Shewanella Oneidensis Strain MR-1 Research Articles

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

The influence of montmorillonite on the mineralogical and magnetic changes in magnetite and maghemite during microbial iron reduction

Magnetic iron minerals, such as magnetite (Fe3O4) and maghemite (γ-Fe2O3), are widely distributed in sediments, holding significant potential for reconstructing paleoenvironments through their magnetic properties. However, the impact of microbial iron reduction on the physicochemical and magnetic characteristics of Fe(III)-containing magnetic minerals has recently been recognized as a crucial but often overlooked factor in paleoenvironmental reconstructions. Although magnetic iron minerals are frequently associated with clay minerals in sedimentary settings, our understanding of the influence of clay minerals on the microbial reduction of Fe(III) in magnetic minerals remains limited. To address this knowledge gap, a series of bioreduction experiments were conducted in a non-growth medium, in which lactate acted as the electron donor, magnetite or maghemite nanoparticles (NPs) served as the electron acceptor, and a typical iron-reducing bacterium, Shewanella oneidensis strain MR-1, acted as the reaction mediator, in the presence or absence of montmorillonite (STx-1b). Our results demonstrated that the addition of montmorillonite STx-1b significantly enhanced the dispersion of magnetite and maghemite NPs, thereby increasing the final extent of microbial iron reduction. Moreover, the degree of microbial iron reduction impacted the crystallography and magnetic properties of magnetite and maghemite. Upon bioreduction, the lattice constant, magnetic susceptibility, and the 10-K magnetic coercivity of these magnetic iron minerals exhibited an increase, accompanied by the presence of more structural defects. Specifically, maghemite-magnetite solid solutions were formed in the maghemite-amended biosystems. These findings highlight the influence of clay minerals in natural settings during microbial iron reduction on the magnetic properties of magnetic iron minerals used for paleoenvironmental reconstructions.

The iron cycling mediated by a single strain Shewanella oneidensis MR-1 and its implication for nitrogen removal

Microbially mediated nitrate-reducing Fe(II) oxidation (NRFO) process has been recognized as an effective route for removing nitrogen in wastewater. However, the need for continuous dose of iron blocks NRFO technology to be scaled up. Microbial iron cycling can improve iron utilization efficiency, but whether it can be mediated by a single strain remains elusive. Here, Shewanella oneidensis strain MR-1 was employed to mediate Fe redox process with the subsequent addition of Fe(III), nitrate, and lactate under anaerobic conditions. Fe(II) kinetic curve showed that strain MR-1 could efficiently reduce Fe(III) with a reduction rate of 2.55 mmol·L−1·d−1, producing a great amount of Fe(II). After nitrate addition, strain MR-1 mediated the NRFO process. Fe(III) reduction and NRFO constituted Fe cycle, indicating that strain MR-1 successfully carried out Fe redox cycling. It is noted that two iron redox cycles were achieved, but the second cycle was weaker. That is, about 86.7 % of total iron was reduced in the first cycle, but only 4.6 % was reduced in the second cycle. As nitrate reduction is the key process for iron cycling, the unsustainability should be related to nitrate reduction inhibition. The possible mechanisms were investigated, revealing that nitrate reduction was inhibited by cell encrustation and nutrient shortage. These findings expand the understanding of microbial iron cycle and provide a new microbial iron cycling model. This will help to improve the biological wastewater treatment technology based on NRFO process.

The Outer Membrane Cytochrome OmcA Is Essential for Infection of Shewanella oneidensis by a Zebrafish-Associated Bacteriophage.

