The genetic basis of nitrogen fixation and carbon metabolism in Azotobacter vinelandii

Abstract

Natural nitrogen fixation is done primarily by prokaryotes that reduce nitrogen gas(N2) to ammonia (NH3) using the enzyme nitrogenase in a process termed biological nitrogen fixation (BNF). BNF is an alternative to industrial fertilizers that could supply crops with the nitrogen they need, either through symbiotic or free-living associations. Engineering these associations requires an understanding of BNF that extends beyond nitrogenase, to the dynamic suite of proteins that support it. Azotobacter vinelandii is a model organism for studying BNF and contains more than 80 suspected BNF genes, many of which have an unknown function or lack experimental data showing direct BNF association. A. vinelandii is known for being a free-living and aerobic nitrogen fixer, making it both convenient for laboratory studies and biotechnologically relevant. Since most research in this organism has focused on nitrogen fixation, there have been few studies on how various carbon sources are metabolized. Characterizing this metabolism in A. vinelandii in the context of nitrogen fixation would help in engineering viable alternatives to Haber-Bosch. Consequently, this research builds on the contextual knowledge of BNF in A. vinelandii by using transposon mutagenesis to identify genes important to growth and/or nitrogen producing phenotypes. First, we used transposon sequencing (Tn-seq) to determine the genome-wide fitness of genes under diazotrophic, non-diazotrophic, and differing carbon sources. We then used the Tn-seq data from growth on the carbon source galactose to identify two galactose dehydrogenases predicted to complete the pathway of galactose metabolism missed by routine genomic annotation algorithms. Lastly, we explored the gene redundancy of NifA in nitrogenase regulation by characterizing two transposon mutagenesis mutants able to support the growth of algae in co-culture. Overall, this thesis expands on the knowledge of BNF in A. vinelandii by providing genome-wide fitness that quantify individual gene contributions to BNF, carbon metabolism and, which also explores gene redundancy in BNF regulation.