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198343 https://hdl.handle.net/2286/R.2.N.198343 http://rightsstatements.org/vocab/InC/1.0/ http://creativecommons.org/licenses/by-nc-sa/4.0 2024-12 31 pages Pham, Vivian Redding, Kevin Vermaas, Willem Barrett, The Honors College School of Life Sciences Text Heliomicrobium modesticaldum is a species of anoxygenic, photoheterotrophic bacteria belonging to the family Heliobacteriaceae. The Heliobacteria have the simplest known photosynthetic apparatus, making them an ideal model to manipulate, for the purpose of gaining a stronger understanding of the evolutionary history of photosynthetic organisms. H. modesticaldum uses photosynthetic electron transfer in the presence of light and grows chemotrophically via pyruvate metabolism in the dark. Previously, it was believed that a ferredoxin-NADP+ oxidoreductase (FNR) was used to catalyze the transfer of electrons from the ferredoxin (Fd) pool to the NADP pool. However, upon sequencing of the H. modesticaldum genome, polypeptides encoded by the genes HM1_0289 (analogous to nfnA) and HM1_0290 (analogous to nfnB) were discovered and are believed to be two subunits of the enzyme NfnAB (NADH-dependent ferredoxin:NADP+ oxidoreductase), which is an electron bifurcating enzyme. The Nfn enzyme is proposed to catalyze the reversible reaction: 2 Fdox + NAD+ + 2 NADPH ⇌ 2 Fdred + NADH + 2 NADP+ in a process called electron bifurcation (in the forward direction) and electron confurcation (in the reverse direction). It was hypothesized that if nfnAB encodes the Nfn enzyme, nfnAB should be essential for H. modesticaldum survival in all growth conditions. This is because Nfn is proposed to catalyze the transfer of electrons from the Fd pool to the NADP+/NADPH pool, a process necessary for the generation of ATP via cyclic electron transfer (in the presence of light) and pyruvate fermentation (in the absence of light). To test this hypothesis, a two-step procedure involving two plasmids was utilized to delete the chromosomal copy of nfnAB. A second copy of the nfnAB gene with several base changes, called the “rescue copy”, was provided on the second plasmid and its transcription was controlled by the xylose operator/repressor system. After completion of the two-step procedure, xylose should be essential for survival of H. modesticaldum. The result was that H. modesticaldum grew relatively equally in the presence and absence of both xylose and Km, leading to inconclusive findings that most likely suggest that the step 2 plasmid integrated into the chromosome. Heliomicrobium modesticaldum NfnAB CRISPR-Cas gene knockout Photosynthesis Deletion of nfnAB from Heliomicrobium modesticaldum to Determine if It Is Essential for Life