The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Microbial reduction and alteration of Fe(III)-containing smectites in the presence of biochar-derived dissolved organic matter

Clay minerals are important components of solid-phase iron pools in soils, and the redox state of structurally-coordinated iron in clay minerals can significantly influence soil physicochemical properties. Due to increasing use of biochar in agriculture, the dissolved organic matter derived from biochar (BDOM) has become an unignorable fraction of dissolved organic carbon in soil systems. However, our knowledge regarding the effect of BDOM on soil biogeochemical processes (particularly on iron redox cycling) is still very limited. To examine the role of BDOM in the microbial reduction of Fe(III) in clays, BDOM was extracted from wheat straw biochar and selectively added to laboratory reactors in which lactate, Fe(III)-containing smectite (montmorillonite SWy-2 or nontronite NAu-2), and a typical iron-reducing bacterium Shewanella oneidensis strain MR-1 were used as the respective electron donor, electron acceptor, and reaction mediator. The results demonstrated that BDOM contained bio-reactive quinone moieties. BDOM amendment increased the initial rate and final extent of Fe(III) bioreduction by strain MR-1. Such promotion effect of BDOM might be ascribed to its quinone moieties functioning as electron shuttles between MR-1 cells and structural Fe(III) in smectite. Structural changes were observed in the bioreduced smectite systems, especially for NAu-2 reactors in which obvious dissolution and illitization occurred. These observations suggest that BDOM is bio-reactive and is able to stimulate the microbial reduction of Fe(III) in clay minerals, and may offer new insights into the effects of biochar application in elemental cycles in soils.

A miniaturized bioelectricity generation device using plant root exudates to feed electrogenic bacteria

This paper presents a miniaturized plant-microbial fuel cell (mPFMC) as an assay tool that can examine different combinations of plants and electrogenic bacteria to maximize bioelectricity generation. The plant is grown in a miniature plant growth chamber (PGC). Root exudates of the plant provide carbon-containing organic matter to support the catalytic reaction of the bacteria inoculated and grown on the carbon cloth anode of a miniature microbial fuel cell (MFC). The mPFMC utilizes a semipermeable hydrophilic filtering membrane to separate the PGC from the MFC. This membrane allows retaining the bacteria inside the MFC for inoculation and growth and prevents them from entering the PGC; however, the membrane still permits the root exudates into the MFC as the sole carbon source to the bacteria grown on the anode of the device. A pilot proof-of-concept study was conducted to validate the mPMFC, by using it to examine electricity generation of the device with different varieties of model organisms, including rice plant cultivars Kitaake and IR24, and bacterial species Shewanella oneidensis strain MR-1 and Pseudomonas aeruginosa strain PA14. The plant cultivars were grown hydroponically without using soils, thus eliminating the effect of soil microorganisms and nutrients on the bioelectricity generation of the device. The present mPFMC has high compactness with the dimensions of 25 × 25 × 29 mm3, consumes only 3 mL volume of plant growth medium and less than 1 mL volume of anolyte and catholyte each, and produces electricity within only several hours after the plants are loaded into the device. The present technology will allow large-scale parallel screening of different combinations of small plants and electrogenic bacteria and their strains for bioelectricity generation.

Reduction of antimony mobility from Sb-rich smelting slag by Shewanella oneidensis: Integrated biosorption and precipitation

The dissimilatory Fe(III)-reducing bacteria play a significant role in the mobility of antimony (Sb) under reducing environment. Sb-rich smelting slag is iron (Fe)-containing antimonic mine waste, which is one of the main sources of antimony pollution. In this study, the soluble antimony reacted with Fe(III) by S. oneidensis (Shewanella oneidensis strain MR-1) was performed in reduction condition, then the dissolution behavior of the Sb-rich smelting slag with S. oneidensis was investigated. The results showed that the released Sb was immobilized by S. oneidensis and the strain adsorbed Sb(III) preferentially. Sb(V) can be reduced by S. oneidensis without aqueous Fe. In the presence of Fe(III), S. oneidensis mediated Sb bio-adsorption and the chemical redox of Sb-Fe occurred simultaneously. Sb was co-precipitated with Fe to form the Sb(V)-O-Fe(III) secondary mineral, which was identified as the bidentate mononuclear edge-sharing structure by extended X-ray absorption fine structure (EXAFS) analysis. These results suggest that S. oneidensis has a positive effect on the immobilization and minimizing toxicity of antimony in anoxic soil and groundwater, which provides a theoretical basis for the treatment of antimony contamination.

Co-existing siderite alleviates the Fe(II) oxidation-induced inactivation of Fe(III)-reducing bacteria.

Abiotic Fe (II) oxidation widely occurs in the natural subsurface environment and engineered dynamic processes, which possibly impacts the growth of indigenous microbes. As previously discovered, the oxidation of aqueous Fe2+ at neutral pH effectively inactivates iron-reducing bacteria Shewanella oneidensis strain MR-1 (MR-1). Herein, the impacts of co-existing iron mineral on the oxidation of aqueous Fe2+ and the subsequent disinfection activity on MR-1 were investigated with siderite selected as a representative iron mineral in the subsurface environment. The oxidation rate of aqueous Fe2+ and the amount of generated OH radical increased as the content of siderite increased, while the MR-1 inactivation was alleviated. An initial concentration of 2.0 × 106 CFU/mL MR-1 was inactivated by about 2.7 orders of magnitude during oxidation of 0.2 mM FeSO4 alone for 30 min, which was reduced to only about 0.6 orders of magnitude in the presence of 4.3 mM co-existing siderite. ROS scavenging results confirmed that the OH radical generated in the bulk solution was not the leading role for the inactivation of MR-1. Morphological changes of the cells observed by SEM demonstrated that the disruption of the cell membrane was alleviated by siderite, which was further supported by the XRD and FTIR spectra. The underlying mechanism was proposed to be the reduced contact time of Fe2+ and MR-1 cells due to the accelerated oxidation. This work provides new insights into the disinfection behavior of heterogeneous Fe (II) oxidation on iron cycling bacterial in the natural environment.

Interplay between iron species transformation and hydroxyl radicals production in soils and sediments during anoxic-oxic cycles

Iron (Fe) redox transformation in soils and sediments plays an essential role in the biogeochemical cycling of associated nutrients and elements. It is documented recently that hydroxyl radicals (OH) can be produced upon oxygenation of Fe(II)-bearing minerals in soils and sediments. As OH induces serious oxidative stress to microbes, it is likely that OH produced upon Fe(II) oxygenation inactivates the coexisting dissimilatory iron-reducing bacteria and subsequently impacts Fe redox transformation. This hidden interplay between Fe species transformation and OH production in soils and sediments during anoxic-oxic cycles remains unexplored. In this study, a field sediment was incubated with 1 × 109 CFU/mL Shewanella oneidensis strain MR-1 and treated for six anoxic-oxic cycles (117-h anoxic and 6-h oxic periods). Fe species transformation, OH production and MR-1 activity variation were measured throughout the six cycles. Fe in the sediments prior to anoxic-oxic cycles mainly existed in Fe-bearing silicates (60–70%) and Fe (hydr)oxides (30–40%). For the anoxic periods, a portion of Fe(III) in sediments was either reduced to structural Fe(II) in (hydr)oxides and silicates or reductively dissolved to aqueous Fe2+. Total Fe(II) produced from Fe(III) bio-reduction increased from 2.46 to 8.24 mM in the first two anoxic periods, but decreased to 0.10 mM in the following four anoxic periods. For the oxic periods, Fe(II) was oxidized along with a production of OH, and the cumulative OH concentrations were around 31 µM in the first three periods but decreased to 6.3 µM in the subsequent three periods. MR-1 was inactivated by about 0.6 order of magnitude in the first two oxic periods, and the inactivation became insignificant for the later oxic periods. The six cycles of anoxic-oxic treatments promoted the transformation of Fe(III) (hydr)oxides into more amorphous phase. A strong interplay between Fe species transformation and OH production occurred in anoxic-oxic cycles. Fe(III) bio-reduction by MR-1 in anoxic periods determined the content and speciation of Fe(II), which governed OH production in the subsequent oxic periods. The generated OH affected MR-1 activity in oxic periods, which further controlled Fe(III) bio-reduction in anoxic periods. The hidden interplay identified herein improves our understanding of the cycling of Fe and associated elements under redox fluctuating environments.

