University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-5-2019 Characterizing Restriction-Modification Systems and DNA Methyltransferases in the Halobacteria Matthew Ouellette University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Ouellette, Matthew, "Characterizing Restriction-Modification Systems and DNA Methyltransferases in the Halobacteria" (2019). Doctoral Dissertations. 2280. https://opencommons.uconn.edu/dissertations/2280 Characterizing Restriction-Modification Systems and DNA Methyltransferases in the Halobacteria Matthew Ouellette, Ph.D. University of Connecticut, 2019 Abstract The Halobacteria are a class of archaeal organisms which are obligate halophiles. Several studies have described the Halobacteria as highly recombinogenic, yet members even within the same geographic location cluster into distinct phylogroups, suggesting that barriers exist which limit recombination. Barriers to recombination in the Halobacteria include CRISPRs, glycosylation, and archaeosortases. Another possible barrier which could limit gene transfer might be restriction-modification (RM) systems, which consist of restriction endonucleases (REases) and DNA methyltransferases (MTases) that both target the same sequence of DNA. The REase cleaves the target sequence, whereas the MTase methylates the site and protects it from cleavage. This dissertation examines the role of DNA methylation and RM systems in the Halobacteria and their impact on halobacterial speciation and genetic recombination. Using the model haloarchaeon, Haloferax volcanii, the genomic DNA methylation patterns (methylome) of an archaeal organism was characterized for the first time. Further investigations via gene deletion were used to identify the DNA methyltransferases responsible for the detected methylation patterns in H. volcanii, and experiments were conducted to determine the impact of RM systems on recombination via cell-to-cell mating. The distribution of MTase and RM system genes throughout the Halobacteria was also examined using a bioinformatics approach. The results indicated that the methylome of H. volcanii consists of two methylated motifs: GCAm6BNNNNNNVTGC, methylated by the Type I RM system RmeRMS, and Cm4TAG, methylated by the orphan MTase HVO_0794. Three other putative MTase genes (HVO_C0040, HVO_A0079, and HVO_A0237) were also identified, but were not observed to contribute to the Matthew Ouellette, University of Connecticut, 2019 methylome. Results from mating experiments indicated that RM systems could potentially limit recombination via cell-to-cell mating in H. volcanii. Furthermore, RM system genes were observed to be patchily distributed among the Halobacteria, whereas orphan MTase gene families were more widespread and well-conserved. The results of these studies provide insight into the life cycle of RM systems in the Halobacteria and how they might contribute to halobacterial diversification and speciation. The RM deletion mutants of H. volcanii also have the potential to be useful in future work examining the role of RM systems and DNA methylation in the Halobacteria. Characterizing Restriction-Modification Systems and DNA Methyltransferases in the Halobacteria Matthew Ouellette B.S., Post University, 2011 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut i Copyright by: Matthew Ouellette 2019 ii APPROVAL PAGE Doctor of Philosophy Dissertation Characterizing Restriction-Modification Systems and DNA Methyltransferases in the Halobacteria Presented by: Matthew Ouellette, B.S. Major Advisor _______________________________________________________________________ R. Thane Papke, Ph.D. Associate Advisor _______________________________________________________________________ Johann Peter Gogarten, Ph.D. Associate Advisor _______________________________________________________________________ Daniel Gage, Ph.D. Associate Advisor _______________________________________________________________________ Kenneth Noll, Ph.D. Associate Advisor _______________________________________________________________________ Michael O’Neill, Ph.D. University of Connecticut 2019 iii This dissertation is dedicated to my father, Michael Ouellette, whose support and encouragement was the greatest thing a son could ask for. iv Acknowledgements I would like to thank my parents, Melanie and Michael Ouellette, and my brother Eric Ouellette. My family has been the greatest blessing in my life, and this work would not have been possible without their love and support. I would also like to thank my major advisor, Thane Papke, whose guidance and creative ideas were essential to shaping my dissertation work and helping me to grow as a researcher. Thank you, also, to Andrea Makkay, Sean Pearson, Jess Lajoie, Scott Chimileski, Nikhil Ram Mohan, and all current and former Papke Lab members, who provided a wonderful, friendly, and supportive work environment. I am also grateful to Artemis Louyakis and Matt Fullmer of the Gogarten Lab for their support, friendship, and bioinformatics expertise. I am also thankful for the support of my associate advisors: Peter Gogarten, Dan Gage, Ken Noll, and Mike O’Neill. v Table of Contents Chapter 1. Introduction.................................................................................................................1 1. Halobacteria .............................................................................................................................1 1.1 Classification and Taxonomy of the Halobacteria .............................................................1 1.2 A Brief History of Halobacteria Discoveries .....................................................................3 1.3 Habitats of Halobacteria .....................................................................................................4 1.4 Physiology of Halobacteria ................................................................................................9 1.5 Genetics of Halobacteria ..................................................................................................13 1.6 Gene Transfer and Recombination in the Halobacteria ...................................................16 2. DNA Methylation and Methyltransferases.............................................................................20 2.1 What is DNA Methylation? ..............................................................................................20 2.2 What are DNA Methyltransferases? .................................................................................21 2.3 Strategies for Detecting DNA Methylation Patterns ........................................................22 3. Restriction-Modification Systems ..........................................................................................24 3.1 What are Restriction-Modification Systems? ...................................................................24 3.2 A Brief History of RM System Discoveries in Bacteria ..................................................25 3.3 Classes of RM Systems ....................................................................................................26 4. Orphan Methyltransferases ....................................................................................................28 4.1 What are Orphan Methyltransferases? .............................................................................28 4.2 DNA Adenine Methyltransferase (Dam) In Escherichia coli ..........................................28 4.3 Cell-Cycle-Regulated Methyltransferase (CcrM) in Caulobacter crescentus .................30 4.4 Campylobacter Transformation System Methyltransferase (CtsM) in Campylobacter jejuni .......................................................................................................................................31 5. DNA Methylation and RM Systems in the Archaea .............................................................32 5.1 RM Systems in Thermophilic Archaea ............................................................................32 5.2 RM Systems in Methanogenic Archaea ..........................................................................35 5.3 RM Systems in the Halobacteria ......................................................................................37 5.4 Domain-wide Investigation of Archaeal DNA Methylation and RM Systems ................38 6. Conclusion ..............................................................................................................................39 References ..................................................................................................................................41 Chapter 2. Dihydroxyacetone metabolism in Haloferax volcanii ............................................58 vi Abstract ......................................................................................................................................59 Introduction ................................................................................................................................59 Materials and Methods ...............................................................................................................60 Strains and Growth Conditions ..............................................................................................60 PCR