The Phages and Phage-like Elements of Rhodobacter capsulatus by © Alexander P. Hynes A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland May 2014 St. John’s Newfoundland and Labrador ABSTRACT Rhodobacter capsulatus is a purple non-sulfur bacterium that produces the gene transfer agent RcGTA. This phage-like particle is capable of transferring ~4-kb of host DNA to other R. capsulatus cells in a process analogous to transduction. The genes known to encode this particle are located in a ~15-kb region called the RcGTA structural gene cluster. As this cluster is larger than the packaging capacity of the particles, RcGTA is non-replicative. In addition to lacking key functions required by a phage, the RcGTA structural gene cluster lacks genes likely to encode functions necessary for its gene transfer activity, such as a cellular release mechanism. The costs and benefits of RcGTA production for R. capsulatus are unclear, making it difficult to explain its persistence in the genome. I investigated R. capsulatus SB1003 prophages to search for other genes involved in RcGTA production and gene transfer activity. I identified two functional prophages, RcapNL and RcapMu, which I characterized and found to be linked to RcGTA production. I also compared the genome-wide transcriptional profiles in a variety of R. capsulatus strains and growth conditions affecting RcGTA production to identify genes consistently co-regulated with the RcGTA structural gene cluster. I found nine such genes at six separate loci. In characterizing several of these genes, I identified a gene required for release of RcGTA by cell lysis, a pair of putative tail fibre-encoding genes, and an additional gene whose product is required for binding to the recipient cell. Finally, I characterized features of RcGTA production by quantitatively determining the packaging frequency of all loci on the R. capsulatus chromosome. Remarkably, while any gene can be transferred, the RcGTA structural gene cluster region was under-represented ii in the particles relative to the genome average. The search for a mechanistic explanation of this anomaly led to the discovery that RcGTA gene expression is elevated in ~3% of the cells in a population, and that these cells undergo lysis to release RcGTA particles. This provided us with the first quantification of the cost of RcGTA production. iii ACKNOWLEDGEMENTS I would like to acknowledge, first and foremost, my supervisor Dr. Andrew S. Lang for his patience, guidance and mentorship. His interest and accessibility have made graduate school engaging, his rigour has driven me to improve, and his continued involvement and support have made this research possible. I also would like to thank my thesis committee for their attention and efforts, despite setbacks and an unnerving propensity for storms, break-ins and other disasters on days of scheduled committee meetings. Of my numerous co-authors, all of whom are acknowledged in more detail in the co-authorship statement, I’d like to make special mention of Dr. Ryan G Mercer, a colleague and friend who made grad school more enjoyable. His feedback was invaluable and, as trailblazer for Lang lab doctoral candidates, his trials, tribulations and thesis served as guideposts for my own. Finally, I must thank my father, Dr. Michael F Hynes. Having a parent in your field is a boon I would wish on every graduate student. His wealth of knowledge, both in our field and of academia in general, has benefited me tremendously. My doctoral program and the research I have performed has been supported by a graduate student fellowship from the Memorial University School of Graduate Studies, a fellowship from Natural Sciences and Engineering Research Council of Canada (NSERC), and support from the Broad Institute’s Marine Virome Sequencing Project with matching contributions from Newfoundland and Labrador Research and Development Corporation (RDC). The research in Dr. Lang’s laboratory was supported by grants from NSERC, RDC and the Canadian Foundation for Innovation (CFI). iv Table of Contents ABSTRACT ........................................................................................................................ ii! ACKNOWLEDGEMENTS ............................................................................................... iv! Table of Contents ................................................................................................................ v! List of Tables ...................................................................................................................... x! List of Figures .................................................................................................................... xi! List of Symbols, Nomenclature or Abbreviations ........................................................... xiii! List of Appendices ........................................................................................................... xvi! The Phages and Phage-like Elements of Rhodobacter capsulatus ..................................... 1! 1! Introduction and Overview ........................................................................................... 1! 1.1! Bacteriophages ........................................................................................................ 1! 1.2! Bacteriophages: discovery & impact ...................................................................... 1! 1.3! Bacteriophage interactions ...................................................................................... 4! 1.3.1! Phage-host interactions .................................................................................... 4! 1.3.2! Phage-phage interactions ................................................................................. 7! 1.3.3! Benefits to the host ........................................................................................... 9! 1.4! Genetic exchange in bacteria ................................................................................ 11! 1.4.1! Transduction .................................................................................................. 12! 1.4.2! Other means of horizontal gene transfer ........................................................ 13! v 1.4.3! The benefits of horizontal gene transfer ........................................................ 14! 1.5! Rhodobacter capsulatus and GTAs ...................................................................... 14! 1.5.1! Rhodobacter capsulatus ................................................................................. 15! 1.5.2! Phages, prophages and bacteriocins in R. capsulatus .................................... 16! 1.5.3! RcGTA: The early years ................................................................................ 18! 1.5.4! RcGTA: In recent years ................................................................................. 20! 1.5.5! RcGTA phylogenetics .................................................................................... 22! 1.5.6! Other GTAs .................................................................................................... 23! 1.6! Research goals ...................................................................................................... 24! Co-Authorship and Contributions ..................................................................................... 27! 2! The Phages of Rhodobacter capsulatus ...................................................................... 30! 2.1! Introduction: .......................................................................................................... 30! 2.2! Experimental procedures ...................................................................................... 32! 2.2.1! Bacteriophage production .............................................................................. 32! 2.2.2! PEG precipitation of phage particles ............................................................. 32! 2.2.3! DNA extraction from phage particles ............................................................ 33! 2.2.4! Separation of phage and RcGTA particles ..................................................... 33! 