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

University Of Notre Dame, Notre Dame IN

Investigators

Abstract

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.