Investigations of Prokaryotic Defense Systems

Bacteria are constantly threatened with infection by mobile genetic elements (MGE) such as bacteriophage and plasmids. Bacteriophage and plasmids require the bacteria's cellular infrastructure to replicate their genomes. Rampant replication can lead to cell death which is one reason why bacteria have developed a diverse array of immune systems to prevent or limit infection. This thesis studies three types of bacterial immune systems, type IV-A CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat –CRISPR associated), type V-A2 CRISPR-Cas systems, and Wadjet systems. The type IV-A system lies adjacent to a dinG-like helicase gene. Research has shown that this system can target plasmids preventing their spread throughout a microbial population. This system is reliant upon the dinG-like gene, but how this system mechanistically prevents plasmid sharing is not understood. The type V-A2 system has been shown to be capable of editing the genome of rice by generating breaks in the DNA. How this editing takes place, and the other biochemical mechanisms of this protein are not understood either. This thesis provides the preliminary framework for studying these putative genome editing tools. Wadjet immune systems prevent the sharing of plasmids between bacteria.These systems share structural similarities with proteins responsible for separating bacterial chromosomes during cellular replication. This thesis contains the foundation for the characterization of the system by cloning genes from native host systems and then recombinantly expressing and purifying proteins.