The Development of Genetic Machinery via an Archaeal Integrating Mobile Genetic Element
By: Matthew Dominguez
Department: Cellular & Molecular 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 in most cultivated archaea.The phylum Nitrososphaera, key players in the global carbon and -nitrogen cycles, are believed to be early relatives to eukaryotes. Like most microorganisms, this phylum contains mobile genetic elements (MGE) that mediate horizontal gene transfer between cells. 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. Recent data from our lab indicates that the plasmid form of the iMGE can be packaged within extracellular vesicles (EVs) released into the culture supernatant. My goal is to assess whether this iMGE can be transferred to other closely related species. Specifically, Nitrosocaldus gerlachensis GBSF is a close relative of HL72 (>95% average nucleotide identity across the entire genome), but does not contain the iMGE. In order to infect GBSF, I used ultrafiltration to purify iMGE-containing EVs from cultures of HL72 grown to stationary phase. Vesicles were then incubated with GBSF cells for 24 hours and grown into several new passages. Successful transfer of the iMGE to GBSF will be tested after several rounds of growth using PCR primer pairs diagnostic for the presence of the iMGE in both plasmid and integrated forms. In parallel to this infection experiment, I am attempting to use the plasmid form of the iMGE to construct a shuttle vector that can be maintained in E. coli. To accomplish this, I purified the plasmid form of the iMGE from vesicles collected as described above. DNA extracted from the EVs was subjected to rolling circle amplification (RCA) and linearized by restriction digest with either BamHI or AvaI. This will allow me to ligate the linearized iMGE to a linear version of the pCCFOS1 E. coli cloning vector, utilizing its origin of replication and chloramphenicol resistance gene to propagate the shuttle vector in E. coli. This plasmid will serve as a foundation for the creation of genetic tools, aimed at manipulating gene expression in this lineage of archaea. Moreover, it could shed light onto a new form of microbial gene transfer, granting us valuable insight on evolution within archaeal communities and possibly early eukaryotic history.