Mixed Conducting Polymers Alter Electron Transfer Thermodynamics to Boost Current Generation from Electroactive Microbes.

Electroactive microbes that can release or take up electrons are essential components of nearly every ecological niche and are powerful tools for the development of alternative energy technologies. Small-molecule mediators are critical for this electron transfer but remain difficult to study and engineer because they perform concerted two-electron transfer in native systems but only individual, one-electron transfers in electrochemical studies. Here, we report that electrode modification with ion- and electron-conductive polymers yields biosimilar, concerted two-electron transfer from Shewanella oneidensis via flavin mediators. S. oneidensis biofilms on these polymers show significantly improved per-microbe current generation and morphologies that more closely resemble native systems, setting a new paradigm for the study and optimization of these electron transfer processes. The unprecedented concerted electron transfer was found to be due to altered mediator electron transfer thermodynamics, enabling biologically relevant studies of electroactive biofilms in the lab for the first time. These important findings pave the way for a complete understanding of the ecological role of electroactive microbes and their broad application in sustainable technologies.

Interface Visualization of Bio-material Interaction Via Cryo-AEM Using the Biosynthesis of Iron-Based Nanoparticles as a Model.

Although interaction between organisms and nonorganisms is vital in environmental processes, it is difficult to characterize at nanoscale resolution. Biosynthesis incorporates intracellular and extracellular processes involving crucial interfacial functions and electron and substance transfer processes, especially on the inorganic-organic interface. This work chooses the biosynthesis of iron-based nanoparticles (nFe) as a model for biomaterial interaction and employs Cryo-AEM (i.e., S/TEM, EELS, and EDS analysis based on sample preparation with cryo-transfer holder system), combined with CV, Raman, XPS, and FTIR to reveal the inorganic-organic interface process. The inorganic-organic interactions in the biosynthesis of iron-based nanoparticles by Shewanella oneidensis MR-1 (M-nFe) were characterized by changes in electron cloud density, and the corresponding chemical shifts of Fe and C EELS edges confirm that M-nFe acquires electrons from MR-1 on the interface. Capturing intact filamentous-like, slightly curved, and bundled structure provides solid evidence of a "circuit channel" for electron transfer between organic and inorganic interface. CV results also confirm that adding M-nFe can enhance electron transfer from MR-1 to ferric ions. A mechanism for the synthesis of M-nFe with MR-1 based on intracellular and extracellular conditions under facultative anaerobic was visualized, providing a protocol for investigating the organic-inorganic interface.

Colony-Based Electrochemistry Reveals Electron Conduction Mechanisms Mediated by Cytochromes and Flavins in Shewanella oneidensis.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Mechanism of Reduction of Aqueous U(V)-dpaea and Solid-Phase U(VI)-dpaea Complexes: The Role of Multiheme c-Type Cytochromes.

The biological reduction of soluble U(VI) complexes to form immobile U(IV) species has been proposed to remediate contaminated sites. It is well established that multiheme c-type cytochromes (MHCs) are key mediators of electron transfer to aqueous phase U(VI) complexes for bacteria such as Shewanella oneidensis MR-1. Recent studies have confirmed that the reduction proceeds via a first electron transfer forming pentavalent U(V) species that readily disproportionate. However, in the presence of the stabilizing aminocarboxylate ligand, dpaea2- (dpaeaH2═bis(pyridyl-6-methyl-2-carboxylate)-ethylamine), biologically produced U(V) persisted in aqueous solution at pH 7. We aim to pinpoint the role of MHC in the reduction of U(V)-dpaea and to establish the mechanism of solid-phase U(VI)-dpaea reduction. To that end, we investigated U-dpaea reduction by two deletion mutants of S. oneidensis MR-1-one lacking outer membrane MHCs and the other lacking all outer membrane MHCs and a transmembrane MHC-and by the purified outer membrane MHC, MtrC. Our results suggest that solid-phase U(VI)-dpaea is reduced primarily by outer membrane MHCs. Additionally, MtrC can directly transfer electrons to U(V)-dpaea to form U(IV) species but is not strictly necessary, underscoring the primary involvement of outer membrane MHCs in the reduction of this pentavalent U species but not excluding that of periplasmic MHCs.

A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1.

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 µA·cm-2 (ΔmtrA/omcA/mtrC), nearly equivalent to that of WT cells (194 µA·cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in ΔcymA and ΔmtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 µA·cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

Accumulation and Pulse Electron Paramagnetic Resonance Spectroscopic Investigation of the 4-Oxidobenzyl Radical Generated in the Radical S-Adenosyl-l-methionine Enzyme HydG.

