Understanding the role of heterodisulfide reductase during nitrogen fixation by Methanosarcina acetivorans

Author

Grant ShelnutFollow

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