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Viewed and Captured at the Electron Microscopy Facility at Miami University on a Zeiss Supra 35 FEG-VP Scanning Electron Microscope Operating at 5 Kv MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Jennifer Marie Hatchel Candidate for the Degree: Doctor of Philosophy ________________________________________________________________________ Dr. Mitchell Balish, Director ________________________________________________________________________ Dr. Eileen Bridge, Reader ________________________________________________________________________ Dr. Kelly Abshire, Reader ________________________________________________________________________ Dr. Xiao-Wen Cheng ________________________________________________________________________ Dr. Richard Moore, Graduate School Representative ABSTRACT STRUCTURE AND FUNCTION OF THE ELECTRON-DENSE CORE IN MYCOPLASMA PNEUMONIAE AND ITS RELATIVES By Jennifer Hatchel Among the mycoplasmas, the human pathogen Mycoplasma pneumoniae is the best- characterized at the cellular level. It has a polar structure, the attachment organelle, which mediates adherence to host cells, is the leading end during gliding motility, and plays a role during cell division. Within the attachment organelle is a detergent-insoluble electron-dense core composed of several proteins, some of which have been identified through the study of cytadherence deficient mutants of M. pneumoniae. Mutants lacking proteins in the electron-dense core are avirulent, suggesting that the core is essential for the proper formation of the attachment organelle, which in turn is essential for virulence. We used scanning electron microscopy (SEM) and time-lapse microcinematography to test the relationship between ultrastructure and gliding motility in M. pneumoniae and some of its close phylogenetic relatives, which vary in ultrastructure, gliding characteristics, host range, and pathogenic potential. Our results show that Mycoplasma amphoriforme, a novel species found in the respiratory tract that is possibly pathogenic to humans, is motile and shares morphological characteristics with its closest relatives, M. pneumoniae and the avian pathogen, Mycoplasma gallisepticum. Using SEM and time- lapse microcinematography, we find that the morphology of seven species of the M. pneumoniae cluster correlates with phylogeny rather than with gliding motility characteristics. We also find that in most species the electron-dense cores have fibers and filaments that remain attached to the base of the core after detergent treatment, but disappear after treatment with DNase, suggesting that they are DNA. It has been hypothesized that the electron-dense core plays a role during cell division, which might utilize protein-DNA interactions between the core and the chromosome. Using fluorescence in situ hybridization (FISH) coupled to immunofluorescence, we attempted to further investigate specific interactions between the oriC region of the chromosome and the electron-dense core in M. pneumoniae. The data suggest that a variable region of the chromosome associates with the base of the electron-dense core, although the FISH protocol still needs optimization. Overall, my work suggests that gliding motility has a major role in the partitioning of the chromosomes in cell division and a minor role in pathogenicity. STRUCTURE AND FUNCTION OF THE ELECTRON-DENSE CORE IN MYCOPLASMA PNEUMONIAE AND ITS RELATIVES 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 Jennifer Marie Hatchel Miami University Oxford, Ohio 2009 Dissertation Director: Dr. Mitchell Balish Table of Contents Page INTRODUCTION………………………………………………… 1 A. Significance of studying M. pneumoniae and its relatives……... 1 B. Phylogeny and cell morphology of M. pneumoniae and relatives. 3 C. Attachment organelle proteins…………………………………... 7 D. Gliding motility and division …………………………………… 12 E. Cell division requires the duplication and migration of the attachment organelle…………………………………………….. 14 F. Hypotheses ………………………………………………………. 15 Chapter 1: Ultrastructure and gliding motility of Mycoplasma amphoriforme, a possible human respiratory pathogen……….. 16 Abstract……………………………………………………………. 17 Introduction………………………………………………………... 18 Materials and Methods…………………………………………….. 20 Results……………………………………………………………... 22 Discussion…………………………………………………………. 39 Chapter 2: Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives…………………………………………………………… 44 Abstract……………………………………………………………. 45 Introduction………………………………………………………... 46 Materials and Methods…………………………………………….. 49 Results……………………………………………………………... 51 Discussion…………………………………………………………. 66 ii Chapter 3: Fluorescence in situ hybridization, as a possible method for characterizing the interaction between the electron-dense core and the chromosome in Mycoplasma pneumoniae..................................... 