Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Luciano Marraffini
Keywords
CRISPR-Cas, phage defense, Csm6, bacterial dormancy, spacer acquisition, reversible immunity
Abstract
Organisms from every kingdom of life constantly need to contend with their parasites to survive and reproduce. Bacteriophages (phages) are the viruses that infect bacteria and often outnumber them in most ecological niches. As a result, prokaryotes have evolved a myriad of defense systems to restrict the propagation of phages as well as other parasitic mobile genetic elements such as plasmids. Genetic loci known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes serve as one such defense system in both bacteria and archaea. A hallmark of CRISPR immunity is its adaptive nature in which memories of prior infections are acquired and stored. CRISPR loci contain clusters of characteristic repeat sequences separated by short "spacer" sequences which are derived from prior invading genetic material and form the basis of these acquired memories. During an immune response, spacer sequences are transcribed into guide RNAs and will recognize complementary sequences on invading genomes. Upon target recognition and binding, guide RNAs direct Cas effector proteins and complexes to the sites of infection where restriction of the phage can take place. The staphylococcal type III-A CRISPR-Cas immune system uses guide RNAs to locate target sequences on nascent viral transcripts instead of on DNA. Due to the transcription dependent nature of target sequence recognition, this response can display two distinct mechanisms of immunity depending on how soon the target is transcribed after infection. When the CRISPR-Cas system is triggered by a target sequence on an early-expressed phage transcript, direct degradation of the associated viral DNA by the DNase domain of Cas10 occurs. This rapid activation of immunity leads to a robust response which cures the host from infection before the phage can replicate. In contrast, when the guide RNA targets a late-expressed transcript, successful defense requires the additional activity of Csm6, a non-specific RNase which cleaves both invader and host RNA in the cell. Csm6 activity results in mass RNA degradation which triggers dormancy in the infected cell. How dormancy protects from infection and whether it can be relieved is not known. Abortive infection mechanisms of immunity are common in prokaryotes and confer defense at the population level – infected cells will sacrifice themselves to inhibit the propagation of the invader, saving uninfected cells in the population from infection. It has long been assumed that Csm6 confers immunity through an abortive infection mechanism. Here my thesis work shows that Csm6 triggers a growth arrest in the host that hinders viral propagation, initially providing immunity at the population level by allowing the continued replication of uninfected cells. My work also demonstrates that Csm6-induced dormancy has selective advantages for cells as it leads to broad immunity against untargeted phages. This selective advantage offers an explanation as to why cells may opt to acquire spacers that rely on Csm6 activity. Finally, my work shows that Csm6-induced dormancy is reversible in a subset of cells through the eventual degradation of the phage DNA, explaining how such spacers are maintained within the population. Collectively, my thesis work addresses a long-standing conundrum in the field and reveals that Csm6 does not operate solely through abortive infection. Instead, the type III-A CRISPR-Cas system has a built-in mechanism that allows for exit from Csm6-induced dormancy which enable the subsistence of spacers that provide broad-spectrum immunity.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Shilton, Amanda K., "Recovering from Dormancy: Fixation of CSM6-Inducing Spacers in the Bacterial Population" (2025). Student Theses and Dissertations. 825.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/825
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy