Forces are invisible yet their presence can be felt anywhere. For large organisms, like humans, the force of gravity exerts a controlling influence on our lives. For smaller organisms, like bacteria, gravity is a trivial force. Rather, intermolecular forces control the world of microbes. This CAREER proposal will explore forces at a range of spatial scales, from binding forces at the peptide-mineral interface to the gravitational attraction that exists between humans and celestial objects. The research portion of this proposal will focus on measuring the energy landscapes of putative mineral-binding proteins and peptides. This will be accomplished by using atomic force microscopy (AFM) to examine the relationship between the transition force (i.e. rupture force) and the rate at which the force is applied to a particular protein-mineral pair. Proteins specimens will include putative mineral-binding proteins (MtrC and OmcA) from the metal reducing bacterium Shewanella oneidensis, as well as phage that express peptides with putative mineral-binding motifs, and artificial membranes doped with mineral-binding proteins. Mineral specimens will include crystalline, thin films of hematite (Fe2O3), goethite (FeOOH), pyrolusite (MnO2), and manganite (MnOOH). Molecular simulations will be used to determine the theoretical stability of putative mineral-binding motifs and the affinity of particular peptides towards specific mineral phases. This proposed work will (1) determine fundamental kinetic and thermodynamic properties such as, the free energy of dissociation, the rate of dissociation, and the distance to the transition barrier, for each peptide-mineral pair (e.g., MtrC-hematite); (2) define the mineral substrate(s) for each protein (MtrC and OmcA) based on fundamental binding elements; (3) define the boundaries of mineral-binding domains within the primary sequences of MtrC and OmcA; (4) determine whether putative mineral-binding domains within MtrC or OmcA are similar or dissimilar to mineral-binding motifs evolved by phage; (5) compare the measured kinetic/thermodynamic data to values predicted by molecular simulations of peptide-mineral bonds; (6) use the binding force-signature of MtrC and OmcA to create affinity maps that show the location of specific proteins on the surface of living cells. Broader impact One of the great strengths of Geological/Earth Science is the breadth of time and space that are encompassed by this discipline. This proposal describes an educational outreach program that teaches students from elementary to graduate school about forces that exist from the nanometer length-scale (e.g., intermolecular forces) to the infinite (e.g., the gravitational force). Students who participate in the proposed outreach programs will come away with a much stronger appreciation of the fundamental forces within our universe and their impact on living organisms. The educational outreach portion of this proposal will create a lecture series on "Forces in Natur" and a complementary lab related to subsurface transport of bacteria. This lecture and lab will be taught every year to about 1500 undergraduate students enrolled in a class called Planet Earth. Education majors enrolled in the Planet Earth class will, in turn, assist the PI as he conducts a one-week summer camp for elementary school students at one of the local, pubic schools. This camp will consist of hands-on experiments related to microbiology, polymers, and forces. On the final day of camp, the students will teach the "teachers", that is, the children will guide the parents/teachers through the same experiments the children performed earlier in the week. The education outreach plan also includes a proposed scientific "test of astrology" that will be run in the university newspaper, which is available to about 50,000 students. This test draws a novel comparison between the gravitational force and the idea that celestial objects exert a force of influence on our lives. Finally, this award will fund three students (two undergraduate and one graduate student) who will participate in the proposed research project described above.
Intellectual Merit: All living organisms possess the ability to sense their surrounding environment. Humans, for example, have evolved senses for vision, hearing, smell, taste and touch. Bacteria, being living organisms too, are no different aside from the mere fact that bacteria lack eyes, ears, nose and a mouth. Despite this apparent setback, bacteria have thrived on Earth for billions of years. Therefore, these "primitive" cells must have evolved ways to sense their surroundings. In this project, the PI investigated a bacterium’s tactile acuity or "sense of touch". This was accomplished by using a combination of instrumental and computational approaches to examine how various bacteria species use protein appendages to interact with solid surfaces. Studies focused primarily on (i) the environmental bacterium Shewanella oneidensis that utilizes special proteins, called cytochromes, to make contact with iron-containing minerals so that it can essentially breath by moving electrons to the iron in the mineral structure; and (ii) the pathogenic bacterium Staphylococcus aureus that uses proteins on its outer cell wall to form infectious biofilms on surfaces such as medical implants in humans. Through these studies, it was discovered that bacteria have evolved special extracellular appendages to allow specific species of bacteria to recognize and bind to specific solid surfaces. Broader Impact: Insight gained by the research, described above, could assist in the creation of new biological nanowires to conduct electricity as well as the development of new materials or surface coatings that resist biofouling. This project also included substantial efforts aimed at outreach, education, and training. Three post-doctoral scholars and nine university students (4 doctoral, 2 masters, and 3 undergraduates) were trained in analytical and computational techniques as part of this grant. All of these students are either employed in academia/federal agencies or are now pursuing higher degrees. The work completed by these students and the PI has been broadly disseminated through numerous national and international conference presentations and peer-reviewed journal publications (e.g., 13 published papers and 2 manuscripts in review). As part of this project, the PI became the primary instructor of two different general science education classes at his university (ENR2100 Introduction to Environmental Science, and ES1100 Planet Earth). The PI completely overhauled these classes focusing on the promotion of science literacy. The PI has taught over 2000 undergraduate students in these two classes since the beginning of this grant. Major accomplishments include several new inquiry-based teaching activities including one that was published in a leading scientific journal (Nature vol. 447, page 528) as well as the creation of an annual Environmental Science Student Symposium for undergraduates. This Symposium was held for the third time on November 25, 2014 in Columbus, Ohio. A total of 769 students presented scientific posters. Each and every poster was reviewed by at least three peers (i.e., peer-review, the cornerstone of science) using a new technology app developed specifically for this event. Student interest in both ENR2100 and ES1100 is stronger than ever as these classes are now oversubscribed every semester. Finally, as a part of this project, the PI developed and taught a series of summer camps for students in grades 6-12. To date, approximately 500 students have taken part in these camps. These camps included (i) a four-day camp in Ohio that focused on geomicrobiology and environmental microbiology (taught annually six times) and (ii) a 10-day international camp in June 2013 that was held on Bonaire Island with a focus on environmental science and biomineralization. Students, and their teachers, learned a variety of field and analytical skills related to microbiology and geochemistry (e.g., proper sample collection techniques, use of optical microscopy, chemical measurements of water quality). Equipment purchased for these outreach activities has expanded the analytical capabilities of the teachers who, with help from the PI and his university advisees, continue to teach these outreach activities to pre-college age students.
