University of Alberta Carotenoid diversity in novel Hymenobacter strains isolated from Victoria Upper Glacier, Antarctica, and implications for the evolution of microbial carotenoid biosynthesis by Jonathan Lee Klassen A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Cell Biotechnology Department of Biological Sciences ©Jonathan Lee Klassen Fall 2009 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission. Examining Committee Dr. Julia Foght, Department of Biological Science Dr. Phillip Fedorak, Department of Biological Sciences Dr. Brenda Leskiw, Department of Biological Sciences Dr. David Bressler, Department of Agriculture, Food and Nutritional Science Dr. Jeffrey Lawrence, Department of Biological Sciences, University of Pittsburgh Abstract Many diverse microbes have been detected in or isolated from glaciers, including novel taxa exhibiting previously unrecognized physiological properties with significant biotechnological potential. Of 29 unique phylotypes isolated from Victoria Upper Glacier, Antarctica (VUG), 12 were related to the poorly studied bacterial genus Hymenobacter including several only distantly related to previously described taxa. Further study of these microorganisms revealed genotypic, phenotypic, morphological and chemotaxonomic divergence from named species and suggested that they likely represent novel Hymenobacter species. These studies also indicated, however, that the systematics of Hymenobacter and related microorganisms is more complex than previously realized, and may exhibit poorly defined species boundaries due to cosmopolitan dispersal, significant rates of horizontal gene transfer and reintroduction of archived genotypes, e.g., from glacial ice. These processes are reflected in the carotenoid composition of Hymenobacter and related organisms, which includes several novel methyl- and xylosyl-derivatives of 2'-hydroxyflexixanthin with distributions indicative of horizontal gene transfer or differential gain and/or loss of terminal biosynthetic pathway steps. These processes have been previously underappreciated in assessments of microbial carotenoid diversity and suggest the need for fine-scale phylogenetic study of carotenoid distribution in other microbial taxa. Further comparative genomics-based evaluation of microbial carotenoid biosynthesis indicated its wide phylogenetic distribution and diversification, controlled by several lineage-specific modes of evolution including horizontal transfer, de novo enzyme evolution followed by differential gene loss, co-evolution with biochemically associated structures and elevated mutation rates. The latter especially interacts with horizontal transfer depending on metabolic pathway topology, exemplified by the evolution of purple bacterial carotenoid biosynthesis. Exploration of VUG microbial diversity, therefore, not only revealed novel taxa and biotechnologically interesting compounds but also spurred broader evaluation of the mechanisms of metabolic pathway evolution applicable to many other taxa and biochemical pathways. Acknowledgements First and foremost, I thank my wife Verity and children Joel and Alice for their love, patience and support throughout my graduate studies. Without your backing, nothing that follows would have occurred. I also thank my parents for their support throughout the long process that was my education. Much thanks and gratitude to my supervisor Dr. Julia Foght for her support throughout this project, especially in encouraging me to pursue my own research interests outside of the original project scope and for the many ways you have taught me so much during my PhD program. Thanks also to all other members of the Microbiology RIG, especially, Dr. P. Fedorak, for your material and intellectual contributions to my research. I especially thank my lab mates past and present for their friendship, listening ears, and excellent technical assistance (after the requisite “you want to do/broke what?!?”): A. Adebusuyi, M. Baker, C. Bentz, S. Cheng, D. Coy, S. Ebert, Dr. B. Hearn, J. Hulecki, Dr. R. Kumaraswamy, Dr. K. Landry, C. Li, X. Lim, Dr. C. Nesbø, T. Penner, A. Scott, K. Semple, Dr. T. Siddique, A. Wong and R. Young. Thanks to Dr. J. Barker and Dr. M. Sharp for their provision of VUG samples. Also appreciated has been the technological assistance of many people at the University of Alberta: Dr. R. McKay (National High Field NMR Centre); W. Moffat (Department of Chemistry Analytical and Instrumental Laboratory); R. Whittal (Department of Chemistry Mass Spectrometry Laboratory); and all of the helpful people in the Department of Biological Sciences Molecular Biology Service Unit. I thank for their provision of bacterial strains: Dr. J. Aislabie (Landcare Research, New Zealand); Prof. H.-J. Busse (University of Vienna, Austria); and C. Fang (China Center for Type Culture Collection, China). This research has been funded by an NSERC post-graduate research scholarship (Doctoral) and NSERC operating grants held by J. Foght, a Province of Alberta Scholarship and a Province of Alberta Graduate Scholarship (Masters), a Walter H. Johns Scholarship and a Mary Louise Imrie Graduate Student Award from the University of Alberta Faculty of Graduate Studies and Research and a Travel Subsidy and teaching support from the Department of Biological Sciences. Table of Contents 1. Introduction....................................................................................................1 1.1. The genus Hymenobacter ......................................................................1 1.2. Carotenoids ............................................................................................4 1.2.1. Structure and nomenclature ...........................................................5 1.2.2. Physiological function...................................................................6 1.2.3. Biotechnological application.......................................................10 1.3. Horizontal gene transfer.......................................................................12 1.4. Thesis overview and research objectives.............................................14 1.5. Additional research conducted outside of the scope of my PhD program ..............................................................................................................15 1.6. Literature cited.....................................................................................15 2. Isolation of Bacteria from Victoria Upper Glacier, Antarctica Glacial Ice .25 2.1. Introduction..........................................................................................25 2.2. Materials and methods .........................................................................27 2.2.1. Study site and sampling ...............................................................27 2.2.2. Bacterial culture and isolation .....................................................28 2.2.3. Bacterial identification using 16S rRNA gene sequencing..........29 2.3. Results..................................................................................................30 2.3.1. Enumeration of cultured heterotrophic bacteria ..........................30 2.3.2. Identification of isolated organisms.............................................31 2.4. Discussion............................................................................................33 2.5. Literature cited.....................................................................................35 3. Genotypic and Phenotypic Characterization of Novel Hymenobacter Species ......................................................................................................................41 3.1. Introduction..........................................................................................41 3.2. Materials and methods .........................................................................42 3.2.1. Strain selection.............................................................................42 3.2.2. Genotypic analysis.......................................................................42 3.2.3. Phenotypic characterization.........................................................44 3.2.4. Fatty acid determination ..............................................................46 3.2.5. Carbon source utilization .............................................................47 3.3. Results..................................................................................................47 3.3.1. Phylogenetic analysis of Hymenobacter species and related strains using the 16S rRNA gene and gyrB.............................................................47 3.3.2. Phenotypic characterization of Hymenobacter species and related VUG strains .................................................................................................51