Effects of the Mobile Genetic Element ICEBs1 on Bacterial Host Fitness Joshua M. Jones B.S. Biochemistry University of Maine, 2014 Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology June 2020 Ⓒ 2020 Massachusetts Institute of Technology. All rights reserved. Signature of Author:……………………………………………………………………………… Joshua M. Jones Microbiology Graduate Program May 6, 2020 Certified by:……………………………………………………………………………………… Alan D. Grossman Professor of Biology Thesis Supervisor Accepted by:……………………………………………………………………………………… Jacquin Niles Associate Professor of Biological Engineering Chair of Microbiology Program 1 Effects of the Mobile Genetic Element ICEBs1 on Bacterial Host Fitness Joshua M. Jones Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for the degree of Doctor of Philosophy ABSTRACT Mobile genetic elements drive bacterial evolution by mediating horizontal gene transfer and by carrying cargo genes that confer important traits to host cells. Traits provided by mobile genetic elements include antibiotic resistance, novel metabolic capabilities, virulence factors, and the ability to form symbioses. Mobile genetic elements, especially Integrative Conjugative Elements (ICEs), are abundant in bacteria. Many do not contain cargo genes with known functions, but some likely carry novel types of cargo genes that provide traits beyond the scope of those currently attributed to mobile elements. In this thesis I describe the characterization of a fitness benefit provided by the mobile genetic element ICEBs1 to its bacterial host, Bacillus subtilis. Activation of ICEBs1 conferred a frequency-dependent selective advantage to host cells during biofilm formation and sporulation. The advantage was due to inhibition of biofilm- associated gene expression and delayed sporulation, which enabled ICEBs1 host cells to exploit their neighbors and grow more prior to sporulation. I identified a single gene within ICEBs1, ydcO, as both necessary and sufficient for the repression of development. Manipulation of host development programs allows ICEBs1 to increase host fitness. These findings highlight that cargo genes can alter existing aspects of physiology rather than providing entirely new traits, broadening our understanding of how mobile genetic elements influence their hosts. Thesis Supervisor: Alan D. Grossman Title: Praecis Professor of Biology; Department Head 2 Acknowledgements I am extremely grateful to Alan for his scientific and personal mentorship during the past five years. Alan helped me learn not only how to do good science and communicate it effectively, but also taught by example how to balance calmness with intensity. Thanks also to my thesis advisory committee members Mike Laub and Jeff Gore for feedback on my projects and encouragement over the years. Thank you to the members of the Grossman lab, past and present. I’m grateful to have worked alongside close friends who are also excellent scientists. I’ll especially miss our brunches and afternoon Muddy meetings. Thank you to all of my friends, both within the MIT community and beyond. I’m extremely fortunate to have a group of friends that are as ridiculously fun as they are caring and supportive. I’m particularly grateful for my family, especially my parents, who have always supported me in my interests and encouraged me to pursue excellence. Finally, a huge thank you to Mónica for being a loving and supportive partner. All the ups and downs of grad school were much better shared with you. 3 Table of Contents Abstract 2 Acknowledgements 3 List of Figures 5 List of Tables 6 Chapter 1 Introduction 7 Chapter 2 A mobile genetic element increases bacterial host fitness by 45 manipulating development Appendix A Genetic screen to isolate ydcO suppressor mutants 88 Appendix B yddI is also important for ICEBs1 host fitness 92 Appendix C Prolonged ICEBs1 induction is detrimental to host cells 96 Chapter 3 Conclusions and Perspectives 105 4 List of Figures Chapter 1 Fig. 1. The three primary forms of HGT in bacteria 10 Fig. 2. The ICE conjugative life cycle 14 Fig. 3. Genetic map of ICEBs1 28 Fig. 4. Regulation of ICEBs1 29 Chapter 2 Fig. 1. The fitness of ICEBs1-containing cells during development 81 depends on their initial frequency in the population Fig. 2. ICEBs1-containing cells delay sporulation in a frequency- 82 dependent manner Fig. 3. The ICEBs1 cell-cell signaling genes, rapI-phrI, are necessary but 83 not sufficient to confer a selective advantage Fig. 4. Expression from Pxis and ydcO are required for the fitness benefit 84 of ICEBs1 Fig. 5. ydcO alone is sufficient to inhibit sporulation and provide a 85 selective advantage Fig. 6. ydcO inhibits expression of genes associated with