Date of Award

Winter 2024

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Louis S. Tisa

Second Advisor

Jessica Ernakovich

Third Advisor

Estelle Hrabak

Abstract

Bacterial species in the family Frankiaceae are soil-dwelling microsymbionts that form a mutualistic symbiosis, termed the actinorhizal symbiosis, with over 220 species of woody dicots. In this relationship, frankia fix atmospheric nitrogen to ammonia, a form usable by plants, while the bacteria benefit from the carbon sources exuded by their actinorhizal hosts. Since nitrogen is typically a limiting nutrient for growth in degraded and contaminated soils, actinorhizal plants establish at these sites as pioneer plant species, initiating a path for phytoremediation. Likely due to their association with plant hosts in contaminated soils, most frankia strains have evolved above average tolerances to a range of heavy metals and possess a significantly larger array of metalloproteins compared to other bacterial species. Species distributed between the four genera of the Frankiaceae family (Frankia, Protofrankia, Parafrankia, and Pseudofrankia) have unique combinations of genes used for metal import, binding, chaperoning, modification, and export. Pseudofrankia inefficax is of particular interest because it has one of the highest tolerances to copper (5.0 mM) among cultured frankia strains. Interestingly, this strain is unable to fix nitrogen but can form nodules (Nod+/Fix-). This makes P. inefficax a useful strain for investigating the role of frankia heavy metal tolerance in symbiosis, since any recorded benefit to plant health cannot be attributed to a supplemental nitrogen supply. Using a range of molecular methods, the work of this dissertation focused on characterizing P. inefficax copper resistance as it relates to 1) the health and survival of a non-typical host plant in the presence of copper and 2) genetic strategies employed for copper tolerance in the free-living vs. symbiotic states. The final goal was to use CRISPR-Cas9 to generate a mutant strain for downstream characterization of the role of copA in P. inefficax copper tolerance.

Download

Share

COinS