Membrane vesicles as a novel strategy for shedding encrusted cell surfaces

Surface encrustation by minerals, which impedes cellular metabolism, is a potential hazard for microbes. The reduction of U(VI) to U(IV) by Shewanella oneidensis strain MR-1 leads to the precipitation of the mineral uraninite, as well as a non-crystalline U(IV) product. The wild-type (WT) strain can produce extracellular polymeric substances (EPS), prompting precipitation of U some distance from the cells and precluding encrustation. Using cryo-transmission electron microscopy and scanning transmission X-ray microscopy we show that, in the biofilm-deficient mutant Delta mxdA, as well as in the WT strain to a lesser extent, we observe the formation of membrane vesicles (MVs) as an additional means to lessen encrustation. Additionally, under conditions in which the WT does not produce EPS, formation of MVs was the only observed mechanism to mitigate cell encrustation. Viability studies comparing U-free controls to cells exposed to U showed a decrease in the number of viable cells in conditions where MVs alone are detected, yet no loss of viability when cells produce both EPS and MVs. We conclude that MV formation is a microbial strategy to shed encrusted cell surfaces but is less effective at maintaining cell viability than the precipitation of U on EPS

Persistence of the Isotopic Signature of Pentavalent Uranium in Magnetite

Uranium isotopic signatures can be harnessed to monitor the reductive remediation of subsurface contamination or to reconstruct paleo-redox environments. However, the mechanistic underpinnings of the isotope fractionation associated with U reduction remain poorly understood. Here, we present a coprecipitation study, in which hexavalent U (U(VI)) was reduced during the synthesis of magnetite and pentavalent U (U(V)) was the dominant species. The measured δ$^{238}$U values for unreduced U(VI) (∼−1.0‰), incorporated U (96 ± 2% U(V), ∼−0.1‰), and extracted surface U (mostly U(IV), ∼0.3‰) suggested the preferential accumulation of the heavy isotope in reduced species. Upon exposure of the U-magnetite coprecipitate to air, U(V) was partially reoxidized to U(VI) with no significant change in the δ$^{238}$U value. In contrast, anoxic amendment of a heavy isotope-doped U(VI) solution resulted in an increase in the δ$^{238}$U of the incorporated U species over time, suggesting an exchange between incorporated and surface/aqueous U. Overall, the results support the presence of persistent U(V) with a light isotope signature and suggest that the mineral dynamics of iron oxides may allow overprinting of the isotopic signature of incorporated U species. This work furthers the understanding of the isotope fractionation of U associated with iron oxides in both modern and paleo-environments

Electron flux is a key determinant of uranium isotope fractionation during bacterial reduction

Uranium isotopic signatures in the rock record are utilized as a proxy for past redox conditions on Earth. However, these signatures display significant variability that complicates the interpretation of specific redox conditions. Using the model uranium-reducing bacterium, Shewanella oneidensis MR-1, we show that the abundance of electron donors (e.g., labile organic carbon) controls uranium isotope fractionation, such that high electron fluxes suppress fractionation. Further, by purifying a key uranium-reducing enzyme, MtrC, we show that the magnitude of fractionation is explicitly controlled by the protein redox state. Finally, using a mathematical framework, we demonstrate that these differences in fractionation arise from the propensity for back-reaction throughout the multi-step reduction of hexavalent uranium. To improve interpretations of observed fractionations in natural environments, these findings suggest that a variable intrinsic fractionation factor should be incorporated into models of uranium isotope systematics to account for differences in electron flux caused by organic carbon availability

Nanoscale mechanism of UO2 formation through uranium reduction by magnetite

Uranium (U) is a ubiquitous element in the Earth’s crust at ~2 ppm. In anoxic environments, soluble hexavalent uranium (U(VI)) is reduced and immobilized. The underlying reduction mechanism is unknown but likely of critical importance to explain the geochemical behavior of U. Here, we tackle the mechanism of reduction of U(VI) by the mixed-valence iron oxide, magnetite. Through high-end spectroscopic and microscopic tools, we demonstrate that the reduction proceeds first through surface-associated U(VI) to form pentavalent U, U(V). U(V) persists on the surface of magnetite and is further reduced to tetravalent UO2 as nanocrystals (~1–2 nm) with random orientations inside nanowires. Through nanoparticle re-orientation and coalescence, the nanowires collapse into ordered UO2 nanoclusters. This work provides evidence for a transient U nanowire structure that may have implications for uranium isotope fractionation as well as for the molecular-scale understanding of nuclear waste temporal evolution and the reductive remediation of uranium contamination

