| NSF Org: |
EAR Division Of Earth Sciences |
| Recipient: |
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| Initial Amendment Date: | January 22, 2015 |
| Latest Amendment Date: | January 22, 2015 |
| Award Number: | 1424968 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Enriqueta Barrera
EAR Division Of Earth Sciences GEO Directorate for Geosciences |
| Start Date: | March 1, 2015 |
| End Date: | February 28, 2018 (Estimated) |
| Total Intended Award Amount: | $70,785.00 |
| Total Awarded Amount to Date: | $70,785.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
10 W 35TH ST CHICAGO IL US 60616-3717 (312)567-3035 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
3300 South Federal Street Chicago IL US 60616-3732 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Geobiology & Low-Temp Geochem |
| Primary Program Source: |
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| Program Reference Code(s): | |
| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.050 |
ABSTRACT
Bacteria are ubiquitous in natural environments. The adherence of metal ions onto the surface of bacterial cells can affect the global cycling of elements, the mobility of metal contaminants, and the effectiveness of contaminant mitigation techniques. Past studies have identified the importance of certain bacterial surface sites in adhering metal ions at unrealistically high metal concentrations. However, recent studies suggest that at environmentally relevant metal concentrations, previously unrecognized sites may be more important. The goal of this study is to better understand the impact of high-affinity, but low abundance, bacterial surface binding sites on metal uptake and reactivity in aquatic systems. Because most metals are present at low concentrations both in natural and contaminated systems, the outcomes of this research could help better understand the environmental fate of heavy metals in natural environments.
An innovative approach will be used to isolate the influence of R-SH sites on bacterial cell envelopes. Specifically, an R-SH-sensitive fluorophore molecule (qBBr) will be used that binds strongly to R-SH sites on the cell envelope. qBBr fluoresces when bound to R-SH sites, and the charge on the molecule prevents it crossing the cell membrane easily; and hence can be used for previously impossible direct determinations of R-SH site concentrations on cell envelopes. In addition, because qBBr binds so strongly to cell envelope R-SH sites, we can use it as a blocking agent in order to isolate proton- and metal-binding reactions with cell envelope R-SH sites. The funded research will, for the first time, directly probe the role of cell envelope thiol sites, and will enable us to study their interactions with metals. Using fluorescence and x-ray absorption spectroscopies, coupled with potentiometric titration and bulk adsorption experiments, the PIs will measure the thiol concentration on cell envelopes of selected bacteria common to most aquatic systems, and to determine how different environmental variables, such as the growth medium and growth conditions (aerobic versus anaerobic) influence the thiol concentrations. The PIs will measure Zn adsorption onto thiol sites, and determine the molecular structures and binding constants of the Zn-thiol complexes on bacterial cell envelopes using sorption and spectroscopy approaches. The detailed measurements that the qBBr approach makes possible have the potential to transform our understanding of how bacteria bind metals under realistic conditions. The results of the proposed research are critical for evaluating the role of these important binding sites on metal speciation and distribution in the environment. The results from this study can be applied not only to contaminant transport modeling, but also to bioremediation engineering and to understanding heavy metal cycling in the environment in general.
The funded research will support a number of outreach activities, including teacher training through Princeton University?s ?Quest? program; the development of a geomicrobiology/environmental chemistry module in South Bend high school science research programs; and teaching a water pollution technology module in South Chicago-area high schools.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
This NSF award has resulted in a total of six peer-reviewed journal articles (four published and two in review), revolving around the role of cellular thiols on Hg and Zn biogeochemistry in natural and contaminated systems. Some of the key findings of our study are as follows.
Our study demonstrates that Hg complexation with intact bacterial cell suspensions, a mechanism which is likely applicable to other chalcophilic metals (e.g. Zn, Cd, and Pb) as well, is strongly dependent on metal loading and that the following conclusions can be drawn: 1) complexation of Hg with cell bound thiols is much more complicated than the formation of a single type of Hg-thiol complex at low Hg:biomass ratios; and 2) Hg can be complexed with cell-bound thiol sites in a variety of stoichiometries depending on the biogeochemical attributes of the ecosystem in question (e.g., the Hg:biomass ratio, the abundance of thiol sites on the bacterial species in question, and whether the species is Hg-methylating or not).
