Title:

Targeted Mutagenesis of Copper Tolerance Genes in Frankia inefficax EuI1c

When associated with bacteria in the genus Frankia, the adverse effects of heavy metal pollution on actinorhizal host plants are mitigated, which aids in phytoremediation performance and survival. Previous studies have shown that the strain Frankia inefficax EuI1c can tolerate up to 5.0 mM of copper, and its resistance mechanisms are hypothesized to mimic those of heavy metal extremophiles. To gain a better understanding of the genetic mechanisms underlying copper tolerance in F. inefficax, two genes that are hypothesized to contribute to this tolerance have been targeted for deletion. Generation of Frankia mutants has historically been limited to non-targeted or transient transformants. Recently, a stable transformation method through conjugation was developed, allowing for CRISPR-Cas9 gene editing in Frankia. Using this method, plasmid constructs are being generated to target two genes in F. inefficax: FraEuI1c_1869, a putative copD gene and FraEuI1c_6307, a putative copA gene. Generation of these mutant F. inefficax strains will allow for the downstream characterization of the mechanisms used by this strain to survive, and subsequently aid the health and survival of actinorhizal hosts, in soils degraded by excess concentrations of heavy metals.

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