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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Aitor, Ml 48106-1346 USA 800-521-0600 UMI UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE SEQUENCE AND ANALYSIS OF ACTINOBACILL US ACTINOMYCETEMCOMITANS A Dissertation SUBMITTED TO THE GRADUATE FACULTY In partial fulfillment of the requirement for the Degree of Doctor of Philosophy By FARES ZOHIRNAJAR Norman, Oklahoma 2002 UMI Number; 3062578 UMI UMI Microform 3062578 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. 00x1346 Ann Arbor, Ml 48106-1346 © Copyright by FARES ZOHIR NAJAR 2002 All Rights Reserved SEQUENCE AND ANALYSIS OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS A Dissertation APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY Acknowledgments I would like to thank my advisory committee, Drs. Bruce Roe, Phillip Klebba, Arm West, Richard Taylor, and John Downard for their support of my efforts to complete my dissertation work. A great deal of gratidute goes to Dr. Paul Cook for all the time and help he provided to help me understand metabolism. A very special thanks and my gratitude goes to Dr. Bruce Roe, my major professor, for his infinite support, encouragement, guidance, and patience throughout my graduate career. I am also thankful for every member of Dr. Roe’s laboratory at the Advanced Center for Genome Technology for their help and support they provided including the wonderful administrative staff, the gel room crew and the fellow graduate students. A special thanks goes to Dr. Doris Kupfer for her help, support, and invigorating scientific discussions beyond the scope of this dissertation, and to Hongshing Lai who’s computer expertise was instrumental and pivotal in providing the annotation needed for the analysis of A. actinomycetemcomitans genes. To Jim White, who wrote and developed various scripts that helped automate and speedup analysis of the massive amount of data generated and for showing me that PERL is the salvation. My thanks also to Steve Kenton who provided an enormous help and insight to the sequence information of A. actinomycetemcomitans genome. To Douglas White for providing me with much computer advice that helped in my final data presentation. And I would like to thank Shaoping Lin for her wonderful insights to the world of PCR. I also would like to thank Dr. Sandra Clifton for the training she provided when I first joined the lab. I would like to thank my family for their love, support, and advise. I simply cannot imagine this work without them on my side. My parents, for constantly pushing me to pursue my higher education. As always, I am forever thankful for them. To my sister, Ranya, who pretended to listen to me while explaining my research to her. To Bassel, my brother, my friend, and my confidant for being there when I needed him. To them I dedicate this dissertation. IV Table of Content List of Tables vil List of Figures ix Abstract xiii Chapter I: Introduction 1 1.1 Background I 1.1.1 DNA and Genes 1 1.1.2 Organization of Prokaryotic Genomes 4 1.1.3 Actinobacillus actinomycetemcomitans and Virulence 5 1.2 Sequencing Strategy 12 1.2.1 Brief History of Sequencing 12 1.2.2 Shotgun Phase of DNA sequencing 17 1.2.3 Sequence Assembly - Phred, Phrap, and Consed 19 1.2.4 Closure Strategies 20 1.2.5 Analysis and Annotation of a DNA sequence 26 Chapter II: Materials and Methods 34 2.1 Actinobacillus actinomycetemcomitans subclone library construction and shotgun sequencing 34 2.1.1. Nebulization 34 2.1.4 Subcloning the fragments and transformation 37 2.1.5 Semi-automated isolation of subclone template DNA for sequencing 39 2.1.7 Removal of unincorporated terminators 42 2.1.8 Sequencing 43 2.2 Gap Closure Phase of Sequencing 46 2.2.1 Primer-walking. Large-insert clones 46 2.2.2 Multiplex polymerase chain reaction (MPCR) 49 2.3 Computer methods and data analysis 51 2.3.1 Sequence analysis 51 Chapter III: Results and Discussion 63 3.1 Sequence statistics and quality 63 3.2 Genome overview 66 3.3 Actinobacillus actinomycetemcomitans metabolism 76 3.3.1 Energy 77 3.3.2 Metabolism of Lipids 88 3.3.3 Amino acid Biosynthesis 91 3.3.4 Nucleotide Metabolism 104 3.3.5 Biosynthesis of Cofactors and Vitamins 111 3.3.6 Macromolecule Metabolism 129 3.3.7 Cell Wall 152 3.3.8 Transport proteins 157 3.3.9 Protein Export and Secretion 162 3.4 Virulence Factors 165 3.4.1 Iron acquisition and utilization 166 3.4.2 Lipopolysacharides and Phosphorylcholine 170 3.4.3 Adhesion factors 171 3.4.4 Invasion 175 3.4.5 Toxins 180 3.4.6 Heat Shock Proteins 183 3.4.7 Proteases 187 3.4.8. Antibiotic Resistance 190 Chapter IV: Conclusion 191 Chapter V: References 191 Appendix-A: A. actionomycetemcomitans ORFs. 192 Appendix-B: Regions of significantly different GC content in A. actinomycetemcomitans. 