Collaborative Research: Highly reactive thiol binding sites on bacterial cell envelopes and their influence on metal speciation in aquatic systems

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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.