Nitrogen is a key nutrient that all living beings need to grow. Nitrogen exists on planet Earth predominantly in gaseous form, inaccessible for assimilation to the vast majority of organisms. Hence fixed nitrogen, either as ammonium, nitrite or nitrate, is competed for vigorously. Most transformations of nitrogen in the Earth's surface are catalyzed by microbes including the interconversion between different fixed nitrogen species. The preferred nitrogen species for growth is ammonium, often a limiting nutrient in ecosystems. This project will detail a new means by which certain microbes "breathe" or respire nitrate and produce ammonium. They do this for the same reason humans breathe oxygen, to conserve energy. Nitrate respiration is not new per se, but the mechanism used by the primary model bacterium for this study, Nautilia profundicola, is novel and may be important for it to making a living at deep-sea hydrothermal vents. Relatives of N. profundicola will also be studied that carry the genes encoding the same pathway and are most often found in association with animals, for example in the oral cavity of humans and in the guts of chickens. The benefit of this pathway to these organisms is currently unclear. By understanding the nitrogen metabolism of these bacteria, we will learn more about how nitrogen moves in the environment and perhaps also how animal-associated bacteria survive in the environment away from their hosts. This project is designed to take advantage of the most recent technological advances in genome sciences, namely high-throughput transcriptomics, and combine it with more traditional physiological approaches to characterize this novel nitrate reduction pathway. This project will ultimately define the key enzymes and intermediates of this pathway and how it is regulated.
Broader Impacts:
The PIs will train several undergraduate students and two graduate students in genome-enabled microbial physiology, enhancing learning through cross-institutional and cross-disciplinary research. All students will be exposed to state of the art high-throughput genomics approaches in addition to traditional microbial physiology and basic nitrogen chemistry. They will be formally trained in the responsible conduct of research through workshops and their professional development will be enhanced by presenting their research in both oral and written form via presentations at national meetings and publications. These activities will be integrated into the strategic plan of the NSF-funded Nitrification Network RCN. Finally, these results will be disseminated to K-12 students via teacher-training workshops and to the public via events like the University of Delaware's Coast Day, with an average annual attendance of over 8,000, and others as opportunities arise for less formal presentations by the PIs.
? Project Team: Dr. Barbara Campbell, PI, Clemson University Dr. Tom Hanson, coPI, University of Delaware Dr. Martin Klotz, coPI, University of North Carolina, Charlotte Nitrate is a compound that naturally occurs in many different environments. It can also be introduced by humans through agricultural fertilization, sewage runoff and other processes. Many microbes use nitrate for growth. Nitrate can be converted to ammonium and used in molecules such as proteins or used by the microbes to capture energy in the absence of oxygen. These transformations are part of the total nitrogen cycle, which may be disturbed by excess input of nitrate from humans. Therefore, it is critical to understand the diverse pathways used by microbes to metabolize nitrate. The PIs were awarded a grant from the NSF to characterize a novel pathway used by bacteria for nitrogen cycling. This pathway is found in bacteria isolated from deep-sea hydrothermal vents and from bacteria that inhabit the human mouth and intestinal tract and may cause human diseases under certain conditions. We hypothesized that this pathway functions to transform nitrite to ammonium through previously unknown or uncharacterized enzymes. The novel aspect of this pathway is that hydroxylamine, a potent mutagen, appears to be an intermediate between nitrite and ammonium. We used a combination of basic microbiological, quantitative molecular, and next generation sequencing approaches to characterize this pathway, called the rHURM pathway. We found that very distantly related bacteria from the same class are able to use this pathway to convert nitrate to ammonium. The bacterium isolated from deep-sea hydrothermal vents, Nautilia profundicola, uses the pathway to convert nitrate to ammonium to make new proteins and cells as well as gain energy from the transformation. Its enzymes can immediately transform toxic hydroxylamine to ammonium. We demonstrated that Campylobacter curvus, isolated from humans, converts nitrate to ammonium solely to gain energy. Our work is important as it allows us to better understand the roles of certain microbes in nitrogen transformations and how the growth of some of these microbes may be influenced by different compounds and under different conditions. This knowledge will result in a better understanding of both the global nitrogen cycle as well as the nitrogen cycle in humans. As part of this funding, the PIs trained several undergraduate and graduate students in basic microbiological and next generation/high throughput sequencing approaches. These students are entering the workforce as skilled STEM personnel, who will or have found jobs in either environmental or biomedical fields. In addition, the PIs and their students have interacted with the general public through outreach efforts with local elementary and middle schools, and public open house annual programs, such as Coast Day on the University of Delaware campus in Lewes, Delaware.
