Esther R. Angert - US grants

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.

One thing birds can do to protect their young from the threat of bacteria in the environment is to transfer protective immune compounds, such as antibodies, directly into their eggs. Although these antibodies are associated with improved offspring growth and immunity, it is not known how levels of egg antibodies are influenced by disease risk. The purpose of this project is to test the hypothesis that tree swallows (Tachycineta bicolor) encountering more bacteria in their nests will have higher levels of antibodies in their eggs. To tackle this question, bacterial communities in swallow nests will be characterized using DNA-sequencing and compared with antibody levels in eggs collected from the same nests. This data set, from across North America, will be the most comprehensive ever collected on bacterial communities associated with any bird species, and the investigators predict that, as in plants and animals, bacterial diversity will be highest at the most southerly sites (e.g., Virginia) and lowest at the most northerly sites (e.g., Alaska). As a consequence, southern birds should have more antibodies in their eggs than northern birds.
In addition to exploring new ground in bacterial biogeography, this study will bring new perspective to the variety of ways in which female birds can protect their offspring. Results from this study will also contribute to a large collaborative study of investigators working to quantify pathogens inhabiting the nests, feathers, feces, blood, and tissues of swallows through the NSF-funded Golondrinas de las Americas network. And sequence data will add to a growing wealth of 16S rRNA gene sequences publically available through GenBank. This project will also contribute to the training of undergraduates working in the field and laboratory. Through this research, students will have the opportunity to share data, ideas, and personal interactions with investigators throughout the hemisphere.

Epulopiscium spp. are among the largest known bacteria with some cigar-shaped cells reaching lengths in excess of 0.6 mm. Epulopiscium spp. and their relatives are intestinal symbionts associated specifically with certain species of tropical reef fish in the surgeonfish family. They are a conspicuous and usual component of the community of microorganisms that inhabit the fish and they contribute to the digestion of polysaccharides consumed by their fish host. The Angert Lab has been using Epulopiscium sp. type B as a model for studies of cell, developmental and evolutionary biology. This organism has been key in providing a better understanding of how a simple bacterial cell can make small changes in its cellular structure and organization to allow it to reach its tremendous size. The current project seeks to improve this experimental system further by completing the genome sequence of Epulopiscium sp. type B. These efforts will yield an important resource for studying the genetic potential of these exceptional bacteria in the context of cell and developmental biology. Gene expression in actively growing populations of Epulopiscium sp. type B will be characterized to identify genes that are essential for supporting cell growth and metabolism. This profile will provide the basis for improving methods to grow these bacteria in laboratory culture.

Broader impacts: This project provides significant educational and training opportunities for postdoctoral and undergraduate research assistants. All trainees will gain experience in molecular biology, microbiology, DNA sequence analysis, and the application of basic bioinformatics tools. Through research presentations, manuscript and proposal preparation, trainees will develop their communication skills. Depending on the project, an individual will gain more extensive experience in bioinformatics, evolutionary biology, and genomics. Such research experiences are essential for the development of a young scientist.

Most life on Earth follows a daily routine guided by predictable environmental cycles (light and dark, temperature changes etc.). Some aspects of the routine are controlled by external cues while some are maintained using circadian rhythms. These internal, self-sustained clocks with a periodicity of about 24 hours allow microbes, animals and plants to anticipate and prepare for daily fluctuations. Recently, self-regulated clocks composed of one or a few proteins have been discovered, suggesting that genes for a robust clock can be readily acquired. Thus far, true bacterial clockworks have been characterized only in phototrophs (that harvest energy from sunlight), but other bacteria would likely benefit from an internal clock. Specifically gut microbes, with a clock that allows them to anticipate changes in host activities, would have an advantage over bacteria without a clock. Shift workers often suffer intestinal discomfort and weight gain. Gut microbes out of sync with their human host may be contributing to these unwanted physiological changes. Uncovering a gut microbe circadian clock would inform targeted remedies to combat undesirable physiological changes that accompany our modern lifestyles.

To date, the complexity of gut microbial communities and lack of any readout for daily processes have hampered the ability to observe regular functions in most gut microbes. To circumvent these issues, wild surgeonfish and their intestinal bacteria will be used as a natural model system to describe daily metabolic and developmental cycles. One group of Firmicutes, Epulopiscium spp. and related epulos, are naturally occurring, abundant intestinal symbionts that produce conspicuous internal offspring once every 24 hours in a predictable process. This provides a meter of everyday physiological changes in natural symbiont populations. Daily physicochemical changes in the intestinal ecosystem will be characterized to uncover regular fluctuations that may be used for entrainment. Symbiont oscillations will be determined by analyses of their temporal transcriptome and the cyclical redox cycle of a widely distributed clock protein. The project provides training opportunities for postdoctoral, graduate and undergraduate researchers. An undergraduate Reciprocal Exchange Program will be developed to build a cooperative learning group that will contribute to the research project while building networks and skills for advanced training. Native Pacific Islanders and other groups that are underrepresented in the sciences will be specifically recruited for these opportunities. Project results will uncover fundamental modes by which gut symbionts coordinate their activities with those of their host.