Determining the mechanism of heritable inactivation of bacterial immunity

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Abstract Bacterial immune systems represent a potent barrier to genetic parasites including viruses (bacteriophages, or phages). Harboring immunity against these often-lethal invaders represents a clear fitness benefit. Unexpectedly, during growth at elevated temperatures (i.e. 42 °C), many Pseudomonas aeruginosa strains inactivate a potent anti-bacteriophage immune system, type I restriction-modification (R-M). Remarkably, the inactivation of restriction endonuclease (iREN) phenotype is heritable and persists for at least 60 generations after return to a low temperature (i.e. 37 or 30 °C), well after the environmental stress has ended. This phenomenon is an uncharacterized example of epigenetic regulation and cellular memory in bacteria. Understanding iREN's mechanism could reveal novel bacterial biology, drug targets, and approaches to potentiate bacteriophage therapeutics. This proposal's objective is to understand the molecular basis of iREN and identify the genes involved in R-M inactivation and memory. This will be accomplished through 1) the use of fluorescently tagged R-M proteins to determine their fate during iREN, and 2) the use of RNA sequencing and a CRISPRi transcriptional repression screen coupled with a phage selection, to collectively observe and perturb operons involved in iREN. These aims are essential to understanding how bacteria mount this regulatory response and how this response persists for 60 generations. Our findings may detail a novel cellular strategy for regulation and epigenetic inheritance, as well as explain how inactivation of immunity could be evolutionarily beneficial to bacteria. This project will be conducted in the Bondy-Denomy lab in the Department of Microbiology and Immunology at UCSF's School of Medicine. The Bondy-Denomy lab is a multicultural and intellectually diverse laboratory where undergraduates, graduate students, and postdoctoral fellows work in approximately a 1:1:1 ratio. This distribution of training levels ensures that graduate students enjoy the opportunity to both find mentorship from various perspectives, as well as develop the leadership skills needed to become a PI by mentoring others. I have been a member of the Bondy-Denomy lab for 2.5 years, during which I have spearheaded a collaborative project under Dr. Bondy-Denomy's supervision, culminating in a first author publication. With two senior graduate students graduating shortly, I am rising to a position of leadership in the lab. The culture of UCSF is guided by our PRIDE values: professionalism, respect, integrity, diversity, and excellence. These values serve as both an ideal and the reality of our nurturing community encouraging an environment of collaboration, mentorship, and rigorous standards for science. By training in this environment, my scientific and leadership skills will be cultivated to become an excellent and responsible scientist at the head of an academic research group.