72 Abstract……………………………………………………………. 73 Introduction………………………………………………………... 74 Materials and Methods…………………………………………….. 78 Results……………………………………………………………… 80 Discussion…………………………………………………………. 85 SUMMARY AND CONCLUDING REMARKS……………….. 90 REFERENCES…………………………………………………… 100 iii List of Tables Page Table 1 Dimensions of the cytoskeleton-like structure of M. 29 amphoriforme and M. pneumoniae Table 2 Gliding motility parameters. (Chapter 1) 36 Table 3 Gliding motility parameters. (Chapter 2) 54 Table 4 Whole cell dimensions (in nm) ± SD, mean of 30 cells. 57 Table 5 Electron-dense core dimensions (in nm) ± SD, mean of 65 30 cells. iv List of figures Page Figure 1 Phylogenetic tree of the Mollicutes and some walled 4 relatives based on 16S rRNA sequences Figure 2 Schematic drawing of the adhesins and the electron- 9 dense core in M. pneumoniae Figure 3 Scanning electron micrographs of mycoplasma cells 23 grown attached to coverslips. Figure 4 Scanning electron micrographs of presumptively 25 dividing M. amphoriforme cells grown attached to coverslips. Figure 5 Scanning electron micrographs of mycoplasma 27 cytoskeletal structures. Figure 6 Consecutive phase-contrast images of M. amphoriforme 31 gliding motility at 5-s intervals at 37°C. Figure 7 Measurement of M. amphoriforme gliding speed by 34 time-lapse microcinematographic analysis. Figure 8 Distribution of mycoplasma velocities about the mean. 37 Figure 9 Phylogenetic tree based on 16S rRNA sequences of 52 mycoplasmas within the M. pneumoniae cluster. v Figure 10 Scanning electron micrographs of mycoplasma cells 55 grown on glass coverslips. Figure 11 Scanning electron micrographs of mycoplasma cells 59 grown on glass coverslips. Figure 12 Scanning electron micrographs of mycoplasma 61 electron-dense cores. Figure 13 Schematic of a typical M. amphoriforme cell. 69 Figure 14 Schematic representation of expected results in a M. 81 pneumoniae cell after FISH with a Cy3-labeled probe and immunofluorescence against the attachment organelle protein, HMW1. Figure 15 FISH using probes against the 16S rRNA gene and the 83 oriC sequences in M. pneumoniae. Figure 16 Comparison of FISH in Caulobacter crescentus and 87 Mycoplasma pneumoniae. vi Acknowledgments The first person I would like to thank is my advisor, Dr. Mitchell Balish. Thank you for your guidance, patience, and support over the last five years. I have learned so much during my time here at Miami, and I am grateful to have had the opportunity to work in your lab. My committee members, Dr. Kelly Abshire, Dr. Eileen Bridge, Dr. Xiao-Wen Cheng, and Dr. Richard Moore have been extremely helpful during this process as well. Thank you for all of your suggestions, for always challenging me to be a better scientist, and for making me think critically about my experiments. Thank you to my lab mates, Dominika and Ryan. I cannot thank you enough for being who you are because it has shaped who I have become. Thank you for being there when I needed someone to talk to, especially at the end. Rachel, even though we didn’t work together in the lab for long, I want to thank you for being my friend. Thanks for encouraging me to be a better person. I wish you well on your journey to completing your degree. Thanks also to Jason, Jason, Jackie, Jianli, Karthik, and Rachael, for making my time here more enjoyable. Thank you to Barb and Darlene for making it easier to work here. I appreciate all the help when ordering items or when the copy machine didn’t like me. Thanks to the rest of the department for being supportive and willing to help. I am glad to have known all of you. To my friends, both here in Ohio and back home in Tennessee, thanks for the support and encouragement. Sara, Matt, Paige, Amanda, Cameron, Lynne, Becky, Dennis, and Frank, thank you for not only being my neighbors, but also for being people I could count on in times of need. I will miss living in our building. I also want to thank Dr. Becky Balish, Annika, and Ian for all that you have done for me. Thanks also to Tommie, Maggie, and Connor for entertaining Alexis while I was at school. I couldn’t have finished without you. THANKS!! Last but not least, I want to thank my family. Those of you in Tennessee, thank you for understanding my decision to go to school in Ohio even though it meant that sometimes I had to be in the lab over holidays instead of being there with you. Thank you for believing in me. Finally, Mark and Alexis, you saw me at the highest of highs and lowest of lows. Thank you for having patience with me and encouraging me through it. I love you!! vii INTRODUCTION The bacterial genus Mycoplasma contains
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