Investigating Nitrogen Fixation and Metabolic Pathways in the Plant Endophyte Gluconacetobacter diazotrophicus

MS graduate Erin Schwister

Advisor: Dr. Brett Barney

Abstract: Plant endophytes can provide growth benefits to their hosts through features such plant hormone production or increased pathogen resistance. Gluconacetobacter diazotrophicus PA1 5 is a plant-growth promoting endophytic bacterium that colonizes crops such as sugarcane, sweet potato, and rice. Among its other plant-growth promoting features, this bacterium performs biological nitrogen fixation (BNF) and has potential biotechnological and agricultural significance. In this work, a deeper understanding of the plant endophyte and nitrogen fixing bacterium G. diazotrophicus is presented through genomic analysis and targeted mutagenesis.

The genome of G. diazotrophicus was first investigated using transposon insertion sequencing (Tn-seq). Tn-seq combines a large-scale library of transposon mutants with next-generation sequencing to determine gene essentiality within a bacterial genome. This Tn-seq study explored gene essentiality relating to nitrogen fixation as well as nutrient metabolism in G. diazotrophicus through rapid characterization across the entire genome. Analysis of the resulting mutant library revealed that G. diazotrophicus has little redundancy in genes critical for nitrogen fixation and regulation. Silencing within the main cluster of nitrogen fixation genes under BNF conditions chiefly resulted in moderate-to-large growth defects, indicating conditional gene essentiality. Genes critical for catabolism of various carbon and nitrogen sources were additionally investigated. Beyond this global analysis study, select genetic manipulations of several key genes in G. diazotrophicus were performed to potentially improve extracellular ammonium production to construct a strain for future 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. This work provides valuable insight into genetic mechanisms critical in G. diazotrophicus and will facilitate further studies into understanding the lifestyle of this plant endophyte.