Mechanism of Attenuation of Uranyl Toxicity by Glutathione in Lactococcus lactis

Abstract

b ABSTRACTBoth prokaryotic and eukaryotic organisms possess mechanisms for the detoxification of heavy metals, and these mechanisms are found among distantly related species. We investigated the role of intracellular glutathione (GSH), which, in a large number of taxa, plays a role in protection against the toxicity of common heavy metals. Anaerobically grown Lactococcus lactis containing an inducible GSH synthesis pathway was used as a model organism. Its physiological condition allowed study of putative GSH-dependent uranyl detoxification mechanisms without interference from additional reactive oxygen species. By microcalorimetric measurements of metabolic heat during cultivation, it was shown that intracellular GSH attenuates the toxicity of uranium at a concentration in the range of 10 to 150 M. In this concentration range, no effect was observed with copper, which was used as a reference for redox metal toxicity. At higher copper concentrations, GSH aggravated metal toxicity. Isothermal titration calorimetry revealed the endothermic binding of U(VI) to the carboxyl group(s) of GSH rather than to the reducing thiol group involved in copper interactions. The data indicate that the primary detoxifying mechanism is the intracellular sequestration of carboxyl-coordinated U(VI) into an insoluble complex with GSH. The opposite effects on uranyl and on copper toxicity can be related to the difference in coordination chemistry of the respective metal-GSH complexes, which cause distinct growth phase-specific effects on enzyme-metal interactions. IMPORTANCEUnderstanding microbial metal resistance is of particular importance for bioremediation, where microorganisms are employed for the removal of heavy metals from the environment. This strategy is increasingly being considered for uranium. However, little is known about the molecular mechanisms of uranyl detoxification. Existing studies of different taxa show little systematics but hint at a role of glutathione (GSH). Previous work could not unequivocally demonstrate a GSH function in decreasing the presumed uranyl-induced oxidative stress, nor could a redox-independent detoxifying action of GSH be identified. Combining metabolic calorimetry with cell number-based assays and genetics analysis enables a novel and general approach to quantify toxicity and relate it to molecular mechanisms. The results show that GSH-expressing microorganisms appear advantageous for uranyl bioremediation.T he natural abundance of uranium and the increasing health risks that originate from its anthropogenic release to the environment have generated a high demand for understanding the molecular mechanisms of uranium toxicity. Its radiotoxicity is of minor ecological relevance compared to its toxicity due to chemical interference with metabolism (1, 2). The latter is caused mostly by the displacement of Ca 2ϩ and Mg 2ϩ from functionally important binding sites in enzymes (3). The water-soluble uranyl cation UO 2 2ϩ is the most commonly found contaminant, which contains uranium in its highest ...

Calorimetrically Determined U(VI) Toxicity inBrassica napusCorrelates with Oxidoreductase Activity and U(VI) Speciation

“…At the same time, handling of radionuclides can be reduced to an absolute minimum. Although microcalorimetry has been shown to also reveal mechanistic details on uranium toxicity when linked to genetic engineering, such data complement but cannot replace studies on uranium-dependent specific enzyme activities (for example, ref ). Instead, the strength of life cell microcalorimetry originates in its quick and noninvasive systemic approach.…”

“…U(VI) Speciation and Metabolic Activity. Microcalorimetry has been used previously for the assessment of U(VI)dependent bacterial activities in both genetically engineered bacteria 11 or communities found in uranium waste heaps. 18 Typically, calorimetric studies have been evaluated based on mathematically well-defined growth models 19 or enthalpy balances 20 to derive per-cell estimates of metabolic rates.…”

“…Correlating molecular information on radionuclide speciation and biomolecular interactions with physiological performance is a major challenge for quantitative radioecology. We have recently shown that the microcalorimetric monitoring of metabolic activity in combination with genetic engineering can identify molecular details of the modulation of uranium toxicity in a living microorganism . However, the application of microcalorimetry to plant metabolism poses unique challenges.…”

“…We have recently shown that the microcalorimetric monitoring of metabolic activity in combination with genetic engineering can identify molecular details of the modulation of uranium toxicity in a living microorganism. 11 However, the application of microcalorimetry to plant metabolism poses unique challenges. In contrast to bacteria, which exhibit exponential growth phases from which division rates can be derived as physiologically meaningful parameters of toxicity; this is not the case with plant cells.…”

Uranium(VI) Complexes of Glutathione Disulfide Forming in Aqueous Solution

“…Most papers investigating GSH and GSSG metal-ion interaction, that is, structures and thermodynamic constants, focused on transition-metal ions, − whereas only a few studies address U­(VI) interaction. ,− Thus, for U­(VI) in aqueous solution in general, and by complementary spectroscopies in particular, the present study allows critical insights into complex structures and speciation. The herein presented results concentrate on GSSG as the ligand, and for discussion and comparison, GSH related results are included or provided as Supporting Information (SI).…”