2023 51 B2

Establishing a Genetic Toolbox for the Archaeon Nitrosocaldus yellowstonensis via an Integrating Mobile Genetic Element

By: Matthew Dominguez

Department: Biology

Faculty Advisor: Dr. José R. De La Torre

Archaea are one of the three fundamental groups of cellular life on Earth. Nevertheless, the biology of these microorganisms remains poorly understood. One reason for this is the lack of useful genetic tools, as only a handful of archaea have complete genetic toolboxes. While believed to be close relatives to eukaryotes, the class Nitrososphaeria, key players in the global carbon and nitrogen cycles, lack these genetic kits. Like most microorganisms, this lineage contains mobile genetic elements (MGE) that can mediate horizontal gene transfer between cells. Metagenomic studies revolving around Nitrosospheria hint that MGEs can serve as a foundation for the creation of said genetic tools, aimed at manipulating gene expression in this lineage of archaea. Nitrosocaldus yellowstonensis HL72, a thermophilic representative cultivated by our laboratory from a hot spring in Yellowstone National Park, contains an integrating mobile genetic element (iMGE) found both as a plasmid and integrated into the chromosome. Before determining whether this iMGE can serve as the backbone for a plasmid vector aimed at manipulating gene expression in these archaea, my goals are to assess its transferability to other closely related species and whether it can express in a new host entirely. Specifically, Nitrosocaldus gerlachensis GBSF, a close relative of N. yellowstonensis HL72 (>95% average nucleotide identity across the entire genome), will undergo electroporation-based protocols to test cell viability for transformation focused procedures. In tandem, I plan to transiently drive expression of super folder GFP (sfGFP) under control of HL72’s S-layer proteins, a highly expressed native promoter, to stand as a precursor for the development and expression of a plasmid vector. These experiments will serve as the foundation for the creation of genetic tools aimed at manipulating gene expression in this lineage of archaea.