Date of Graduation

7-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Daniel Lessner

Committee Member

Paul Adams

Second Committee Member

Timothy Kral

Third Committee Member

Mack Ivey

Fourth Committee Member

David McNabb

Keywords

Archaea, Bioinformatics, Eukaryotes, Genetic Analysis, Iron-sulfur cluster, Methanogen, Nitrogen fixation

Abstract

Iron-sulfur (Fe-S) clusters are among the oldest cofactors on the planet, used by proteins in almost all forms of life on Earth to carry out processes ranging from energy transfer to DNA replication. Among the organisms believed to use these Fe-S proteins more extensively than almost any others are the methanogens, an ancient lineage of archaeal microbes that produce methane as a required product of their metabolism. Methane, the primary component of commercial natural gas, is both a potent greenhouse gas and an important fossil fuel. It can also be renewably produced as a biofuel. Biogenic methane is almost entirely a product of archaea carrying out methanogenesis, a metabolic process with an absolute requirement for Fe-S clusters at multiple steps. They are also believed to be the lineage in which nitrogen fixation began, an activity that uses multiple Fe-S proteins to convert nitrogen gas into ammonia, which can be used by microbes and plants for growth. Despite this heavy reliance on Fe-S proteins, we know very little about how Fe-S clusters are generated in these methanogens. This process is well understood in bacteria and eukaryotes, but what little we know about archaeal Fe-S biogenesis suggests that the mechanisms in these organisms may not work the way they do in the other two domains. This dissertation presents several different but related projects undertaken to gain a deeper understanding of Fe-S cluster biogenesis in methanogenic archaea. Methanogens contain homologs of proteins known to function in Fe-S cluster biogenesis in bacteria and eukaryotes, including components of the ISC and SUF systems, as well as an ApbC Fe-S carrier protein. To test the hypothesis that these proteins have similar roles in methanogens, I employed biochemical and genetic approaches to assess the function of these homologs in the model methanogen Methanosarcina acetivorans. The primary avenue of investigation was a characterization of heterologously expressed putative ISC-type Fe-S cluster biogenesis proteins, and an assessment of the effects of deleting the genes encoding them, from M. acetivorans. Another subject of study was the putative Fe-S carrier protein ApbC, whose gene I also deleted. Finally, I demonstrated the feasibility of a CRISPR/Cas9 genome editing system that was recently developed for M. acetivorans by deleting the genes for a putative SUF-type Fe-S biogenesis system. My results demonstrate that M. acetivorans contains a functional ISC system but that the system is not essential. However, the results support the ISC system playing an important role in nitrogen fixation, potentially supplying Fe-S clusters to nitrogenase, but only when cysteine is the sulfur source. ApbC is also not essential but seems to be important for growth with certain sulfur sources, especially the thiosulfate ion. Deletion of SUF failed to produce any phenotype indicating SUF is not the primary Fe-S cluster biogenesis system in methanogens. Taken together, these results suggest that despite the evidence from bacteria and eukaryotes, and counter to the conclusions a bioinformatic approach would indicate, the ISC, ApbC, and perhaps even SUF cluster biogenesis systems individually appear surprisingly dispensable in methanogens.

Additional Files
Appendix 1.xlsx (25 kB)
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