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 (-).

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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 their encouragement, support, and willingness to help whenever I needed it. It was a true pleasure having such wonderful people at my disposal for guidance and support. I truly appreciate our many conversations regarding science and everything else. To my best friend Kirk Atwood and his wonderful wife Jessica, Robert and Donna O’Neal, Samuel Bernstein, Jeffrey Alborn and his lovely wife Jennifer, Ross Whelpton, Andrew Troy, Kirsten Frye, Zachary Bracken, Matthew Puz, Laura Prylinski, Cecile Dixon, Corrie Cogley, Jennifer Selm and her wonderful family, Gloria Actis, Chris and Kara Hall, Mike Steupe, Jane Zaenkert, and my many other friends, I love you all very much and I couldn’t have gotten this far without you. To my brother Peter and my parents, Peter and Nancy Relich, I can never repay you for everything that you’ve done for me. In fact, words cannot accurately describe my gratitude and love for you. Thank you for nurturing my interests and fostering my love of all of the little critters that we share the world with. I am truly blessed to share my life with you and I can’t imagine a better family to have! Also, I thank my extended family and my cousins Kory Klukan and Christy Smith for being so wonderful and supportive. I owe a great deal of gratitude to my aunt Deborah Bush for supplying me with all of the motivation, encouragement, and tools necessary for a very young microbiologist to get started and stick with it. To my grandparents, Ford and Nan Shankle and Shirley Relich, I owe the world to you. Thank you for helping to shape me into the person who I have become. To my parents-in-law Richard and Colleen Hartle, my brothers-in-law and their families, I owe special thanks for all of the love, encouragement, and support that you all have given me time and time again. To all of my professional friends and acquaintances, I owe so much as well. To my brother in clinical microbiology, Dr. Larry Gray, whose advice and friendship has brought me many laughs and valuable connections, I extend my deepest and most sincere thanks. I look forward to working with you in the future, brother! To Dr. Douglas Smith, Dr. Kate Eggleton, Dr. Terry Morrow, Dr. Bill Barnes, Dr. Clifford Vogan, Dr. Iddo Friedberg, Dr. Xiao-Wen Cheng, Dr. Luis Actis, Mrs. Robin Sutter, Mr. Matt Duley, Mr. Terry Knepshield, Ms. Amy Cribbs, the staff from the Cowansville Area Health Center,

ix Mr. Robert Dahlman, Mr. Stephen Johnson, Mr. Dan Bruner, the laboratory staff from Clarion Hospital, the microbiology laboratory staff from Armstrong County Memorial Hospital, and the laboratory staff from McCullough-Hyde Memorial Hospital I thank you so much for all of your help and support. To the love of my life and soulmate, Amanda Relich, you are my rock and life support system. There will never be a way that I can fully thank you for all of the sacrifices that you’ve made to ensure that I have everything that I could ask for, and then some. Without you in my life and the endless love and support that you give me, I could not have made it this far. Last, but certainly not least, I would like to thank my son, Samuel, for becoming the best recent addition to my life. I never thought that I could love anything as much as I love you, Sam; I love you with all of my heart, buddy! I dedicate all that I do from here on to you and any siblings that you may have. To everyone whose names do not appear in these acknowledgments, I haven not forgotten you, my fingers are just tired. I sincerely thank you as well for making all of this possible!

x INTRODUCTION

The ability to move affords many cells the opportunity to acquire nutrients, evade unfavorable environmental conditions, and disseminate (Spormann, 1999). But, despite being a common attribute of eukaryotic and prokaryotic cells, the molecular bases for motility differ across taxa (McBride, 2001; Vincensini et al., 2011). In the context of bacterial movement, flagellum-mediated propulsion through liquids or across surfaces and pilus-mediated twitching are perhaps the most commonly considered forms of locomotion. However, many bacteria are capable of gliding motility, which is characterized by cells smoothly moving across surfaces, typically along the long axis of the cell body (McBride, 2001). For motile species belonging to the genus Mycoplasma, gliding occurs by disparate mechanisms from those known about in other bacteria. Mycoplasma gliding is not mediated by pili or polysaccharide secretions as with myxobacterial social motility and cyanobacterial gliding, respectively (Spormann, 1999; McBride, 2001). In mycoplasmas, propulsive forces appear to be generated by multiple different means amongst species, including extracellular motility proteins, and in most species, a differentiated tip structure houses the gliding machinery. For the most part, the exact mechanisms underlying motility in gliding mycoplasmas are unknown and there appear to be several routes of gliding evolution in these bacteria. The research presented within this dissertation examines how a protein common to species within the Mycoplasma pneumoniae phylogenetic cluster is involved in gliding by use of orthologous gene replacement between the closely related species M. pneumoniae and Mycoplasma genitalium as well as analysis of M. pneumoniae transformants that produce varying low amounts of this protein. Also, we describe a novel cytoskeleton and gliding in a recently described rod-shaped species, Mycoplasma insons, which is a member of a different cluster. The observations made in this organism challenge the common hypothesis that gliding in Mycoplasma species occurs only in association with a differentiated tip structure.

1

A. Description of the genus Mycoplasma. Organisms belonging to the class Mollicutes, genus Mycoplasma, trivially called mycoplasmas, are cell wall-less bacteria that have undergone evolutionary genome reduction from a gram-positive ancestor (Razin et al., 1998). As a consequence, these organisms possess the smallest genomes of any cell type capable of axenic growth and are limited with regard to biosynthetic capability; nearly all of the genes necessary for amino acid and cofactor synthesis have been lost (Rottem & Naot, 1998). For cultivation in the laboratory, Mycoplasma species must be provided with a rich growth medium, which is typically supplemented with a variety of nutrients, including cholesterol, a component of their cell membranes (Rottem, 2002). In nature, mycoplasmas parasitize vertebrate animals to acquire nutrients that they cannot synthesize de novo. The relationships that these bacteria have with their hosts are largely benign, but some species are opportunists or important pathogens. With regard to their role as pathogens, some Mycoplasma species are significant causes of morbidity and mortality in humans and animals. The lack of a cell wall in these organisms precludes visualization by techniques traditionally used to examine walled bacteria, such as Gram staining. Mycoplasmas are much smaller than most other bacteria, and the resolution provided by many light microscopes is typically insufficient for analysis of their cell morphology; techniques such as phase-contrast light microscopy and electron microscopy are more appropriate for such examinations. High-resolution images show that most mycoplasmas are coccoid and highly pleomorphic, a result of lacking a rigid cell wall, but most motile Mycoplasma species are flask- or spindle-shaped. The polarity observed in these species is conferred by a membrane protrusion commonly referred to as the attachment organelle, especially in the members of the Mycoplasma pneumoniae phylogenetic cluster. The attachment organelle morphologies and related properties of these species are distinct (Hatchel & Balish, 2008), and the mechanisms governing these differences are unknown, but we propose that they are analogous to those of the corresponding structure in the fish pathogen Mycoplasma mobile.

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B. Mycoplasma species as agents of human and animal disease. Human and animal diseases caused by Mycoplasma species are well documented, and the public health and economic impacts of which can be staggering. For example, in the case of bovine mastitis associated with Mycoplasma bovis, some estimates put the annual financial losses to the dairy industry over $100 million in the United States alone (Maunsell et al., 2011). Many other pathogenic mycoplasmas are known to be associated with diseases of cattle, pigs, crocodiles, goats, avians, and many other animals, both those of agricultural significance and those found in the wild (Nicholas, 1998). Of the species that cause disease in humans, Mycoplasma pneumoniae (Eaton et al., 1944) is perhaps the best known and most well studied. M. pneumoniae is the etiological agent in approximately 30% of community-acquired pneumonia cases per year in some areas (Kannan et al., 2010). Tracheobronchitis and primary atypical, or “walking,” pneumonia are the major diseases associated with M. pneumoniae and outbreaks of these diseases occur with some frequency, especially among institutionalized individuals or those living in close quarters (Kleemola & Jokinen, 1992; Gray et al., 1997; Felkin et al., 1999; Hyde et al., 2001). The pulmonary infections caused by M. pneumoniae have been implicated in exacerbation of asthma in children (Waites & Talkington, 2004) as well as non-pulmonary pathologies, such as diseases affecting the skin, central nervous system, and eyes (Salzman et al., 1992; Talkington et al., 2001; Waites & Talkington, 2004; Tsiodras et al., 2005; Harr & French, 2010; Guo et al., 2011; Waites et al., 2008). Post-infectious sequelae have also been reported, and include acute psychosis (Banerjee & Petersen, 2009), hemolytic anemia (Cherry, 1993), and Guillan-Barré syndrome (Sharma et al., 2011). Another important human pathogen, Mycoplasma genitalium, is a significant cause of sexually transmitted infections, being second only to Chlamydia trachomatis with regard to prevalence in cases of infectious urethritis according to some studies (McGowin & Anderson-Smits, 2011). Infection with this organism has also been implicated in cases of pelvic inflammatory disease, cervicitis, and infertility (McGowin & Anderson-Smits, 2011). Transmission of pathogenic mycoplasmas among humans and animals appears to occur by mechanisms commonly associated with transmission of many other infectious agents. These routes include inhalation of infectious aerosols and handling of

