Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2018 Investigating Genetic (IN)Compatibility Between Temperate Phages and CRISPR-CAS Systems in Staphylococcus Aureus Gregory W. Goldberg Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons INVESTIGATING GENETIC (IN)COMPATIBILITY BETWEEN TEMPERATE PHAGES AND CRISPR-CAS SYSTEMS IN STAPHYLOCOCCUS AUREUS A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Gregory W. Goldberg June 2018 © Copyright by Gregory W. Goldberg 2018 INVESTIGATING GENETIC (IN)COMPATIBILITY BETWEEN TEMPERATE PHAGES AND CRISPR-CAS SYSTEMS IN STAPHYLOCOCCUS AUREUS Gregory W. Goldberg, Ph.D. The Rockefeller University 2018 Prokaryotic organisms employ various mechanisms for defending against parasitism by viruses and other mobile genetic elements. One form of defense comprises the adaptive immune systems derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and CRISPR-associated (cas) genes. CRISPR-Cas immune systems enable the acquisition of heritable resistance to specific mobile genetic elements on the basis of nucleic acid sequence recognition, but do not necessarily discriminate between target elements which are burdensome and those which are beneficial. My thesis is concerned with the consequences of CRISPR-Cas immunity directed at a particular breed of bacterial DNA viruses, known as temperate phages, which cause both harmful (lytic) and benign (lysogenic) infections under different conditions. Initial studies investigating prokaryotic CRISPR-Cas immunity seemed to indicate that functional, DNA-targeting systems cannot stably co-exist with their target elements in vivo. For example, in studies where immunity was directed at temperate phages, DNA- targeting CRISPR-Cas systems were found to prevent both lysogenic and lytic infections except when targeting was altogether abrogated via mutation or inhibition of the CRISPR-Cas system. The first part of my thesis work includes in vivo experiments which challenged the generality of this view, with regard to the different types of DNA- targeting CRISPR-Cas systems. Namely, I demonstrated that a staphylococcal branch of the ‘type III’ CRISPR-Cas systems is capable of tolerating lysogenic infections by specific temperate phages which are otherwise targeted during lytic infections. I further established that the capacity for conditional temperate phage tolerance results from a transcription-dependent targeting modality which was not anticipated for this particular DNA-targeting type III system. In contrast, I observed only the expected genetic escape outcomes when temperate phages were targeted by a ‘type II’ CRISPR- Cas system with a transcription-independent (Cas9-based) DNA targeting modality. These findings laid the groundwork for subsequent studies of CRISPR-Cas immunity to phages in Staphylococcus aureus hosts, and guided my colleagues towards in vitro characterization of the type III system’s transcription-dependent targeting mechanism. CRISPR-Cas systems have been identified in about 50% of sequenced bacterial genomes, and the factors which influence this distribution are still not fully understood. My description of conditional tolerance by a staphylococcal, type III CRISPR-Cas system illustrated that, in principle, these particular systems could stably co-exist with their temperate phage target elements in lysogenic hosts while maintaining their ability to protect against lytic infections. During the second part of my thesis work, I set out to define additional phenotypic consequences for the lysogenized lineages of S. aureus which maintain conditional tolerance, in an effort to better understand how this phenomenon might influence the distribution and stability of type III systems among natural isolates. Notably, I found that the maintenance of certain temperate-phage- targeting systems can incur fitness costs in lysogenic populations. I showed, furthermore, that these costs are potentially greater if more than one temperate phage is targeted in populations of double lysogens, but that they can be alleviated by mutations which do not abrogate phage targeting during lytic infections. Collectively, these findings imply that long-term maintenance of type III systems in natural populations of lysogens might require additional evolutionary fine-tuning, particularly among lineages which are prone to multiple infection. This thesis is dedicated to my family and dear friends iii ACKNOWLEDGMENTS I am deeply grateful for the opportunities, training, and resources I was provided during my time at The Rockefeller University. It was an inspiring and formative experience for me, which would not have been possible if not for