Date of Graduation

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Lessner, Daniel J.

Committee Member

Henry, Ralph L.

Second Committee Member

Thallapuranam, Suresh

Third Committee Member

Ivey, D. Mack

Fourth Committee Member

Pinto, Ines

Keywords

Biological sciences; Archaea; Iron-sulfur clusters; Methanogen; Methanosarcina acetivorans; Oxidative stress; Rna polymerase

Abstract

Methanogens are archaea possessing a conserved metabolic pathway which produces methane. Many of the enzymes in the methanogenesis pathway are Fe-S proteins, meaning methanogens are sensitive to conditions which disrupt Fe-S clusters. Molecular oxygen is capable of disrupting Fe-S clusters through oxidation of the iron atoms. Furthermore, reduced iron can facilitate the production of reactive oxygen species (ROS), meaning methanogens must possess antioxidant mechanisms. Detection and eradication of ROS is important for all cells, due to the potentially fatal consequences of unchecked oxidation. This dissertation presents two separate projects investigating mechanisms the model methanogen Methanosarcina acetivorans possess for dealing with ROS. One project investigated the roles two [4Fe-4S] clusters present in RNA polymerase (RNAP) subunit D play in assembly and activity of RNAP; to determine if a mechanism exists for linking sensitivity of the clusters to oxygen to RNAP function. My data shows that both clusters and the cluster binding domain play an important role in assembly of RNAP downstream of D-L heterodimer formation, preventing optimal assembly of at least subunits B’ and A’’ when the clusters are absent. Cluster one plays a more critical role in this process compared to cluster two. Coupled with experimental evidence that the clusters are oxygen sensitive, this provides support for the hypothesis that the clusters regulate RNAP assembly in response to redox state of the cell. The second project investigated two putative catalase genes present in the M. acetivorans genome. Experimental evidence showed neither catalase was functional. Engineering of a M. acetivorans strain to express functional catalase from Escherichia coli increased the tolerance of M. acetivorans to H2O2, but not oxygen during growth in standard conditions. Catalase does not appear to be an important component in the oxidative stress response of M. acetivorans.

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