The radical S-adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN- ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB•), which is the precursor to the HydG p-cresol byproduct. Native HydG only generates a small amount of 4-OB•, limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB• is significantly increased in reactions using HydG variants, in which the "dangler Fe" to which CO and CN- bind is missing or substituted by a redox-inert Zn2+ ion. This allows for the detailed characterization of 4-OB• using high-field EPR and electron nuclear double resonance spectroscopy to extract its g-values and 1H/13C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB• models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB•. Overall, our results depict a clearer electronic structure of the transient 4-OB• radical and provide new insights into the radical SAM chemistry of HydG.

Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF.

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.

Identification dehydratase domains from Schizochytrium sp. and Shewanella sp. and distinct functions in biosynthesis of fatty acids.

Polyunsaturated fatty acid (PUFA) synthase is a special and effective enzyme for PUFA synthesis, and dehydratase (DH) domain played a crucial role in it. In this work, we compared four different DH domains from different strains (Schizochytrium sp. HX-308 and Shewanella sp. BR-2) and different gene clusters. First bioinformatics analysis showed that DH1, 2 and DH3 were similar to FabA and PKS-DH, respectively, and all of them got a hot-dog structure. Second, four DH domains were expressed in Escherichia coli that increased biomass. Especially, Schi-DH1,2 presented the highest dry cell weight of 2.3 g/L which was 1.62 times of that of control. Fatty acids profile analysis showed that DH1,2 could enhance the percentage of unsaturated fatty acids, especially DH1,2 from Schizochytrium sp., while DH3 benefited for the saturated fatty acid biosynthesis. Furthermore, five kinds of fatty acids were added to the medium to study the substrate preferences. Results revealed that DH1,2 domain preferred to acting on C16:0, while DH3 domain trended acting on C14:0 and C15:0, which illustrated DH from different clusters do have specific substrate preference. Besides, DH expression could save the cell growth inhibition by mid-chain fatty acids. This study provided more information about the catalysis mechanism of polyunsaturated fatty acid synthase and might promote the modification study based on this enzyme.

Electrochemical Characteristics of Shewanella loihica PV-4 on Reticulated Vitreous Carbon (RVC) with Different Potentials Applied.

The current output of an anodic bioelectrochemical system (BES) depends upon the extracellular electron transfer (EET) rate from electricigens to the electrodes. Thus, investigation of EET mechanisms between electricigens and solid electrodes is essential. Here, reticulated vitreous carbon (RVC) electrodes are used to increase the surface available for biofilm formation of the known electricigen Shewanella loihica PV-4, which is limited in conventional flat electrodes. S. loihica PV-4 utilizes flavin-mediated EET at potential lower than the outer membrane cytochromes (OMC), while at higher potential, both direct electron transfer (DET) and mediated electron transfer (MET) contribute to the current output. Results show that high electrode potential favors cell attachment on RVC, which enhances the current output. DET is the prevailing mechanism in early biofilm, while the contribution of MET to current output increased as the biofilm matured. Electrochemical analysis under starvation shows that the mediators could be confined in the biofilm. The morphology of biofilm shows bacteria distributed on the top layer of honeycomb structures, preferentially on the flat areas. This study provides insights into the EET pathways of S. loihica PV-4 on porous RVC electrodes at different biofilm ages and different set potential, which is important for the design of real-world BES.

Combining two optimized and affordable methods to assign chemoreceptors to a specific signal.

Chemotaxis allows bacteria to detect specific compounds and move accordingly. This pathway involves signal detection by chemoreceptors (MCPs). Attributing a chemoreceptor to a ligand is difficult because there is a lot of redundancy in the MCPs that recognize a single ligand. We propose a methodology to define which chemoreceptors bind a given ligand. First, an MCP is overproduced to increase sensitivity to the ligand(s) it recognizes, thus promoting accumulation of cells around an agarose plug containing a low attractant concentration. Second, the ligand-binding domain (LBD) of the chemoreceptor is fused to maltose-binding protein (MBP), which facilitates purification and provides a control for a thermal shift assay (TSA). An increase in the melting temperature of the LBD in the presence of the ligand indicates that the chemoreceptor directly binds it. We showed that overexpression of two Shewanella oneidensis chemoreceptors (SO_0987 and SO_1056) promoted swimming toward an agarose plug containing a low concentration of chromate. The LBD of each of the two chemoreceptors was fused to MBP. A TSA revealed that only the LBD from SO_1056 had its melting temperature increased by chromate. In conclusion, we describe an efficient approach to define chemoreceptor-ligand pairs before undertaking more-sophisticated biochemical and structural studies.

