Intellectual Merit: Using Epulopiscium as a model, one of the Angert lab's long term research goals is to address a fundamental problem faced by all large cells; how does a cell with a small surface-to-volume ratio overcome constraints imposed on it by the diffusion coefficients of bioactive molecules?
With cigar-shaped cells reaching 600 µm x 80 µm, Epulopiscium spp. are the largest heterotrophic bacteria described to date. Despite its size, Epulopiscium is able to support a high level of activity. The presence of large amounts of repetitive DNA located at the periphery of the cytoplasm and a highly invaginated cell membrane may be significant adaptations to maintain large cell size in Epulopiscium. The amplification and distribution of genomic resources help support a robust metabolism while expansion of the cell membrane and membrane associate transporters may enhance exchange with the environment and facilitate movement of molecules within the cytoplasm. In this project Dr. Angert and her colleagues seek to further characterize these features and determine 1) the timing and location of DNA replication in the cell, 2) how multiple chromosomes with differing fates are positioned and managed and 3) the impact of intracellular membranes on molecular diffusion. Continued efforts toward culturing Epulopiscium spp. are planned. Maintaining Epulopiscium outside of its host will allow for in vivo experiments and ultimately the development of genetic tools.
Broader Impacts: These projects provide significant educational and training opportunities for graduate, undergraduate and high school students interested in the sciences. All students will gain experience in basic molecular biology, microbiology and microscopic techniques. Depending on the project, students will gain more extensive experience in areas such as genomics, microscopy, phylogenetic analyses or nanobiotechnology, to name a few. Such laboratory research experience is essential to the development of a young scientist.
Epulopiscium provides an exciting model for conveying basic concepts of the bacterial cell. These cells are visually appealing and can be seen with the unaided eye. The fact that Epulopiscium spp. are so large, but not pathogenic, makes them a good representative of the microbial world not only for biology students but for the general public as well. To facilitate information flow, a website featuring Epulopiscium has been established. Students at all levels are involved in the development and maintenance of the site. This project instills in students the responsibility of all researchers to disseminate information to the public and it will enhance the ability of the students to communicate their work and its significance to a diverse audience. In addition, members of the Angert Lab assist with a Cornell Institute for Biology Teachers Summer Workshop called The Microbial World.
Using Epulopiscium as a model, one of the Angert lab’s long term research goals is to address a fundamental problem faced by all large cells; how does a cell, with a small surface area relative to its cytoplasmic volume, overcome constraints imposed on it by the diffusion coefficients of bioactive molecules? Cells rely on diffusion to encounter nutrients, move metabolites throughout the cell and disperse biomolecules. Diffusion is a highly efficient means of moving molecules but only over short distances. Eukaryotic cells have broken free of some diffusion-dictated constraints in part by having a compartmentalized and organized subcellular structure. They use a cytoskeleton and motor proteins to move vesicles within the cell, and membrane bound organelles to organize specific functions. Once thought of as structurally "simple" and therefore limited to high surface-to-volume ratios (and very small cell sizes), it is now apparent that bacterial cells are highly organized and share features with eukaryotes, including a dynamic cytoskeleton. Although most bacteria are small, notable exceptions have been identified. Epulopiscium spp. are giants in the bacterial world. Somehow they are able to maintain a high level of metabolic activity despite extraordinarily low surface-to-volume ratios. We have begun to identify cellular features in Epulopiscium, in particular highly repetitive DNA and an extensive intracellular membrane system, which may allow it to overcome size limitations. Throughout this project, we studied aspects of the subcellular organization of Epulopiscium, which has provided insights into the fundamental cellular issue of getting molecules to distinct locations within a cell in a timely fashion. Through the funded research we discovered that although Epulopiscium has a genome about the size of most bacterial genomes, an individual cell contains a large number of copies of the genome (tens of thousands of copies). We found that these copies are evenly spaced around the periphery of the cytoplasm and located just under the cell membrane. The geometric arrangement and spacing of genome copies is maintained over a large size range of Epulopiscium cells. We also discovered that genome copies follow one of two fates. A small number of the copies is passed on to offspring cells while most of the copies are dedicated to support the metabolism of the mother cell and developing offspring. These supportive (somatic) genomes are never directly inherited by an offspring but replicate during times of offspring growth. Eventually, these somatic genome copies are dismantled as the mother cell dies. This dictate of different roles for subsets of chromosomes appears to be a common theme in biology. In addition, the arrangement of genetic resources just under the cell membrane in large cells allows for a rapid response to environmental changes and these resources may be scaled up in cells with high metabolic demands. Finally, we investigated diffusion of small molecules in these enormous bacteria to see if the cells have developed a means of working with diffusion to enhance movement of molecules in the cell membrane or cytoplasm. We found that diffusion of small molecules appeared to be no different in large bacterial cells compared to more normal-sized bacteria. The grant supported the training of one postdoctoral research associate, three graduate students and nine undergraduates. These kinds of hands-on laboratory research training opportunities are essential for the development of a young scientist. The postdoctoral trainee is now a tenure-track assistant professor, and the graduate trainees have gone on to start careers in higher education or research and development. All of the undergraduates are attending graduate or medical school or have jobs in major engineering or biotechnology firms. Five of the students, who did laboratory research as undergraduates, are racial minorities that are typically underrepresented in the sciences or are the first member of their immediate family to earn a bachelors degree, five are female. Furthermore, this grant supported projects of two college professors who worked in the Angert lab during their sabbatical leaves. This exposure allowed the professors to learn new methods that they could then take back and use on research projects initiated at their home institutions.
