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

细菌在地球表面无处不在。细菌利用细胞壁上的特定化学部位从水中吸收必需元素。然而，这些地点也会吸附金属毒素，影响它们在环境中的命运。最近，在细菌细胞上发现了一种新的结合位点——巯基位点。这些位点与有毒金属结合强烈，它们可能对自然环境中的细菌最重要，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的丰度和分布的因素，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操纵细菌产生更有效地从污染的水或污水中去除有毒金属污染物的细胞的能力。该项目将涉及与南本德当地高中科学研究项目的外展，并由高中生参与研究。在这些高中科学研究项目中，将创建并教授生物地球化学模块，每年将有2-3名高中生进行一些实验。金属在细菌上的吸附可以影响环境中金属的命运，并可以控制金属对细菌的生物利用度。以前的研究已经发现，在细菌的细胞包膜内和细胞外聚合物（EPS）分子上广泛存在巯基金属结合位点。这些位点的丰度很低，但由于巯基位点对这些元素具有极高的亲和力，这些位点可能在大多数环境中典型的低金属负载条件下对细菌吸附亲铜元素（如Hg、Cd、Se、As等）起主导作用。控制巯基位点的浓度和分布的因素是未知的，它们在自然系统中的浓度也是未知的。资助的研究将确定使用实验室实验来验证以下假设：1)一系列因素，如电子供体，碳源和生长过程中的氧气浓度影响细菌表面巯基位点的浓度。这一假设将通过电位滴定法进行测试，以确定细菌上结合位点的浓度，在有和没有巯基阻断的情况下进行，在有和没有从细胞中去除EPS的情况下进行。2)细菌包膜巯基位点位于细菌表面的含巯基蛋白上，这些蛋白在细胞表面的表达是可以控制的。为了验证这一假设，将使用电位滴定法和荧光法测量在细胞包膜内特定蛋白质表达增强或减少的菌株的巯基结合位点浓度，并测试这些参数是否与细菌包膜内蛋白质浓度一致。3)富有机质土壤样品中天然有机物和细菌上的巯基结合位点占这些环境中总结合位点的重要组成部分。将收集一系列天然样品，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。这项资助的研究将提高我们对地下水和地表水系统中重金属的环境命运控制的理解，并可能导致对这些水域的生物修复方法的改进。细菌代谢过程会影响汞、镉、砷和硒等元素的形态形成，要对这些过程进行生物修复策略的优化，需要对细菌通过金属-巯基结合进行吸附有透彻的了解，并有可能控制这种吸附。拟议的研究项目将涉及与南本德当地高中科学研究项目的外展和协调，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，他们经营着非常成功的科学研究项目。地球微生物学/环境化学模块将在这些高中科学研究项目中创建和教授（一个由项目pi讲授的四堂课的短期课程，由圣母大学研究生进行演示），每年将招募2-3名高中生进行一些资助的实验。该奖项反映了美国国家科学基金会的法定使命，并通过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，被认为值得支持。

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.