3 contaminated fomites, as for M. pneumoniae (Waites & Talkington, 2004), transfer of infectious organisms by direct contact, as for the pig pathogen Mycoplasma hyopneumoniae (Villarreal et al., 2011), and sexual contact, as for M. genitalium (McGowin & Anderson-Smits, 2011). Successful colonization of hosts requires the use of cellular adhesins to stick to the host mucosal epithelium, a surface on which most pathogenic and non-pathogenic species remain. For M. pneumoniae, and potentially all of the species belonging to the M. pneumoniae phylogenetic cluster, initial adherence of the bacterial cells to the host requires the attachment organelle and its associated adhesins. The property of gliding motility is thought to play a role in pathogenicity (Miyata, 2010), perhaps as a means for dissemination. Pathology associated with mycoplasma infections is typically associated with elicitation of a robust and damaging immune response and the production of secreted factors (Waites & Talkington, 2004), including hydrogen peroxide, a hemolytic by- product of glycerol metabolism of M. pneumoniae (Somerson et al., 1965; Low et al., 1968; Miles et al., 1991; Hames et al., 2009), and the community-acquired respiratory distress syndrome toxin, and ADP-ribosylating toxin identified in M. pneumoniae (Kannan & Baseman, 2006). Phase variation most likely contributes to persistence of this organism within hosts (Waites & Talkington, 2004). For eradication of mycoplasma infections, antimicrobial chemotherapy is almost always effective. The drugs of choice are typically tetracycline or macrolides (Clyde, 1993; Yanagihara et al., 2009) and alternatives include fluoroquinolones and doxycycline (Winn et al., 2006). An increasing rate of resistance to certain antibiotics has been identified in several Mycoplasma species, including M. pneumoniae and M. genitalium, revealing a need for more effective treatments and prophylaxis (Taylor-Robinson & Bébéar, 1997; Li et al., 2010; Miyashita et al., 2010; Shimada et al., 2010; Miyashita et al., 2011; Chironna et al., 2011; Yew et al., 2011).

C. Comparisons of bacterial motility with emphasis on gliding motility in Mycoplasma species. Many bacteria can utilize one or more flagella to propel themselves through liquids in a form of motility called swimming. However, some organisms, including Proteus

4 mirabilis and Serratia marcescens, utilize flagella for a moist-surface-based form of motility called swarming (Harshey, 1994), in addition to swimming. Twitching, a form of bacterial movement across surfaces, often utilizes the extension and retraction of type IV pili, the effects of which tug cells across surfaces. This form of motility is common to many bacteria, including the ubiquitous opportunistic pathogen Pseudomonas aeruginosa (Miller et al., 2008). Among organisms that move by gliding, there is no single means. For adventurous (A)-motility in the non-pathogenic bacterium Myxococcus xanthus, gliding is proposed to be mediated by bipolar nozzle-like structures that extrude a polysaccharide-containing slime (Wolgemuth et al., 2002) and proton motive force- driven rotation of helices composed of the A-motility protein AgmU (Nan et al., 2011). Distortion of the M. xanthus cell surface caused by the rotation AgmU helices creates pressure waves in the polysaccharide slime, which causes cells to move (Nan et al., 2011). To change directions, M. xanthus cells close the nozzle at one pole and secrete slime from the nozzle at the opposite pole (Shapiro et al., 2002). Proposed mechanisms for gliding motility of the Cytophaga-Flavobacterium group of bacteria invoke the cytoskeleton and other structures. One model suggests that proteins anchored in the cell membrane of these organisms harvest the proton motive force, which propels outer membrane proteins along peptidoglycan-anchored tracks. The temporary attachment of the outer membrane proteins to the surface causes movement of the cell (Lapidus & Berg, 1982). Other mechanisms have been proposed to explain gliding in these organisms as well, including generation of outer membrane waves (Duxbury et al., 1980) and movement of periplasmic or cytoplasmic structures (Burchard, 1984). Several Mycoplasma species are known to glide and almost all of these organisms have a differentiated tip structure, or attachment organelle, leading many to believe that motility does not occur in species lacking attachment organelles. However, we have discovered that the rod-shaped species Mycoplasma insons is capable of gliding motility, which challenges this notion because this organism lacks an obvious attachment organelle (May et al., 2007). We have yet to uncover the mechanisms governing movement in this species, but we speculate that it is driven by means not dissimilar to those in other species. For M. pneumoniae, Mycoplasma mobile, and other species, the attachment organelle is both the leading end of the cell during gliding and is known to be

5 the compartment that houses the motor activity (Bredt, 1968; Hasselbring & Krause, 2007; Miyata, 2010). Despite the ubiquity of this structure in these species, the attachment organelle-associated properties and morphologies are distinct, even among very closely related species (Fig. 1). Perhaps the best-understood system for motility generation in Mycoplasma species so far is that which governs gliding in the fish pathogen M. mobile, a distant relative of M. pneumoniae. In this organism, a surface-exposed attachment organelle protein, Gli349, which resides in the region between the tip of the attachment organelle and cell body, is believed to drive motility in concert with other proteins comprising the motor (Fig. 2; Miyata, 2010). Incubation of cells with an antibody generated against Gli349 inhibits motility and eventually cells detach from the substrate (Uenoyama et al., 2004). Electron and atomic force microscopy of purified Gli349 revealed the morphology of this protein in multiple conformations and as having several regions, including a foldable hinge region that connects two short linear regions, a long filament, and an oval “foot” (Adan-Kubo et al., 2006; Miyata, 2007). Analysis of Gli349 by biochemical methods indicates that the Gli349 C-terminal “foot” has sialyl oligosaccharide binding activity, implicating this region in substrate binding (Uenoyama et al., 2009). The N- terminal region of Gli349 associates with Gli521, a protein that bridges Gli349 to the intracellular ATPase P42 and the protein Gli123 (Miyata, 2010). The model for these proteins in the process of gliding in M. mobile is energy harvested from ATP hydrolysis is transduced through Gli521 to Gli349, which undergoes conformational changes that pulls cells along a the substratum that is composed of sialylated proteins (Miyata, 2010). Gliding motility has also been characterized for the putative human pathogen Mycoplasma penetrans. This species possesses a polar tip structure as well, but the cytoskeleton is distinct from those of both M. mobile and M. pneumoniae. Preliminary data suggests that the energy for motility in M. penetrans is not derived from ATP hydrolysis, proton motive force, or sodium motive force (Jurkovic & Balish, unpublished). Conceivably, transmembrane adhesins or extracellular motility proteins produce the locomotive forces from harvesting energy from substrate hydrolysis, or perpetuation of movement is generated by a Brownian ratchet type mechanism.

6

Fig. 1. Scanning electron micrographs of two attachment organelle-possessing mycoplasmas, M. pneumoniae (left) and its closest genetic relative, M. genitalium (right). These two motile species possess distinct attachment organelles located at the leading-end of the cells (white arrow). Bar, 500 nm.

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Fig. 2. Schematic of Mycoplasma mobile gliding machinery. Energy produced from ATP hydrolysis by protein P42 is predicted to be transmitted to the gliding leg protein Gli349, which is thought to undergo sequential attachment-and-release cycles, pulling cells across a sialyl oligosaccharide-coated surface (sialylgalactose). The protein Gli521 is proposed to transmit the propulsive force generated within the cell to Gli349. From Miyata, 2010.