a number of outstanding individuals that I had the fortune of interacting with. First, I’d like to sincerely thank my advisor, Dr. Luciano Marraffini, for years of support and patience, and for establishing a truly vibrant and innovative research program at Rockefeller, centered on the biology of CRISPR-Cas systems in bacteria. I was entirely unfamiliar with this intriguing subject prior to reading about his newly formed lab in 2011, and ultimately drawn to the David Rockefeller Graduate program because of it. I am further grateful that, shortly after rotating with him, Dr. Marraffini warmly entertained my wishes to forego additional rotations and pursue doctoral research in his lab. It was here that I worked closely with him on many occasions, and came to know his many remarkable qualities. Dr. Marraffini is a superbly clever and devoted scientist, and I will always be inspired by his courage, wise regard for pragmatism, and ability to foster a work environment where diverse people could come together to freely share their ideas and passion for science, and yet also cultivate their own individuality. I am also indebted to the members of my thesis committee, Drs. Charlie Rice and Vincent Fischetti, for their thoughtful suggestions, commentary, and genuine encouragement over the years—it truly was an indispensable part of my experience here. I’d further like to thank my external examiner, Dr. Bruce Levin, for graciously agreeing to fulfill this role, and for the cheerful, personal encouragement he offered me after learning of my early work in the lab. Likewise, I am indebted to Dean Sidney Strickland, Emily Harms, Marta Delgado, Kristen Cullen, Cristian Rosario, Stephanie Fernandez, Andrea Morris, and any other members of the Dean’s Office I might be missing, for maintaining a lively, warm, and welcoming place in Founder’s Hall for graduate students over the years, and for running a truly superb graduate program at The Rockefeller University. Additionally, I’d like to sincerely thank Dr. Chad Euler (formerly of the Fischetti Laboratory, now Assistant Professor at CUNY Hunter), not only for his expert handling of the mouse work during our brief collaboration, but also for years of collegial interactions at joint lab meetings and Rockefeller-related events. iv Next, I’d like to thank all the members of the Marraffini Lab, past and present, for the countless scientific discussions we shared, and for humoring my often loquacious demeanor therein. In particular, I’d like to highlight those who contributed directly to this thesis at some point or another. Dr. Wenyan Jiang, first and foremost, deserves a very special thanks for his inspiring work ethic and dedication, and for his seemingly countless contributions to the lab. Wenyan took initiative on so many occasions, and in so doing opened key research avenues for me and other members of the lab. Among his invaluable and timely contributions to this work in particular was the cloning of our first plasmid-borne CRISPR-Cas system, pWJ30β, from which all of my type III plasmids are originally derived. Wenyan was also an excellent bay-mate throughout our 5 years together in the lab, and I am extremely grateful for the technical training and feedback he provided me on numerous occasions. I am also extremely grateful to Dr. David Bikard for his generally learned and resourceful contributions to the lab, in addition to the technical training he provided me. Dr. Bikard’s lively feedback truly helped to strengthen the rigor of my early work, and I will always appreciate that. I would like to sincerely thank Dr. Poulami Samai for inspiring me with her patient and devoted approach to both science and laboratory work, in addition to her expert knowledge of nuclease biology. Dr. Samai, I should also mention, ultimately provided a skillful in vitro demonstration that the type III-A CRISPR-Cas complex we study cleaves the non-template strand of target DNA in a transcription-dependent fashion, as well as its RNA transcripts. These were monumental and reassuring findings for the field, and utterly invaluable to the confident progression of our continued work on type III-A systems in the lab. Inbal Maniv and Dr. Asma Hatoum-Aslan were instrumental in getting the lab on its feet from the very beginning, and assisted in my technical training on numerous occasions. On both accounts I am truly indebted to them. Inbal in particular had a range of designated responsibilities around the lab, and managed to keep it in order for many years before moving on to her doctoral work in Israel. I can’t help but mention that she also trained me on my very first top agar experiment… Many