Preventing Hydrogen Disposal Increases Electrode Utilization Efficiency by Shewanella oneidensis

Many bacteria use hydrogen anaerobically as both a source and sink for electrons; consuming hydrogen when it is plentiful and producing it when concentrations are low enough to allow proton reduction. While this can increase an organism’s competitiveness, hydrogen uptake or excretion can also make it difficult to control electron flux to a specific product. For example, when Shewanella oneidensis strain MR-1 is used to oxidize organic molecules and recover electrons in microbial electrochemical devices, small changes in ambient hydrogen concentrations could dramatically alter the efficiency of electron capture at the anode due to this organism’s respiratory flexibility. When new three-electrode reactor designs created to minimize oxygen intrusion during anodic growth were tested with lactate-oxidizing S. oneidensis, current production decreased significantly in reactors vented to remove hydrogen produced at the counter electrode, suggesting a role for hydrogen uptake or disposal when cells used electrodes as electron acceptors. A ∆hydA∆hyaB mutant lacking both hydrogenases reversed this trend, and nearly doubled current production rates. This increase was shown to be due to the efficiency of lactate oxidation, as 90% of electrons were recovered as electricity in the ∆hydA∆hyaB mutant compared to only 50% for wild type. Experiments with Fe(III) oxide provided additional evidence that S. oneidensis generates hydrogen reducing equivalents during reduction of insoluble electron acceptors, while experiments with cells incubated with Fe(III) citrate showed increased survival of wild-type compared to ∆hydA∆hyaB in stationary phase. Together these data show how the multiple routes of electron disposal of S. oneidensis, while beneficial under changing conditions, limits the efficiency of electron recovery in electrochemical systems, and demonstrates a simple approach to increasing current production rates in systems where hydrogen is being captured as a product.

Attenuation of Fe(III)-reducing bacteria during table fluctuation of groundwater containing Fe2+

Groundwater table fluctuation during natural and anthropogenic processes can facilitate the interaction between oxygen (O2) from the unsaturated zone and ferrous iron (Fe2+) from the saturated zone. In light of previous findings that Fe(III)-reducing bacteria can be killed by the reactive oxidants produced from Fe2+ oxidation under static oxic conditions, we hypothesize that Fe(III)-reducing bacteria will be attenuated during groundwater table fluctuations. To test this hypothesis, this study explored the variations of cell numbers of Shewanella oneidensis strain MR-1 (MR-1), a typical strain of Fe(III)-reducing bacteria, together with dissolved oxygen (DO) and Fe2+, at different points during controlled groundwater table fluctuations in a sand column. The results showed that, during the rise of the water table, O2 in the pore air was entrapped by the deoxygenated groundwater, and Fe2+ in the groundwater was oxidized by the entrapped O2. In this process, 1.40–2.42 orders of magnitude of viable MR-1 cells were killed at different points in the column. Further investigation proposed that the death of MR-1 is caused by the production of intracellular reactive oxidants, such as O2•− and OH•, from the oxidation of adsorbed/absorbed Fe2+ instead of by bulk reactive oxidants, such as OH• and Fe(IV), produced from the oxidation of aqueous Fe2+. The findings here provide new insights for Fe biogeochemical cycling in the redox-dynamic zone.

Transport and retention of Shewanella oneidensis strain MR1 in water-saturated porous media with different grain-surface properties

Bacterium Shewanella oneidensis strain (MR1), a facultative microorganism that plays critical role in contaminant transformation and degradation, was used as an example to decipher the transport and retention of microorganisms in water-saturated porous media with different grain-surface properties and under different ionic compositions (i.e. Na+, Ca2+, and Mg2+). Dolomite and quartz sands, which contained different surface charge properties, were used as the representative minerals. Dolomite was selected because its surface charges are significantly affected by solution composition. The mobility of MR1 in the dolomite column was lower than that in the quartz column, because the lower energy barrier between MR1 and dolomite than that between MR1 and quartz, resulting in the larger retention of MR1 in the dolomite column. The breakthrough curves were well simulated by the two sites kinetic model with HYDRUS-1D. The maximum concentration of attached bacteria (Smax) were positively correlated to the ionic strength regardless of mineral types. The values of Smax were about 1.1–4.0 times larger in the MR1-dolomite system than that in the MR1-quartz system under different ionic strength conditions. The retention of the MR1 on dolomite surfaces in the presence of divalent cations Ca2+ is significantly higher than that on quartz surfaces primarily due to the larger electrostatic attraction energy between the MR1 and dolomite grains. The findings demonstrate that the porous media with the lower negative charge has the higher capacity for the retention and deposition of MR1, potentially affecting the transport of MR1 and other bacteria in the subsurface.

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. Here, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 106 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.

Redox Structures of Humic Acids Derived From Different Sediments and Their Effects on Microbial Reduction Reactions.

Herein, we investigated the chemical, electrochemical, and spectroscopic characteristics of humic acids (HAs) extracted from sediments of different origin [Ling Qiao river, Xi Xi wetland, Qi Zhen lake (QZ), and Hu Zhou pond in Zhejiang province, China], paying particular attention to their role in the enhancement of nitrate and FeOOH reduction. Notably, the highest C/N ratio (16.16), O/C ratio (1.89), and Fe content (11.57 g kg-1 sample) were observed for HAs extracted from QZ sediment. Cyclic voltammetry analyses confirmed that all HAs contained redox-active groups and exhibited redox potentials between -0.36 and -0.28 V vs. the standard hydrogen electrode. All HAs showed similar Fourier transform infrared spectra with variable absorption intensity, the spectra verified the presence of aromatic C=C, C–H, and C=O of quinone ketones group in HAs. Electron spin resonance suggested that quinone moieties within HAs are the redox-active centers. All HAs promoted the microbial reduction of nitrate and amorphous FeOOH by Shewanella oneidensis strain MR-1, achieving high nitrate reduction extents of 79–98.4%, compared to the biotic and abiotic control values of 29.6 and 0.006%, respectively. The corresponding extents of Fe(II) production equaled 43.25–60.5%, exceeding those of biotic and abiotic controls (28.5 and 0.005%, respectively). In addition to the highest C/N, O/C ratio, and Fe content, HA extracted from QZ sediment also exhibited the highest nitrate and FeOOH reduction performances. Although the proportion of organic redox-active carbon is small, the potential electron-mediating ability is not ignorable. HAs are redox active for enhancing microbial reduction of nitrate and amorphous FeOOH regardless of the location or texture of parent sediments, implying their great potential for acting as redox mediator in enhancing multiple microbial reduction, thereby affecting various biogeochemical processes (i.e., iron cycle, nitrogen cycle, etc.) as well as in situ remediation in anaerobic environment.

Impact of Fe(II) oxidation in the presence of iron-reducing bacteria on subsequent Fe(III) bio-reduction

The interplay of Fe(II) oxidation and Fe(III) bio-reduction occurs widely in both natural and engineered redox-dynamic systems. This study aimed to unravel the impact of Fe(II) oxidation by O2 in the presence of iron-reducing bacteria on subsequent Fe(III) bio-reduction. Mixed solutions of Fe2+ (0.1–0.5 mM) and Shewanella oneidensis strain MR-1 (MR-1, 2.0 × 107 CFU/mL) at neutral pH were first exposed to laboratory air for Fe(II) oxidation and bacterial inactivation, and then the resultant Fe(III) suspensions were switched to anoxic conditions for bio-reduction by the surviving bacteria. In the oxidation step, the coexisting MR-1 was inactivated by 0.8–1.71 orders of magnitude within 60 min. In the subsequent bio-reduction step, the resultant Fe(III) was bio-reduced by the surviving MR-1. Bio-reduction of the resultant Fe(III) by the surviving MR-1 was 1.8–2.5 times faster than that of the Fe(III) that was produced from Fe2+ oxidation without MR-1 by fresh MR-1 cells at 2.0 × 107 CFU/mL. Although MR-1 inactivation during Fe(II) oxidation may inhibit Fe(III) bio-reduction, the increase in bio-availability of the resultant Fe(III) and the residual reactivity of dead cells led to net enhancement of bio-reduction under the tested conditions. Lepidocrocite was the sole Fe(III) mineral that was produced from Fe2+ oxidation without MR-1, while 19% ferrihydrite was produced from Fe2+ oxidation in the presence of MR-1. The formation of low-crystallinity ferrihydrite accounts for the increase in bio-availability of the Fe(III) minerals. The findings of this study highlight an important but overlooked impact underlying the interplay of Fe(II) oxidation and Fe(III) bio-reduction.