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With regard to M. pneumoniae and its relatives comprising the M. pneumoniae phylogenetic cluster (Fig. 3), the mechanisms underlying motility generation and speed calibration are largely unknown as well. Cryo-electron microscopic examination of M. pneumoniae has demonstrated conformational differences in the core components of the attachment organelles of whole cells (Henderson & Jensen, 2006). These observations prompted Henderson & Jensen (2006) to propose that the conformational changes of the attachment organelle core might lead to movement of M. pneumoniae cells in an inchworm-like fashion. In contrast to this proposition, other research implicates the transmembrane adhesins that are localized to the attachment organelle in gliding. The work of Hasselbring and colleagues (2005) identifies a necessity for the transmembrane adhesin P30 in gliding. Briefly, in M. pneumoniae mutants lacking P30 or possessing truncated P30, gliding does not occur or it occurs at a reduced speed, respectively (Hasselbring et al., 2005). Similarly, experiments performed by Seto and colleagues (2005a) in which M. pneumoniae cells are treated with antibodies to the primary adhesin P1 demonstrate the necessity for this protein in gliding; gliding slows and cells eventually detach. Because all of the members of the M. pneumoniae phylogenetic cluster that have been analyzed contain attachment organelles and orthologs to most M. pneumoniae attachment organelle proteins and substructures, we can speculate that motility in these organisms is driven by mechanisms comparable to those in M. pneumoniae. In an attempt to identify motility-generating mechanisms different from those characterized in M. mobile and M. pneumoniae, we examined a novel rod-shaped Mycoplasma species isolated from iguanas, Mycoplasma insons. To our surprise, M. insons was capable of slow gliding despite the absence of a discernable polar tip structure. We also uncovered a novel cytoskeleton in this organism. The preliminary observations suggest that motility in the absence of an attachment organelle is possible and that the propulsive mechanisms in this species could be completely different from what are known about in other motile species.

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Fig. 3. Tree of the Mycoplasma pneumoniae phylogenetic cluster based on 16S rRNA gene sequence analysis. Bar indicates 0.1 substitutions per site. From Hatchel & Balish, 2008.

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D. The composition of the attachment organelle of M. pneumoniae. The attachment organelle of M. pneumoniae and many of its close relatives is composed of many novel proteins, largely structural proteins and proteins involved in adherence to surfaces. Despite being the subject of study for some time, not much is known about how the attachment organelle functions in any of the activities with which it is associated. However, analysis of spontaneously arising M. pneumoniae mutants lacking various attachment organelle proteins provides some insight into the necessity for this prostheca in adherence and gliding. Mutations in the genes encoding some of these proteins have deleterious effects on the ability of M. pneumoniae to attach to and/or glide along surfaces. Hasselbring and colleagues (2007) analyzed M. pneumoniae cells possessing mutations in a gene, MPN311, encoding the attachment organelle protein P41 and have demonstrated that when this attachment organelle-anchoring protein is absent, attachment organelles detach from cells and glide away, while the cell body remains stationary. In their place, new attachment organelles form, but they too are destined to detach and glide away. These data directly implicate the attachment organelle of M. pneumoniae as the site at which the motile force acts. Detergent extraction and subsequent visualization of cells by scanning electron microscopy reveals that the area occupying the attachment organelle of M. pneumoniae phylogenetic cluster members contains an electron-dense, rod-shaped core composed of three distinct components (Hatchel & Balish, 2008). At the distal end of the core is a terminal button that is connected by a bipartite rod to a bowl-shaped base at the proximal end. With regard to composition of the core components, the terminal button most likely contains the proteins P65 (Balish & Krause, 2005), a surface-exposed protein (Proft et al., 1995) that is destabilized in the absence of the adhesin P30 (Jordan et al., 2001), and HMW3 (Stevens & Krause, 1992). Electron cryotomography of M. pneumoniae reveals that the rod consists of two parallel pieces (Henderson & Jensen, 2006; Seybert et al., 2006) and is speculated to be largely composed of the coiled-coil protein HMW2 (Bose et al., 2009), which is stabilized by the protein HMW1 (Balish et al., 2001). The base of the core contains the proteins P24, P28, and P41 (Krause et al., 1997; Jordan et al., 2001; Seto & Miyata, 2003). The protein P200 is known to be a part of the attachment organelle and appears to be required for gliding (Jordan et al., 2007). The J-domain protein, TopJ

14

(Cloward & Krause, 2010), appears to function as a co-chaperone for attachment organelle assembly and is thus a key constituent of this apical structure. Besides core components, the transmembrane adhesins P1 (Hu et al., 1977) and P30 (Baseman et al., 1982; Morrison-Plummer et al., 1986) also localize to the attachment organelle. P1 is known to complex with two other proteins, B and C, which, in addition to being located at other parts of the cell, are concentrated at the attachment organelle (Baseman et al., 1982; Feldner et al., 1982; Seto et al., 2002; Nakane et al., 2011). P1 is known as the primary adhesin of M. pneumoniae and has orthologs in other species within the M. pneumoniae phylogenetic cluster (Balish et al., unpublished), but is unique to these organisms. For example, in M. genitalium, the P1 ortholog MgPa is also an adhesin (Hu et al., 1987), as is the Mycoplasma gallisepticum P1 ortholog GapA (Goh et al., 1998). P1 is required for adherence and virulence (Baseman et al., 1982), and experiments using anti-P1 antibodies added to M. pneumoniae cells causes slowing of gliding motility and eventual detachment (Seto et al., 2005a). The other adhesin, P30, is a 274-amino acid, bitopic membrane protein that is localized to the tip of the attachment organelle of M. pneumoniae (Dallo et al., 2006). Orthologs of this protein have been identified in every species of the M. pneumoniae phylogenetic cluster examined (Balish et al., unpublished). P30 has three functional domains: I, II, and III (Chang et al., 2011). Domain I consists of a 72-amino acid intracellular stretch, which is followed by a transmembrane domain, then the beginning of the extracellular region (domain II), and finally a C-terminal region (domain III) that includes 13 proline-rich repeats (Chang et al., 2011). Examination of spontaneously arising hemadsorption-negative M. pneumoniae mutants indicates a requirement for the P30 adhesin in the process of gliding motility (Hasselbring et al., 2005). In the mutant M. pneumoniae II-3, a frameshift mutation within the P30-encoding gene, MPN453, leads to instability of the N-terminal half of P30, which accounts for lack of detectable protein (Balish & Krause, 2005). M. pneumoniae II-3 is capable of neither adherence nor motility and cells exhibit profound pleomorphy (Romero-Arroyo et al., 1999; Hasselbring et al., 2005). These data indicate a relationship between gliding motility and cell division; in the absence of the protein P30, cells cannot glide, and consequentially, they do not divide appropriately. In the revertant II-3R, a pair of mutations restores P30; however, 17 amino

15 acids within the extracellular domain are different from those in the wild-type protein (Hasselbring et al., 2005). II-3R is capable of adherence and gliding, but gliding speed is greatly diminished (Hasselbring et al., 2005). Likewise, a 48-amino acid truncation in the mutant M. pneumoniae II-7 allows for adherence of these cells, but motility is also diminished (Hasselbring et al., 2005). Because of the apparent necessity for P30 in M. pneumoniae gliding motility, it was an attractive protein for our studies described here.

E. The importance of studying motility in M. pneumoniae and other motile species. The high prevalence of mycoplasma infections coupled with an increasing incidence of resistance to antibiotics in clinical isolates suggests a need for improved treatment options and effective prophylaxis. The P30 adhesin of M. pneumoniae appears essential for gliding and, interestingly, for pathogenicity; in the absence of P30, cells are immotile and nonpathogenic. Based on this knowledge, it seems not only reasonable, but also rather essential, that investigations are undertaken to understand the importance of this protein in the cell biology and pathogenicity of M. pneumoniae, as well as its relatives within the M. pneumoniae phylogenetic cluster. By studying P30 and other mechanisms associated with the life cycle of M. pneumoniae it could be possible to identify novel targets for both antimicrobial chemotherapy and vaccines. In addition to the public health concerns about this and related organisms, a general understanding of the diversity of motility-generating mechanisms in Mycoplasma species is important to the field of bacterial cell biology. As such, this knowledge may prove useful for the development of techniques to study as yet unknown bacteria. Based on experiments from many groups around the world, it is apparent that the underpinnings of motility have taken multiple routes of evolution in Mycoplasma species. A general understanding of how these mechanisms may unveil implications in the fields of biotechnology and nanotechnology. Hiratsuka and colleagues (2006) have described a microrotary motor powered by the gliding motility of Mycoplasma mobile. It may be possible to harness the power of mycoplasma gliding in the future as an alternate means for performance of work.

