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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Covalent DNA Modifications in Phage and Bacterial Dynamics Alexandra Bryson University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Microbiology Commons Recommended Citation Bryson, Alexandra, "Covalent DNA Modifications in Phage and Bacterial Dynamics" (2016). Publicly Accessible Penn Dissertations. 1627. https://repository.upenn.edu/edissertations/1627 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1627 For more information, please contact [email protected]. Covalent DNA Modifications in Phage and Bacterial Dynamics Abstract The microorganisms on and in the human body play a significant role in health and disease; however, little is known about how the interactions between these complex communities affect our wellbeing. This study examines how bacteria and phage interact through bacterial nucleases that restrict infection, such as restriction enzymes and CRISPR systems, and the covalent DNA modifications that neutralize them. Multiple targeted nucleases equip bacteria with an innate immune response against phage, and CRISPR systems provide an adaptive immune response. I report three main studies. 1) To study the human gut microbiome and virome (comprised predominately of phage), we collected fecal samples from a healthy individual over four years. From the fecal samples, total bacterial DNA and DNA from purified virus like particles (VLPs) were sequenced using Illumina and Pacific iosB cience single-molecule real-time (SMRT) sequencing to yield information about genome sequences and covalent modifications. Using computational methods we identified seven bacterial contigs and one phage contig with CRISPR arrays targeting phage contigs. This suggests that both bacteria and phage use CRISPR systems to compete with other phage. 2) Covalent DNA modifications are known to block the nuclease activity of restriction enzymes, however it was unknown if they can block the nuclease activity of CRISPR systems. To address this, we test if the CRISPR-Cas9 system could target wild type T4 phage and two T4 mutants. Wild type T4 modifies all its cytosines to glycosylated hydroxymethylcytosine (glc-HMC), and the two mutant T4 phage contain either hydroxymethylcytosine (HMC) or unmodified cystosines (C). These tests confirmed that glc-HMC and HMC in high concentrations can block CRISPR-Cas9. 3) To explore interactions between bacteria and phage further, we used covalent DNA modification data to link bacteria and phage pairs from the human gut microbiome, based on the idea that phage and bacterial DNAs in the same cell have been exposed to the same DNA modifying enzymes and thus share modification patterns. Overall, 443 modified motifs were shared between phage and bacteria, suggesting many possible phage-host pairs. In our data, 73% of phage genomes and 56% of bacterial genomes contained motifs that were completely modified, highlighting how ubiquitous and important the roll of DNA modifications are. These data allowed us to begin to specify the extent and types of interactions between phage and bacteria in longitudinal data. This work explores the complex interactions between bacteria and phage, a crucial step in understanding how these organisms contribute to human health and disease. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Frederic D. Bushman Keywords CRISPR, DNA modifications, Human Microbiome, Human Virome, Phage Dynamics, T4 Phage This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1627 Subject Categories Microbiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1627 COVALENT DNA MODIFICATIONS IN PHAGE AND BACTERIAL DYNAMICS Alexandra Lynn Bryson A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation _____________________ Frederic Bushman W. M. Measey Professor Chair, Department of Microbiology Graduate Group Chairperson _____________________ Dan Kessler, Associate Professor of Cell and Developmental Biology Dissertation Committee Mark Goulian, Professor of Biology Mecky Pohlschröder, Professor of Biology Rahul Kohli, Assistant Professor of Medicine Elizabeth Grice, Assistant Professor of Dermatology Dedication To Rick Bushman, the Bushman Lab members, and the MVP matriculating class of 2011. ii ABSTRACT COVALENT DNA MODIFICATIONS IN PHAGE AND BACTERIAL DYNAMICS Alexandra Lynn Bryson Dr. Frederic Bushman’s Laboratory The microorganisms on and in the human body play a significant role in health and disease; however, little is known about how the interactions between these complex communities affect our wellbeing. This study examines how bacteria and phage interact through bacterial nucleases that restrict infection, such as restriction enzymes and CRISPR systems, and the covalent DNA modifications that neutralize them. Multiple targeted nucleases equip bacteria with an innate immune response against phage, and CRISPR systems provide an adaptive immune response. I report three main studies. 1) To study the human gut microbiome and virome (comprised predominately of phage), we collected fecal samples from a healthy individual over four years. From the fecal samples, total bacterial DNA and DNA from purified virus like particles (VLPs) were sequenced using Illumina and Pacific Bioscience single-molecule real-time (SMRT) sequencing to yield information about genome sequences and covalent modifications. Using computational methods we identified seven bacterial contigs and one phage contig with CRISPR arrays targeting phage contigs. This suggests that both bacteria and phage use CRISPR systems to compete with other phage. 2) Covalent DNA modifications are known to block the nuclease activity of restriction enzymes, however it was unknown if they can block the iii nuclease activity of CRISPR systems. To address this, we test if the CRISPR- Cas9 system could target wild type T4 phage and two T4 mutants. Wild type T4 modifies all its cytosines to glycosylated hydroxymethylcytosine (glc-HMC), and the two mutant T4 phage contain either hydroxymethylcytosine (HMC) or unmodified cystosines (C). These tests confirmed that glc-HMC and HMC in high concentrations can block CRISPR-Cas9. 3) To explore interactions between bacteria and phage further, we used covalent DNA modification data to link bacteria and phage pairs from the human gut microbiome, based on the idea that phage and bacterial DNAs in the same cell have been exposed to the same DNA modifying enzymes and thus share modification patterns. Overall, 443 modified motifs were shared between phage and bacteria, suggesting many possible phage-host pairs. In our data, 73% of phage genomes and 56% of bacterial genomes contained motifs that were completely modified, highlighting how ubiquitous and important the roll of DNA modifications are. These data allowed us to begin to specify the extent and types of interactions between phage and bacteria in longitudinal data. This work explores the complex interactions between bacteria and phage, a crucial step in understanding how these organisms contribute to human health and disease. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................. VIII LIST OF FIGURES ................................................................................................ II CHAPTER 1: INTRODUCTION ............................................................................ 1 1.1 THE HUMAN GUT VIROME AND MICROBIOME ................................................................................................ 1 1.2 PHAGE INFLUENCE ON BACTERIAL COMMUNITIES ....................................................................................... 4 1.3 RESTRICTION-MODIFICATION SYSTEMS ....................................................................................................... 5 1.4 CRISPR ........................................................................................................................................................... 7 1.5 COVALENT DNA MODIFICATIONS IN PHAGE .............................................................................................. 11 1.6 REFERENCES ..................................................................................................................................................... 11 CHAPTER 2: RAPID EVOLUTION OF THE HUMAN GUT VIROME ................ 19 2.1 CONTRIBUTIONS .............................................................................................................................................. 19 2.2 ABSTRACT ......................................................................................................................................................... 19 2.3 INTRODUCTION ................................................................................................................................................ 20 2.4 RESULTS ............................................................................................................................................................ 21 2.4.1 Sample Collection, Viral Purification, and DNA Sequencing.

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