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Diffusion of electrons over distances on the order of 100 μm has been observed in crystals of a small tetraheme cytochrome (STC) from Shewanella oneidensis [J. Huang et al. J. Am. Chem. Soc. 142, 10459–10467 (2020)]. Electron transfer between hemes in adjacent subunits of the crystal is slower and more strongly dependent on temperature than had been expected based on semiclassical electron-transfer theory. We here explore explanations for these findings by molecular-dynamics simulations of crystalline and monomeric STC. New procedures are developed for including time-dependent quantum mechanical energy differences in the gap between the energies of the reactant and product states and for evaluating fluctuations of the electronic-interaction matrix element that couples the two hemes. Rate constants for electron transfer are calculated from the time- and temperature-dependent energy gaps, coupling factors, and Franck–Condon-weighted densities of states using an expression with no freely adjustable parameters. Back reactions are considered, as are the effects of various protonation states of the carboxyl groups on the heme side chains. Interactions with water are found to dominate the fluctuations of the energy gap between the reactant and product states. The calculated rate constant for electron transfer from heme IV to heme Ib in a neighboring subunit at 300 K agrees well with the measured value. However, the calculated activation energy of the reaction in the crystal is considerably smaller than observed. We suggest two possible explanations for this discrepancy. The calculated rate constant for transfer from heme I to II within the same subunit of the crystal is about one-third that for monomeric STC in solution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Microorganisms can exist in both suspended and attached forms in natural and engineered systems. While extensive research has been performed to investigate the bioreduction kinetics of iron oxides by suspended microorganisms, the kinetics of iron oxide reduction by biofilms has not been well studied. This study investigated the rates of ferrihydrite bioreduction by biofilms of Shewanella oneidensis MR-1 with variable concentration ratios of electron donor to acceptor, and different biofilm thickness. Results indicated that the bioreduction rate of ferrihydrite by biofilms increased with increasing biofilm surface area and was faster than that by suspended microorganisms for the same biomass amount, apparently as a result of the biofilm surface area facilitating the direct contact between ferrihydrite and microorganisms for electron transfer. Increasing biofilm thickness decreased bioreduction rate for the same total biomass as a result of the decrease of the ratio of the biofilm surface area to total biomass. The bioreduction rate increased with increasing electron donor and ferrihydrite inputs, consistent with the saturation type of kinetic models. The different rates of ferrihydrite reduction in the biofilm system resulted in different rates of Fe(II) production as a function of electron donor, acceptor, and biofilm surface area, which in turn affected the type and concentrations of the secondary iron minerals, such as lepidocrocite and goethite. A kinetic model was developed that integrates biofilm surface area, and electron donor and acceptor concentrations that well described the measured rates of ferrihydrite bioreduction. The results emphasized the importance of the biofilm surface area in understanding and modeling iron redox biogeochemistry and iron-related biogeochemical processes. [ABSTRACT FROM AUTHOR]

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens. [ABSTRACT FROM AUTHOR]

Hexavalent chromium (Cr(VI)) could be sequestrated by soils via microbial reduction to Cr(III) and association with minerals, however, quantitative understanding of metal reducing bacteria on the coupled kinetics of Cr and Fe minerals is still lacking. Here, microbial-mediated ferrihydrite transformation and reductive sequestration of Cr(VI) were investigated with Shewanella oneidensis MR-1 under varying Cr/Fe ratios. Quantitative results depicted that the contents of magnetite from ferrihydrite transformation decreased from 70 % to 22 % after 288 h as Cr/Fe ratios increased from 0 to 8 × 10−3, and reductive transformation rates increased with decreasing Cr/Fe ratio. Elemental mapping and line scan analyses at nano-scale revealed that Cr(VI) was evenly combined within fresh ferrihydrite, and a part of produced Cr(III) was doped into the crystal lattice of magnetite and goethite. Cr may be sequestrated by secondary Fe minerals via structural substitution, surface complexation, and physical encapsulation. Microscopy-based results exhibited that more Fe minerals adsorbed on the surface of cell and less cells were lysed at low Cr/Fe ratios. Confocal laser scanning microscopy depicted that less cells were alive at high Cr/Fe ratios. The amplification of extracellular electron transfer-associated genes was downregulated as Cr/Fe ratios increased. The Cr/Fe ratios could affect cell activity, the combination with Fe minerals, and Mtr gene expression, thus controlling the reductive sequestration of Cr(VI) and phase transformation of ferrihydrite. A kinetic model has been established by combining elementary reductions, and it could well describe Cr(VI) reduction and Cr(III) incorporation under various Cr/Fe ratios during dissimilatory Fe reduction process. These findings could broaden our knowledge of quantifying the migration and transport of Cr in anoxic Cr-contaminated soil environments, and could be useful for simulating Cr dynamic behaviors in the natural soil, aquatic, and sediment environments. [ABSTRACT FROM AUTHOR]