16

F. Hypotheses. We propose that the mechanisms underlying motility in Mycoplasma species have evolved differently, but overall motile species use either transmembrane adhesins or extracellular proteins to mediate locomotion, the energy from which being derived from either intracellular structures or substrate hydrolysis. Regarding the research described here, we hypothesized that, in M. pneumoniae and its close relatives, the quality and quantity of the P30 adhesin impact gliding motility speed. To demonstrate this, we used orthologous gene replacement and an inducible-promoter system to examine the role of the transmembrane adhesin P30 in mycoplasma attachment organelle biology. We also tested the hypothesis that mycoplasma motility requires a tip structure by analyzing M. insons, a rod-shaped species isolated from iguanas. In chapter 1, we hypothesized that the inherent differences between P30 orthologs of M. pneumoniae and M. genitalium confer species-specific cellular attributes, including morphology and gliding motility characteristics. To test this, we transformed a well- characterized P30 null M. pneumoniae mutant, II-3, with the M. genitalium gene encoding its ortholog, P32 (Reddy et al., 1995), and examined several transformants by scanning electron microscopy, immunoblot analysis, indirect immunofluorescent localization, and evaluation of gliding motility parameters. In chapter two, we hypothesized that the concentration of the P30 adhesin of M. pneumoniae positively correlated with the speed at which M. pneumoniae cells glide. To investigate this, we transformed M. pneumoniae II-3 cells with the P30 gene from wild type M. pneumoniae under the control of a promoter which allowed for acquisition of transformants producing variable low levels of P30. We examined multiple transformants as for chapter one. In chapter three, we focused on characterizing the rod-shaped M. insons, which lacks a differentiated tip structure, using techniques amenable to many other mycoplasmas, including M. pneumoniae and its relatives. We characterized the motility of this organism and hypothesized that a novel cytoskeleton underlay gliding. To examine the cytoskeleton, we used detergent extraction and scanning electron microscopy. Finally, we hypothesized that perhaps the P30 adhesin, which localizes to the tip of the M. pneumoniae attachment organelle, might provide insight into both polarity- and motility- generating mechanisms in M. insons. We tested this hypothesis by introducing a

17 fluorescent protein-P30 fusion construct into M. insons cells in order to visualize P30 localization within cells. The data for these experiments appear in appendix A.

18

CHAPTER 1

Insights into the Function of Mycoplasma pneumoniae Protein P30 from Orthologous Gene Replacement

Ryan F. Relich and Mitchell F. Balish Accepted to the Journal Microbiology

19

Summary

The attachment organelles of the bacterial species belonging to the Mycoplasma pneumoniae phylogenetic cluster are required for host cytadherence, gliding motility, and virulence. Despite being closely related, these bacteria possess distinct cellular morphologies and gliding characteristics. The molecular bases for most attachment organelle phenotypes, including shape and ability to power motility, are obscure. The attachment organelle-associated P30 protein of M. pneumoniae is implicated in both adherence and motility, with mutations altering cell morphology, adherence, gliding, and virulence. To test whether the P30 alleles of different mycoplasma species confer species-specific attachment organelle properties, we created an M. pneumoniae strain in which the Mycoplasma genitalium P30 ortholog, P32, was substituted for the native P30. Selected clones were visualized by scanning electron microscopy to assess morphology and by indirect immunofluorescence microscopy to localize P32. Cytadherence capability and gliding motility were assessed by hemadsorption assay and phase-contrast microcinematography, respectively. Cell and attachment organelle morphologies were indistinguishable from wild-type M. pneumoniae as well as M. pneumoniae II-3 expressing a C-terminally 6X-His tagged P30 construct. P32 was localized to the tip of the attachment organelle of transformant cells. Although a specific role for P30 in species-specific phenotypes was not identified, this first test of orthologous gene replacement in different mycoplasma species demonstrates that the differences in the M. pneumoniae and M. genitalium proteins contribute little if anything to the different attachment organelle phenotypes between these species.

20

Introduction

Mycoplasmas are cell wall-less bacteria that belong to the class Mollicutes. By virtue of reductive evolution, these organisms have the smallest genomes of any self-replicating cells capable of axenic growth. In nature, these organisms parasitize host cells for nutrients due to limited biosynthetic capabilities, and in the laboratory, they must be provided with a rich growth medium (Razin et al., 1998). Absence of a cell wall imparts pleomorphy to many of these organisms; however, many species of the genus Mycoplasma appear flask-shaped. Polarity is conferred by a differentiated tip structure (Hatchel & Balish, 2008), the attachment organelle, which mediates primary attachment of these organisms to surfaces such as host epithelia. Attachment organelles are required for host colonization and virulence in the human respiratory and genitourinary tract pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively (Razin & Jacobs, 1992; Ueno et al., 2008; Waites & Talkington, 2004). Unlike many of their walled counterparts, mycoplasmas lack locomotory appendages such as flagella and pili. Instead, the attachment organelle provides the means necessary for an adherence- dependent form of locomotion called gliding motility. The speeds at which species within the M. pneumoniae phylogenetic cluster glide are different (Hatchel & Balish, 2008), implying that some component of the motor apparatus regulates speed. Interestingly, the failure of an M. pneumoniae mutant that moves about as fast as M. genitalium to successfully colonize a normal human bronchial epithelial cell culture (Jordan et al., 2007) suggests that these species-specific speeds are calibrated to other species-specific properties. Triton X-100 (TX)-insoluble components of the attachment organelles of each of the species of the M. pneumoniae cluster are visible by electron microscopy (Göbel et al., 1981; Meng & Pfister, 1980; Hatchel & Balish, 2008). These structures prominently include an electron-dense core (Biberfeld & Biberfeld, 1970), which is a bipartite rod (Henderson & Jensen, 2006; Seybert et al., 2006) with a terminal button located at the end that is distal to the cell body, and a base, which physically interacts with the cell chromosome, at the proximal end (Hatchel & Balish, 2008). Scanning electron micrographs of TX-extracted M. pneumoniae and several of its close relatives

21 demonstrate that core substructures are distinct across species, leading to differences in core length, width, and curvature, and conferring distinct morphological properties to the attachment organelle of each species (Hatchel & Balish, 2008). In particular, M. pneumoniae has a straight attachment organelle that is 290 nm in length, whereas that of M. genitalium is only 170 nm long and curved approximately 20°, with a more prominent terminal knob. The attachment organelle of M. pneumoniae and its close relatives is composed of many novel proteins (Balish & Krause, 2005; Balish, 2006), including structural proteins such as HMW1 (Stevens & Krause, 1991), HMW2 (Krause et al., 1982), HMW3 (Stevens & Krause, 1992), and P65 (Jordan et al., 2001; Proft et al., 1995), and proteins involved in adherence to host cells, such as P1 (Baseman et al., 1982, Feldner et al., 1982; Hu et al., 1982) and P30 (Morrison-Plummer et al., 1986). The specific relationships between any of these proteins and either attachment organelle morphology or the process of gliding motility are largely obscure. However, analysis of M. pneumoniae cells containing a transposon that disrupts the gene encoding attachment organelle protein P41 indicates clearly that the motor activity for gliding is contained within the attachment organelle (Hasselbring & Krause, 2007). Henderson and Jensen (2006) have proposed that the electron-dense core drives motility, undergoing conformational changes that move the cells in an inchworm-like fashion. Other evidence suggests that adhesins localized to the attachment organelle may be responsible for gliding motility. Gliding motility and glass binding of M. pneumoniae cells treated with a monoclonal anti-P1 antibody are negatively impacted in an antibody concentration-dependent manner, whereas the antibody minimally affects non-gliding cells (Seto et al., 2005), suggesting a role for the P1 adhesin in gliding. In addition, a spontaneously occurring hemadsorption-negative mutant of M. pneumoniae, II-3, can neither attach to substrates nor glide because of a frameshift mutation in the gene encoding the 30-kilodalton attachment organelle protein P30 (Hasselbring et al., 2005; Romero-Arroyo et al., 1999). Also, decreased stability of the attachment organelle protein P65 is evident in these cells (Jordan et al., 2001). An altered stretch of 17 amino acids in P30 as well as truncated versions of P30 allow for very slow motility (Hasselbring et al., 2005; Chang et al., 2011).

