilatory iron reduction. It forms a complex together with the periplasmic decaheme c-type cytochrome MtrA and the outer membrane decaheme c-type cytochrome MtrC. Consequently, mtrB deletion mutants are unable to reduce ferric iron. The data presented here show that this inability can be overcome by a mobile genomic element with the ability to activate the expression of downstream genes and which is inserted within the SO4362 gene of the SO4362-to-SO4357 gene cluster. This cluster carries genes similar to mtrA and mtrB and encoding a putative cell surface DMSO reductase. Expression of SO4359 and SO4360 alone was sufficient to complement not only an mtrB mutant under ferric citrate-reducing conditions but also a mutant that furthermore lacks any outer membrane cytochromes. Hence, the putative complex formed by the SO4359 and SO4360 gene products is capable not only of membrane-spanning electron transfer but also of reducing extracellular electron acceptors.

Analysis of structural MtrC models based on homology with the crystal structure of MtrF

The outer-membrane decahaem cytochrome MtrC is part of the transmembrane MtrCAB complex required for mineral respiration by Shewanella oneidensis. MtrC has significant sequence similarity to the paralogous decahaem cytochrome MtrF, which has been structurally solved through X-ray crystallography. This now allows for homology-based models of MtrC to be generated. The structure of these MtrC homology models contain ten bis-histidine-co-ordinated c-type haems arranged in a staggered cross through a four-domain structure. This model is consistent with current spectroscopic data and shows that the areas around haem 5 and haem 10, at the termini of an octahaem chain, are likely to have functions similar to those of the corresponding haems in MtrF. The electrostatic surfaces around haem 7, close to the β-barrels, are different in MtrF and MtrC, indicating that these haems may have different potentials and interact with substrates differently.