Bioelectrochemical systems (BESs) are an innovative technology for the efficient degradation of antibiotics. Shewanella oneidensis (S. oneidensis) MR-1 plays a pivotal role in degrading sulfamethoxazole (SMX) in BESs. Our study investigated the effect of BES conditions on SMX degradation, focusing on microbial activity. The results revealed that BESs operating with a 0.05 M electrolyte concentration and 2 mA/cm2 current density outperformed electrolysis cells (ECs). Additionally, higher electrolyte concentrations and elevated current density reduced SMX degradation efficiency. The presence of nutrients had minimal effect on the growth of S. oneidensis MR-1 in BESs; it indicates that S. oneidensis MR-1 can degrade SMX without nutrients in a short period of time. We also highlighted the significance of mass transfer between the cathode and anode. Limiting mass transfer at a 10 cm electrode distance enhanced S. oneidensis MR-1 activity and BES performance. In summary, this study reveals the complex interaction of factors affecting the efficiency of BES degradation of antibiotics and provides support for environmental pollution control. [ABSTRACT FROM AUTHOR]

Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements, referred to as phage satellites, exploits phages to disseminate their own genetic material. Here, we isolated a novel member of the family Inoviridae, Shewanella phage Dolos, along with an autonomously replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment and, thus, acts as a phage satellite. pDolos can disseminate further genetic material encoding, e.g., resistances or fluorophores to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus and the wide distribution of phages of this group, we speculate that similar phage-satellite systems are common among bacteria. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of S. oneidensis was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of S. oneidensis. • Efficient synthesis of 5-ALA solely relying on the S. oneidensis endogenous C5 pathway was achieved for the first time. • Employ a modular assembly strategy to build production, antioxidant, and inhibition modules for tuning the metabolic flux. • Rational metabolic engineering enhances the 5-ALA yield of S. oneidensis by 123 times. [ABSTRACT FROM AUTHOR]

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the 'phosphorylated' state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis. Regulation of siderophore biosynthesis in Shewanella occurs at both transcriptional and post-transcriptional levels by two TCSs, one of which is composed of an orphan phosphorylation-independent RR functioning via a novel mechanism. [ABSTRACT FROM AUTHOR]

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyiardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products. Key points: •Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production. •Deletion of ackA/pta redirects carbon flux towards TCA cycle. •Heterologous expression of cadA and acnB enables itaconic acid production. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products. Key points: •Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production. •Deletion of ackA/pta redirects carbon flux towards TCA cycle. •Heterologous expression of cadA and acnB enables itaconic acid production. [ABSTRACT FROM AUTHOR]

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death. [ABSTRACT FROM AUTHOR]

Hexabromocyclododecane (HBCD) is a widely used brominated flame retardant; however, it is a persistent organic pollutant as well as affects the human thyroid hormones and causes cancer. However, the degradation of HBCD has received little attention from researchers. Due to its bioaccumulative and hazardous properties, an appropriate strategy for its remediation is required. In this study, we investigated the biodegradation of HBCD using Shewanella oneidensis MR-1 under optimized conditions. The Box-Behnken design (BBD) was implemented for the optimization of the physical degradation parameters of HBCD. S. oneidensis MR-1 showed the best degradation performance at a temperature of 30 °C, pH 7, and agitation speed of 115 rpm, with an HBCD concentration of 1125 μg/L in mineral salt medium (MSM). The strain tolerated up to 2000 μg/L HBCD. Gas chromatography-mass spectrometry analysis identified three intermediates, including 2-bromo dodecane, 2,7,10-trimethyldodecane, and 4-methyl-1-decene. The results provide an insightful understanding of the biodegradation of HBCD by S. oneidensis MR-1 under optimized conditions and could pave the way for further eco-friendly applications. Key points: • HBCD biodegradation by Shewanella oneidensis • Optimization of HBCD biodegradation by the Box-Behnken analysis • Identification of useful metabolites from HBCD degradation [ABSTRACT FROM AUTHOR]

Extracellular electron transfer (EET) empowers electrogens to catalyse the bioconversion of a wide range of xenobiotics in the environment. Synthetic bioengineering has proven effective in promoting EET output. However, conventional strategies mainly focus on modifications of EET‐related genes or pathways, which leads to a bottleneck due to the intricate nature of electrogenic metabolic properties and intricate pathway regulation that remain unelucidated. Herein, we propose a novel EET pathway‐independent approach, from an energy manipulation perspective, to enhance microbial EET output. The Controlled Hydrolyzation of ATP to Enhance Extracellular Respiration (CHEER) strategy promotes energy utilization and persistently reduces the intracellular ATP level in Shewanella oneidensis, a representative electrogenic microbe. This approach leads to the accelerated consumption of carbon substrate, increased biomass accumulation and an expanded intracellular NADH pool. Both microbial electrolysis cell and microbial fuel cell tests exhibit that the CHEER strain substantially enhances EET capability. Analysis of transcriptome profiles reveals that the CHEER strain considerably bolsters biomass synthesis and metabolic activity. When applied to the bioconversion of model xenobiotics including methyl orange, Cr(VI) and U(VI), the CHEER strain consistently exhibits enhanced removal efficiencies. This work provides a new perspective and a feasible strategy to enhance microbial EET for efficient xenobiotic conversion. [ABSTRACT FROM AUTHOR]

Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis is a good model for investigating the bactericidal effects of chemical agents. In this study, the effects of tricarbonyldichlororuthenium dimer (CORM-2) on the aerobic and anaerobic growth of S. oneidensis were studied, and its bactericidal mechanisms were elucidated. CORM-2 (5 µM) significantly inhibited aerobic and anaerobic respiration. Growth potential tests showed that mutants Δ katB , Δ oxyR, Δ ccmF, and Δ crp and mutants that deleted each cytochrome c-encoding gene exhibited sensitivity to CORM-2 similar to that of the wild-type strain MR-1. Enzymatic analyses showed that cytochrome oxidase, catalase and cysteine desulfurase were activated by CORM-2, while nitrite reductase, nitrate reductase, sulfite reductase, thiosulfate reductase, peroxidase and NADH dehydrogenase were inhibited by CORM-2, and no significant changes were found in the activities of superoxide dismutase and aconitase. Promoter activity assays showed that cco , cyd , cox , katB , sirA and psrA were repressed by CORM-2. Sulfide alleviated the bactericidal effect of CORM-2 and protected NADH dehydrogenase from CORM-2 inhibition. Thus, oxidative stress and cytochrome oxidase inactivation are not the primary reasons for the bactericidal effect of CORM-2, and NADH dehydrogenase is the main target in S. oneidensis. The bactericidal effect of CORM-2 is caused by the toxicity of Ru2+, not by carbon monoxide. [Display omitted] • The first paper to investigate the effect of CORM-2 on the growth of S. oneidensis. • Cytochrome oxidase, catalase and cysteine desulfurase are activated by CORM-2. • Oxidative stress is not the major bactericidal reason of CORM-2 in S. oneidensis. • NADH dehydrogenase is the main target of CORM-2 in S. oneidensis. • The bactericidal effect of CORM-2 is caused by Ru2+, not by CO. [ABSTRACT FROM AUTHOR]

This study employs a bibliography study method to evaluate 472 papers focused on Shewanella oneidensis biofilms. Biofilms, which are formed when microorganisms adhere to surfaces or interfaces, play a crucial role in various natural, engineered, and medical settings. Within biofilms, microorganisms are enclosed in extracellular polymeric substances (EPS), creating a stable working environment. This characteristic enhances the practicality of biofilm-based systems in natural bioreactors, as they are less susceptible to temperature and pH fluctuations compared to enzyme-based bioprocesses. Shewanella oneidensis , a nonpathogenic bacterium with the ability to transfer electrons, serves as an example of a species isolated from its environment that exhibits extensive biofilm applications. These applications, such as heavy metal removal, offer potential benefits for environmental engineering and human health. This paper presents a comprehensive examination and review of the biology and engineering aspects of Shewanella biofilms, providing valuable insights into their functionality. [ABSTRACT FROM AUTHOR]

Bacterial genomes are a huge reservoir of genes encoding J‐domain protein co‐chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J‐domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C‐terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J‐domain proteins that (i) are found in several environmental, mostly aquatic, β‐ or ɣ‐proteobacteria and (ii) contain a conserved PX7W motif in their C‐terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7W motif determines the strong AtcJ–AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low‐temperature growth and, given the variation in the atc operons, possibly for other biological functions. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR-1 (S. oneidensis MR-1) has been shown to benefit from microbial electrosynthesis (MES) due to its exceptional electron transfer efficiency. In this study, genes involved in both extracellular electron uptake (EEU) and intracellular CO 2 conversion processes were examined and regulated to enhance MES performance. The key genes identified for MES in the EEU process were mtrB, mtrC, mtrD, mtrE, omcA and cctA. Overexpression of these genes resulted in 1.5–2.1 times higher formate productivity than that of the wild-type strains (0.63 mmol/(L·μg protein)), as 0.94–1.61 mmol/(L·μg protein). In the intracellular CO 2 conversion process, overexpression of the nadE, nadD, nadR, nadV, pncC and petC genes increased formate productivity 1.3-fold–3.4-fold. Moreover, overexpression of the formate dehydrogenase genes fdhA1, fdhB1 and fdhX1 in modified strains led to a 2.3-fold–3.1-fold increase in formate productivity compared to wild-type strains. The co-overexpression of cctA, fdhA1 and nadV in the mutant strain resulted in 5.59 times (3.50 mmol/(L·μg protein)) higher formate productivity than that of the wild-type strains. These findings revealed that electrons of MES derived from the electrode were utilized in the energy module for synthesizing ATP and NADH, followed by the synthesis of formate in formate dehydrogenase by the combinatorial effects of ATP, NADH, electrons and CO 2. The results provide new insights into the mechanism of MES in S. oneidensis MR-1 and pave the way for genetic improvements that could facilitate the further application of MES. • Regulating genes related to EEU and intracellular CO 2 conversion processes improved formate productivity in the MES of S. oneidensis MR-1. • The co-overexpression of cctA , fhdA1 , and nadV in S. oneidensis MR-1 increased the productivity of formate by 5.59 times higher than that of the WT strain. • Distinct functional genes between S. oneidensis MR-1 EEU and EET were discovered. • Further information about the MES process of S. oneidensis MR-1 was revealed. [ABSTRACT FROM AUTHOR]

