Chemolithoautotrophic bacteria obtain energy by oxidizing inorganic compounds such as sulfide, ammonia, and iron, and use this energy to power carbon dioxide and bicarbonate fixation. These organisms are of great ecological and environmental relevance; for example, they are the base of the food web at hydrothermal vents, occupy critical roles in the global nitrogen cycle, and acidify areas impacted by mine tailings. Some of their habitats have chronically or episodically low concentrations of carbon dioxide and bicarbonate, which puts organisms with an enhanced ability to grow under 'low carbon' conditions at a distinct advantage. It is reasonable to predict that many chemolithoautotrophs have extensive adaptations to cope with 'low carbon' conditions. The hydrothermal vent chemolithoautotroph Thiomicrospira crunogena has recently been demonstrated to act as a 'carbon dioxide vacuum'. It is capable of growing rapidly in the presence of vanishingly low concentrations of bicarbonate and carbon dioxide, and can somehow pack its cells with bicarbonate, to the point where the concentration of bicarbonate inside the cells is 100X higher than outside. Furthermore, T. crunogena's genome has recently been sequenced, which will greatly facilitate the discovery of the genes responsible for its 'carbon dioxide vacuuming' ability.
The overall objective of this project is to physiologically and genetically characterize T. crunogena's ability to 'pump' vast quantities of bicarbonate and carbon dioxide into its cells. To meet this objective, both traditional physiological methods and cutting-edge molecular tools will be utilized. Chemolithoautotrophs catalyze processes of profound ecological and geochemical importance; developing an understanding of how they cope with an obvious environmental stressor will substantially enhance our capability to predict their activities in situ, and how they may be impacted by increasing concentrations of atmospheric carbon dioxide.
Broader impacts These research objectives rely heavily on genome data manually annotated by the undergraduate and graduate students enrolled in K. Scott's Genomics class. Spurred by the extraordinary enthusiasm of the students enrolled in this course, and by the growing importance of genome-level analyses in biological research, the principal investigator intends to expand genomics education to the secondary level. The objectives of the educational component are 1. To create summer workshops in microbial genomics for secondary science educators. Five of the twenty educators enrolling per summer would be selected from schools with predominantly minority enrollment. 2. To create a web-based interface allowing secondary students to interact with the PI and her graduate and undergraduate researchers to learn more about microbial genomics. 3. To incorporate QRT-PCR into an upper-level Microbial Physiology Lab.
This integrated approach to research and education will yield fundamental insights into chemolithoautotroph physiology, provide target genes for future analyses of environmental samples and key insights into the interpretation of genomic data from other microorganisms, and prepare secondary students to join the genomics revolution.
PROJECT OUTCOMES Intellectual merit of the project activities Microorganisms (including chloroplasts) that fix carbon dioxide (CO2) introduce carbon into food webs and therefore sustain all other forms of life. Some of this CO2 fixation is stimulated by chemical energy, instead of light energy, and the organisms that catalyze this process are referred to as chemolithoautotrophs. The uptake of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO3-2) is necessary for the growth of chemolithoautotrophs, as they sustain food webs at hydrothermal vents, nitrify, acidify areas impacted by mine tailings, and catalyze other processes of ecological and geochemical importance. Some of these habitats have chronically or episodically low concentrations of DIC, which put organisms with adaptations to acquiring DIC at an advantage. Prior to this study, DIC uptake by chemolithoautotrophs had not been characterized. The chemolithoautotrophic bacterium Thiomicrospira crunogena and related organisms sustain life at deep-sea hydrothermal vents and other habitats in the ocean where chemical energy is present as reduced sulfur compounds (such as sulfide). Therefore, we deemed it important to understand CO2 uptake and fixation as a way to understand the factors that might influence the communities of organisms that live there. Research outcomes Eleven peer-reviewed publications have resulted from our work, with three more in prep and more forthcoming. Four invited presentations, and seven others at national and international meetings, detailed the outcomes of the study. This work tracked the involvement of all of the genes contained in the T. crunogena genome in responding to low CO2 concentrations, implicated the roles of the multiple carbonic anhydrase enzymes in growth under low CO2 concentrations, and also resulted in genetic manipulation of T. crunogena to create mutant strains to highlight the roles of different genes in CO2 uptake. This work also stimulated comparisons to other organisms, including symbiotic bacteria from the deep-sea vent tubeworm Riftia pachyptila as well as the subtidal clam Solemya velum. A second chemolithoautotroph (Sulfurimonas denitrificans) also had its genome sequenced, and was a particularly interesting comparison to T. crunogena, as it uses a completely different biochemistry for carbon fixation. This project also spurred interest in how T. crunogena synthesizes the organic compounds necessary to make all of its building blocks (amino acids, nucleotides), as key steps appear to be missing from central carbon metabolism. Furthermore, it caused us to re-evaluate the notion that the Calvin cycle is the most expensive carbon-fixing pathway; based on our analyses, it will be necessary for us to re-evaluate our notions of what makes a particular carbon fixing pathway more or less of an advantage in different habitats. Broader impacts resulting from the project activities The expansion of bioinformatics and functional genome tools to undergraduate (and secondary!) classes was a raging success. The bioinformatics tools used for the molecular component of the work detailed above were taught to, and utilized by, undergraduate students enrolled in my Genomics class each fall (2004 to present). Two manuscripts on curriculum development were published in peer-reviewed publications, one manuscript was published using analyses conducted by a class of undergraduate students (who were listed as authors on the paper), and one manuscript in preparation is the result of four years of analysis by undergraduate students. Bioinformatic tools and approaches were also presented to high school teachers in two summer workshops, to encourage them to incorporate simple online tools into their biology curricula, as these provide facile examples of how evolution works. The functional genomics approaches (e.g., knockout mutants, gene expression studies) needed for the physiological component of the study were successfully incorporated into an undergraduate level Microbial Physiology lab (2006 to present). The methods to incorporate bioinformatics and functional genomics into undergraduate curricula were communicated to faculty from other postsecondary institutions at workshops I co-chaired that were supported by the American Society for Microbiology, and the Joint Genome Institute. A PhD student and two Masters students centered their thesi on T. crunogena physiology, with two giving particular emphasis toward CO2 uptake by T. crunogena.
