Determination of the Controls on Bacterial Cell Surface Sulfhydryl Binding Site Concentrations

地球表面到处都有细菌。细菌通过利用细胞壁上的特定化学位点从水中吸收必需元素。然而，这些位点也吸附金属毒素，影响它们在环境中的命运。最近，在细菌细胞上发现了一种称为巯基位点的新型结合位点。这些位点强烈结合有毒金属，它们可能是自然环境中细菌最重要的位点，但这些位点在自然系统中的丰度尚不清楚。该项目将研究控制细菌上巯基位点丰度和分布的因素，以便模型可以预测在现实条件下有多少有毒金属会与细菌结合。 这项研究还可能导致一种操纵细菌产生细胞的能力，这种细胞在从污染的沃茨或污水中去除有毒金属污染物方面更有效。 该项目将涉及外展与当地高中科学研究计划在南本德，印第安纳州的学校，并参与高中学生的研究。在这些高中科学研究计划中将创建和教授一个地球化学模块，每年有2-3名高中生将进行一些实验。金属吸附到细菌上可以影响金属在环境中的命运，并且可以控制金属对细菌的生物利用度。先前的研究已经确定了细菌细胞包膜内和胞外聚合物（EPS）分子上广泛存在的巯基金属结合位点。这些位点的丰度较低，但这些位点可能主导亲硫元素的吸附（例如，汞、镉、硒、砷等）在大多数环境设置的典型的低金属负载条件下，由于巯基位点对这些元素的极高亲和力，控制巯基位点的浓度和分布的因素是未知的，因为它们在自然系统中的浓度。资助的研究将确定使用实验室实验来测试以下假设：1）一系列因素，如电子供体，碳源和生长过程中的氧浓度影响细菌表面巯基位点的浓度。将使用电位滴定测量来测试该假设，以确定细菌上结合位点的浓度，在存在和不存在巯基封闭以及存在和不存在从细胞中去除EPS的情况下进行。 2)细菌细胞包膜的巯基位点位于细菌表面的巯基蛋白上，并且这些蛋白在细胞表面上的表达可以被控制。为了检验这一假设，将使用电位滴定和荧光方法测量细胞包膜内特定蛋白表达增强或减弱的细菌菌株的巯基结合位点浓度，并测试这些参数是否与细菌细胞包膜内的蛋白浓度一致。3)巯基结合位点的天然有机物和细菌在有机丰富的土壤样品代表了在这些环境中的总结合位点的重要组成部分。将采集一系列天然样品，并使用与检验假设1相同的电位滴定法测量总浓度和巯基结合位点浓度。 资助的研究将提高我们对地下水和地表水系统中重金属环境命运控制的理解，并可能导致这些沃茨的生物修复方法的改进。优化的生物修复策略，涉及细菌的代谢过程，影响汞，镉，砷，硒等元素的形态需要一个彻底的了解，并有可能控制，通过金属-巯基结合在细菌上的吸附。拟议的研究项目将涉及与印第安纳州南本德当地高中科学研究项目的外联和协调，以及高中学生参与资助的研究。这两名PI与南本德学校的两名高中教师关系密切，他们开展了非常成功的科学研究项目。将在这些高中科学研究项目中创建和教授地球微生物学/环境化学模块（由项目PI提供的4个讲座和圣母大学研究生进行的演示的短期课程），以及2-每年将招募3名高中生进行一些资助的实验。这个奖项反映了NSF的法定使命，并被认为是值得支持的，使用基金会的知识价值和更广泛的影响审查标准进行评估。

Bacteria are present everywhere on the Earth's surface. Bacteria take in essential elements by adsorbing them from water using specific chemical sites on their cell walls. However, these sites also adsorb metal toxins, affecting their fate in the environment. Recently, a new type of binding site called sulfhydryl sites was discovered on bacterial cells. These sites bind toxic metals strongly and they may be the most important for bacteria in natural settings, but the abundance of these sites in natural systems is unknown. This project will investigate the factors that control the abundance and distribution of sulfhydryl sites on bacteria so that models can predict how much toxic metal will bind to bacteria under realistic conditions. This research could also lead to an ability to manipulate bacteria to produce cells that are more effective in removing toxic metal pollutants from contaminated waters or sewage. The project will involve outreach with local high school science research programs in South Bend, IN schools, and participation by high school students in the research. A biogeochemistry module will be created and taught in these high school science research programs, and 2-3 high school students each year will conduct some of the experiments.Metal adsorption onto bacteria can affect the fate of metals in the environment, and can control metal bioavailability to bacteria. Previous studies have identified the widespread presence of sulfhydryl metal binding sites both within bacterial cell envelopes and on extracellular polymeric substance (EPS) molecules. These sites are low in abundance, yet the sites likely dominate the adsorption of chalcophile elements (e.g., Hg, Cd, Se, As, etc.) onto bacteria under the low metal loading conditions typical of most environmental settings due to the extremely high affinity of sulfhydryl sites for these elements. The factors that control the concentration and distribution of sulfhydryl sites are unknown, as are their concentrations in natural systems. The funded research will determine use lab experiments to test the following hypotheses: 1) A range of factors such as electron donor, carbon source, and oxygen concentration during growth affect the concentration of bacterial surface sulfhydryl sites. This hypothesis will be tested with potentiometric titration measurements to determine the concentration of binding sites on the bacteria, conducted with and without sulfhydryl blocking and with and without EPS removal from the cells. 2) Bacterial cell envelope sulfhydryl sites are located on sulfhydryl-bearing proteins at the bacterial surface, and the expression of these proteins on the cell surface can be controlled. To test this hypothesis, sulfhydryl binding site concentrations will be measured for bacterial strains that are produced with either enhanced or diminished expression of specific proteins within the cell envelope, using potentiometric titration and fluorescence approaches and testing whether these parameters vary consistently with protein concentration within the bacterial cell envelopes. 3) Sulfhydryl binding sites on natural organic matter and bacteria in organic-rich soil samples represent a significant component of the total binding sites in these environments. A range of natural samples will be collected, and total and sulfhydryl binding site concentrations will be measured using the same potentiometric titration approaches used to test Hypothesis 1. The funded research will improve our understanding of the controls on the environmental fate of heavy metals in ground and surface water systems, and may lead to improved bioremediation approaches for those waters. Optimization of bioremediation strategies involving bacterial metabolic processes that affect the speciation of elements such as Hg, Cd, As, and Se requires a thorough understanding of, and potentially an ability to control, adsorption through metal-sulfhydryl binding on bacteria. The proposed research project will involve outreach and coordination with local high school science research programs in South Bend, IN schools, and participation by high school students in the funded research. The two PIs have close ties with two high school teachers in South Bend schools who run highly successful science research programs. A geomicrobiology/environmental chemistry module will be created and taught in these high school science research programs (a short-course of 4 lectures given by the project PIs and demonstrations run by University of Notre Dame graduate students), and 2-3 high school students will be recruited each year to conduct some of the funded experiments.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.