YiiP from Shewanella oneidensis is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+. [ABSTRACT FROM AUTHOR]

Biohybrid systems can combine inorganic light‐harvesting materials and whole‐cell biocatalysts to utilize solar energy for the production of chemicals and fuels. Whole‐cell biocatalysts have an intrinsic self‐repair ability and are able to produce a wide variety of multicarbon chemicals in a sustainable way with metabolic engineering. Current whole‐cell biohybrid systems have a yet undefined electron transfer pathway between the light‐absorber and metabolic enzymes, limiting rational design. To enable engineering of efficient electron transfer pathways, covalent biohybrids consisting of graphitic nitrogen doped carbon dots (g‐N‐CDs) and the outer‐membrane decaheme protein, MtrC from Shewanella oneidensis MR‐1 are developed. MtrC is a subunit of the MtrCAB protein complex, which provides a direct conduit for bidirectional electron exchange across the bacterial outer membrane. The g‐N‐CDs are functionalized with a maleimide moiety by either carbodiimide chemistry or acyl chloride activation and coupled to a surface‐exposed cysteine of a Y657C MtrC mutant. MtrC∼g‐N‐CD biohybrids are characterized by native and denaturing gel electrophoresis, chromatography, microscopy, and fluorescence lifetime spectroscopy. In the presence of a sacrificial electron donor, visible light irradiation of the MtrC∼g‐N‐CD biohybrids results in reduced MtrC. The biohybrids may find application in photoinduced transmembrane electron transfer in S. oneidensis MR‐1 for chemical synthesis in the future. [ABSTRACT FROM AUTHOR]

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments. [ABSTRACT FROM AUTHOR]

Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We identified 18 gene ontologies and 13 gene operons that make up key systems that affect biosorption. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO_3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO_4685, a capsular polysaccharide (CPS) synthesis gene, disruption of which results in an up to 79% increase in biosorption; and nusA, a transcriptional termination/anti-termination protein, disruption of which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO_3183 produce small but significant increases (≈ 1%) in relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Modeling suggests that while these changes to lanthanide biosorption selectivity are small, they could already reduce the length of repeated enrichment process by up to 27%. This broad exploratory study begins to elucidate how genetics affect REE-biosorption by S. oneidensis, suggests new areas of investigation for better mechanistic understanding of the membrane chemistry involved in REE binding, and offer potential targets for improving biosorption and separation of REE by genetic engineering. [ABSTRACT FROM AUTHOR]

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer. [ABSTRACT FROM AUTHOR]

Interfacing bacteria as biocatalysts with an electrode provides the basis for emerging bioelectrochemical systems that enable sustainable energy interconversion between electrical and chemical energy. Electron transfer rates at the abiotic‐biotic interface are, however, often limited by poor electrical contacts and the intrinsically insulating cell membranes. Herein, we report the first example of an n‐type redox‐active conjugated oligoelectrolyte, namely COE‐NDI, which spontaneously intercalates into cell membranes and mimics the function of endogenous transmembrane electron transport proteins. The incorporation of COE‐NDI into Shewanella oneidensis MR‐1 cells amplifies current uptake from the electrode by 4‐fold, resulting in the enhanced bio‐electroreduction of fumarate to succinate. Moreover, COE‐NDI can serve as a "protein prosthetic" to rescue current uptake in non‐electrogenic knockout mutants. [ABSTRACT FROM AUTHOR]

Interfacing bacteria as biocatalysts with an electrode provides the basis for emerging bioelectrochemical systems that enable sustainable energy interconversion between electrical and chemical energy. Electron transfer rates at the abiotic‐biotic interface are, however, often limited by poor electrical contacts and the intrinsically insulating cell membranes. Herein, we report the first example of an n‐type redox‐active conjugated oligoelectrolyte, namely COE‐NDI, which spontaneously intercalates into cell membranes and mimics the function of endogenous transmembrane electron transport proteins. The incorporation of COE‐NDI into Shewanella oneidensis MR‐1 cells amplifies current uptake from the electrode by 4‐fold, resulting in the enhanced bio‐electroreduction of fumarate to succinate. Moreover, COE‐NDI can serve as a "protein prosthetic" to rescue current uptake in non‐electrogenic knockout mutants. [ABSTRACT FROM AUTHOR]

