Date of Graduation

8-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Lessner, Daniel J.

Committee Member

Ivey, D. Mack

Second Committee Member

Pinto, Ines

Third Committee Member

Fan, Chenguang

Fourth Committee Member

Du, Yuchun

Keywords

Iron-sulfur clusters; Methanogens; Biogenesis; Methanogenesis

Abstract

Iron-sulfur (Fe-S) clusters, the ancient cofactors found in proteins across all life forms, play vital roles in numerous cellular processes. Fe-S clusters exhibit structural diversity, ranging from simple to complex forms. Methanogens are anaerobic archaea that produce methane as a metabolic by-product through methanogenesis, a process dependent on numerous Fe-S proteins. Notably, methanogens are capable of fixing nitrogen (diazotrophy) facilitated by the enzyme complex called nitrogenase, which contains both simple and complex Fe-S clusters. While methanogens possess the largest number of Fe-S proteins, the factors involved in Fe-S cluster biogenesis remain largely unknown. Bacteria possess three distinct and well-characterized Fe-S cluster biogenesis systems - ISC, SUF, and NIF, with the SUF and ISC systems functioning in general Fe-S cluster biogenesis and the NIF system specific to Fe-S cluster biogenesis in nitrogenase. All methanogens encode homologs of SufBC, the core Fe-S cluster scaffold component of the SUF system and some species also encode homologs of the core components of the ISC system (IscS and IscU). No species contains homologs of the NIF system components, despite many methanogens containing nitrogenase. The SUF system is the most ancient of all Fe-S cluster biogenesis systems and since it is present in all methanogens, it has been postulated to serve as the primary and nitrogenase-specific Fe-S cluster biogenesis system in methanogens. This dissertation investigates the biogenesis and roles of Fe-S proteins during nitrogen fixation by Methanosarcina acetivorans, a model methanogen that contains all three types of nitrogenase. In chapter one, the recently developed CRISPRi-dCas9 and CRISPR-Cas9 systems were used to investigate the role of SufBC in M. acetivorans. Findings from gene repression and deletion studies reveal that the SUF system is not essential to M. acetivorans, is not the primary Fe-S cluster biogenesis system, nor is it required for nitrogenase Fe-S cluster biogenesis. In chapter two, the role of NifB in the biosynthesis of the complex Fe-S clusters in nitrogenases was investigated. NifB is a radical S-adenosy-L-methionine (SAM) enzyme required for the insertion of carbide into FeMo-co, FeV-co, and FeFe-co during the maturation of Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. Surprisingly, results for gene deletion studies revealed that nifB is essential to the viability of M. acetivorans and that NifB serves a distinct function apart from nitrogenase maturation, a result not seen in bacteria. In chapter three, potential factors involved in the electron transfer to nitrogenases were investigated. Specifically, a low-potential flavodoxin (FldA) and the Fe-S protein complex heterodisulfide reductase (HdrABC) were tested for the involvement in diazotrophy using the CRISPRi repression system. The absence of any observable phenotype in the fldA and hdrABC repression strains suggests that neither of these genes are involved in electron transfer to nitrogenase. Overall, these findings shed light on the roles of SufBC and NifB in the biosynthesis of simple and complex Fe-S clusters and provide new insights into the factors involved in electron transfer to nitrogenase during methanogenesis.

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