22

To test the relationship between gliding speed and sequence features of P30, we undertook an effort to complement the M. pneumoniae P30 null mutant II-3 with P32 from M. genitalium (Reddy et al., 1995), which is approximately 43% identical to P30 (Fig. 4). We introduced a 6X-His epitope-tagged P32-encoding construct, as well as a non-tagged construct, into mutant II-3. We subsequently examined several transformants in terms of attachment organelle morphology, cytadherence ability, and gliding motility parameters, as well as P65 stabilization. Our results rule out a specific role in gliding motility for P30 / P32 sequence differences between M. pneumoniae and M. genitalium, but clearly demonstrate the potential for the use of orthologous gene complementation among mycoplasma species to test gene / protein function.

23

Materials and Methods

Strains and growth conditions. M. pneumoniae wild-type strain M129, P30 null mutant M. pneumoniae II-3, M. genitalium wild-type strain G37, and M. pneumoniae II-3 transformants were grown in plastic tissue-culture flasks in SP-4 broth (Tully et al., 1979) at 37°C until mid-exponential phase (phenol red indicator was orange; 3 – 5 d) in an ambient air atmosphere. For assays requiring plating of strains, cells were grown on SP-4 containing 1% (w/v) Noble agar (Becton, Dickinson and Company, Franklin Lakes, NJ). To prepare motility stocks, the protocol detailed by Hatchel et al. (2006) was used. For selection and propagation of transformants only, 18 µg gentamicin ml-1 was included in all media.

Genomic DNA isolation, PCR, and cloning. Mid-exponential phase SP-4 broth- cultures, with or without gentamicin, were harvested by centrifugation for 20 min at 17, 400 x g. Cell pellets were washed 3 times with phosphate buffered saline (PBS; 150 mM

NaCl / 3.2 mM NaH2PO4 / 13.6 mM Na2HPO4, pH 7.2) and genomic DNA was extracted using the Blood and Body Fluid Spin Protocol of the QIAamp DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA). Purified genomic DNA was quantitated spectrophotometrically, diluted with ultrapure water, and stored at -20°C until use. The P30- or P32-encoding genes of M. pneumoniae M129 and M. genitalium G37, respectively, as well as the P21-encoding genes immediately upstream of these genes, designated P21MP and P21MG, were amplified using the primers listed in Table 1. To make polyhistidine-tagged P30 and P32 proteins, six histidine codons were engineered in frame into primers that were used to amplify the 3’ end of the gene, resulting in production of P30His and P32His. Following PCR, amplicons were cloned using the TA cloning vector pCR®2.1 (TA Cloning® Kit; Invitrogen, Carlsbad, CA). Next, clones were screened by restriction analysis and sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and capillary electrophoresis on an Applied Biosystems 3130xl genetic analyzer at the Miami University Center for Bioinformatics and Functional Genomics. Inserts were subcloned from pCR®2.1 plasmids into the Tn4001-containing suicide vector pMT85 (Zimmermann

24

& Herrmann, 2005) for transformation of mycoplasmas. These plasmids were designated pOO12, pOO17, and pOO24 (Table 1). To generate pOO30 (Table 1), site-directed mutagenesis was used to introduce a

ClaI restriction site in the P21-P32His intergenic region of the construct contained in the plasmid pOO15 (progenitor of pOO17; not shown) using the QuikChange II® site- directed mutagensis kit (Agilent Technologies, Inc., Santa Clara, CA) and the primer

ClaMUT (Table 1). Next, ClaI and MfeI were used to remove the P32His-encoding gene, these restriction sites were blunted, and the plasmid was recircularized. This modification allowed for retention of the original EcoRI site, which was exploited to facilitate subcloning of this construct into pMT85 for subsequent transformation of M. pneumoniae II-3. To generate pOO37 (Table 1), site-directed mutagenesis was used to reintroduce the

® original translational stop codon back into P30His of the pCR 2.1 progenitor of pOO24 (pOO21; not shown). The resulting construct was next subcloned into pMT85. Electroporation was used for transformation of M. pneumoniae II-3 cells and was performed as described by Hedreyda et al. (1993). Transformants were selected on gentamicin-containing SP-4 agar and multiple gentamicin-resistant colonies were picked to control for transposon insertion site positional effects in transformants (Hedreyda et al., 1993). To ensure genetic homogeneity of transformants, colonies were grown in selective broth, filtered through a 0.22-µm syringe filter, and then plated onto selective agar. This filter-cloning approach was carried out a total of 3 times. Transformants were next grown in 10 ml selective broth and aliquots were frozen at -80°C for further use.

Hemadsorption assay. To assess the cytadherence capability of the mycoplasmas examined in this study, the hemadsorption assay was performed on plate cultures following 7 d of incubation as described elsewhere (Sobeslavsky et al., 1968); however, sheep blood in Alsever’s solution was used, and an additional PBS wash was also done. Colonies were visualized with an inverted microscope to determine whether colonies hemadsorbed sheep red blood cells.

Immunoblot analysis. Cell pellets were prepared from 50-ml SP-4 broth cultures with or without 18 µg gentamicin ml-1 by centrifugation for 20 min at 17,400 x g at 4°C,

25 followed by 3 washes with PBS. Pellets were resuspended in 150 mM NaCl / 20 mM Tris HCl, pH 7.5 and protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL). For P30 and polyhistidine immunoblots, cellular material equivalent to 5 µg of protein was used, and for P65 immunoblotting, 2.5 µg of protein was used. In all cases, lysates were electrophoresed through 12% SDS-polyacrylamide gels (Laemmli, 1970) and transferred to nitrocellulose membranes overnight at room temperature for immunoblotting (Towbin et al., 1979). Blots were blocked for 2 h at room temperature in 5% (w/v) skim milk and blotted with a 1:250 dilution of P30 antiserum (Romero-Arroyo et al., 1999), a 1:1000 dilution of anti- P65 (Proft et al., 1995), or a 1:1000 dilution of anti-6X-His (Immunology Laboratory Consultants, Inc., Newberg, OR). Membranes were subsequently probed with alkaline phosphatase-conjugated goat anti-rabbit IgG (Fc) (Promega, Madison, WI). Between each antibody incubation step, membranes were washed 5 times for 5 min each with Tris- buffered saline containing Tween-20 (50 mM Tris base / 150 mM NaCl / 0.05% (v/v) Tween 20, pH 7.5). Protein bands were detected with a solution of nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indoylphosphate. To demonstrate a lack of cross-reactivity of the anti-6X-His antibody with non-transformed M. pneumoniae, total cell lysate of M. pneumoniae wild-type strain M129 was probed with the anti-6X-His antibody along with a positive control, M. pneumoniae 24-A.

Immunofluorescence microscopy. In order to localize P30His and P32His within cells, a modified version of the protocol detailed by Jordan et al. (2001) was used. Briefly, 200- µl aliquots of cells were allowed to adhere to glass coverslips for 2 h at 37°C. Following incubation and fixation, cells were probed with 2 µg anti-6X-His antibody ml-1 (Immunology Consultants Laboratories, Newberg, OR) follwed by a 1:100 dilution of a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Coverslips were rinsed once with sterile water before being mounted on glass microscope slides using VECTASHIELD (Vector Laboratories, Burlingame, CA). Cells were examined using epifluorescence illumination with a Leica DM IRB inverted microscope (Leica Microsystems, Weilburg, Germany). Images were taken and manipulated with SPOT v4.1.1 software (Diagnostic Instrument, Inc., Sterling Heights,

26

MI) and Adobe Photoshop software (Adobe, San Jose, CA) to reduce background fluorescence while maintaining P30His and P32His focus integrity.