The staggering increase in pollution associated with a sharp tightening in global energy demand is a major concern for organic substances. Renewable biofuel production through simultaneous waste reduction is a sustainable approach to meet this energy demand. This study co-fermentation of dairy whey and SCB was performed using mixed and pure bacterial cultures of Salmonella bongori, Escherichia coli, and Shewanella oneidensis by dark fermentation process for hydrogen production. The maximum H2 production was 202.7 ± 5.5 H2/mL/L, 237.3 ± 6.0 H2/mL/L, and 198 ± 9.9 H2/mL/L obtained in fermentation reactions containing dairy whey, solid and liquid hydrolysis of pretreated sugarcane bagasse as mono-substrates. The H2 production was greater in co-substrate by 347.3 ± 18.5 H2/mL/L under optimized conditions (pH 7.0, temperature 37 °C, substrate concentration 30:50 g/L) than expected in mono-substrate conditions, which confirms that co-fermentation of different substrates enhances the H2 potential. Fermentation medium during bio-H2 production under GC analysis has stated that using mixed cultures in dark fermentation favored acetic acid and butyric acid. Co-substrate degradation produces ethyl alcohol, benzoic acid, propionic acid, and butanol as metabolic by-products. The difference in the treated and untreated substrate and carbon enrichment in the substrates was evaluated by FT-IR analysis. The present study justifies that rather than the usage of mono-substrate for bio-H2 production, the co-substrate provided highly stable H2 production by mixed bacterial cultures. Fabricate the homemade single-chamber microbial fuel cell to generate electricity. [ABSTRACT FROM AUTHOR]

Microbial reduction of Se(IV) to Se(0) is extremely important for the detoxification of selenium oxyanion, but the reductive mechanism is still controversial. In this study, the reduction of Se(IV) by Shewanella oneidensis MR-1 and Shewanella putrefaciens was systematically investigated. The results showed that excessive Se(IV) severely inhibited the growth of microorganisms and Se(IV) reduction. The extracellular accumulation of Nano-Se(0) particles was shown to be approximately 250–650 nm in diameter. Riboflavin facilitated Se(IV) reduction, while rotenone, as an electron inhibitor, could completely inhibit Se(IV) reduction, validating the extracellular electron transport process for biosynthesized Nano-Se(0). Importantly, the amorphous Nano-Se(0) synthesized by S. oneidensis MR-1 were mixtures of helical chains and Se 8 rings, while those synthesized by S. putrefaciens were mainly composed of helical chains. FTIR results demonstrated that Nano-Se(0) particles were wrapped by some proteins, organic substances and lipids, exhibiting superior dispersibility and biocompatibility. Biogenic Nano-Se(0) particles can also be considered for technological applications in various fields. [Display omitted] • S. oneidensis MR-1 and S. putrefaciens can reduce Se(IV) to Nano-Se(0) particles. • Se(IV) reduction is achieved through extracellular electron transfer. • Riboflavin exhibited enhancement to Se(IV) reduction at high doses. • Nano-Se(0) produced by S. oneidensis MR-1 is well-dispersed than that of S. putrefaciens. [ABSTRACT FROM AUTHOR]

Electrochemically active bacteria (EAB) are capable of electrically interacting with electrodes, enabling their application in bioelectrochemical systems (BESs). As the performance of BES is related to the metabolic activities of EAB, the development of methods to control their metabolic activities is important to facilitate BES applications. A recent study found that the EAB Shewanella oneidensis MR-1 uses the Arc system to regulate the expression of catabolic genes in response to electrode potentials, suggesting that a methodology for electrical control of gene expression in EAB, referred to as electrogenetics, can be developed by using electrode potential-responsive, Arc-dependent transcriptional promoters. Here, we explored Arc-dependent promoters in the genomes of S. oneidensis MR-1 and Escherichia coli to identify electrode potential-responsive promoters that are differentially activated in MR-1 cells exposed to high- and low-potential electrodes. LacZ reporter assays using electrode-associated cells of MR-1 derivatives revealed that the activities of promoters located upstream of the E. coli feo gene (P feo) and the MR-1 nqrA2 (SO_0902) gene (P nqr2) were significantly increased when S. oneidensis cells were exposed to electrodes poised at +0.7 V and −0.4 V (versus the standard hydrogen electrode), respectively. Additionally, we developed a microscopic system for in situ monitoring of promoter activity in electrode-associated cells and found that P nqr2 activity was persistently induced in MR-1 cells associated with an electrode poised at −0.4 V. Our results indicate that these electrode potential-responsive promoters enable efficient regulation of gene expression in EAB, providing a molecular basis for the development of electrogenetics. [Display omitted] [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. [ABSTRACT FROM AUTHOR]

