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Dissertation V3 MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Ryan Ford Relich Candidate for the Degree: Doctor of Philosophy Dr. Mitchell F. Balish, Director Dr. Kelly Z. Abshire, Reader Dr. Joseph M. Carlin, Reader Dr. Gary R. Janssen Dr. John Z. Kiss, Graduate School Representative ABSTRACT GLIDING MOTILITY MECHANISMS IN DIVERGENT MYCOPLASMA SPECIES by Ryan Ford Relich Bacteria belonging to the Mycoplasma pneumoniae phylogenetic cluster possess polarity that is conferred by a differentiated tip structure called the attachment organelle. Among the species comprising this cluster, all but one have been experimentally demonstrated to exhibit a contact-dependent form of motility categorized as gliding, a process that is mediated by the attachment organelle. The subcelluar structures within the attachment organelle are conserved in all of these species; however, the morphology and gliding speed of each are distinct. The reasons for these phenotypic disparities are unknown, but we propose that an adhesin common to all of these species, called P30 in M. pneumoniae, contributes many of the species-specific differences, and the concentration of this protein at the attachment organelle tip dictates gliding speed. To test our hypotheses, we examined several phenotypes of an M. pneumoniae P30 null mutant, II-3, expressing a P30 ortholog, P32, from the closely related species Mycoplasma genitalium, which is phenotypically distinct from M. pneumoniae. Although these experiments did not identify a role for P30 in species-specific phenotypes, P32 was demonstrated to be a functional surrogate for P30 in M. pneumoniae. These data also comprise the first report of successful orthologous gene replacement in mycoplasmas, a technique that is potentially amenable for the study of other aspects of mycoplasma biology. We next examined phenotypes of M. pneumoniae II-3 cells expressing native P30 under the control of the M. pneumoniae ldh promoter, which gave rise to several transformant strains expressing variable low levels of P30. These data indicated a positive correlation between the concentration of P30 and the speed at which cells glide. We also used techniques for the analysis of M. pneumoniae and its relatives to examine the rod-shaped mycoplasma, Mycoplasma insons. We were able to characterize a novel cytoskeleton and gliding motility in this species, although, we were not able to define the bases for polarity or motility generation. Overall, the work described herein provides insight into the biology of mycoplasmas and their motility, as well as description of novel experimental approaches for studying these unique microorganisms. GLIDING MOTILITY MECHANISMS IN DIVERGENT MYCOPLASMA SPECIES A DISSERTATION Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Ryan Ford Relich Miami University Oxford, Ohio 2011 Dissertation Director: Dr. Mitchell F. Balish Table of Contents Page INTRODUCTION………………………………………………….………...... 1 A. Description of the genus Mycoplasma……………………………… 2 B. Mycoplasma species as agents of human and animal disease……… 3 C. Comparisons of bacterial motility with emphasis on gliding motility in Mycoplasma species……………………………. 4 D. The composition of the attachment organelle of Mycoplasma pneumoniae phylogenetic members…………………. 14 E. The importance of studying motility in M. pneumoniae and other motile species…………………………………………… 16 F. Hypotheses………………………………………………………… 17 CHAPTER 1: Insights into the Function of Mycoplasma pneumoniae Protein P30 from Orthologous Gene Replacement. Summary..…………………………………………………………….. 20 Introduction…………………………………………………………… 21 Materials and Methods………………………………………………... 24 Results………………………………………………………………… 28 Discussion…………………………………………………………….. 45 CHAPTER 2: Gliding Speed Positively Correlates with the Amount of the Attachment Organelle Protein P30 in Mycoplasma pneumoniae. Summary..…………………………………………………………….. 50 Introduction…………………………………………………………… 51 Materials and Methods……………………………………………....... 54 Results………………………………………………………………… 59 Discussion…………………………………………………………….. 73 ii CHAPTER 3: Novel Cellular Organization in a Gliding Mycoplasma, Mycoplasma insons. Summary..……………………………………………………………. 78 Introduction…………………………………………………………... 79 Materials and Methods……………………………………………….. 81 Results………………………………………………………………... 82 Discussion……………………………………………………………. 90 APPENDIX A: Transformation of Mycoplasma insons with a P30GFP Construct in an Attempt to Define Polarity………………………………. 92 CONCLUDING REMARKS and FUTURE DIRECTIONS……………. 100 REFERENCES……………………………………………………………… 106 iii List of Tables Page Table 1 Bacterial strains, plasmids and primers used in this study. 