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Evidence of Mobility in the 3-Chlorobenzoate Degradative

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Evidence of Mobility of the 3-Chlorobenzoate Degradative Genes in a Pristine Soil Isolate, Burkholderia phytofirmans OLGA172 by Soulbee Jin A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology University of Toronto © Copyright by Soulbee Jin, 2010. Evidence of Mobility of the 3-Chlorbenzoate Degradative Genes in a Pristine Soil Isolate, Burkholderia phytofirmans OLGA172 Soulbee Jin Master of Science Ecology and Evolutionary Biology University of Toronto 2010 Abstract The genome of B. phytofirmans OLGA172 has been sequenced by Next Generation sequencing methods, Illumina Solexa and Genome Analyzer and Roche 454 GS-FLX. Through various bioinformatic and molecular work, over 42 kbp of its genome surrounding its 3CBA degradative genes was assembled and annotated. The most important method used here was the synteny method, which implies homology between the genes, and descent from a common ancestor (Guttman, 2008). The conserved gene order between B. phytofirmans PsJN, B. xenovorans LB400, and OLGA172 was used as a confirmation of annotation through BLASTn, enabled closing of the gaps in NextGen sequencing data, and allowed prediction of genes further downstream. Though the whole genome may not have been assembled, a very significant region that carries a concentrated area of mobile genetic elements (MGE) has been found to surround the degradative genes, tfdC IDIEIFI in OLGA172. This thesis details the sequence evidence that, upon examination of closely related strains, OLGA172 and its related strain from pristine soils may be the ancestral chlorobenzoate degraders. ii Acknowledgments I would like to thank Nicole Ricker (Ph.D. candidate), Jackie Goordial, Cindy Bongard, and especially Dr. Roberta Fulthorpe for all their effort and time. I really could not have asked for a better supervisor over the years of my research at U of T. She was always there to answer my countless emails, phone calls, and any small questions I had along the way. She was always available when I needed her, not only for the research advice, but also for personal issues, always providing me with thoughtful advice and guidance. She has become more of a friend to me and I would really like to thank her for that. Jackie, Nicole, and Cindy helped me so much in clarifying many issues surrounding this thesis as well as a great source of moral support. They have helped me keep composed and calm throughout the stress and anxiety that comes with fast approaching deadlines. I would like to thank each one of them and as it would have been very difficult to complete this project without them. All of their input and comments has made a positive impact on me, and it is really great to have such good friends as my lab mates. Lastly, I would like to thank all of my family and friends for all their support, as well as Second Cup for providing me with great coffee and a nice place to write. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vii List of Figures .............................................................................................................................. viii List of Abbreviations and Acronyms .............................................................................................. x List of Appendices ......................................................................................................................... xi Chapter 1 ......................................................................................................................................... 1 1 Introduction to B. phytofirmans OLGA172 and General Literature Review. ............................ 1 1.1 Background information ..................................................................................................... 1 1.2 Mobile Genetic Elements .................................................................................................... 4 1.3 Chloro-aromatic degraders from contaminated/industrial sites .......................................... 9 1.4 Spontaneous phenotypic instability in other strains .......................................................... 11 1.5 Hypotheses and Structure of the Thesis ............................................................................ 12 Chapter 2 ....................................................................................................................................... 13 2 Next Generation Sequencing .................................................................................................... 13 2.1 Methods ............................................................................................................................. 15 2.1.1 Purification and DNA extraction of B. phytofirmans OLGA172.......................... 15 2.1.2 Confirming the identity and purity of OLGA172 DNA ........................................ 16 2.1.3 Next Generation Sequencing and preliminary assembly ...................................... 18 2.1.4 GC content and overall Genome Matches ............................................................. 19 2.1.5 Multiple sequence alignment and phylogenetic trees ........................................... 21 2.2 Results ............................................................................................................................... 21 2.2.1 Isolation and confirmation of OLGA172 .............................................................. 21 iv 2.2.2 Total genome homology and GC content and analysis ......................................... 22 2.2.3 Summary of Data Generated by Solexa Genome Analyzer and 454 GS-FLX ..... 24 2.2.4 There are no evidence of plasmids in OLGA172 .................................................. 25 2.2.5 Gene annotation ..................................................................................................... 26 2.2.6 Sequence analysis of the integrases ...................................................................... 28 2.3 Discussion ......................................................................................................................... 31 2.3.1 Advantages and Disadvantages of NextGen Sequencing ..................................... 31 2.3.2 Possible HGT of the tfd catabolic operon in OLGA172 ....................................... 34 Chapter 3 ....................................................................................................................................... 38 3 Extension of regions flanking the catabolic operon ................................................................. 38 3.1 Methods ............................................................................................................................. 40 3.1.1 Sequence linkage via PCR based on synteny analysis .......................................... 40 3.1.2 Linkage of sequences via Thermal Asymmetric InterLaced PCR (TAIL PCR) ... 43 3.1.3 Secondary structure analysis and GC content calculation .................................... 45 3.2 Results ............................................................................................................................... 46 3.2.1 OLGA172 shares conserved gene order with PsJN and LB400 ........................... 46 3.2.2 TAIL PCR ............................................................................................................. 49 3.2.3 Sequence extension beyond RIT BphO1 ................................................................ 50 3.2.4 Secondary structure analysis and GC content calculation .................................... 51 3.3 Discussion ......................................................................................................................... 53 3.3.1 Shared homology between OLGA, PsJN, and LB400 .......................................... 53 3.3.2 Confirmation of “junkyard” presence in OLGA172 ............................................. 54 3.3.3 DNA sequencing interrupted by secondary structure ........................................... 55 Chapter 4 ....................................................................................................................................... 57 4 Analysis of OLGA172 and related strains from pristine soils. ................................................ 57 v 4.1 Methods ............................................................................................................................. 57 4.1.1 The ability of OLGA172 and its related strains from pristine soils to break down and grow in the presence of CBA ............................................................... 57 4.1.2 Lysis of cells and amplification of CBA degradative genes ................................. 58 4.1.3 Phylogenetic analysis ............................................................................................ 61 4.2 Results ............................................................................................................................... 61 4.2.1 Growth of OLGA172 and its related pristine isolates on CBA and their possession of degradative genes ...........................................................................
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