SEM. Strains were grown on glass coverslips in a 24-well tissue culture plate until mid- exponential phase (phenol red indicator was orange). To avoid disrupting non-adherent microcolonies and cells of M. pneumoniae II-3 and M. pneumoniae 30-C that settled onto the surface of coverslips and adherent cells of other strains, broth was gently aspirated from each well. Cells were next fixed to the coverslips with a solution of 1.5% glutaraldehyde / 1% formaldehyde / 0.1 M sodium cacodylate, pH 7.2, for 30 min at room temperature. Next, coverslips were rinsed with 0.1 M sodium cacodylate, pH 7.2 for 30 min and were subsequently dehydrated in increasing concentrations of ethanol (from 25 to 100% (v/v)). Coverslips were critical-point dried and gold sputter-coated as previously described (Hatchel et al., 2006). Cells were examined using a Zeiss Supra 35 FEG-VP scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) at the Miami University Center for Advanced Microscopy and Imaging.

Time-lapse microcinematography. Fifty-microliter motility stocks were inoculated into 750 µl SP-4 broth containing 3% (w/v) gelatin and 18 µg gentamicin ml-1 for transformants. Samples were next syringe-triturated 7 times through a 25-gauge needle and passed into chamber slides (Thermo Fisher Scientific, Rochester, NY). Inoculated chamber slides were incubated for 3 h at 37°C. Phase-contrast microcinematography was performed as previously reported (Hatchel et al, 2006; Relich et al., 2009) using 1-s intervals between frames for 27 consecutive frames. Motility parameters were assessed using images manipulated with both SPOT v4.1.1 and Adobe Photoshop software. Mean gliding speeds of strains were compared using a one-way analysis of variance with Tukey multiple comparisons. A significance level of 0.05 (family-wise error rate for multiple comparisons) was used for all analyses.

27

Results

Construct engineering and transformation of M. pneumoniae II-3. The Tn4001- containing plasmid pMT85 (Zimmermann & Herrmann, 2005) was used to introduce constructs for production of M. pneumoniae P30 and its M. genitalium ortholog, P32, into the P30 mutant M. pneumoniae II-3 (Krause et al., 1982) using established protocols (Hedreyda et al., 1993). Mutant II-3 has a frameshift mutation near the middle of the gene, but antisera against polypeptides corresponding to the region upstream of the frameshift fail to detect a truncated protein (Romero-Arroyo et al, 1999; Chang et al., 2011; data not shown), suggesting that II-3 is a null mutant. The relevant transformants are listed in Table 1. Attempts to drive production of P32 from the promoter in Tn4001 were unsuccessful (data not shown). Therefore, to produce P30 or P32 in M. pneumoniae II-3, we engineered constructs that included the native promoter region (Waldo et al., 1999) and the gene located between the promoter and the P30- (MPN453) or P32-encoding gene (MG318), designated as the P21MP- (MPN454) or P21MG- (MG319) encoding gene, (Fig. 5a-d). To enable detection of P32 by a commercially available antibody, we created a P32-encoding construct that carried a C-terminal 6X-His tag (Fig. 5b). To eliminate the possibility of tag-associated artifacts, parallel constructs without 6X-His tags were also introduced into M. pneumoniae II-3. To confirm that the 6X-His antibody did not cross- react with M. pneumoniae, whole-cell lysate from the parental strain was blotted in parallel with the positive control M. pneumoniae 24-A. No bands were detected (Fig. 6), confirming that all signal from this antibody could be attributed to P30His or P32His. P30 was detected by immunoblot analysis in wild-type M. pneumoniae and the II-

3 transformants containing the P30His and P30 constructs, strains 24-A and 37-D, respectively. P30 was not detected in either the P32His- or P32-producing strains (Fig. 7a), indicating a lack of reversion to the wild-type P30 allele. The 6X-His epitope tag was detected in all strains containing tagged constructs, but not in strains lacking such constructs (Fig. 7b).

28

P21MG does not complement phenotypes associated with P30 loss. To control for possible phenotypes conferred by P21MG or by additional P21MP, we created a plasmid, pOO30, which contains a derivative of the pOO17 insert and lacks the P32His-encoding gene (Fig. 7c). M. pneumoniae pOO30 transformant 30-C, which was representative of several transformants, was non-cytadherent (Table 1) and its cellular morphology was indistinguishable from II-3 (Fig. 8). Like mutant II-3, transformant 30-C did not attach to the flask during growth. Also, full stabilization of P65 was not evident (Fig. 7c). Overall,

P21MG did not complement any aberrant phenotype of mutant II-3, eliminating the possibility that our results were meaningfully impacted by the presence of this protein or its M. pneumoniae ortholog in our experimental constructs.

M. genitalium P32 complements P30 loss in mutant II-3. As a result of lacking P30, M. pneumoniae II-3 is non-cytadherent and possesses morphology quite distinct from that of wild-type M. pneumoniae. Rather than having a straight and narrow attachment organelle at the anterior end of the cell and a long trailing filament at the posterior end, II-3 cells exhibit extensive pleomorphy and most cells appear ovoid with one or more tip structures protruding from the cell body (Fig. 8; Romero-Arroyo et al., 1999). In addition, when grown in broth, II-3 cells form aggregates that are suspended in the medium. In contrast, wild-type M. pneumoniae cells are cytadherent and are dispersed individually on the surface of culture vessels (Fig. 8) and associated with attached microcolonies. The correlation between the absence of P30 production and the loss of both cytadherence and wild-type morphology has been well documented (Hasselbring et al., 2005; Romero- Arroyo et al., 1999). To determine the extent to which P32 could complement these phenotypes, HA assays and SEM were performed to gauge cytadherence and morphology, respectively. Also, we examined the site of P32His and P30His localization by epifluorescence microscopy. All P30- or P32-producing transformants grew attached to the surface of the flask, and they were all cytadherent, as indicated by the adherence of sheep erythrocytes to colonies (Table 1 and data not shown). The presence of the 6X-His tag did not interfere with hemadsorption and all colonies evaluated were indistinguishable from one another. SEM of P30- or P32-producing transformants revealed adherent cells with straight and

29 slender attachment organelles, relatively thin bodies, and long trailing filaments, which are all characteristic features of wild-type M. pneumoniae cells and distinct from wild- type M. genitalium cells (Fig. 8). When examined by indirect immunofluorescence microscopy using a 6X-His primary antibody for detection, P32His and P30His were found at discrete foci at the tips of the attachment organelles of transformed cells (Fig. 9). Together, these data suggest that P32 is trafficked to the same intracellular site as P30 within M. pneumoniae, and that P32 fully complements wild-type M. pneumoniae morphology and does not confer the curvature of the attachment organelle characteristic of M. genitalium. These data do not support a specific role for P30 in differential morphogenesis of the attachment organelle across species.

The attachment organelle protein P65 is stabilized in P32 and P32His-expressing transformants. The protein P65 is localized within the attachment organelle of M. pneumoniae and is unstable in the absence of P30 (Jordan et al., 2001). To determine whether P32 could restore P65 stabilization, transformant lysates were immunoblotted with a polyclonal P65 antiserum (Proft et al., 1995). Transformants expressing P32 produced more P65 than was present in mutant II-3 cells (Fig. 7c), suggesting stabilization of P65.

P32-expressing transformants glide at speeds similar to that of wild-type M. pneumoniae. The mean gliding speeds of both the P30- and P32-expressing transformants were similar to the mean gliding speed of the wild-type M. pneumoniae strain M129, although a small statistical difference, independent of the origin of the P30/P32 allele, was evident (Table 2). We suspect that this difference could be attributed to somewhat lower levels of P30 or P32 produced in transformants. Giving credence to this possibility are recent observations suggesting that low levels of P30 production by M. pneumoniae II-3 transformants correlate with slower gliding speed (in press). However, the 6X-His tag does not interfere with the function of P30 or P32 in gliding, since all transformants glided at similar speeds (Table 2).

30

Table 1. Bacterial strains, plasmids and primers used in this study.

