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Insights Into the Genomic and Metabolic Adaptations of Termite INSIGHTS INTO THE GENOMIC AND METABOLIC ADAPTATIONS OF TERMITE ASSOCIATED VERRCUCOMICROBIA STRAINS USING FUNCTIONAL AND COMPARATIVE GENOMICS by Malini Kotak Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON DECEMBER 2016 Copyright © by Malini Kotak 2016 All Rights Reserved II Acknowledgements I would like to express my sincere gratitude to Dr. Jorge L. M. Rodrigues for being my mentor and providing opportunities, encouragement, guidance and support throughout my doctoral studies. I thank him for believing in me and pushing me beyond what I thought were my limits. Next, I would like to thank Dr. Demuth for agreeing to be my supervisor, and giving me an opportunity to finish my dissertation. I especially thank him for patiently answering all my questions regarding evolutionary and computational biology. I would like to thank Dr. Chrz for letting me use his lab, and for thought provoking conversations about microbial physiology. I am also thankful to Dr. Grover and Dr. Christensen for finding time amidst their busy schedule to guide me. Needless to mention, science is a collaborative endeavor and I would like to express my immense gratitude to all the erstwhile scientists who have worked hard to bring human knowledge to where it stands. I am humbled to have had the chance to be able to contribute to this collaborative effort. Completing my Ph. D. studies would have been very difficult without support form the Biology Department personnel including Linda, Gloria, Paulette, Sherri, Sufera, Kim, Melissa and Anya. I am very grateful to Jantiya, Lita, Nit, Noom, Fon, and Luis for helping me with my research and being excellent labmates. A special thanks to Wadud for everything, starting from letting me use his pipettes to listeining to my endless rants. Thanks are due to Shweta, Debanjana, Utpal, Catherine, Diwash, Elisa, Fabio, Cori and my Micro team for making my stay in the US even more memorable. III I would like to thank Rasika, Prashanth, Bhushan, Dhvani and Dhananjay for being my family in the US and helping me make great lifetime memories. A special thanks to Prashanth for long insightful conversations on everything science/non-science, and for pushing me to be a better person. Finally, I would like to thank my parents, sisters and Adwait for their unconditional love and support throughout my graduate studies. November 15, 2016 IV Abstract INSIGHTS INTO THE GENOMIC AND METABOLIC ADAPTATIONS OF TERMITE ASSOCIATED VERRCUCOMICROBIA STRAINS USING FUNCTIONAL AND COMPARATIVE GENOMICS Malini Kotak, Ph. D. University of Texas at Arlington, 2016 Supervising Professor: Dr. Jeff Demuth The symbiotic gut organisms of termites play an important role in lignocellulose digestion and other related metabolic processes. Previous studies on Diplosphaera colitermitum TAV2, a Verrucomicrobium isolated from the hindgut of Reticulitermes flavipes, revealed its microaerophilic nature and involvement in important ecophysiological processes in the termite gut. In the present study, a detailed account of genomic sequences of 3 additional strains is provided, and the genomes of all 5 TAV strains isolated so far are compared, to gain a comprehensive understanding of these organisms. Our comparative genomic study shows that these strains represent two distinct species, and despite significant number of genes being conserved across the genomes, synteny of genomes between species is lost. We found important ecological attributes like microaerophilic physiology and the ability to digest lignocellulose to be conserved at the genomic level in these strains, suggesting their common roles in O2 scavenging and lignocellulose digestion. However, high numbers of genes associated with functional category transcription were present in species-specific genomes, suggesting differential regulation of gene expression to be a potential primary feature distinguishing these otherwise V highly similar genomes. Our study identified signatures of adaptive variation in enzymes of the electron transport chain, which might be linked to microaerophilic physiology. Additionally, several membrane proteins/enzymes involved in recombination and repair, nitrogen metabolism and lignocellulose degradation were found undergoing adaptive evolution, reflecting the evolutionary mechanisms used by TAV strains to survive in the termite gut. Furthermore, the transcriptome of strain TAV2 under aerobic (20% O2) and hypoxic conditions (2% O2) was studied to understand its microaerophilic physiology. Putative CAZymes that degrade lignocellulose and other polysaccharides like pectin represent the majority of up-regulated genes. Genes associated with functional categories like oxidative stress protection, motility, amino acid metabolism, coenzyme metabolism were down-regulated. Additionally, changes in electron transport chain and putative oxygen sensing signal transducing proteins in the TAV2 strain were identified. In addition to corroborating our previous findings, these results reveal the metabolic adaptations used by TAV2 to survive in hypoxic conditions, and will serve as a foundation for future studies to understand the microaerophilic physiology of TAV strains. VI TABLE OF CONTENTS Acknowledgements ................................................................................................................. III Abstract ...................................................................................................................................... V List of Figures .......................................................................................................................... XI List of Tables ......................................................................................................................... XII Introduction................................................................................................................. 1 1.1 Termites ............................................................................................................................... 2 1.2 Lignocellulose structure ..................................................................................................... 2 1.3 Termite gut structure and digestive strategy ................................................................... 3 1.4 Symbionts and their lignocellulose-degrading activities in hindgut. ............................. 4 1.4.1 Cellulases ............................................................................................................................................................... 5 1.4.2 Hemicellulases .................................................................................................................................................... 5 1.4.3 Lignin modifying enzymes ............................................................................................................................. 6 1.5 Other activities in the hindgut ........................................................................................... 7 1.5.1 Fermentation ....................................................................................................................................................... 7 1.5.2 Reductive acetogenesis ................................................................................................................................... 7 1.5.3 Methanogenesis .................................................................................................................................................. 8 1.5.4 Nitrogen metabolism ........................................................................................................................................ 8 1.6 Effect of oxygen on the hindgut activity ........................................................................... 8 1.7 Termite Associated Verrucomicrobia (TAV) strains ........................................................ 9 1.7.1 Preliminary Genomic Characterization .................................................................................................. 10 1.7. 2 Transcriptomic and proteomic studies with strain TAV2 ............................................................. 11 1.7.3 Phylogenetic Characterization ................................................................................................................... 12 1.8 Aims of current investigation ........................................................................................... 12 1.9 References .......................................................................................................................... 14 Complete genome sequence of the Opitutaceae bacterium strain TAV5, a potential facultative methylotroph of the wood-feeding termite Reticulitermes flavipes. Error! Bookmark not defined. 2.1 Abstract .............................................................................................................................. 21 VII 2.2 Introduction ....................................................................................................................... 21 2.3 Nucleotide sequence accession number ........................................................................... 23 2.4 References .......................................................................................................................... 23 Draft genome sequences of strains TAV3 and TAV4 (Verrucomicrobial:
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