Understanding gene regulatory network function during stress response adaptation of an archaeal extremophile
Intellectual merit: Although life science research has entered the post-genomic era, we still understand little about the diversity of microbial life on earth. Information is particularly lacking on microbial extremophiles, which thrive at the limits of life, in deep-sea hydrothermal vents under high pressure and temperature, saturated salt lakes, and polar icecaps. Many of these organisms are members of the third domain of life, the archaea. Although archaea contribute substantially to global carbon and energy cycles, they remain understudied because they are difficult to culture and genetically manipulate. How do these microorganisms cope with an extreme and changing environment? How do they alter their genetic programs and metabolic pathways to adapt and survive changes in their unique habitats on earth? These questions are particularly relevant today, as climate change rapidly alters the conditions that support life across the globe. The impact of such changes on the microbial communities responsible for global carbon and energy cycling is unclear, but is expected to have enormous implications for human society. To address these issues, the long-term goal of the proposed research is to understand how organisms maintain homeostasis in the face of fluctuating environmental conditions. Central to this process are gene regulatory networks (GRNs) composed of groups of regulatory proteins that switch genes on and off in response to environmental stimuli. Upon sensing a change in the environment, GRNs promote the production proteins that repair damage, restore the cell to a healthy state and prepare it for future stress conditions. The Halobacterium salinarum studied in this research thrives in high salt environments. This organism is a stress response specialist, capable of surviving in the Great Salt Lake during strong daily fluctuations of light, heat, oxygen, and nutrients at extreme salt concentrations. In response, the organism shifts its metabolism between four light- and oxygen-dependent energy-generating modes. The aim of the proposed work is to determine how the organism uses GRNs,or gene circuitries, to adapt to the dynamic alterations in light, oxygen, and nutrients to ensure survival. This research will employ an innovative systems biology approach, which combines cutting-edge high throughput experimental techniques with computational and statistical modeling. Halobacterium is a good model system for studying archaeal extremophiles because it is easy to culture and genetically manipulate. What we learn about the genetic circuitry of Halobacterium will be readily applicable toward mapping the genetic circuits of other archaea. Moreover, it will provide a deeper understanding of the dynamics of microbial GRNs and expression patterns in response to changing environmental conditions. More generally, the outcome of this research will lay the foundation for understanding microbial energy production and global carbon cycling in response to climate change.
Broader impacts: The research will contribute to education, training, and outreach at the high school, undergraduate, and graduate levels. Specifically, students at all levels will have training opportunities at the interface of mathematics and biology. First, the PI and lab members will teach a week-long mini course for students from North Carolina School of Science and Math, a public high school in Durham, NC that draws the top students each year from each of the congressional districts in North Carolina. Second, outreach research opportunities associated with the project will be provided for undergraduates from North Carolina universities that serve underrepresented groups through established summer programs at Duke (e.g. North Carolina Central University, Fayetteville State University). Third, a graduate student funded on the project will receive next generation interdisciplinary training at the interface of mathematics and biology.
INTELLECTUAL MERIT Motivation. Although life science research has entered the post-genomic era, we still understand little about the diversity of microbial life on earth. Information is particularly lacking on microbial extremophiles, which thrive at the limits of life. Extremophiles can be found in such inhospitable environments as saturated salt lakes, polar icecaps, and deserts. Many of these organisms are members of the third domain of life, the archaea. Although archaea are important for many environmental and industrial applications, they remain understudied because they are difficult to culture in the lab and manipulate genetically. How do these microorganisms cope with an extreme and changing environment? How do they turn genes on and off (gene expression) to adapt and survive changes in their unique habitats on earth? Activities. To address these questions, the goal of this project was to understand how stress response regulatory proteins, or transcription factors (TFs), work together regulate gene expression in response to a changing environment to allow archaea to survive. We are conducting this research in an organism living in saturated salt lakes, where wide variations in combinations of extreme conditions occur daily (e.g. UV irradiation, heavy metal exposure, nutrient limitation). As a result, the organism, called Halobacterium salinarum, has evolved a robust tolerance to oxidative damage and flexible metabolic capabilities. The hypothesis of the project has been that the coordinated action of a large web, or network, of TFs orchestrates the coupled, dynamic response of functionally related sets of genes. This project undertook an interdisciplinary approach spanning the fields of microbiology, systems biology, and mathematical modeling. Major outcomes. This project has accomplished the three objectives and has resulted in 4 publications, with 2 papers under review. Underlined names denote undergraduate contributions. Names with asterisks denote graduate student contributions. The PI's name is bolded. Publications 1. Tonner, P.D*., Pittman, A.M.C., Gulli, J.G., Sharma, K., Schmid, A.K. 2015. A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea. PLoS Genetics. 11(1):1004912. 2. Todor, H.*, Dulmage, K.*, Gillum, N., Bain, J.R., Muehlbauer, M.J., Schmid, A.K. 2014. A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum. Mol Microbiol. 93(6):1172-82. 3. Todor, H.*, Sharma, K., Pittman, A.M.C, Schmid, A.K. 2013. Protein-DNA binding dynamics predict transcriptional responses to nutrients in archaea. Nucleic Acids Research. (18):8546-58. 4. Sharma, K., Gillum, N., Boyd, J.L.*, Schmid, A.K. 2012. The RosR transcription factor is required for gene expression dynamics in response to extreme oxidative stress in a hypersaline-adapted archaeon. BMC Genomics 13:351. Papers under review 1. Darnell, C. and Schmid, A.K. Systems biology approaches to defining archaeal regulatory networks. Methods. 2. Dulmage, K.*, Todor, H.*, Schmid, A.K. Growth-phase specific modulation of gene expression by an archaeal histone. Impact. Taken together, the results of this research demonstrate that regulatory proteins of ancient evolutionary ancestry in archaea provide mechanistic links between various stress responses as well as between the regulatory network and its effects on cell physiology (e.g. transcriptional regulation, metabolic activity, growth rate, and cell morphology). These results have made significant progress in understanding gene network function, how it may be integrated with cell physiology, and how the network may evolve in response to stress throughout the tree of life. This project has contributed substantially to the fields of microbiology, systems biology, and mathematical modeling. BROADER IMPACTS Goals. The broader impact goals of this research were to: (1) foster undergraduate learning and training; and (2) facilitate high school student learning and training. Both of these training opportunities were targeted to the interface between mathematics and biology. Central to these goals was increasing participation in hands-on research activities of underrepresented groups. Activities. To accomplish the first goal, undergraduate students from underrepresented groups and HBCU’s were recruited to the PI’s lab to conduct research. To accomplish the second goal, the PI, in collaboration with teachers and an interdisciplinary team of graduate students, taught a weeklong high school science inquiry immersion course called "mini-term" on microbial growth and stress response each year. Major outcomes and impact. The research integrated trainees into cutting-edge research to answer open questions in the fields of microbiology and mathematical modeling. Both high school and undergraduate students have contributed directly to the research data resulting from this project. Specifically, 16 participants at the high school, undergraduate, and graduate levels have been trained. 11 of these trainees are members of underrepresented groups in STEM fields. These training efforts have been successful based on several metrics, including co-authorship on publications, retention in STEM fields (9 of the16 students graduated and remain in STEM careers), and teaching 48 high school students in the mini-term course. The words of high school students sum up the impact of this project on their careers: "You expanded our insight on future career paths and are helping keep science and math alive in school."