U(VI) reduction by spores of Clostridium acetobutylicum

Vegetative cells of Clostridium acetobutylicum are known to reduce hexavalent uranium (U(VI)). We investigated the ability of spores of this organism to drive the same reaction. We found that spores were able to remove U(VI) from solution when H, was provided as an electron donor and to form a U(IV) precipitate. We tested several environmental conditions and found that spent vegetative cell growth medium was required for the process. Electron microscopy showed the product of reduction to accumulate outside the exosporium. Our results point towards a novel U(VI) reduction mechanism, driven by spores, that is distinct from the thoroughly studied reactions in metal-reducing Proteobacteria. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved

Biogeochemical controls on the product of microbial U(VI) reduction

Biologically mediated immobilization of radio-nuclides in the subsurface is a promising strategy for the remediation of uranium-contaminated sites. During this process, soluble U(VI) is reduced by indigenous microorganisms to sparingly soluble U(TV). The crystalline U(IV) phase uraninite, or UO2, is the preferable end-product of bioremediation due to its relatively high stability and low solubility in comparison to biomass-associated nonuraninite U(IV) species that have been reported in laboratory and under field conditions. The goal of this study was to delineate the geochemical conditions that promote the formation of nonuraninite U(IV) versus uraninite and to decipher the mechanisms of its preferential formation. U(IV) products were prepared under varying geochemical conditions and characterized with X-ray absorption spectroscopy (XAS), scanning transmission X-ray microscopy (STXM), and various wet chemical methods. We report an increasing fraction of nonuraninite U(IV) species with decreasing initial U concentration. Additionally, the presence of several common groundwater solutes (sulfate, silicate, and phosphate) promote the formation of nonuraninite U(IV). Our experiments revealed that the presence of those solutes promotes the formation of bacterial extracellular polymeric substances (EPS) and increases bacterial viability, suggesting that the formation of nonuraninite U(IV) is due to a biological response to solute presence during U(VI) reduction. The results obtained from this laboratory-scale research provide insight into biogeochemical controls on the product(s) of uranium reduction during bioremediation of the subsurface

The product of microbial uranium reduction includes multiple species with U(IV)-phosphate coordination

Until recently, the reduction of U(VI) to U(IV) during bioremediation was assumed to produce solely the sparingly soluble mineral uraninite, UO2(s). However, results from several laboratories reveal other species of U(IV) characterized by the absence of an EXAFS U-U pair correlation (referred to here as noncrystalline U(IV)). Because it lacks the crystalline structure of uraninite, this species is likely to be more labile and susceptible to reoxidation. In the case of single species cultures, analyses of U extended X-ray fine structure (EXAFS) spectra have previously suggested U(IV) coordination to carboxyl, phosphoryl or carbonate groups. In spite of this evidence, little is understood about the species that make up noncrystalline U(IV), their structural chemistry and the nature of the U(IV)-ligand interactions. Here, we use infrared spectroscopy (IR), uranium L-III-edge X-ray absorption spectroscopy (XAS), and phosphorus K-edge XAS analyses to constrain the binding environments of phosphate and uranium associated with Shewanella oneidensis MR-1 bacterial cells. Systems tested as a function of pH included: cells under metal-reducing conditions without uranium, cells under reducing conditions that produced primarily uraninite, and cells under reducing conditions that produced primarily biomass-associated noncrystalline U(IV). P X-ray absorption near-edge structure (XANES) results provided clear and direct evidence of U(IV) coordination to phosphate. Infrared (IR) spectroscopy revealed a pronounced perturbation of phosphate functional groups in the presence of uranium. Analysis of these data provides evidence that U(IV) is coordinated to a range of phosphate species, including monomers and polymerized networks. U EXAFS analyses and a chemical extraction measurements support these conclusions. The results of this study provide new insights into the binding mechanisms of biomass-associated U(IV) species which in turn sheds light on the mechanisms of biological U(VI) reduction. (C) 2014 Elsevier Ltd. All rights reserved