UCSF

Cells coexist with and are often outnumbered by a long and diverse list of molecular parasites that utilize cellular resources to replicate. Viruses are particularly deleterious, as their replicative cycle often culminates in cellular lysis. To prevent resource-costly and potentially lethal infection by bacterial viruses (bacteriophages, phages), bacteria contain many different anti-phage immune systems. In this thesis, I discuss two examples of bacterial immune system failure in Pseudomonas aeruginosa. First, I discuss the mechanism by which ΦKZ, a jumbophage, resists all tested DNA-targeting immune systems. ΦKZ resisted 4 different CRISPR-Cas and 2 different restriction-modification (R-M) DNA-targeting systems in vivo but succumbed to the RNA-targeting CRISPR-Cas13. Fluorescently-labeled DNA nucleases were excluded from accessing ΦKZ‘s DNA, because the phage assembles a nucleus-like compartment. A fusion of EcoRI to a phage-encoded recombinase trafficked the restriction enzyme into the DNA compartment and enabled immune activity. ΦKZ is the most immune evasive phage studied to date and our work revealed the mechanism, DNA segregation. Second, I discuss an epigenetic state in which an R-M system is inactivated for greater than 60 generations after cellular replication at 43 °C. Though first discovered more than 50 years ago, this phenomenon has not been further investigated and its molecular mechanism remains unknown. I determined that inactivation of restriction occurs post-translationally and requires bacterial replication in liquid culture at greater than 42 °C. Preliminary results suggest that protein aggregation may be involved in inactivation of the restriction enzyme. Together, the studies in this thesis explain 2 interesting cases of bacterial immune system failure, which occur by fascinating mechanisms.