Electrogens are very common in nature and becoming a contemporary theme for research as they can be exploited for extracellular electron transfer. Extracellular electron transfer is the key mechanism behind bioelectricity generation and bioremediation of pollutants via microbes. Extracellular electron transfer mechanisms for electrogens other than Shewanella and Geobacter are less explored. An efficient extracellular electron transfer system is crucial for the sustainable future of bioelectrochemical systems. At present, the poor extracellular electron transfer efficiency remains a decisive factor in limiting the development of efficient bioelectrochemical systems. In this review article, the EET mechanisms in different electrogens (bacteria and yeast) have been focused. Apart from the well-known electron transfer mechanisms of Shewanella oneidensis and Geobacter metallireducens, a brief introduction of the EET pathway in Rhodopseudomonas palustris TIE-1, Sideroxydans lithotrophicus ES-1, Thermincola potens JR, Lysinibacillus varians GY32, Carboxydothermus ferrireducens, Enterococcus faecalis and Saccharomyces cerevisiae have been included. In addition to this, the article discusses the several approaches to anode modification and genetic engineering that may be used in order to increase the rate of extracellular electron transfer. In the side lines, this review includes the engagement of the electrogens for different applications followed by the future perspective of efficient extracellular electron transfer. [ABSTRACT FROM AUTHOR]

Living bio-nano systems for artificial photosynthesis are of growing interest. Typically, these systems use photoinduced charge transfer to provide electrons for microbial metabolic processes, yielding a biosynthetic solar fuel. Here, we demonstrate an entirely different approach to constructing a living bio-nano system, in which electrogenic bacteria respire semiconductor nanoparticles to support nanoparticle photocatalysis. Semiconductor nanocrystals are highly active and robust photocatalysts for hydrogen (H2) evolution, but their use is hindered by the oxidative side of the reaction. In this system, Shewanella oneidensis MR-1 provides electrons to a CdSe nanocrystalline photocatalyst, enabling visible light-driven H2 production. Unlike microbial electrolysis cells, this system requires no external potential. Illuminating this system at 530 nm yields continuous H2 generation for 168 h, which can be lengthened further by replenishing bacterial nutrients. [ABSTRACT FROM AUTHOR]

The effect of the polarity of the electrical stimulation of the external circuit of the bioelectrochemical systems, as well as the immobilization of Shewanella oneidensis MR-1 cells containing the DyP peroxidase gene on the rate of discoloration of dyes of different types, was found. For the crystal violet triphenylmethane dye, the maximum decolorization rate by suspended S. oneidensis MR-1 cells of 2.05 ± 0.07 μM/h was noted when connecting a 1.2 V direct polarity DC voltage source. One of the minimum rates was observed with reverse polarity of the connection. For cells immobilized on the anode, the rate was higher, reaching 2.91 ± 0.09 μM/h and did not decrease with increasing substrate concentration. The lowest values were also noted for the reverse connection of the voltage source. For the azo dye congo red, the maximum rate was found for a source with direct connection and an open circuit (0.26 ± 0.01 and 0.29 ± 0.02 μM/h, respectively); the minimum value was 0.11 ± 0.02 μM/h for a reverse connection. For crystal violet decolorization products, a significant decrease in the intensity of the main absorption peak at 590 nm band was found, with no notable hypsochromic shift. The qualitative changes in the decolorization product composition were indicated by the appearance, with a direct polarity of the ionistor connection, of a new absorption maximum in the region of 360 nm. The results may be of interest for the development of new methods of bioelectrochemical cleaning. [ABSTRACT FROM AUTHOR]

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 ± 11.42 µm 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 (3736 mW 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. [ABSTRACT FROM AUTHOR]

Background: 1,3-Propanediol (1,3-PDO) is a platform compound, which has been widely used in food, pharmaceutical and cosmetic industries. Compared with chemical methods, the biological synthesis of 1,3-PDO has shown promising applications owing to its mild conditions and environmental friendliness. However, the biological synthesis of 1,3-PDO still has the problem of low titer and yield due to the shortage of reducing powers. Results: In this study, Klebsiella sp. strain YT7 was successfully isolated, which can synthesize 11.30 g/L of 1,3-PDO from glycerol in flasks. The intracellular redox regulation strategy based on the addition of electron mediators can increase the 1,3-PDO titer to 28.01 g/L. Furthermore, a co-culturing system consisting of strain YT7 and Shewanella oneidensis MR-1 was established, which can eliminate the supplementation of exogenous electron mediators and reduce the by-products accumulation. The 1,3-PDO yield reached 0.44 g/g and the final titer reached 62.90 g/L. The increased titer and yield were attributed to the increased redox levels and the consumption of by-products. Conclusions: A two-bacterium co-culture system with Klebsiella sp. strain YT7 and S. oneidensis strain MR-1 was established, which realized the substitution of exogenous electron mediators and the reduction of by-product accumulation. Results provided theoretical basis for the high titer of 1,3-PDO production with low by-product concentration. [ABSTRACT FROM AUTHOR]