Deletion of PBP1a/LpoA complex compromises cell envelope integrity in Shewanella oneidensis.

High molecular weight penicillin-binding proteins (PBPs) are responsible for the biosynthesis of peptidoglycan. In Escherichia coli, PBP1a and PBP1b form multienzyme peptidoglycan-synthesizing complexes with outer membrane lipoproteins LpoA and LpoB, respectively. The two complexes appear to be largely redundant, although their distinct physiological roles remain unclear. PBP1a/LpoA and PBP1b/LpoB also exist in Shewanella oneidensis strain MR-1, but effects of the two complexes on aerobic growth and β-lactam resistance are quite different. In this study, the phenotypes of strains lacking a certain complex in S. oneidensis were compared. Deletion of PBP1a/LpoA caused aberrant cell morphology (including branches and bulges), enhanced sensitivity to various envelope stresses and outer membrane permeability. On the contrary, strains lacking PBP1b/LpoB displayed phenotypes similar to the wild type.

Survival of Anaerobic Fe2+ Stress Requires the ClpXP Protease.

Shewanella oneidensis strain MR-1 is a versatile bacterium capable of respiring extracellular, insoluble ferric oxide minerals under anaerobic conditions. The respiration of iron minerals results in the production of soluble ferrous ions, which at high concentrations are toxic to living organisms. It is not fully understood how Fe2+ is toxic to cells anaerobically, nor is it fully understood how S. oneidensis is able to resist high levels of Fe2+ Here we describe the results of a transposon mutant screen and subsequent deletion of the genes clpX and clpP in S. oneidensis, which demonstrate that the protease ClpXP is required for anaerobic Fe2+ resistance. Many cellular processes are known to be regulated by ClpXP, including entry into stationary phase, envelope stress response, and turnover of stalled ribosomes. However, none of these processes appears to be responsible for mediating anaerobic Fe2+ resistance in S. oneidensis Protein trapping studies were performed to identify ClpXP targets in S. oneidensis under Fe2+ stress, implicating a wide variety of protein targets. Escherichia coli strains lacking clpX or clpP also display increased sensitivity to Fe2+ anaerobically, indicating Fe2+ resistance may be a conserved role for the ClpXP protease system. Hypotheses regarding the potential role(s) of ClpXP during periods of high Fe2+ are discussed. We speculate that metal-containing proteins are misfolded under conditions of high Fe2+ and that the ClpXP protease system is necessary for their turnover.IMPORTANCE Prior to the evolution of cyanobacteria and oxygenic photosynthesis, life arose and flourished in iron-rich oceans. Today, aqueous iron-rich environments are less common, constrained to low-pH conditions and anaerobic systems such as stratified lakes and seas, digestive tracts, subsurface environments, and sediments. The latter two ecosystems often favor dissimilatory metal reduction, a process that produces soluble Fe2+ from iron oxide minerals. Dissimilatory metal-reducing bacteria must therefore have mechanisms to tolerate anaerobic Fe2+ stress, and studying resistance in these organisms may help elucidate the basis of toxicity. Shewanella oneidensis is a model dissimilatory metal-reducing bacterium isolated from metal-rich sediments. Here we demonstrate a role for ClpXP, a protease system widely conserved in bacteria, in anaerobic Fe2+ resistance in both S. oneidensis and Escherichia coli.

MgtE Homolog FicI Acts as a Secondary Ferrous Iron Importer in Shewanella oneidensis Strain MR-1.