271 VI List of Tables Table 1.1. Different forms of DNA. 2 Table 1.2. Known virulence factors inact/>Jom>'ce/emcoOT//flnj. 7 Table 1.3. General characteristics of prokaryotic signal peptides. 30 Table 3.1. Sequencing statistics of ^4. acr/>io/n>'ce/emcoff»//a/jj. 64 Table 3.2. Enzymes involved in glycolysis. 78 Table 3.3. Pentosephosphate pathway. 80 Table 3.4. Enzymes involved in anaerobic pyruvate metabolism. 80 Table 3.5. Enzymes involved in aerobic pyruvate metabolism. 81 Table 3.6. Enzymes involved in citric acid cycle. 82 Table 3.7. Fermentation. 85 Table 3.8. Fatty acid biosynthesis . 89 Table 3.9. Phospholipid biosynthesis. 91 Table 3.10. Amino acid synthesis. 92 Table 3.11. Branched-chain amino acids biosynthsis. 95 Table 3.12. Biosynthesis of lysine, threonine, and methionine. 97 Table 3.13. Biosynthesis of serine, glycine and cysteine. 98 Table 3.14. Biosynthesis of aromatic amino acid biosynthesis. 100 Table 3.15. Biosynthesis of arginine and proline. 101 Table 3.16. Biosynthesis of histidine. 103 Table 3.17. Biosynthesis of pyrimidines. 107 Table 3.18. Biosynthesis of purines. 110 Table 3.19. Biosynthesis of riboflavin. 113 Table 3.20. Biosynthesis of folate. 114 Table 3.21. Transport of thiamine. 117 Table 3.22. Biosynthesis of pantothenate. 121 Table 3.23. Biosynthesis of biotin. 122 Table 3.24. Biosynthesis of NAD. 128 Table 3.25. Restriction-Modification genes. 132 Table 3.26. Proteins involved in DNA repair. 139 vu Table 3.27. Proteins involved in transcription. 140 Table 3.28. RNA modification enzymes. 144 Table 3.29. Proteins involves in protein synthesis. 150 Table 3.30. List of Proteases. 151 Table 3.31. First stage of peptidoglycan synthesis. 152 Table 3.32. Enzymes involved in stages 2 and 3 of peptidoglycan synthesis. 153 Table 3.33. Biosynthesis of Lipid A and KDO. 157 Table 3.34. Transport proteins. 158 Table 3.35. Cation and anion transporters. 159 Table 3.36. The PTS system in A. actinomycetemcomitans. 161 Table 3.37. Biosynthesis of heme. 169 Table 3.38. The dTDP rhamanose pathways. 179 Table 3.39. Heat shock protein genes. 187 Table 3.40. Putative virulence factors. 190 vni List of figures Figure 1.1. The structure of DNA. 3 Figure 1.2. The central dogma. 4 Figure 1.3. 5,7-dichloro rhodamine (d-rhodamine) terminators used for sequencing. 15 Figure 1.4. Bigdye terminator (attached to thiamine). 16 Figure 1.5. The nebulizer. 18 Figure 1.6. Repetitive elements and misassembly. 21 Figure 1.7. Mpcr and gap closure. 24 Figure 1.8. The structure of 7-deaza-dGTP. 25 Figure 1.9. Overview of sequence analysis. 32 Figure 2.1. Agarose image of nebulized genomic DNA. 37 Figure 2.2. Solid-phase oligonucleotide synthesis. 48 Figure 2.3. An illustration of protected nucleoside-3'-phosphoramidites. 49 Figure 2.4. The main window of Artemis. 53 Figure 2.5. Displaying Blast output of marked ORFs in Artemis. 54 Figure 2.6. Text file of the metabolic schema. 55 Figure 2.7. Output of the keyword search script get EC. 56 Figure 2.8. Output of MOTIF and COGnitor programs. 58 Figure 2.9. Excel file generated ORFs information. 59 Figure 2.10. Signal? output. 60 Figure 2.11. The typical output file form tRNAscan. 61 Figure 2.12. Summary of the analysis and annotation process used for A. actinomycetemcomitans. 62 Figure 3.1. Contigs distribution. 65 Figure 3.2. Metabolic overview. 66 Figure 3.3. Homology profile of A. actinomycetemcomitans. 67 Figure 3.4. Codon usage in A. actinomycetemcomitans. 68 Figure 3.5. Codon usage of both A. actinomycetemcomitans and H. influenzae. 69 IX « Figure 3.6.
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