In this paper, we presented a novel microbial fuel cell (bMFC) structure, with a bipolar membrane separating the anode and cathode chambers. A bipolar membrane divides the bMFC into anode and cathode chambers. The bipolar membrane comprises anion and cation exchange layers. The anode chamber side has the cation exchange layer, while the cathode chamber side has the anion exchange layer. The anode chamber of the bMFC was loaded with Shewanella oneidensis MR-1 and lactic acid, while the cathode chamber was loaded with pure water and iron (III) hydroxide. The bMFC generated electrons for 20 days at a maximum current density of 30 mA/m2 and the ohmic resistance value was estimated to be 500 Ω. During the operation of the bMFC, both the anode and cathode chambers kept anaerobic conditions. There was no platinum catalyst in the cathode chamber, which is required for the reaction of protons with oxygen. Therefore, oxygen could not serve as an electron acceptor in the bMFC. We considered a bMFC mechanism in which protons produced by S. oneidensis react with hydroxide ions, the counter anions of Fe3+, inside the bipolar membrane to produce water. In other words, the electron acceptor in bMFC would be Fe3+. [ABSTRACT FROM AUTHOR]

The high electrical conductivity and low dimensionality of graphene is essential for the development of novel lightweight bioanodes for new-generation energy technologies. However, the integration of graphene in biointerfaces presents a formidable challenge, especially because the surface energy of graphene is not compatible with living matter. Here we propose a sustainable chemical control method to reach the demanded surface hydrophilicity and conductivity of graphene nanowalls to form a lightweight, graphene-based, sponge bioanode. The few-nanometer–thick conductive graphene nanowalls create biocompatible hydrophilic microconfinements to harvest the biomass density of electrogenic Shewanella Oneidensis MR-1. The graphene-based bioanode shows a stable and rapid response with a steady-state biocurrent density of 135.35 mA m−2 realized within a few hours. Our novel and sustainable graphene-based material provides a revolutionary energy opportunity for the establishment of new energy-related graphene industries as well as facilitates many startups. A facile and chemical-free method was leveraged to produce the graphene-based bioanode. Balancing the hydrophilicity and conductivity of the graphene nanowalls was achieved by simply tuning its reduction temperature and was the first time, revealed to play an important role in the performance of the microbial electrode. A steady-state bio-current density of 135.35 mA m−2 was realized within a few hours and with long-term duration. [Display omitted] [ABSTRACT FROM AUTHOR]

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems. Inevitably, researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates. Base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1. Since multiplexed application of base editing is still limited by its low throughput procedure, we thus here develop a rapid and efficient multiplex base editing system in S. oneidensis. Four approaches to express multiple gRNAs were assessed firstly, and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster. Then, a smart scheme was designed to deliver one-pot assembly of multiple gRNAs. 3, 5, and 8 genes were deactivated using this system with editing efficiency of 83.3%, 100% and 12.5%, respectively. To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette, different promoters, handles, and terminators were screened. This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain. The maximum power density of the multiplex engineered strain HRF(8BE) in microbial fuel cells was 1108.1 mW/m2, which was 21.67 times higher than that of the wild-type strain. This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella, and may provide valuable insights in fundamental and applied research of extracellular electron transfer. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis MR‐1 is a promising chassis organism for microbial electrosynthesis because it has a well‐defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH‐dependent reaction in the cytoplasm, reduction of acetoin to 2,3‐butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3‐butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR‐1 should focus on optimizing flux through NADH dehydrogenases. [ABSTRACT FROM AUTHOR]

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (,10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria. [ABSTRACT FROM AUTHOR]

In this study, we presented the results of experiments designed to harvest energy from a bioreactor where exoelectrogenic Shewanella oneidensis bacteria were inoculated in the electrolyte solution containing a carbon source. In the absence of metal electrodes, external electrical circuit, proton-exchange membrane (PEM) or any possible sources of electric potential in the reactor, we associate the electric potential gradients with the capability to transport electrons through the conductive nanowires. Shewanella oneidensis bacteria produce the nanowires in the environment with limited electron acceptors. The nanowires facilitate electron transfer from the oxidizing zone (lower part of the reactor) and the reducing zone (upper part of the reactor), generating electric potential difference. Transmission electron microscopy (TEM) imaging revealed nanowire formation in the electrolyte solution. After inoculation with the liquid medium amended with a carbon source, open-circuit voltages were produced in the range of 380–470 mV. A scaling-up approach was demonstrated by connecting two reactors in series, producing a maximum of 960 mV. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis has 2 functional chemosensory systems named Che1 and Che3, and 27 chemoreceptors. Che3 is dedicated to chemotaxis while Che1 could be involved in RpoS post-translational regulation. In this study, we have shown that two chemoreceptors Aer2so and McpAso, genetically related to the Che1 system, form distinct core-signaling units and signal to Che1 and Che3, respectively. Moreover, we observed that Aer2so is a cytoplasmic dynamic chemoreceptor that, when in complex with CheA1 and CheW1, localizes at the two poles and the centre of the cells. Altogether, the results obtained indicate that Che1 and Che3 systems are interconnected by these two chemoreceptors allowing a global response for bacterial survival. [ABSTRACT FROM AUTHOR]

