BEING Aquifex aeolicus: UNTANGLING A HYPERTHERMOPHILE‘S CHECKERED PAST by Robert J.M. Eveleigh Submitted in partial fulfillment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia December 2011 © Copyright by Robert J.M. Eveleigh, 2011 DALHOUSIE UNIVERSITY DEPARTMENT OF COMPUTATIONAL BIOLOGY AND BIOINFORMATICS The undersigned hereby certify that they have read and recommend to the Faculty of Graduate Studies for acceptance a thesis entitled ―BEING Aquifex aeolicus: UNTANGLING A HYPERTHERMOPHILE‘S CHECKERED PAST‖ by Robert J.M. Eveleigh in partial fulfillment of the requirements for the degree of Master of Science. Dated: December 13, 2011 Co-Supervisors: _________________________________ _________________________________ Readers: _________________________________ ii DALHOUSIE UNIVERSITY DATE: December 13, 2011 AUTHOR: Robert J.M. Eveleigh TITLE: BEING Aquifex aeolicus: UNTANGLING A HYPERTHERMOPHILE‘S CHECKERED PAST DEPARTMENT OR SCHOOL: Department of Computational Biology and Bioinformatics DEGREE: MSc CONVOCATION: May YEAR: 2012 Permission is herewith granted to Dalhousie University to circulate and to have copied for non-commercial purposes, at its discretion, the above title upon the request of individuals or institutions. I understand that my thesis will be electronically available to the public. The author reserves other publication rights, and neither the thesis nor extensive extracts from it may be printed or otherwise reproduced without the author‘s written permission. The author attests that permission has been obtained for the use of any copyrighted material appearing in the thesis (other than the brief excerpts requiring only proper acknowledgement in scholarly writing), and that all such use is clearly acknowledged. _______________________________ Signature of Author iii TABLE OF CONTENTS List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii Abstract ....................................................................................................................... viii List of Abbreviations Used ................................................................................................ ix Acknowledgements ............................................................................................................. x Chapter 1 Introduction ...................................................................................................... 1 1.1 Introduction to Phylogenomics ........................................................................ 1 1.2 Overview of Phylogenomics ............................................................................ 4 1.2.1 Introduction to Phylogenetic Inference ........................................................... 4 1.2.2 Phylogenetic Incongruence and Assumptions .............................................. 14 1.2.3 Phylogenomic Inference ............................................................................... 18 1.2.4 The Unresolved Tree of Life......................................................................... 26 Chapter 2 Materials and Methods ................................................................................... 32 2.1 Genome Retrieval .......................................................................................... 32 2.2 Putative Cluster Determination ...................................................................... 32 2.3 Alignment Preparation and Phylogenetic Inference ...................................... 33 2.4 Determination of the Cohesion Within the Aquificae Phylum ...................... 34 2.4.1 Ranked Phylogenetic Profiling Approach .................................................... 34 2.4.2 Tree-based Cohesion Approach .................................................................... 35 2.5 Assessment of Relationship between Aquificae and Other Lineages ............ 35 2.5.1 Phylogenetic Profile Construction ................................................................ 35 2.5.2 Phylogenetic and Bipartition Analysis.......................................................... 36 2.6 Functional Classification of Clusters ............................................................. 36 2.6.1 Clusters of Orthologous Groups (COGs) ...................................................... 36 2.6.2 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ................. 37 2.7 Recombination Analysis ................................................................................ 38 Chapter 3 Results ............................................................................................................ 40 3.1 Affinities within the Aquificae ...................................................................... 40 3.2 Cohesion of the Aquificae Phylum ................................................................ 40 3.3 Affinities of Aquificae with Key Groups ....................................................... 46 iv 3.4 Affinities of Aquificae with Other Groups .................................................... 46 3.5 Affinities of Each Individual Aquificae Genome .......................................... 47 3.6 System-level Analysis .................................................................................... 48 3.6.1 Phylogenetic Profile Analysis of Functional Groups.................................... 50 3.6.2 Ribosomal Structure and Biogenesis ............................................................ 54 3.6.3 Cell Motility: Flagellar Assembly ................................................................ 58 3.6.4 Lipopolysaccharide (LPS) Biosynthesis ....................................................... 59 3.6.5 Energy Metabolism: Oxidative Phosphorylation .......................................... 61 Chapter 4 Discussion ...................................................................................................... 67 Chapter 5 Conclusion ..................................................................................................... 72 Appendix A Supplementary Materials ............................................................................. 92 v List of Tables Table 3.1: Phyletic pattern breakdown of all 2433 Aquificae COGs at the parent level classification ........................................................................................................... 522 Table 3.2: Phyletic pattern breakdown of four biological subsystems of interest identifying the number of ubiquitous, inclusive and exclusive R, E and T profiles. ................................................................................................................................. 522 Table A.1: The species distribution of 774 genomes, 53 Archaea and 721 Bacteria, categorized by domain, phylum, class and number of thermophiles used for phylogenomic analysis. ........................................................................................... 921 Table A.2: Functional breakdown of all child categories of a) Cellular processes and signaling, b) Information storage and processing, c) Metabolism and d) Poorly characterized. ............................................................................................................ 92 vi List of Figures Figure 1.1: Two alternative hypotheses concerning the closest phylogenetic partners of the Aquificae ............................................................................................................. 27 Figure 3.1: Overlap in homologous gene content among the three sequenced members of phylum Aquificae.................................................................................................... 411 Figure 3.2: Summary of all phylogenetic profiles of all Aquificae subsets, subdivided into the core AHS subset and the variable subset ............................................................ 42 Figure 3.3: Bootstrap distributions for the pairings of different Aquificae among the a) 237 cohesive and b) 107 non-cohesive maximum likelihood trees of the AHS core subset where A=Aquifex, H=Hydrogenobaculum and S=Sulfurihydrogenibium ... 444 Figure 3.4: Phyletic breakdowns for the lineage-restricted subsets (A-only, H-only, S- only in grey) and inclusive Aquificae subsets (in color) ........................................ 499 Figure 3.5: Relative support for the affinities of Aquificae with Archaea (blue), ε- Proteobacteria (purple), Thermotogae (red) evaluated with the variable preference index (VPI).............................................................................................................. 533 Figure 3.6: Linear arrangement of six ribosomal operons: L11+ rif (blue), str (orange), S10 (red), spc (green) and α (purple) and their respective gene order in Aquifex, Hydrogenobaculum and Sulfurihydrogenibium. ..................................................... 577 vii Abstract Lateral gene transfer (LGT) is an important factor contributing to the evolution of prokaryotic genomes. The Aquificae are a hyperthermophilic bacterial group whose genes show affiliations to many other lineages, including the hyperthermophilic Thermotogae, the Proteobacteria, and the Archaea. Previous phylogenomic analyses based on the concatenation of genes thought to be recalcitrant to LGT suggest that the Aquificae are sister to Thermotogae, but many phylogenies