Date of Graduation
5-2023
Document Type
Thesis
Degree Name
Bachelor of Arts
Degree Level
Undergraduate
Department
Biological Sciences
Advisor/Mentor
Lessner, Daniel
Committee Member/Reader
Paré, Adam
Committee Member/Second Reader
Striegler, Susanne
Committee Member/Third Reader
Clay, Matt
Abstract
Methanosarcina acetivorans is a methanogenic archaeon (methanogen) that is important for several processes in our biosphere. A feature of interest within this methanogen is the enzyme nitrogenase which catalyzes the reduction of unusable dinitrogen gas (N2) into a usable form of nitrogen, ammonia (NH3). How electrons are transferred within M. acetivorans to nitrogenase during N2 reduction is unknown. Heterodisulfide reductase (Hdr) is a key enzyme known to reduce the electron carrier protein ferredoxin, which is a direct electron donor to nitrogenase in bacteria. I hypothesized that cytoplasmic HdrA2B2C2 functions in electron transfer to nitrogenase and is therefore critical to the process of N2 fixation by M. acetivorans. To test this hypothesis, I used a CRISPRi-dCas9 system to knockdown hdrA2 and hdrC2B2 of M. acetivorans. Specifically, I integrated dCas9-containing plasmids with guide RNAs (gRNA) into experimental cell strains of M. acetivorans; some strains contained a plasmid with gRNA targeting hdrA2 while other strains contained a plasmid with gRNA targeting hdrC2B2. Targeting these two gene clusters for repression separately helped to elucidate the importance of each one’s expression in N2 fixation. Once I harvested the cell strains, ensured proper plasmid integration, and ensured proper gene repression due to dCas9, I conducted growth studies. I compared the growth of hdrA2 and hdrC2B2 knockdown strains with one another and with control strains when forced to fix N2 and when not. My findings resulted in a better understanding of the role (or lack thereof) of cytoplasmic Hdr in N2 fixation. Better understanding of N2 fixation has environmental implications. If we can implement an N2-fixing mechanism into plants, for example, the need for fertilizer, a pollutant, will be reduced.
Keywords
methanogens; nitrogen fixation; heterodisulfide reductase; CRISPRi-dCas9; gene repression; nitrogenase
Citation
Shelnut, G. (2023). Understanding the role of heterodisulfide reductase during nitrogen fixation by Methanosarcina acetivorans. Biological Sciences Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/biscuht/87
