A STUDY ON THE PHYLOGENETICS OF GENE TRANSFER: FROM PATHWAYS TO KINGDOMS A THESIS SUBMITTED TO THE GRADUATE DIVISION OF UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MOLECULAR BIOSCIENCES AND BIOENGINEERING May 2008 By Aren Ewing Thesis Committee: Gemot Presting, Chairperson Anne Alvarez Daniel Rubinoff We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Masters of Science in Molecular Biosciences and Bioengineering. Thesis Committee 2 Acknowledgments With much gratitude I would like to thank my advisor Dr. Gemot Presting. He provided and outstanding workplace and lab, with the freedom to explore our interests and the facility to follow them. During my time in the Presting lab, I also met some outstanding people and scientists. I would like to thank Thomas Wolfgruber, Anupma Sharma, Kevin Schneider, Jeffery Lai, and Normon Wang. They have provided great support and friends over the years. I would also like to thank my committee who has stuck in there with me for the completion of this degree. It was a struggle to take what was once a PhD project and convert to a MS and while making it as excellent as possible. I would also like to thank my girlfriend Hideko Kasahara for her support and encouragement during the writing of this thesis. 3 Table of Contents Acknowledgments ........................................................................................................................... 3 Table of Contents ............................................................................................................................ 4 List of Tables .................................................................................................................................. 5 List of Figures ................................................................................................................................. 6 Abstract ........................................................................................................................................... 8 Chapter I: Introduction ................................................................................................................... 9 Objectives ................................................................................................................................. 17 Chapter 2 ....................................................................................................................................... 20 Phylogenetic profiling of the Arabidopsis thaliana proteome .................................................. 20 Introduction .................................................................................................. ,........................ 20 Methods ................................................................................................................................. 24 Results ................................................................................................................................... 28 Discussion ............................................................................................................................. 48 Conclusion ............................................................................................................................ 63 Chapter 3 ....................................................................................................................................... 64 Elucidation ofXanthomonas species relationships using comparative genomics .................... 64 Introduction ........................................................................................................................... 64 Methods ................................................................................................................................. 73 Results ................................................................................................................................... 77 Discussion ............................................................................................................................. 89 Conclusion ............................................................................................................................ 96 Thesis Conclusion ......................................................................................................................... 98 1: Pathways evolve as a group as demonstrated with phylogenetic profiling ...................... 98 2: There is a dominant phylogenetic tree which can identifY consistent relationships between closely related species in the genus Xanthomonas . ................................................ 99 3: Lateral gene transfer occurred from a plant to bacteria and possibly confers a selective advantage for the infection of host plants ........................................................................... 100 Appendix ..................................................................................................................................... 101 Evaluating functionality of a putative horizontally transferred gene in Xanthomonas axonopodis pv. citri ........... ........ ............... ........ ....... ........ ...... ...................... ............... ............ 10 1 Introduction ..... ........ ....... ......... ...................... ....... ...................... ........ .............. ........ ........... 10 1 Summary ........................ ....................... ....................... ....... ....... ......................................... 125 References. ............................................. ....... .............................. ......................... ........ ....... ........ 126 4 List of Tables Table 1.1: Distribution of matches with E values from Ie-03 to le-18 ....................................... 32 Table 2.1: Gene counts summed by trees and generation method ................................................ 78 Table 2.2: Agreement on topology by model ............................................................................... 81 Table 2.3: Annotation group and method agreement for each tree topology ............................... 83 Table 2.4: List of 44 Xanthomonas genes shared by 14 organisms in iteration 3 ........................ 88 Table 3.1: Presence of insert sequenced and corresponding match to known LTG! sequence. 110 Table 3.2: List of 16 Xanthomonads ........................................................................................... 112 Table 3.3: LTG 1 presence vs host range .................................................................................... 122 Table 3.4: Infection Experiments ................................................................................................ 123 5 List of Figures Figure 1.1: Distribution of Arabidopsis gene matches in 170 non-plant genomes ..................... 30 Figure 1.2: Greedy outlier removal of pathway members .......................................................... 35 Figure 1.3: Phylogenetic profile clusters of (A) 11 isoprenoid, (B) 24 tryptophan and (C) 25 heme/chlorophyll biosynthesis genes ................................................................................... 40 Figure 1.4: Amino acid biosynthesis genes cluster together ....................................................... 44 Figure 1.5: The heme/chlorophyll biosynthesis pathway ........................................................... 46 Figure 2.1: Three monophyletic Xanthomonas trees ................................................................... 80 Figure 2.2: Consensus Tree for 229 Genes .................................................................................. 85 Figure 2.3: tRNA methyltransferase expanded 14 organism tree ............................................... 87 Figure 3.1: Xanthomonas region comparison ............................................................................ 108 Figure 3.2: Alignment of DUF239 to 28 known plant homologs .............................................. 109 Figure 3.3: PCR using Genomic DNA ...................................................................................... 112 Figure 3.4: Genomic Southern blot ofXanthomonas isolates ................................................... 113 Figure 3.5: Verification of Southern blot for integrity of Genomic DNA re-probed 16s rDNA 113 Figure 3.6: Knockout plasmid pAC3 ......................................................................................... 116 Figure 3.7: Deletion suicide plasmid pUFR080 ......................................................................... 117 Figure 3.8: Insertion plasmid pAC3 ........................................................................................... 119 Figure 3.9: Insertion suicide plasmid pUFR080 ........................................................................ 120 6 Abbreviations Alignment matrix: A conceptual matrix created when comparing two sequences, with 1 sequences on each axix. BLAST: Basic Local Alignment Search Tool BLOSSUM: Stands for (BLOcks of Amino Acid SUbstitution Matrix) primarily used to score alignments of evolutionary divergent protein sequences. Blossum62 is a matrix calculated for proteins with a 62% sequence identity. Gene Set: (Chapter 2) the group of genes that were most similar between all genomes compared based on the query genome. For the first phase of project this consists of 4 genes 3 Xanthomonas