sporulation 86 initiation and biofilm formation Appendix C Fig. 1. ICEBs1 induction incurs a frequency-dependent fitness cost to host 101 cells Fig. 2. ICEBs1 induction is detrimental during stationary phase 102 Fig. 3. ICEBs1 replication and conjugation gene expression contribute to 103 growth and stationary phase defects 5 List of Tables Chapter 2 Table 1. Frequency of transconjugants generated in biofilm matings 80 Table 2. B. subtilis strains used 87 Appendix A Table 1. ydcO suppressor mutants 91 Appendix B Table 1. B. subtilis strains used 94 Appendix C Table 1. B. subtilis strains used 104 6 Chapter 1 Introduction 7 Overview Bacteria are able to evolve rapidly in part due to their ability to acquire new genetic material through horizontal gene transfer. Mobile genetic elements are important drivers of horizontal gene transfer, as they encode genes to transfer themselves between cells. Mobile genetic elements often encode “cargo genes” that provide novel traits to the host, notably antibiotic resistance genes. In this thesis, I describe the characterization of a fitness benefit provided by a mobile genetic element, ICEBs1, to its bacterial host Bacillus subtilis. We found that a single ICEBs1 gene, ydcO, provided a fitness benefit by interfering with the host’s developmental pathway that controls biofilm formation and sporulation. When ICEBs1 gene expression is induced in the context of a growing biofilm, ydcO enables cells with ICEBs1 to express costly biofilm-associated genes at lower levels and delay sporulation, both of which contribute to a growth advantage. Introduction to Horizontal Gene Transfer in Bacteria Horizontal gene transfer (HGT) is the acquisition of DNA from non-parental origin. Horizontal gene transfer has been documented in all kingdoms of life, but is by far the most frequent and best characterized in bacteria, where it is a major driving force of evolution (de la Cruz and Davies, 2000; Soucy et al., 2015). Bacteria can acquire foreign DNA directly from their surrounding environment and from other cells that are not part of a parent-offspring relationship (Thomas and Nielsen, 2005). Horizontal gene transfer exposes bacteria to diverse genetic material, promoting evolution on a rapid timescale. In many cases, horizontally acquired segments of DNA make up a substantial fraction of the genome in bacteria, and they are often 8 responsible for important differences among otherwise closely related organisms (de la Cruz and Davies, 2000; Gogarten and Townsend, 2005; Koonin and Wolf, 2008; Ochman et al., 2000). There are three major, widely recognized mechanisms of horizontal gene transfer in bacteria: conjugation, transduction, and transformation (Fig. 1) (Soucy et al., 2015; Thomas and Nielsen, 2005). Conjugation and transduction are DNA transfer processes mediated by self-transmissible mobile genetic elements (MGEs), which are segments of DNA with the ability to move between cells (Frost et al., 2005). MGEs encode genes that, when expressed inside a bacterial host cell, provide the means of transfer. The biology of mobile genetic elements and their functions will be discussed in detail below. The third main mechanism of HGT is transformation, which is the acquisition of DNA present in the environment (Johnston et al., 2014). The ability to take in DNA for transformation is called genetic competence. Not all bacteria are known to possess this ability, and among most of those that do, it is positively regulated by conditions such as starvation and stress (Claverys and Martin, 2003; Johnston et al., 2014). Beyond these canonical forms of horizontal gene transfer, there are less well-characterized mechanisms whose contributions to microbial evolution are not well understood. Gene transfer agents (GTAs) are phage-like particles that transfer random fragments of chromosomal DNA (Lang et al., 2012). DNA and other molecules can be transferred by nano-tube bridges between adjacent cells (Dubey and Ben-Yehuda, 2011) and by membrane vesicles released from cells (Domingues and Nielsen, 2017). In Archaea, transient cell fusions can lead to exchange of plasmids and recombination between chromosomes (Naor and Gophna, 2012). 9 Conjugation Transduction Transformation Direct cell-to-cell DNA transfer DNA transfer through Uptake of DNA in the through secretion system viral particles environment Figure 1. The three primary forms of HGT in bacteria. Conjugation is mediated by integrative conjugative elements (ICEs) and conjugative plasmids. Transduction is mediated by bacteriophages. Organisms that can become genetically competent can undergo transformation with DNA present outside of the cell. Mobile genetic elements Mobile genetic elements