Seaweed-associated heterotrophic bacteria: new paradigm of prospective anti-infective and anticancer agents.

Ever since the development of the first antibiotic compound with anticancer potential, researchers focused on isolation and characterization of prospective microbial natural products with potential anti-infective and anticancer activities. The present work describes the production of bioactive metabolites by heterotrophic bacteria associated with intertidal seaweeds with potential anti-infective and anticancer activities. The bacteria were isolated in a culture-dependent method and were identified as Shewanella algae MTCC 12715 (KX272635) and Bacillus amyloliquefaciens MTCC 12716 (KX272634) based on combined phenotypic and genotypic methods. Further, the bacteria were screened for their ability to inhibit drug-resistant infectious pathogens and prevent cell proliferation of human liver carcinoma (HepG2) and breast cancer (MCF7) cell lines, without affecting the normal cells. Significant anti-infective activity was observed with bacterial cells and their organic extracts against broad-spectrum multidrug-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa with minimum inhibitory concentration ≤ 3.0 µg mL-1 as compared to the antibiotic agents' chloramphenicol and ampicillin, which were active at ≥ 6.25 mg mL-1. The extracts also exhibited anticancer activity in a dose-responsive pattern against HepG2 (with IC50, half maximal inhibitory concentration ~ 78-83 µg mL-1) and MCF7 (IC50 ~ 45-48 µg mL-1) on tetrazolium bromide screening assay with lesser cytotoxic effects on normal fibroblast (L929) cell lines (IC50 > 100 µg mL-1). The results revealed that seaweed-associated heterotrophic bacteria could occupy a predominant role for a paradigm shift towards the development of prospective anti-infective and anticancer agents.

Macrocyclic polyketides with siderophore mode of action from marine heterotrophic Shewanella algae: Prospective anti-infective leads attenuate drug-resistant pathogens.

AIMS: Biotechnological and chemical characterization of previously undescribed homologous siderophore-type macrocyclic polyketides from heterotrophic Shewanella algae Microbial Type Culture Collection (MTCC) 12715 affiliated with Rhodophycean macroalga Hypnea valentiae of marine origin, with significant anti-infective potential against drug-resistant pathogens. METHODS AND RESULTS: The heterotrophic bacterial strain in symbiotic association with intertidal macroalga H. valentiae was isolated to homogeneity in a culture-dependent method and screened for bioactivities by spot-over-lawn assay. The bacterial organic extract was purified and characterized by extensive chromatographic and spectroscopic methods, respectively, and was assessed for antibacterial activities with disc diffusion and microtube dilution methods. The macrocyclic polyketide compounds exhibited wide-spectrum of anti-infective potential against clinically significant vancomycin-resistant Enterococcus faecalis (VREfs), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Klebsiella pneumonia with minimum inhibitory concentration of about 1-3 µg ml-1 , insomuch as the antibiotics chloramphenicol and ampicillin were active at ≥6·25 µg ml-1 . The studied compounds unveiled Fe3+ chelating activity, which designated that their prospective anti-infective activities against the pathogens could be due to their siderophore mechanism of action. In support of that, the bacterium exhibited siderophore production on bioassay involving the cast upon culture agar plate, and the presence of siderophore biosynthetic gene (≈1000 bp) (MF 981936) further corroborated the inference. In silico molecular modelling with penicillin-binding protein (PBP2a) coded by mecA genes of MRSA (docking score -11·68 to -12·69 kcal mol-1 ) verified their in vitro antibacterial activities. Putative biosynthetic pathway of macrocyclic polyketides through stepwise decarboxylative condensation initiated by malonate-acyl carrier protein further validated their structural and molecular attributes. CONCLUSIONS: The studied siderophore-type macrocyclic polyketides from S. algae MTCC 12715 with significant anti-infective potential could be considered as promising candidates for pharmaceutical and biotechnological applications, especially against emerging multidrug-resistant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study exhibited the heterotrophic bacteria in association with intertidal macroalga as propitious biological resources to biosynthesize novel antibacterial agents.

Chemical mining of heterotrophic Shewanella algae reveals anti-infective potential of macrocyclic polyketides against multidrug-resistant pathogens.

Heterotrophic Gamma-proteobacterium Shewanella algae MTCC 12715, associated with an intertidal red algae Hypnea valentiae, presented broad-spectra of antibacterial activities against pathogenic bacteria bringing about nosocomial infection. Bioassay-guided fractionation of the bacterial crude extract resulted in two undescribed macrocyclic polyketide analogs, with anti-infective activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis (MIC 3.1-5.0 µg/mL). In order to identify the polyketide biosynthetic machinery termed type-I polyketide synthase (pks-I) encoding biologically active secondary metabolites in this strain, the ketosynthase-coding regions of DNA with ≈700 bp size, were amplified, and the partial sequence was submitted in the GenBank (accession number MH157093). The titled compounds were classified under macrocyclic polyketides bearing dodecahydropyrano-trioxacyclooctadecine-dione and trioxo-octadecahydro-1H-benzo[o]tetraoxacyclopentacosine-carboxylate functionalities. Structure-activity correlation analysis displayed that hydrophobic descriptor of the studied compounds could play a prominent role in its anti-infective property against the opportunistic pathogens. Further, in silico molecular docking studies were performed in the allosteric sites of penicillin-binding protein (PBP2a) coded by mecA genes of MRSA, and the best binding pose for each compound (docking score -8.47 kcal/mol and -9.58 kcal/mol, respectively) could be correlated with their in vitro antibacterial activities. The pks-I assisted biosynthetic pathway of macrocyclic polyketides through step-wise decarboxylative condensation initiated by malonate-acyl carrier protein corroborated their structural attributes. Chemical mining of the studied macroalgae-associated heterotrophic bacterium thus revealed the promising antagonistic properties of macrocyclic polyketides isolated from Shewanella algae MTCC 12715 against multidrug-resistant pathogens.

Bacterial extracellular electron transfer: a powerful route to the green biosynthesis of inorganic nanomaterials for multifunctional applications.

Synthesis of inorganic nanomaterials such as metal nanoparticles (MNPs) using various biological entities as smart nanofactories has emerged as one of the foremost scientific endeavors in recent years. The biosynthesis process is environmentally friendly, cost-effective and easy to be scaled up, and can also bring neat features to products such as high dispersity and biocompatibility. However, the biomanufacturing of inorganic nanomaterials is still at the trial-and-error stage due to the lack of understanding for underlying mechanism. Dissimilatory metal reduction bacteria, especially Shewanella and Geobacter species, possess peculiar extracellular electron transfer (EET) features, through which the bacteria can pump electrons out of their cells to drive extracellular reduction reactions, and have thus exhibited distinct advantages in controllable and tailorable fabrication of inorganic nanomaterials including MNPs and graphene. Our aim is to present a critical review of recent state-of-the-art advances in inorganic biosynthesis methodologies based on bacterial EET using Shewanella and Geobacter species as typical strains. We begin with a brief introduction about bacterial EET mechanism, followed by reviewing key examples from literatures that exemplify the powerful activities of EET-enabled biosynthesis routes towards the production of a series of inorganic nanomaterials and place a special emphasis on rationally tailoring the structures and properties of products through the fine control of EET pathways. The application prospects of biogenic nanomaterials are then highlighted in multiple fields of (bio-) energy conversion, remediation of organic pollutants and toxic metals, and biomedicine. A summary and outlook are given with discussion on challenges of bio-manufacturing with well-defined controllability.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

The degradation activities for three seaweed polysaccharides of Shewanella sp. WPAGA9 isolated from deep-sea sediments.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

Enhanced Light-Driven Hydrogen Production by Self-Photosensitized Biohybrid Systems.

Storage of solar energy as hydrogen provides a platform towards decarbonizing our economy. One emerging strategy for the production of solar fuels is to use photocatalytic biohybrid systems that combine the high catalytic activity of non-photosynthetic microorganisms with the high light-harvesting efficiency of metal semiconductor nanoparticles. However, few such systems have been tested for H2 production. We investigated light-driven H2 production by three novel organisms, Desulfovibrio desulfuricans, Citrobacter freundii, and Shewanella oneidensis, self-photosensitized with cadmium sulfide nanoparticles, and compared their performance to Escherichia coli. All biohybrid systems produced H2 from light, with D. desulfuricans-CdS demonstrating the best activity overall and outperforming the other microbial systems even in the absence of a mediator. With this system, H2 was continuously produced for more than 10 days with a specific rate of 36 µmol gdcw-1 h-1 . High apparent quantum yields of 23 % and 4 % were obtained, with and without methyl viologen, respectively, exceeding values previously reported.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility.

Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.

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.