31 Table 2 Gliding motility parameters of wild-type and transformant 32 strains. iv List of figures Page Figure 1 Scanning electron micrographs of two attachment organelle- 7 possessing mycoplasmas, M. pneumoniae (left) and its closest genetic relative, M. genitalium (right). Figure 2 Schematic of Mycoplasma mobile gliding machinery. 9 Figure 3 Tree of the Mycoplasma pneumoniae phylogenetic cluster 12 based on 16S rRNA gene sequence analysis. Figure 4 Comparison of P30 orthologs from M. pneumoniae strain 33 M129 and M. genitalium strain G37. Figure 5 Constructs generated for this study. 35 Figure 6 Immunoblot confirmation that the 6X-His antibody does 37 not cross react with any proteins in non-transformed M. pneumoniae. Figure 7 Immunoblot analysis for the demonstration of P30, P30His, 39 P32His, and P65. Figure 8 Morphology of strains used in this assay. 41 Figure 9 P30His and P32His localize to the attachment organelle tip, 43 the site of localization of native P30. Figure 10 Immunoblot analysis of P30His in the transformant 63 M. pneumoniae 36-D grown in the presence of 1% added glycerol (+) or no added glycerol (-). v Figure 11 Hemadsorption analysis of wild-type M. pneumoniae strain 65 M129, P30 null mutant II-3, and the 36-series transformants. Figure 12 Immunolocalization of P30His in transformant M. pneumoniae 67 36-C, a representative for all other M. pneumoniae 36-series transformants. Figure 13 Immunoblot analysis of various attachment organelle proteins 69 in the M. pneumoniae 36-series transformants. Figure 14 Scanning electron microscopy of M. pneumoniae 36-series 71 transformants expressing different low levels of P30. Figure 15 Scanning electron micrographs of whole cells of M. insons 84 attached to a glass coverslip. Figure 16 Consecutive phase-contrast images of M. insons in a chamber 86 slide at ~5-s intervals at 37°C. Figure 17 Scanning electron micrographs of M. insons attached to glass 88 coverslips and extracted with TX. Figure A1 Detection of MPN453 fragment in Mycoplasma insons 39-series 96 transformants using PCR. Figure A2 Absence of P30 expression in the Mycoplasma insons 39-series 98 transformants. vi I dedicate this work to all of the people who have positively impacted my life; because of your encouraging words and actions, I have been able to follow my dreams. vii ACKNOWLEDGMENTS Graduate school has been one of most rewarding, yet trying, times of my life. The success that I have enjoyed so far would not be possible without the endless encouragement, love, and kindness that I have received from so many wonderful people. I am eternally grateful and forever indebted to each and every one of these individuals. I would first like to thank my advisor, Dr. Mitchell Balish, for his unending support, incalculable contributions of knowledge and expertise, his witty sense of humor that has brightened so many days, and his friendship. I will always cherish our many scientific and non-scientific conversations, some of which ended in “ah-ha” moments, but most others in fits of laughter. To that end, I hope that my future scientific career affords me the chance to work with you again, Mitch. I also thank the other Dr. Balish for her friendship; your kind and down-to-earth personality is always refreshing and was needed on many occasions. To my lab siblings Dominika Jurkovic, Jennifer Hatchel, Rachel Pritchard, and Steven Distelhorst, a special place in my heart will always be there for each of you. The four of you have helped me through so much and I can’t imagine what the last six years of my life would have been like without you in it. I hope that our friendships last a lifetime. To the many Balish lab undergraduate students, specifically Jordan Norton, Aaron Friedberg, Ryan Brady, Christine Whalin, Chelsea Yanda, Kendall Sibbing, and Jack McChesney, I thank you for the many opportunities that you have given me to share my love of microbiology and for challenging me to be a better mentor and scientist. To all of my fellow graduate students in the Department of Microbiology at Miami, I wish you the best of luck and I deeply thank you for all that you have done for me. Thanks to Barb Stahl and Darlene Davidson for all of your help and entertaining conversations. I would like to thank Bill Penwell and his beautiful wife Mary, Jason Clark, Jason Hayes, Jianli Xue, Racheal Desmone, Jackie Giliberti, Jay Brock, Jennifer Seabaugh, Jennifer Gaddy and her lovely family, Shomita Mathew, Karthik Krishnan, Daniel Jung, and many, many other graduate students for your friendship, support, and for the knowledge and passion for science that you’ve each shared with me. viii I would like to thank my dissertation committee, Dr. Kelly Abshire, Dr. Joe Carlin, Dr. Gary Janssen, and Dr. John Kiss for
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