TN5 MUTAGENESIS REVEALS POTENTIAL MECHANISMS OF METABOLIC PARASITISM OF APPLE FRUIT BY ERWINIA AMYLOVORA
Open Access
- Author:
- Finley, Melissa Lauren
- Graduate Program:
- Plant Pathology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 25, 2017
- Committee Members:
- Tim McNellis, Thesis Advisor/Co-Advisor
Cristina Rosa, Committee Member
Seogchan Kang, Committee Member
Edward Dudley, Committee Member - Keywords:
- plant pathology
phytopathology
phytobacteriology
bacterial metabolism
erwinia
erwinia amylovora
pathogenicity
parasitism
tn5 mutagenesis
auxotroph
auxotrophy
amino acids
amino acid auxotrophy
bacterial parasitism
nutrient acquisition - Abstract:
- The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, a destructive disease of apples, pears, and other Rosaceae species. This study seeks to further elucidate the trophic aspects of the host-pathogen parasitic interaction and which metabolic pathways are required for pathogenicity. Auxotrophic mutants of E. amylovora were generated via Tn5 transposon mutagenesis followed by plating mutagenized bacterial cells on a selective minimal media on which auxotrophs could not grow. Forty-seven confirmed auxotrophic mutants were then inoculated onto immature ‘Gala’ apple fruits in order to evaluate their pathogenicity. The mutated genes were identified by Sanger sequencing of E. amylovora DNA flanking the Tn5 insertion in each auxotrophic mutant. Characterization of transposon insertion sites showed that the following biosynthetic pathways or cellular functions were disrupted: amino acid biosynthesis (19), nucleotide biosynthesis (12), sulfur metabolism (5), nitrogen metabolism (2), survival protein biosynthesis (2), exopolysaccharide biosynthesis (3), and a selection of uncharacterized proteins (4) (the number of mutants of each type is listed in parentheses). We hypothesize that if an auxotrophic mutant is able to cause disease, the mutant must be able to derive the missing metabolites from host tissues. If an auxotrophic mutant is not able to cause disease, this suggests that it cannot derive the missing metabolites from their host. It was determined that the disruption of amino acid biosynthesis, such as for the production of arginine, leucine, and methionine, and nucleotide biosynthesis, such as for the production of purines and pyrimidines, resulted in reduction or elimination of pathogenicity. These two groups of mutants are unable to obtain sufficient amounts of the missing metabolic products from the host tissue in order to complement their metabolism and grow normally. Conversely, mutants with disrupted sulfur metabolism remained pathogenic, indicating that these mutants were able to obtain sufficient amounts of sulfur and sulfur metabolites from the host tissue. In addition, mutants defective in several survival proteins and exopolysaccharide biosynthesis were identified during screening as possible auxotrophs, and they displayed reduced or absent disease expression. The question of why these mutants are auxotrophs is still being investigated. In summary, this genetic study revealed new details of the profile of pathogen-accessible metabolites in colonizing the host tissues and furthered understanding of which metabolic pathways are needed for disease development.