Schwister, Erin2021-09-242021-09-242021-07https://hdl.handle.net/11299/224501University of Minnesota M.S. thesis. July 2021. Major: Bioproducts/Biosystems Science Engineering and Management. Advisor: Brett Barney. 1 computer file (PDF); viii, 94 pages.Reduction of the reliance upon synthetic fertilizers is critical for a more sustainable agricultural future. Plant endophytes, especially those that engage in biological nitrogen fixation, provide a potential route towards this end. In this work, a deeper understanding of the plant endophyte and nitrogen fixing bacterium Gluconacetobacter diazotrophicus is presented through the following four chapters. The first chapter provides contextual background on this important plant endophyte and its potential for use in agriculture. Following this introduction, a large-scale transposon library of G. diazotrophicus is explored in the second and third chapters through highthroughput sequencing in a transposon insertion sequencing (Tn-seq) study. This Tn-seq study explores gene essentiality relating to nitrogen fixation as well as nutrient metabolism in G. diazotrophicus through rapid characterization across the entire genome. Lastly, the fourth chapter describes genetic manipulations of several key genes in G. diazotrophicus, performed to improve extracellular ammonium production to construct a strain for potential use as a biofertilizer. This work provides a deeper understanding of the genetic mechanisms essential to nitrogen fixation in G. diazotrophicus as well as those essential for growth under varied environmental conditions. As a nitrogen-fixing plant endophyte, further exploration of G. diazotrophicus as a potential alternative to synthetic fertilization brings us closer to a more sustainable agricultural future.enGluconacetobacter diazotrophicusMetabolismNitrogen FixationTn-seqThesis or Dissertation