2.2.5! Pulsed field gel electrophoresis (PFGE) ........................................................ 34! 2.2.6! DNA sequencing ............................................................................................ 34! 2.2.7! Bioinformatics ................................................................................................ 35! 2.2.8! Proteomics ...................................................................................................... 36! 2.2.9! Plaque assays ................................................................................................. 36! vi 2.2.10! Phage & prophage DNA end characterization ............................................. 37! 2.2.11! Phage knockouts and curing RcapNL .......................................................... 37! 2.2.12! GTA activity assays ..................................................................................... 38! 2.3! Results ................................................................................................................... 39! 2.3.1! Phage detection .............................................................................................. 39! 2.3.2! Phage discovery ............................................................................................. 42! 2.3.3! Genomics & Proteomics of RcapNL ............................................................. 42! 2.3.4! RcapNL cos sites ............................................................................................ 48! 2.3.5! RcapNL prophage .......................................................................................... 48! 2.3.6! RcapNL integration ........................................................................................ 49! 2.3.7! Host range ...................................................................................................... 51! 2.3.8! Confirming phage-knockout strains ............................................................... 51! 2.3.9! RcapH ............................................................................................................ 52! 2.3.10! Linking phage production and RcGTA activity ........................................... 54! 2.4! Discussion ............................................................................................................. 54! 3! The Non-Contiguous Genome of RcGTA, the Rhodobacter capsulatus Gene Transfer Agent ............................................................................................................................. 59! 3.1! Introduction ........................................................................................................... 59! 3.2! Experimental procedures ...................................................................................... 61! 3.2.1! Strains and plasmids ...................................................................................... 61! 3.2.2! Microarray analyses ....................................................................................... 64! 3.2.3! RcGTA activity bioassays .............................................................................. 65! vii 3.2.4! Detection of RcGTA major capsid protein by western blotting .................... 65! 3.2.5! Assays of RcGTA binding to cells ................................................................. 66! 3.2.6! DNA extraction from RcGTA particles ......................................................... 66! 3.2.7! Protein purification ........................................................................................ 66! 3.2.8! Electrophoretic mobility shift assay ............................................................... 67! 3.2.9! Polysaccharide lyase activity assays .............................................................. 67! 3.3! Results ................................................................................................................... 68! 3.3.1! Identification of genes with transcript level changes according to changes in RcGTA production .................................................................................... 68! 3.3.2! Identification of genes with roles in RcGTA activity .................................... 72! 3.3.3! Roles of identified proteins in RcGTA function ............................................ 72! 3.4! Discussion ............................................................................................................. 77! 4! DNA Packaging Bias and Differential Expression of Gene Transfer Agent Genes Within a Population During Production and Release of the Rhodobacter capsulatus Gene Transfer Agent, RcGTA ...................................................................................... 84! 4.1! Introduction ........................................................................................................... 84! 4.2! Experimental procedures ...................................................................................... 86! 4.2.1! Bacterial strains, growth conditions, and plasmids ........................................ 87! 4.2.2! RcGTA DNA isolation .................................................................................. 89! 4.2.3! Microarray analyses ....................................................................................... 89! 4.2.4! RcGTA activity bioassays .............................................................................. 90! 4.2.5! Western blots targeting the RcGTA major capsid protein ............................. 91! viii 4.2.6! RcGTA gene expression in single cells ......................................................... 92! 4.2.7! Bioinformatic analyses ................................................................................... 92! 4.2.8! Treatments of RcGTA DNA for ligation experiments .................................. 92! 4.3! Results ................................................................................................................... 93! 4.3.1! Genome-wide quantification of DNA packaged in RcGTA particles ........... 93! 4.3.2! Transfer rates of genes from different locations in the R. capsulatus genome .......................................................................................................... 96! 4.3.3! RcGTA gene expression in single cells ......................................................... 98! 4.3.4! Identification of the putative RcGTA lysis gene ......................................... 100! 4.3.5! Bioinformatic analysis of the RcGTA terminase ......................................... 103! 4.3.6! Characterization of the ends of the DNA molecules in RcGTA particles ........................................................................................................ 104! 4.4! Discussion ........................................................................................................... 105! 5! Summary ................................................................................................................... 111! 6! Bibliography ............................................................................................................. 119! ix List of Tables Table 2-1: Primers and gene disruption sites used in this study ....................................... 35! Table 2-2: Strains tested for presence of a detectable phage band. .................................. 41! Table 2-3: RcapNL predicted ORFs and their best phage BLAST hits, where available ..................................................................................................................... 44! Table 2-4: Screening R. capsulatus natural isolates and phage-knockouts for the presence of RcapNL by PCR and the ability to serve as a host for RcapNL. ............ 51! Table 3-1: Strains and experimental plasmids used in this study ..................................... 62! Table 3-2: Primers and associated restriction sites used in mutant construction, complementation and protein expression ................................................................... 63! Table 3-3: R. capsulatus genes co-regulated in transcriptome comparisons .................... 70! Table 4-1: R. capsulatus strains and experimental plasmids used in this study ............... 86! x