The transport of metals into and out of cells is necessary for the maintenance of appropriate intracellular concentrations. Metals are needed for incorporation into metalloproteins but become toxic at higher concentrations. Many metal transport proteins have been discovered in bacteria, including the Mg2+ transporter E (MgtE) family of passive Mg2+/Co2+ cation-selective channels. Low sequence identity exists between members of the MgtE family, indicating that substrate specificity may differ among MgtE transporters. Under anoxic conditions, dissimilatory metal-reducing bacteria, such as Shewanella and Geobacter species, are exposed to high levels of soluble metals, including Fe2+ and Mn2+ Here we characterize SO_3966, which encodes an MgtE homolog in Shewanella oneidensis that we name FicI (ferrous iron and cobalt importer) based on its role in maintaining metal homeostasis. A SO_3966 deletion mutant exhibits enhanced growth over that of the wild type under conditions with high Fe2+ or Co2+ concentrations but exhibits wild-type Mg2+ transport and retention phenotypes. Conversely, deletion of feoB, which encodes an energy-dependent Fe2+ importer, causes a growth defect under conditions of low Fe2+ concentrations but not high Fe2+ concentrations. We propose that FicI represents a secondary, less energy-dependent mechanism for iron uptake by S. oneidensis under high Fe2+ concentrations.IMPORTANCEShewanella oneidensis MR-1 is a target of microbial engineering for potential uses in biotechnology and the bioremediation of heavy-metal-contaminated environments. A full understanding of the ways in which S. oneidensis interacts with metals, including the means by which it transports metal ions, is important for optimal genetic engineering of this and other organisms for biotechnology purposes such as biosorption. The MgtE family of metal importers has been described previously as Mg2+ and Co2+ transporters. This work broadens that designation with the discovery of an MgtE homolog in S. oneidensis that imports Fe2+ but not Mg2+ The research presented here also expands our knowledge of the means by which microorganisms have adapted to take up essential nutrients such as iron under various conditions.

The Detoxification and Degradation of Benzothiazole from the Wastewater in Microbial Electrolysis Cells.

In this study, the high-production-volume chemical benzothiazole (BTH) from synthetic water was fully degraded into less toxic intermediates of simple organic acids using an up-flow internal circulation microbial electrolysis reactor (UICMER) under the hydraulic retention time (HRT) of 24 h. The bioelectrochemical system was operated at 25 ± 2 °C and continuous-flow mode. The BTH loading rate varied during experiments from 20 g·m−3·day−1 to 110 g·m−3·day−1. BTH and soluble COD (Chemical Oxygen Demand) removal efficiency reached 80% to 90% under all BTH loading rates. Bioluminescence based Shewanella oneidensis strain MR-1 ecotoxicity testing demonstrated that toxicity was largely decreased compared to the BTH wastewater influent and effluent of two control experiments. The results indicated that MEC (Microbial Electrolysis Cell) was useful and reliable for improving BTH wastewater treatment efficiency, enabling the microbiological reactor to more easily respond to the requirements of higher loading rate, which is meaningful for economic and efficient operation in future scale-up.

A Ferrous Iron Exporter Mediates Iron Resistance in Shewanella oneidensis MR-1.

Shewanella oneidensis strain MR-1 is a dissimilatory metal-reducing bacterium frequently found in aquatic sediments. In the absence of oxygen, S. oneidensis can respire extracellular, insoluble oxidized metals, such as iron (hydr)oxides, making it intimately involved in environmental metal and nutrient cycling. The reduction of ferric iron (Fe(3+)) results in the production of ferrous iron (Fe(2+)) ions, which remain soluble under certain conditions and are toxic to cells at higher concentrations. We have identified an inner membrane protein in S. oneidensis, encoded by the gene SO_4475 and here called FeoE, which is important for survival during anaerobic iron respiration. FeoE, a member of the cation diffusion facilitator (CDF) protein family, functions to export excess Fe(2+) from the MR-1 cytoplasm. Mutants lacking feoE exhibit an increased sensitivity to Fe(2+). The export function of FeoE is specific for Fe(2+), as an feoE mutant is equally sensitive to other metal ions known to be substrates of other CDF proteins (Cd(2+), Co(2+), Cu(2+), Mn(2+), Ni(2+), or Zn(2+)). The substrate specificity of FeoE differs from that of FieF, the Escherichia coli homolog of FeoE, which has been reported to be a Cd(2+)/Zn(2+) or Fe(2+)/Zn(2+) exporter. A complemented feoE mutant has an increased growth rate in the presence of excess Fe(2+) compared to that of the ΔfeoE mutant complemented with fieF. It is possible that FeoE has evolved to become an efficient and specific Fe(2+) exporter in response to the high levels of iron often present in the types of environmental niches in which Shewanella species can be found.