Cellular Mercury Coordination Environment, and Not Cell Surface Ligands, Influence Bacterial Methylmercury Production.

The conversion of inorganic mercury (Hg(II)) to methylmercury (MeHg) is central to the understanding of Hg toxicity in the environment. Hg methylation occurs in the cytosol of certain obligate anaerobic bacteria and archaea possessing the hgcAB gene cluster. However, the processes involved in Hg(II) biouptake and methylation are not well understood. Here, we examined the role of cell surface thiols, cellular ligands with the highest affinity for Hg(II) that are located at the interface between the outer membrane and external medium, on the sorption and methylation of Hg(II) by Geobacter sulfurreducens. The effect of added cysteine (Cys), which is known to greatly enhance Hg(II) biouptake and methylation, was also explored. By quantitatively blocking surface thiols with a thiol binding ligand (qBBr), we show that surface thiols have no significant effect on Hg(II) methylation, regardless of Cys addition. The results also identify a significant amount of cell-associated Hg-S3/S4 species, as studied by high energy-resolution X-ray absorption near edge structure (HR-XANES) spectroscopy, under conditions of high MeHg production (with Cys addition). In contrast, Hg-S2 are the predominant species during low MeHg production. Hg-S3/S4 species may be related to enhanced Hg(II) biouptake or the ability of Hg(II) to become methylated by HgcAB and should be further explored in this context.

High Energy Resolution-X-ray Absorption Near Edge Structure Spectroscopy Reveals Zn Ligation in Whole Cell Bacteria.

Identifying the zinc (Zn) ligation and coordination environment in complex biological and environmental systems is crucial to understand the role of Zn as a biologically essential but sometimes toxic metal. Most studies on Zn coordination in biological or environmental samples rely on the extended X-ray absorption fine structure (EXAFS) region of a Zn K-edge X-ray absorption spectroscopy (XAS) spectrum. However, EXAFS analysis cannot identify unique nearest neighbors with similar atomic number (i.e., O versus N) and provides little information on Zn ligation. Herein, we demonstrate that high energy resolution-X-ray absorption near edge structure (HR-XANES) spectroscopy enables the direct determination of Zn ligation in whole cell bacteria, providing additional insights lost from EXAFS analysis at a fraction of the scan time and Zn concentration. HR-XANES is a relatively new technique that has improved our understanding of trace metals (e.g., Hg, Cu, and Ce) in dilute systems. This study is the first to show that HR-XANES can unambiguously detect Zn coordination to carboxyl, phosphoryl, imidazole, and/or thiol moieties in model microorganisms.

The role of cysteine and sulfide in the interplay between microbial Hg(ii) uptake and sulfur metabolism.

Biogenic thiols, such as cysteine, have been used to control the speciation of Hg(ii) in bacterial exposure experiments. However, the extracellular biodegradation of excess cysteine leads to the formation of Hg(ii)-sulfide species, convoluting the interpretation of Hg(ii) uptake results. Herein, we test the hypothesis that Hg(ii)-sulfide species formation is a critical step during bacterial Hg(ii) uptake in the presence of excess cysteine. An Escherichia coli (E. coli) wild-type and mutant strain lacking the decR gene that regulates cysteine degradation to sulfide were exposed to 50 and 500 nM Hg with 0 to 2 mM cysteine. The decR mutant released ∼4 times less sulfide from cysteine degradation compared to the wild-type for all tested cysteine concentrations during a 3 hour exposure period. We show with thermodynamic calculations that the predicted concentration of Hg(ii)-cysteine species remaining in the exposure medium (as opposed to forming HgS(s)) is a good proxy for the measured concentration of dissolved Hg(ii) (i.e., not cell-bound). Likewise, the measured cell-bound Hg(ii) correlates with thermodynamic calculations for HgS(s) formation in the presence of cysteine. High resolution X-ray absorption near edge structure (HR-XANES) spectra confirm the existence of cell-associated HgS(s) at 500 nM total Hg and suggest the formation of Hg-S clusters at 50 nM total Hg. Our results indicate that a speciation change to Hg(ii)-sulfide controls Hg(ii) cell-association in the presence of excess cysteine.

Spectroscopic and Microscopic Evidence of Biomediated HgS Species Formation from Hg(II)-Cysteine Complexes: Implications for Hg(II) Bioavailability.