Our results illustrate that B. subtilis and S. oneidensis MR-1 cells show similar Hg complexation behavior with cell bound thiols, albeit, the transition from Hg-S2 to Hg-S3 occurs at lower Hg loadings for B. subtilis due to lower thiol abundance compared to S. oneidensis MR-1. In the case of S. oneidensis MR-1, Hg forms the Hg-S3 complex below aqueous Hg concentrations of 0.5 µM, but forms Hg-S2 and Hg-S complexes at higher Hg concentrations. In contrast, under the same Hg concentration conditions, the Hg methylating species G. sulfurreducens forms only Hg-S2 and Hg-S complexes without a detectable Hg-S3 complex. This difference in surface complexation of Hg on the G. sulfurreducens cells was not caused by the lack of sufficient thiols on G. sulfurreducens, which has the highest abundance of thiols among the three species examined.
Although a definitive reason for the inconsistent behavior of G. sulfurreducens cell envelope compared to those of S. oneidensis MR-1 and B. subtilis is beyond the scope of our study, these differences could provide insights about Hg cell surface complexes for methylating vs. non-methylating species. We hypothesize that the differences in the membrane protein chemistry (Hg transporters) and Hg uptake mechanism of G. sulfurreducens inhibits G. sulfurreducens to form Hg-S3 type complexes unlike other two species examined. Our hypothesis is strengthened by a previous study which shows that aqueous Hg-S2 complexes enhances Hg(II) uptake and subsequent methylation by G. sulfurreducens while aqueous Hg-S3 complexes inhibit the same (Schaefer and Morel, 2009). In order to form Hg-S3 complexes within cell envelopes, cell surface proteins must contain at least 3 thiol sites in close proximity to each other. Although G. sulfurreducens exhibits the highest concentration of thiols among the examined bacterial species, the thiol site density (i.e. sites/A2) of G. sulfurreducens must not be high enough to make tridentate Hg-S3 complex. These results suggest that the cell envelope S-amino acid containing proteins are significantly different between G. sulfurreducens and S. oneidensis MR-1, specifically their density and reactivity, which are critical in Hg binding, transport and possibly uptake.
Differences in abundance and density of thiol sites on cells of different bacterial species, and the corresponding stoichiometry of Hg-thiol complexes, could also explain the observed differences in passive oxidation of Hg(0) mediated by cell bound thiols (Colombo et al., 2014). These cell surface bound Hg-S complexes also form readily in the presence of other strongly competing ligands, such as Cl- and NOM (which also contains thiols), and were found to be stable in aqueous solutions at room temperature for over a period of 2 months (Dunham-Cheatham et al., 2014; Dunham-Cheatham et al., 2015). While the cell envelope-bound Hg-thiol complexes constitute the pool of Hg(II) transported inside the cell for Hg-methylation in the case of G. sulfurreducens, Hg-S3 complexes in the non-methylating bacterial species B. subtilis and S. oneidensis MR-1 would likely stay as Hg-Sn complexes until the amino acid residue is oxidized. Given the high thermodynamic stability of Hg-S3 complexes, they are not expected to be released back into the aqueous phase as thiol complexes. Alternatively, they could slowly transform into inorganic Hg-sulfide (e.g. meta-cinnabar) nanoparticles under sulfidic environments. It has been recently shown that Hg forms colloidal meta-cinnabar when reacted with DOM in the presence of sulfide, presumably via reaction with thiols in the DOM (Gerbig et al., 2011). It remains to be determined if thiols present within bacterial cell envelopes could also mediate the formation of meta-cinnabar, limiting the bioavailability of Hg for microbial processes (Zhang et al., 2012). Since bacteria are ubiquitous in all natural systems, and their cell envelope-bound reactive thiol site concentrations often exceed the aqueous concentrations of Hg in many natural and contaminated settings, this study suggests that cell envelope-bound thiol sites play a key role in the speciation, fate and bioavailability of Hg in aquatic and terrestrial ecosystems.
Last Modified: 08/06/2018
Modified by: Bhoopesh Mishra
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