Author ORCID Identifier:
Date of Graduation
7-2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy in Biology (PhD)
Degree Level
Graduate
Department
Biological Sciences
Advisor/Mentor
Lessner, Daniel
Committee Member
Alrubaye, Adrian
Second Committee Member
Pinto, Inés
Third Committee Member
Ivey, Mack
Fourth Committee Member
Du, Yuchun
Keywords
Alternartive nitrogenases; CRISPR-Cas9; Methanosarcina acetivorans; nif operon; NifEN; Nitrogen fixation
Abstract
Biological nitrogen fixation (diazotrophy) is catalyzed by nitrogenase, a metalloenzyme that converts atmospheric dinitrogen (N₂) into ammonia. All diazotrophs produce a molybdenum (Mo)-dependent nitrogenase, whereas some also encode alternative vanadium (V)- and Fe-only nitrogenases that function when Mo is unavailable. The methanogenic archaeon Methanosarcina acetivorans is one of the few archaeal species known to encode Mo-, V-, and Fe-only nitrogenases. Previous studies showed that expression of the nif operon is required for alternative nitrogenase usage, but the basis for this requirement is unknown. The objective of this study was to determine why the nif operon is required for alternative nitrogenase usage in M. acetivorans. To examine the role of the nif operon, the complete nifHI1I2DKEN operon was deleted from M. acetivorans using CRISPR-Cas9, and engineered complementation strains expressing the complete nif operon or truncated versions of the nif operon were constructed. Deletion of the nif operon abolished diazotrophic growth under Mo + Fe, V + Fe, and Fe-only conditions, indicating that the nif operon is required for diazotrophy. Complementation with the complete nif operon restored diazotrophic growth, whereas complementation with a version lacking nifEN did not, indicating that NifEN is required for alternative nitrogenase usage. In addition, functional Mo-nitrogenase was produced in NH₄Cl-grown cells expressing the engineered nif operon, showing that nitrogenase production can be uncoupled from nitrogenase-dependent growth. Deletion of the nif operon also impaired non-diazotrophic growth, revealing an unexpected role for the nif operon outside nitrogen fixation. Unexpectedly, prolonged incubation of the DJL200 Δnif strain permitted recovery of diazotrophic growth under all three metal conditions, demonstrating that alternative nitrogenase usage can eventually be re-established despite the continued absence of the nif operon. DJL200V, DJL200Fe, and DJL200Mo produced V-nitrogenase and Fe-nitrogenase, demonstrating that alternative nitrogenase production can occur in the absence of the nif operon. Whole-genome sequencing suggested that recovery in DJL200V and DJL200Fe resulted from physiological acclimation rather than stable genetic change. In contrast, DJL200Mo contained a four-copy tandem amplification of the anf gene cluster, including the anfI1I2DGK and the adjacent anfO, anf3, and vnfH genes, suggesting that recovery of DJL200Mo may have involved a distinct genetic adaptation. DJL200V remained responsive to Mo, indicating that recovery does not eliminate the inhibitory effect of Mo on alternative nitrogenase usage. Overall, the work in this dissertation identifies NifEN as a critical factor required for alternative nitrogenase usage and provides new insight into the physiological and genetic mechanisms by which alternative nitrogenase usage can be re-established following deletion of the nif operon.
Citation
Trujillo Peralta, M. (2026). Molecular Characterization of the Role of the nif Operon in Methanosarcina acetivorans. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/6388