Strain, plasmid, or primer Description Source / Reference

Bacterial strains Escherichia coli DH5α Used for plasmid propagation Laboratory stock Escherichia coli XL-1 Blue Used for mutagenesis Agilent Technologies Mycoplasma genitalium G37 Wild-type strain, HA+ Laboratory stock Mycoplasma pneumoniae M129 Wild-type strain, HA+ Laboratory stock Mycoplasma pneumoniae II-3 P30 –, host for cloning, HA– Laboratory stock Mycoplasma pneumoniae 12-A M. pneumoniae II-3 + M. genitalium G37 This study P32 operon, HA+ Mycoplasma pneumoniae 17-B M. pneumoniae II-3 + M. genitalium G37 This study + P32His operon, HA Mycoplasma pneumoniae 24-A M. pneumoniae II-3 + M. pneumoniae This study + M129 P30His operon, HA Mycoplasma pneumoniae 30-C M. pneumoniae II-3 + M. genitalium This study G37 P32 promoter region + P21 gene, HA– Mycoplasma pneumoniae 37-D M. pneumoniae II-3 + M. pneumoniae This study M129 P30 operon, HA+

Plasmids pCR®2.1 TA cloning vector Invitrogen pMT85 Suicide vector; contains Tn4001 Laboratory stock / Zimmerman and Herrmann, 2005 pOO12 pMT85 + M. genitalium G37 P32 This study operon pOO17 pMT85 + M. genitalium G37 P32His This study operon pOO24 pMT85 + M. pneumoniae M129 P30His This study operon pOO30 pMT85 + M. genitalium G37 P32 This study promoter region and P21 gene pOO37 pMT85 + M. pneumoniae M129 P30 operon This study

Amplification and Mutagenic Primers†‡ MGP21Bam TAGAGGATCCTAAAGCTCTATAGTT This study AAT RFRECORI AAAGAATTCTAAATTAGGGTTTTAA This study ACC RFRECORIHis TTTGAATTCTCTTTAGTGATGATGGTGG This study TGATGACCACCATAGGGTTTTAAACC MPN454Bam AAGGGATCCAAGCTGTAAGTGGGGAATT This study AAAGC MPN453EcoHis AGCGAATTCCTTTTAGTGATGATGGTGG This study TGATGACCACCATAGCGTTTTGGTGG ClaIMUT* GGAACTAATTAAGTATCGATAAAGATGG This study AGTT A_41_T* GGTGGTGATGACCACCTTAGCGTTTTGG This study TGGAA A_41_T-antisense* TTCCACCAAAACGCTAAGGTGGCATCA This study CCACC

† Restriction enzyme recognition sites are underlined. ‡ Polyhistidine epitope tag-encoding bases are in bold. * Primers used for site-directed mutagenesis.

31

Table 2. Gliding motility parameters of wild-type and transformant strains. Gliding speed Percentage of Percentage of frames Strain (nm s-1) ± SD Range moving cells (total containing moving cells n = 100 (nm s-1) cells analyzed) (total frames = 2600)

M. genitalium str. G37 111 ± 26 53 – 192 87 (575) 94 M. pneumoniae str. M129 269 ± 62 129 – 503 45 (463) 82 M. pneumoniae str. II-3 ------M. pneumoniae str. 12-A 237 ± 52 111 – 352 43 (255) 71 M. pneumoniae str. 17-B 236 ± 57 114 – 406 43 (553) 75 M. pneumoniae str. 24-A 252 ± 66 118 – 396 39 (788) 78 M. pneumoniae str. 30-C ------M. pneumoniae str. 37-D 237 ± 68 116 – 404 39 (554) 67

32

Fig. 4. Comparison of P30 orthologs from M. pneumoniae strain M129 and M. genitalium strain G37. Shaded amino acids are conserved between the two proteins. The highest amount of conservation occurs in the regions corresponding to the transmembrane domains. Amino acid sequences were aligned using ClustalX software.

33

M. pne P30 MKLPPRRKLKLFLLAWMLVLFSALIVLATLILVQHNNTELTEVKSELSPLNVVLHAEED- M. gen P32 MELNGFLRYKKLFIVLALLFTTILIVSLSLLAFAL------VVKTNGSELGVVFHQTEDN

M. pne P30 TVQIQGKPITEQAWFIPTVAGCFGFSALAIILGLAIGLPIVKRKEKRLLEEKERQEQLAE M. gen P32 TTVIQGRSIVEQPWFIPTVAGSFGFSALAIILGLAIGLPIVKRKEKRLLEEKERQEQIAE

M. pne P30 QLQRISAQQEEQ-QALEQQAAAEAHAEAEVEPAPQPVPVPPQPQVQINFGP----RTGFP M. gen P32 QLQRISDQQEQQTVEIDPQQSQAQPSQPQVQQPLQPQFQQRVPLLRPAFNPNMQQRPGFN

M. pne P30 PQPGMAPRPGMPPHPGMAP---RPGFPPQPGMAPRPGMP-PHPGMAPRPGFPP--QPGMA M. gen P32 -QPNQQFQPHNNFNPRMNPNMQRPGFNPN--MQQRPGFNQPNQQFQPHNNFNPRMNPNMQ

M. pne P30 PRPGM-PPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR---- M. gen P32 -RPGFNQPHPNQFAQPNN-FNPNMQQRPGFNPNMQQRPNPSQLMPKGGLK

34

Fig. 5. Constructs generated for this study. PCR was used to amplify the genes of interest from genomic DNA of either wild-type M. pneumoniae strain M129 or wild-type M. genitalium strain G37. Amplicons were cloned in the TA cloning vector pCR®2.1, sequenced, and subcloned into the Tn4001-containing vector pMT85 for delivery of the constructs into M. pneumoniae II-3 or M. pneumoniae M129. (a) construct in pOO12; (b) construct in pOO17; (c) construct in pOO30; (d) construct in pOO24. The construct in pOO37 is a derivative of that which is in pOO24; it contains the original MPN453 translational stop codon (the position of which is indicated by the vertical arrow) and does not include the 6X-His epitope tag.

35

36

Fig. 6. Immunoblot confirmation that the 6X-His antibody does not cross react with any proteins in non-transformed M. pneumoniae. Wild-type M. pneumoniae strain M129 total cell lysate was probed with the 6X-His antibody alongside the transformant M. pneumoniae 24-A. Molecular weight markers indicated at left.

37

38

Fig. 7. Immunoblot analysis for the demonstration of P30, P30His, P32His, and P65. Whole-cell lysates of wild-type M. pneumoniae strain M129, M. pneumoniae II-3, and the transformant strains M. pneumoniae 30-C, 12-A, 17-B, 24-A, and 37-D were probed with anti-P30 (a), anti-6X-His (b), or anti-P65 (c) antibodies. Molecular weight markers indicated at left.

39

40

Fig. 8. Morphology of strains used in this assay. SEM reveals that transformants containing either P30 or P32, with or without the C-terminal 6X-His tag, are indistinguishable from wild-type M. pneumoniae M129. Strains are indicated in the upper left of each panel, with G37 indicating M. genitalium. White arrowheads indicate attachment organelles and the black arrowhead indicates a trailing filament. Scale bar, 1 µm.

41

42

Fig. 9. P30His and P32His localize to the attachment organelle tip, the site of localization of native P30. Whole cells grown on glass coverslips (red channel) were probed with a monoclonal anti-6X-His antibody and a FITC-conjugated secondary antibody (green channel). Strains are indicated in the upper left of each panel, except that NC indicates wild-type cells processed with only secondary antibody as a negative control. Scale bar, 2 µm.

43

44

Discussion

The mechanisms underlying differences in gliding speed and other attachment organelle properties in members of the M. pneumoniae phylogenetic cluster are not understood. For this reason, we chose to investigate the contribution of the P30 family of transmembrane attachment organelle proteins by using orthologous gene replacement, a technique that has not previously been reported using mycoplasmas. This approach allowed for evaluation of phenotypes conferred by the M. genitalium P32 protein to the P30 null mutant M. pneumoniae II-3, allowing us to address aspects of the specific role of P30 in attachment organelle function. We focused on whether the substantial sequence differences between the two orthologs (Fig. 4) contributed in any observable way to the distinct attachment organelle phenotypes of the two organisms. Transformants with P32 constructs were cytadherent and morphologically indistinguishable from wild-type M. pneumoniae cells. Moreover, gliding speeds and stabilization of attachment organelle protein P65 in transformants producing P32 and

P32His were very similar to wild-type M. pneumoniae and M. pneumoniae II-3 producing

P30 and P30His, although there was in some transformants a subtle decrease in speed that could be attributable to the somewhat reduced amount of P30 or P32 present in these cells. Like P30, P32 localized to the attachment organelle, suggesting that they are equivalent substrates for the unknown trafficking mechanism responsible for M. pneumoniae cell polarity. These data suggest that the sequence differences between P30 and P32 are not responsible for the obvious morphological differences seen in the attachment organelles of M. pneumoniae and M. genitalium (Fig. 8; Hatchel & Balish, 2008). Overall, these analyses indicate that P32 functions in M. pneumoniae about as well as P30. Conceivably, the high similarity between P30 and P32 in and near the transmembrane domain provides the basis for the ability of P32 to substitute for P30, supporting an important role for this region in protein function, as suggested by other studies (Chang et al., 2011). Additionally, P30 and P32 have in common a proline-rich repeat region at the C-terminus, although the repeats themselves are different and less regular in P32. The fact that alterations to this region in P30 lead to significant

