Uncovering an Alternative Electron Transfer Pathway to Nitrogenase

University of Minnesota M.S. thesis.July 2020. Major: Microbial Engineering. Advisor: Kathryn Fixen. 1 computer file (PDF); vi, 54 pages.Rhodopseudomonas palustris is a metabolically versatile bacterium capable of fixing nitrogen from the atmosphere to ammonium using the enzyme nitrogenase. In wild-type R. palustris, the electron-bifurcating FixABCX complex is essential for electron transfer to nitrogenase. It takes electrons from NADH and reduces the low-potential electron carrier proteins Fer1 and FldA, which can directly donate electrons to nitrogenase. A suppressor mutant (∆fixC *) was isolated that restores the ability of R. palustris to fix nitrogen even when FixABCX is inactive. This strain encodes sixteen point mutations. A large part of this project was establishing which of the mutations the ∆fixC* strain acquired were necessary and/or sufficient to recover the cell’s ability to fix nitrogen without the FixABCX complex. One of the mutations produced a variant Fer1, and this mutation is essential for nitrogen fixation in ∆fixC*. We also found FldA, a secondary donor in the wild-type strain, does not appear to donate electrons to nitrogenase in ∆fixC*. This led to the hypothesis that the variant Fer1 interacts with an alternative electron donor. To determine the identify of this donor, a randomized loss of function approach using Tn5 mutagenesis was used to isolate mutants defective in electron transfer to nitrogenase. Characterization of these mutants is ongoing, but from this work, we hope to understand how R. palustris re-wired electron transfer to nitrogenase. This work will help us understand how we can divert more electrons to nitrogenase and potentially increase the production of valuable products by nitrogenase