Manganese (Mn) and Fe indicator of reduction in soils (IRIS) devices are low‐cost, convenient tools for identifying reducing conditions in soils. Because Mn is reduced at similar redox potentials as nitrate, there is considerable interest in using Mn IRIS tools for understanding microbial reduction of Mn as a surrogate for processes such as denitrification. However, the sensitivity of these devices to differences in Mn‐reducing capacity has not been empirically investigated. Here we have found that the rate of birnessite paint removal from Mn IRIS films exposed to a twofold dilution series of the Mn‐reducing bacterium Shewanella oneidensis is directly proportional to the number of S. oneidensis cells added. Thus, regularly monitored birnessite IRIS sensors are capable of indicating twofold differences in Mn reduction in soil and can be used to measure relative Mn reduction rates over time in a single location or compare and contrast Mn reduction rates across soil types. Core Ideas: Manganese indicator of reduction in soils film sensitivity was determined using a Shewanella oneidensis dilution series.Manganese indicator of reduction in soils films are sensitive tools for measuring Mn reduction rates in soil.Timing of Mn indicator of reduction in soils film deployment and observation is critical for maximum sensitivity. [ABSTRACT FROM AUTHOR]

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0–250 μM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases. [ABSTRACT FROM AUTHOR]

Mutant uridine phosphorylase genes from Shewanella oneidensis MR-1 (S. oneidensis) were constructed by site-directed mutagenesis and strains-producers of the corresponding recombinant (F5I and F5G) proteins were obtained on the basis of Escherichia coli cells. The mutant proteins were purified and their physicochemical and enzymatic properties were studied. It was shown that the N-terminal fragment of uridine phosphorylase plays an important role in the thermal stabilization of the enzyme as a whole. The role of the aminoacid (a.a.) residue phenylalanine (F5) in the formation of thermotolerance of uridine phosphorylases from gamma-proteobacteria was revealed. [ABSTRACT FROM AUTHOR]

Bacterial reduction of Se(IV) is a significant component of the global selenium cycle, and hence affects the fate and transport of selenium in both natural and contaminated environments. However, it is unknown whether bacterially-produced exudates are capable of reducing selenium independent of bacterial cells. In this study, we measured the rate and extent of Se(IV) reduction by exudates from three bacterial species, and we determined the importance of exudate sulfhydryl sites by conducting parallel experiments after treatment of the exudates with a sulfhydryl-specific blocking molecule. We also conducted experiments with whole cell biomass for each of the three bacterial species to determine the importance of exudate-only reduction relative to whole biomass-promoted reduction. Under our experimental conditions, exudates from Bacillus subtilis and Pseudomonas putida remove Se(IV) from solution after an approximately 20–24 h lag period, but exudates from Shewanella oneidensis are ineffective at Se(IV) removal, except when the cells are grown in the presence of Se(IV). For both B. subtilis and P. putida , pretreatment blocking of the sulfhydryl sites on the exudate molecules dramatically decreases the rate and extent of Se(IV) removal, strongly suggesting that sulfhydryl groups on the exudate molecules play a key role in the Se(IV) reduction mechanism. The behavior of S. oneidensis exudates indicates that up-regulation of the Se(IV) reduction mechanism can occur in response to the Se content of the medium in which the cells grow. Our results demonstrate the capacity of some bacterial exudates to reduce Se(IV), and suggest that the activity of bacterial exudates should be accounted for when modeling selenium cycling in natural and engineered environments. [ABSTRACT FROM AUTHOR]

The peptidoglycan (PG) layer of bacterial cells is essential for maintaining the cell shape and survival of cells; therefore, the synthesis of PG needs to be spatiotemporally controlled. While it is well established that PG synthesis is mediated posttranslationally through interactions between PG synthases and their cognate partners, much less is known about the transcriptional regulation of genes encoding these synthases. Based on a previous finding that the Gram‐negative bacterium Shewanella oneidensis lacking the prominent PG synthase exhibits impaired cell wall integrity, we performed genetic selections to isolate the suppressors. We discovered that disrupting the sspA gene encoding stringent starvation protein A (SspA) is sufficient to suppress compromised PG. SspA serves as a transcriptional repressor that regulates the expression of the two types of PG synthases, class A penicillin‐binding proteins and SEDS/bPBP protein complexes. SspA is an RNA polymerase‐associated protein, and its regulation involves interactions with the σ70‐RNAP complex and an antagonistic effect of H‐NS, a global nucleoid‐associated protein. We also present evidence that the regulation of PG synthases by SspA is conserved in Escherichia coli, adding a new dimension to the current understanding of PG synthesis and its regulation. [ABSTRACT FROM AUTHOR]