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The Characterization of PG0228 in Porphyromonas Gingivalis W83 Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2011 The Characterization of PG0228 in Porphyromonas gingivalis W83 Courtney Schlenker Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Physiology Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/219 This Thesis is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. THE CHARACTERIZATION OF PG0228 IN PORPHYROMONAS GINGIVALIS W83 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Physiology and Biophysics at Virginia Commonwealth University. by COURTNEY DANIELLE SCHLENKER Bachelor of Science in Biology, University of Toledo, 2007 Director: JANINA P. LEWIS, PH.D. ASSOCIATE PROFESSOR, PHILIPS INSTITUTE OF OCMB SCHOOL OF DENTISTRY Virginia Commonwealth University Richmond, Virginia May, 2011 ii ACKNOWLEDGEMENTS Most importantly, I would like to thank Dr. Lewis for this exciting project and giving me the opportunity to work in her lab. Her mentorship and constant support have made pursuing a Masters degree a challenging yet rewarding process. No matter how busy she was, she always managed to find the time to address any concerns or questions I might have had. In addition to Dr. Lewis, I would like to thank the other members of my committee, Dr. Rife and Dr. Qiao, for their insight and guidance on this project. A special thank you goes to Cecilia Anaya-Bergman for her mentorship during my research rotation last year. I would especially like to thank Sai Yanamandra, Dr. Karl Thompson, and Dr. Tiana Wyant for their time, patience, guidance, and advice this past year and a half. Finally, I must thank the rest of the lab including Kandice Klepper, Anuya Paranjape, Tyrone King, Evys Collazo, Cheyanne Warren, and Huan Mo for their support and friendship. Lastly, I would like to thank my parents and family for giving me the unconditional love, support, and confidence to go out and chase my dreams. You have been there to guide me through life’s many roadblocks, and through your example, you have shown me the power of hard work and dedication. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ...............................................................................................................ii LIST OF TABLES .............................................................................................................................v LIST OF FIGURES ..........................................................................................................................vi LIST OF ABBREVIATIONS ............................................................................................................viii ABSTRACT .......................................................................................................................................x INTRODUCTION .............................................................................................................................1 1.1. Periodontal Disease ................................................................................................1 1.2. Oral Microflora .......................................................................................................4 1.3. Porphyromonas gingivalis ......................................................................................5 1.4. Gene Regulation in Porphyromonas gingivalis ......................................................6 1.5. Regulatory small RNAs (sRNAs) ...........................................................................7 1.6. Sm Family of RNA-Associated Proteins ................................................................9 1.7. Hfq Protein ..............................................................................................................10 1. MATERIALS AND METHODS .......................................................................................................21 2.1. Project Aims............................................................................................................21 2.2. Bacterial Strains and Plasmids ................................................................................25 2.3. Growth Conditions and Media ................................................................................25 2.4. Generation of P. gingivalis Mutant Strains.............................................................29 2.5. Generation of Δ0228 Mutant Porphyromonas gingivalis Strain ............................29 2.6. Generation of 0228-HaLo Porphyromonas gingivalis Strain .................................35 2.7. Growth Curve of Δ0228 Mutant .............................................................................39 2.8. Microarray Analysis of Δ0228 Mutant ...................................................................39 2.9. Survival of Δ0228 Mutant Strain with Eukaryotic Cells ........................................45 2.10. Immunoprecipitation with 0228-HaLo Tagged Strain ............................................46 2.11. Bioinformatics.........................................................................................................51 RESULTS .........................................................................................................................................52 3.1. Sequence Alignment with PG0228 .........................................................................52 3.2. Generation of Δ0228 Mutant Strain ........................................................................55 3.3. Generation of 0228-HaLo tagged Porphyromonas gingivalis Strain .....................62 3.4. Evaluation of Δ0228 Growth Compared to Wild Type W83 .................................71 3.5. Differential Expression Using Microarray Analysis...............................................74 3.6. Analysis of Δ0228’s Ability to Survive with Host Cells ........................................83 DISCUSSION ...................................................................................................................................88 iv 4.1. Δ0228 Strain ...........................................................................................................88 4.2. Differential Gene Expression ..................................................................................90 4.3. RNA Binding to PG0228 ........................................................................................94 4.4. Evaluation of Phenotypic Changes in Δ0228 .........................................................96 4.5. Alternate Hypothesis ...............................................................................................98 4.6. Future Studies .........................................................................................................99 LITERATURE CITED .....................................................................................................................101 VITA ..................................................................................................................................................109 v LIST OF TABLES Table 1: Primer Sequences ................................................................................................................28 Table 2: Optical Density of Δ0228 Compared to W83 ....................................................................72 Table 3: Upregulated Genes Detected in Δ0228 Strain ...................................................................76 Table 4: Downregulated Genes Detected in Δ0228 Strain ...............................................................77 Table 5: Transcription Profile of nusA .............................................................................................80 Table 6: Transcription Profile of luxR ..............................................................................................81 Table 7: Transcription Profile of luxS ..............................................................................................82 Table 8: Colonies Found on the Blood Agar Plates after HN4 Invasion .........................................86 vi LIST OF FIGURES Figure 1: Periodontal Diseases .........................................................................................................3 Figure 2: Examples of Hfq Regulation in E. coli .............................................................................13 Figure 3: Structure Summary of Hfq from Pseudomonas aeruginosa .............................................16 Fig. 4a: Presence or Absence of an hfq gene in Completed Genomes of Bac. Pathogens .............19 Fig. 4b: Summary of Phenotypes of hfq Mutants of Pathogenic Bacteria ......................................20 Figure 5: Project Aims ......................................................................................................................24 Figure 6: pCR2.1 TOPO Vector .......................................................................................................26 Figure 7: pFC20K (HaLoTag 7) T7 SP6 Flexi Vector ....................................................................27 Figure 8: Schematic Diagram of Δ0228 Strain Generation .............................................................34 Figure 9: Schematic Diagram of 0228-HaLo Strain Generation .....................................................38
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