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

Biological Reduction of a U(V)-Organic Ligand Complex

Metal-reducing microorganisms such as Shewanella oneidensis MR-1 reduce highly soluble species of hexavalent uranyl (U(VI)) to less mobile tetravalent uranium (U(IV)) compounds. The biologically mediated immobilization of U(VI) is being considered for the remediation of U contamination. However, the mechanistic underpinnings of biological U(VI) reduction remain unresolved. It has become clear that a first electron transfer occurs to form pentavalent (U(V)) intermediates, but it has not been definitively established whether a second one-electron transfer can occur or if disproportionation of U(V) is required. Here, we utilize the unusual properties of dpaea2– ((dpaeaH2═bis(pyridyl-6-methyl-2-carboxylate)-ethylamine)), a ligand forming a stable soluble aqueous complex with U(V), and investigate the reduction of U(VI)–dpaea and U(V)–dpaea by S. oneidensis MR-1. We establish U speciation through time by separating U(VI) from U(IV) by ion exchange chromatography and characterize the reaction end-products using U M4-edge high resolution X-ray absorption near-edge structure (HR-XANES) spectroscopy. We document the reduction of solid phase U(VI)–dpaea to aqueous U(V)–dpaea but, most importantly, demonstrate that of U(V)–dpaea to U(IV). This work establishes the potential for biological reduction of U(V) bound to a stabilizing ligand. Thus, further work is warranted to investigate the possible persistence of U(V)–organic complexes followed by their bioreduction in environmental systems

Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

Monoterpenes (C10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of Escherichia coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP production but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes. Biotechnol. Bioeng. 2017;114: 1703-1712. © 2017 Wiley Periodicals, Inc

Insights into the microbial reduction of pentavalent and hexavalent uranium species by Shewanella oneidensis MR-1

Decades of uranium-related activities such as ore mining, nuclear power generation, weapon manufacture, storage of nuclear wastes, have contributed to the release of U in the environment. U occurrence is concerning, since in surface environments, U is typically found in the U(VI) oxidation state, as uranyl, complexed with various ligands, and is highly soluble. Therefore, this toxic metal is likely to leech into soils and contaminate surface waters and groundwaters. In addition, owing to the long half-lives of the predominant isotopes 238U and 235U, U is considered as a persistent contaminant over geological timescales. For several decades, efforts have focused on the development of in situ remediation solutions to tackle U pollution in the subsurface, and limit its mobility. With this aim, interest in the metabolic potential for U(VI)-respiration by dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, has seen a significant increase. This metabolically-versatile bacterium has been reported to reduce mobile U(VI) to typically insoluble crystalline and amorphous U(IV). The reduction of U(VI) in S. oneidensis MR-1 is coupled to the oxidation of an electron donor, which feeds electrons into an electron transport chain, extending from the cytoplasm to the outer-membrane of the cells. The microbially mediated reduction of U(VI) to U(IV) is the result of a two-step process. It is assumed that one electron is first transferred to U(VI) to form a pentavalent U(V) intermediate, followed by the abiotic disproportionation of two U(V) atoms into U(IV) and U(VI). However, evidence for this mechanism is limited to experimental systems rich in carbonate, which permits the rapid disproportionation of U(V). Thus, it remains unclear whether a second, biologically-mediated, electron transfer to U(V) is possible under conditions in which disproportionation is limited. To explore this, a novel U(V)-dpaea complex, that is stable in water at pH 7, was utilized to investigate the second step of the reduction mechanism. Here, we observed that U(V) can be biologically reduced by an additional one-electron transfer, resulting in the accumulation of U(IV) without the need for disproportionation. To improve our understanding of the molecular mechanism of U-dpaea reduction, we incubated mutant strains of S. oneidensis MR-1, lacking (i) only outer-membrane c-type cytochromes or (ii) all c-type cytochromes, with solid phase U(VI)-dpaea and aqueous U(V)-dpaea. We determined that U(VI)-dpaea reduction proceeds via the initial dissolution of the solid phase and that U(V)-dpaea reduction is mediated by outer-membrane c-type cytochromes. In particular, in vitro reactions between the purified outer-membrane c-type cytochrome MtrC and U(V)-dpaea demonstrated that MtrC can directly transfer electrons to U(V)-dpaea. Finally, we sought to determine the factors that influence electron transfer kinetics between DMRB and U. To this end, we reacted U(VI) coordinated by various aminocarboxylate ligands with purified MtrC. Here, U speciation significantly impacted reduction rates and appeared to be related to the binding strength of the U-MtrC interaction, i.e., hydrogen bonding versus electrostatic. All together, these findings provide further insights in the reduction mechanism of U by DMRB, and underline the importance of U speciation in controlling the pathway and rate of electron transfer

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

Abstract 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