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Viewed on a Zeiss Supra 35 FEG-VP Scanning Electron Microscope MIAMI UNIVERSITY The Graduate School CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Dominika Angelika Jurkovic Candidate for the Degree: Doctor of Philosophy ______________________________________________ Dr. Mitchell F. Balish, Director ______________________________________________ Dr. Eileen Bridge, Reader ______________________________________________ Dr. Gary R. Janssen, Reader _____________________________________________ Dr. Luis Actis _____________________________________________ Dr. David G. Pennock, Graduate School Representative ABSTRACT STRUCTURE, ORGANIZATION, AND FUNCTION OF THE TERMINAL ORGANELLE IN MYCOPLASMA PENETRANS by Dominika Angelika Jurkovic Bacteria utilize cytoskeletal elements to confer both morphological and functional polarity, which are necessary for growth and survival in their native environments. Some bacteria utilize polar appendages for attachment and motility to and within a host. Additionally, some bacteria utilize functional polarity for the subcellular localization of certain molecules to a particular area of bacterium. In most bacteria the cell wall plays a critical role in generating polarity, which poses a problem for the members of the Mycoplasma genus, whose members lack cell walls. Despite reductive evolution from gram-positive bacteria, mycoplasmas harbor a wide variety of morphological and functional complexity. Polarized species of mycoplasmas utilize polar organelles conferred by unique cytoskeletal elements utilized in attachment and motility, two processes associated with pathogenic species. One of these, Mycoplasma penetrans, is a potential opportunist usually isolated from human immunodeficiency (HIV)-infected individuals, proposed to play a role in AIDS progression. Previously, it has been demonstrated that M. penetrans attach to epithelial cells by a polar tip structure, yet nothing is known about the components involved in the architecture and functioning of the tip structure. We used time-lapse microcinematography to characterize gliding motility in both M. penetrans and its relative, Mycoplasma iowae, a poultry pathogen. Gliding speeds observed among strains in both species correlated positively with cytadherence differences by hemadsorption assay, suggesting that attachment and motility are both mediated by the same cellular components. During cell division, attachment to a surface and motility were critical for cytokinesis. We used scanning electron microscopy (SEM) and electron cyro-tomography to characterize the internal cellular organization and of the Triton X-100 (TX)-insoluble, cytoskeletal structure underlying the terminal organelle. The results from the different microscopy techniques are consistent with the cytoskeletal structure being artifactually dehydrated during processing for SEM, suggesting the cytoskeletal structure to be a proteinaceous gel found in varying amounts at both cell poles. Based on the importance motility plays in proper cell division, and the internal composition and organization of M. penetrans, we propose a cell cycle model in which a pole develops by accumulating enough TX-insoluble material in order to function in attachment and/or motility, and therefore power cytokinesis. To further identify the components of the TX-insoluble structure, we sought to identify proteins enriched in the TX-insoluble fraction compared to whole cells. We identified two proteins, MYPE1560 and MYPE1570, two of six proteins with similar features found in a putative transcriptional unit. Our observations of morphology and organization of M. penetrans and the TX-insoluble material demonstrate a novel cellular organization for generating polarity. We observed continued motility in the presence of an ATP depletion agent, a proton motive force inhibitor, and a sodium motive force inhibitor, raising the possibility that M. penetrans does not use chemically-derived energy source for motility. However, analysis of gliding under different temperature and pH conditions led us to conclude that thermal energy plays an important role in gliding motility. We propose a model for M. penetrans gliding motility wherein thermal energy is converted to unidirectional motility by means of a Brownian ratchet. Additionally, we provide evidence supporting the hypothesis that terminal organelles in distantly related mycoplasma species have evolved independently of each other, rather than diverging from a common ancestor. STRUCTURE, ORGANIZATION, AND FUNCTION OF THE TERMINAL ORGANELLE IN MYCOPLASMA PENETRANS 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 Dominika Angelika Jurkovic Miami University Oxford, OH 2012 Dissertation Director: Dr. Mitchell F. Balish TABLE OF CONTENTS INTRODUCTION......................................................................................................................... 1 A. Mycoplasma penetrans ............................................................................................................... 1 B. Bacterial polarity: morphology and function ............................................................................. 1 C. Polarity in the Mycoplasma genus ............................................................................................. 2 D. Mycoplasma cytoskeletons ........................................................................................................ 6 E. Proposed mechanism of gliding motility in Mycoplasma genus .............................................. 12 F. Significance of studying M. penetrans ..................................................................................... 15 G. Hypotheses ............................................................................................................................... 15 CHAPTER 1: Conserved terminal organelle morphology and function in Mycoplasma penetrans and Mycoplasma iowae .............................................................................................. 19 Abstract ......................................................................................................................................... 20 Introduction ................................................................................................................................... 21 Materials and Methods .................................................................................................................. 22 Results ........................................................................................................................................... 24 Discussion ..................................................................................................................................... 42 CHAPTER 2: Structure and composition of the terminal organelle cytoskeleton of Mycoplasma penetrans ................................................................................................................ 45 Abstract ......................................................................................................................................... 46 Introduction ................................................................................................................................... 47 Materials and Methods .................................................................................................................. 48 Results ........................................................................................................................................... 52 Discussion ..................................................................................................................................... 64 CHAPTER 3: Analysis of the energy source for Mycoplasma penetrans gliding motility... 76 Abstract ......................................................................................................................................... 77 Introduction ................................................................................................................................... 78 i Materials and Methods .................................................................................................................. 79 Results ........................................................................................................................................... 80 Discussion ..................................................................................................................................... 88 SUMMARY AND CONCLUDING REMARKS ..................................................................... 93 REFERENCES .......................................................................................................................... 105 ii LIST OF TABLES Table 1 Gliding motility parameters and Triton X-100 insoluble 26 structure dimensions of M. penetrans and M. iowae. iii LIST OF FIGURES Figure 1 Cell cycle of Caulobacter crescentus. 4 Figure 2 Range of morphologies within the Mycoplasma genus. 8 Figure 3 Polarity-generating cytoskeletons of Mycoplasma genus. 10 Figure 4 Schematic of M. mobile gliding machinery. 14 Figure 5 Transmission electron microscopy of M. penetrans cells 17 attached to epithelial cells. Figure 6 Consecutive phase-contrast images of M. penetrans. 28 Figure 7 Distribution of mycoplasma gliding velocities. 30 Figure 8 Colony HA of M. penetrans and M. iowae. 32 Figure 9 SEM of M. penetrans and M. iowae cells. 35 Figure
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