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

细菌在自然环境中无处不在。金属离子附着在细菌细胞表面会影响元素的全球循环、金属污染物的迁移以及污染物缓解技术的有效性。过去的研究已经确认了某些细菌表面位置在不切实际的高金属浓度下附着金属离子的重要性。然而，最近的研究表明，在与环境相关的金属浓度下，以前未被识别的位置可能更重要。这项研究的目的是更好地了解高亲和力但低丰度的细菌表面结合位点对水生系统中金属摄取和反应的影响。由于大多数金属在自然和受污染的系统中都以低浓度存在，本研究的结果有助于更好地了解重金属在自然环境中的环境命运。一种创新的方法将被用于分离R-SH位点对细菌细胞膜的影响。具体地说，将使用对R-SH敏感的荧光团分子(QBBR)，该分子与细胞被膜上的R-SH位点强烈结合。当qBBr结合到R-SH位置时会发出荧光，分子上的电荷阻止它很容易地穿过细胞膜；因此可以用于以前不可能直接测定细胞膜上R-SH位置的浓度。此外，由于qBBR与细胞膜R-SH位点结合很强，我们可以用它作为阻断剂来分离与细胞膜R-SH位点的质子和金属结合反应。这项资助的研究将首次直接探索细胞包膜硫醇位点的作用，并使我们能够研究它们与金属的相互作用。利用荧光和X射线吸收光谱，结合电位滴定和整体吸附实验，PI将测量大多数水生系统常见的选定细菌细胞膜上的硫醇浓度，并确定不同的环境变量，如生长介质和生长条件(有氧和厌氧)如何影响硫醇浓度。PI将测量锌在硫醇位置上的吸附，并使用吸附和光谱方法确定细菌细胞膜上锌-硫醇络合物的分子结构和结合常数。QBBR方法使详细的测量成为可能，有可能改变我们对细菌在现实条件下如何结合金属的理解。拟议的研究结果对于评估这些重要结合部位对环境中金属形态和分布的作用至关重要。这项研究的结果不仅可以应用于污染物迁移模型，还可以应用于生物修复工程和了解环境中重金属的一般循环。资助的研究将支持一系列外联活动，包括通过普林斯顿大学？S？Quest？进行的教师培训。项目；在南本德高中的科学研究方案中开发地球微生物学/环境化学模块；在南芝加哥地区的高中教授水污染技术模块。

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.