Browsing by Subject "Tn-seq"

Now showing 1 - 4 of 4
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    The genetic basis of nitrogen fixation and carbon metabolism in Azotobacter vinelandii
    (2022-06) Knutson, carolann
    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.
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    Investigating Nitrogen Fixation and Metabolic Pathways in the Plant Endophyte Gluconacetobacter diazotrophicus
    (2021-07) Schwister, Erin
    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.
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    Meddling with Marinobacter: Microbial Interactions with the Environment
    (2018-06) Bonis, Benjamin
    Despite inhabiting a wide variety of biomes and conditions, relatively little work has been devoted to the genus Marinobacter beyond isolation and initial characterization. The broad range of tolerances displayed by the Marinobacter suggest a metabolically diverse clade with great potential for biotechnological use and environmental study. In addition to hydrocarbon degradation and the synthesis of wax esters, many strains have been found to influence the redox state of metals and oxidize cathodes. Ubiquitously found and readily cultivated, a surfeit of genomic sequences and metadata are available for this genus, though little effort devoted to curation during deposition. Here we utilize 71 public and private Marinobacter genomes to curate the taxonomic and phylogenetic status of these strains, and use comparative genomics to assay the core and pangenome content and how it relates to the demonstrated plasticity of this genus. We find a number of strains erroneously assigned or lacking species identifiers, and offer an updated taxonomy. We also find a deeply branching clade of psychrophilic Marinobacter, suggesting a dearth in the continuity of sequence space of the genus. Due to their extensive distribution and abundance, of particular interest regarding the Marinobacter is the contribution to elemental cycling through extracellular redox chemistry. Utilizing a novel species of Marinobacter isolated from the Soudan Iron Mine in northern Minnesota, we develop a model system to better interrogate the interactions of Marinobacter with the environment. Characterization, development of a robust genetic system, and the establishment of Marinobacter subterrani strain JG233 as an Fe(II)-oxidizing bacterium additionally provides a model organism for the assessment of environment-microbe interactions. Using the techniques optimized in Marinobacter subterrani, genetic systems were developed for an additional 6 strains of Marinobacter to assess essential gene profiles across a genus using transposon mutant libraries. Comparative essential gene profiling using strains from diverse biomes of isolation provides the foundational tools to begin assessment of the effect of environment on gene content, regulation, and conditional essentiality.
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    Mycobacterium tuberculosis Erdman ATCC 35801 arrayed Himar1 transposon library isolated using MtbYM rich medium.
    (2022-08-18) Tischler, Anna D; Block, Alisha; Palani, Nagendra; Brokaw, Alyssa; Zhang, Leanne; Namugenyi, Sarah; Beckman, Kenneth; tischler@umn.edu; Tischler, Anna D; University of Minnesota Tischler Lab
    We created a Himar1 transposon library in Mycobacterium tuberculosis Erdman ATCC 35801 strain containing ~8000 mutants arrayed in 80 tube racks in a 96-well format. The transposon library was isolated on MtbYM rich medium that allowed recovery of auxotrophic mutants that would not be viable on standard Middlebrook M. tuberculosis culture medium. Using orthogonal pooling and transposon sequencing (Tn-seq), we mapped the location of Tn mutants in the library. The library was split into two groups of 40 racks. For each set of 40 racks, pools were created of all mutants in each row (rows A-H), column (columns 1-12) and rack (racks 1-40 or 41-80) to generate 60 Tn-seq samples for Illumina sequencing. Two different mapping methods, the heuristic Straight Three strategy and the probabilistic Knockout Sudoku algorithm, were used to map the arrayed library with good agreement between both mapping methods. This repository contains Illumina sequencing data for all rack, column, and row pools used to map the entire 80 rack arrayed library.