We investigated the chemistry of Hg(II) during exposure of exponentially growing bacteria ( Escherichia coli, Bacillus subtilis, and Geobacter sulfurreducens) to 50 nM, 500 nM, and 5 µM total Hg(II) with and without added cysteine. With X-ray absorption spectroscopy, we provide direct evidence of the formation of cell-associated HgS for all tested bacteria. The addition of cysteine (100-1000 µM) promotes HgS formation (>70% of total cell-associated Hg(II)) as a result of the biodegradation of added cysteine to sulfide. Cell-associated HgS species are also detected when cysteine is not added as a sulfide source. Two phases of HgS, cinnabar (α-HgS) and metacinnabar (ß-HgS), form depending on the total concentration of Hg(II) and sulfide in the exposure medium. However, α-HgS exclusively forms in assays that contain an excess of cysteine. Scanning transmission electron microscopy images reveal that nanoparticulate HgS(s) is primarily located at the cell surface/extracellular matrix of Gram-negative E. coli and G. sulfurreducens and in the cytoplasm/cell membrane of Gram-positive B. subtilis. Intracellular Hg(II) was detected even when the predominant cell-associated species was HgS. This study shows that HgS species can form from exogenous thiol-containing ligands and endogenous sulfide in Hg(II) biouptake assays under nondissimilatory sulfate reducing conditions, providing new considerations for the interpretation of Hg(II) biouptake results.

Cysteine Addition Promotes Sulfide Production and 4-Fold Hg(II)-S Coordination in Actively Metabolizing Escherichia coli.

The bacterial uptake of mercury(II), Hg(II), is believed to be energy-dependent and is enhanced by cysteine in diverse species of bacteria under aerobic and anaerobic conditions. To gain insight into this Hg(II) biouptake pathway, we have employed X-ray absorption spectroscopy (XAS) to investigate the relationship between exogenous cysteine, cellular metabolism, cellular localization, and Hg(II) coordination in aerobically respiring Escherichia coli (E. coli). We show that cells harvested in exponential growth phase consistently display mixtures of 2-fold and 4-fold Hg(II) coordination to sulfur (Hg-S2 and Hg-S4), with added cysteine enhancing Hg-S4 formation. In contrast, cells in stationary growth phase or cells treated with a protonophore causing a decrease in cellular ATP predominantly contain Hg-S2, regardless of cysteine addition. Our XAS results favor metacinnabar (ß-HgS) as the Hg-S4 species, which we show is associated with both the cell envelope and cytoplasm. Additionally, we observe that added cysteine abiotically oxidizes to cystine and exponentially growing E. coli degrade high cysteine concentrations (100-1000 µM) into sulfide. Thermodynamic calculations confirm that cysteine-induced sulfide biosynthesis can promote the formation of dissolved and particulate Hg(II)-sulfide species. This report reveals new complexities arising in Hg(II) bioassays with cysteine and emphasizes the need for considering changes in chemical speciation as well as growth stage.

The molecular structure of aqueous Hg(II)-EDTA as determined by X-ray absorption spectroscopy.

The widespread presence and persistence of ethylenediaminetetraacetic acid (EDTA) in aquatic environments can lead to the formation of metal-EDTA complexes that influence metal bioavailability and mobility. Recently, the Hg(II)-EDTA complex was observed to slightly promote the biouptake of Hg(II) to the bacterium Escherichia coli and to undergo a relatively quick ligand exchange reaction with thiols at the bacterial cell membrane. The reactivity of a metal complex depends on its molecular structure; however, the molecular structure of aqueous Hg(II)-EDTA has yet to be reported. Here, we use X-ray absorption spectroscopy to determine the molecular structure of aqueous Hg(II)-EDTA. Our results suggest that aqueous Hg(II)-EDTA displays distorted octahedral geometry with one water molecule in the coordination sphere.

Chemical interactions between Nano-ZnO and Nano-TiO2 in a natural aqueous medium.

The use of diverse engineered nanomaterials (ENMs) potentially leads to the release of multiple ENMs into the environment. However, previous efforts to understand the behavior and the risks associated with ENMs have focused on only one material at a time. In this study, the chemical interactions between two of the most highly used ENMs, nano-TiO2, and nano-ZnO, were examined in a natural water matrix. The fate of nano-ZnO in Lake Michigan water was investigated in the presence of nano-TiO2. Our experiments demonstrate that the combined effects of ZnO dissolution and Zn adsorption onto nano-TiO2 control the concentration of dissolved zinc. X-ray absorption spectroscopy was used to determine the speciation of Zn in the particulate fraction. The spectra show that Zn partitions between nano-ZnO and Zn2+ adsorbed on nano-TiO2. A simple kinetic model is presented to explain the experimental data. It integrates the processes of nano-ZnO dissolution with Zn adsorption onto nano-TiO2 and successfully predicts dissolved Zn concentration in solution. Overall, our results suggest that the fate and toxicity potential of soluble ENMs, such as nano-ZnO, are likely to be influenced by the presence of other stable ENMs, such as nano-TiO2.