45 compromise of function (Jordan et al., 2007; Chang et al., 2011) makes it surprising that the divergent corresponding region in P32 is fully substitutable. These results suggest that the overall structure of the domain is similar in P30 and P32, and loss of repeat elements is deleterious to this structure. The specific role of P30 in the process of cellular gliding is not yet known, except that our evidence suggests that its activity is not rate-limiting in terms of gliding speed. We envision a scenario in which the M. pneumoniae gliding machinery is at least partly analogous to that of M. mobile (Fig. 2), in which an adhesin, Gli349 (Adan-Kubo et al, 2006; Nakane et al., 2011), is proposed to undergo conformational changes with respect to the substrate, directly generating movement; these changes are driven ultimately by an ATPase and proximately by proteins like Gli521 that bridge the adhesin and the ATPase (Miyata, 2010). One possibility for P30 function is that it is analogous to Gli349, which is supported by its known adherence function (Morrison-Plummer et al., 1986). Another possibility is that P30 interacts with and promotes the activity of proteins that directly drive motility themselves. In M. mobile, the protein Gli521 is proposed to transfer the propulsive force to Gli349 (Miyata, 2010). By analogy, P30 might perform this role in M. pneumoniae, with some other attachment organelle protein like the major adhesin P1 in the role of Gli349. In neither case is P30 rate-limiting, leaving other components of the M. pneumoniae gliding apparatus responsible for species-specific differences in speed, possibly including other proteins involved in adherence, like P1, as well as any unidentified proteins that provide energy to drive conformational changes. As an additional possibility, the number of P30 molecules, rather than the individual activity of each one, might control gliding speed, as suggested by the slight reduction in speed in transformants producing slightly less P30His or P32His (in press). P30 sequence differences also appear not to have a specific role in attachment organelle morphology, although MG217, the ortholog of the P30-associated protein P65, is linked to attachment organelle curvature in M. genitalium (Burgos et al., 2008). The work presented here demonstrates for the first time the usefulness of orthologous gene transformation as a means for assessing protein function in mycoplasmas. Experiments to further characterize the contribution of P30 proteins from

46 other related Mycoplasma species as well as the roles of other attachment organelle proteins is currently underway using methods adapted from this research.

47

Acknowledgements

This work was supported by Public Health Service grant R15 AI073994 from the National Institutes of Health and by the Miami University Doctoral-Undergraduate Opportunities in Scholarship program. We thank Dr. Duncan Krause (University of Georgia) for the generous gift of anti-P30 antiserum, Mr. Michael Hughes (Miami University) for help with statistical analysis of gliding motility, and Dr. Iddo Friedberg (Miami University) for advice about protein comparison software. We would also like to thank the Miami University Center for Bioinformatics and Functional Genomics and the Miami University Center for Advanced Microscopy and Imaging for use of their equipment and facilities.

48

CHAPTER 2

Gliding Speed Positively Correlates with the Amount of the Attachment Organelle Protein P30 in Mycoplasma pneumoniae

Ryan F. Relich and Mitchell F. Balish Submitted to the Journal Microbiology

49

Summary

The human pathogen Mycoplasma pneumoniae is cytadherent and exhibits gliding motility. Cytadherence and gliding motility are both conferred by a differentiated tip structure called the attachment organelle. This polar prostheca contains a detergent- insoluble electron-dense core and several transmembrane proteins that function in adherence and gliding motility. The transmembrane adhesin P30 is crucial for both cytadherence and gliding motility, but the nature of its contribution to gliding motility is unclear. We used the M. pneumoniae ldh promoter to drive production of C-terminally 6X-His-tagged P30 in the M. pneumoniae P30 null mutant II-3 at levels lower than wild- type and obtained several transformants producing different levels of P30 that exhibited reduced adherence to surfaces. Scanning electron microscopy indicated that, below a threshold level of P30, cells assume an aberrant morphology closely resembling that of mutant II-3. Analysis of cellular gliding properties by phase-contrast microcinematography demonstrated a successive reduction of gliding speed in transformants producing decreasing amounts of P30. These effects are independent of the attachment organelle protein P65, which is fully stabilized in all transformants. Together, these data suggest a strong positive correlation between the amount of P30 produced by M. pneumoniae and gliding speed, from which we infer that P30 plays a significant role in the control of gliding motility speed in M. pneumoniae and its relatives.

50

Introduction

The human pathogen Mycoplasma pneumoniae is an important cause of pulmonary disease, responsible for up to 30% of all cases of community-acquired pneumonia (Kannan et al., 2010). This organism is also implicated in the exacerbation of asthma and can cause extrapulmonary diseases, including central nervous system and dermatological manifestations (Waites & Talkington, 2004, Waites et al., 2008; Esposito et al., 2011). Transmission of M. pneumoniae appears to occur most commonly by inhalation of infectious aerosols expelled by infected persons (Waites & Talkington, 2004). Once inside a new host, M. pneumoniae adheres to mucosal epithelial cell surfaces of the respiratory tract, a step that is essential for the persistence of this organism and an attribute that is critical to its pathogenicity (Waites & Talkington, 2004). The elaboration of secreted factors, including hydrogen peroxide and the community-acquired respiratory distress syndrome toxin, appears to play roles in pathology subsequent to adherence of M. pneumoniae to the respiratory epithelium (Kannan & Baseman, 2006; Waites et al., 2008). A characteristic of M. pneumoniae associated with cytadherence is a surface- dependent form of motility that is categorized as gliding (Jordan et al., 2007). Gliding motility is a well-characterized phenotype of M. pneumoniae and appears to play a role in the normal cell division process of both M. pneumoniae and its close relative, Mycoplasma genitalium (Hasselbring et al., 2006; Lluch-Senar et al., 2010). Both cytadherence (Collier & Clyde, 1971) and gliding motility (Hasselbring & Krause, 2007) are conferred by a differentiated polar tip structure called the attachment organelle. Duplication of this prostheca occurs early in cytokinesis and precedes division of the cell body (Bredt, 1968; Seto et al., 2001). Analysis of attachment organelles of M. pneumoniae and several of its close relatives by electron microscopy following extraction with the nonionic detergent Triton X-100 reveals the presence of a detergent-insoluble, electron-dense core (Biberfeld & Biberfeld, 1970; Meng & Pfister, 1980; Göbel et al., 1981; Hatchel & Balish, 2008). Various forms of immunocytochemistry indicate that the attachment organelle of M. pneumoniae is the location of several proteins that function in cytadherence and/or gliding motility (Krause & Balish, 2004; Kenri et al., 2004; Jordan

51 et al., 2007; Cloward & Krause, 2009). Some, like P1 and P30, are implicated as adhesins (Hu et al., 1977; Morrison-Plummer et al., 1986), with proteins B and C likely having related function (Waldo & Krause, 2006). Another protein, TopJ, appears to serve as a co-chaperone in attachment organelle assembly (Cloward & Krause, 2009). However, the specific functions of most of the rest of the attachment organelle proteins remain to be elucidated, including HMW1, HMW2, HMW3, P200, and P65, for all of which general structural roles are inferred (Balish & Krause, 2006). The attachment organelle is both the leading end of the cell during gliding motility (Bredt, 1968) and houses the gliding motor for locomotion (Hasselbring & Krause, 2007). Interestingly, different relatives of M. pneumoniae glide at different characteristic speeds over a 60-fold range, despite having similar attachment organelle organization (Hatchel et al., 2008). The hypothesis that gliding speed is both important for virulence and in some way calibrated to each species is exemplified by a mutant strain of M. pneumoniae that glides at a speed similar to that of the pathogen M. genitalium but is unable to successfully colonize an epithelial cell monolayer (Jordan et al., 2007). The mechanism by which M. pneumoniae glides, including factors responsible for its gliding speed, is unknown. Although models invoking movements of the electron- dense core have been proposed (Hegermann et al., 2002; Henderson & Jensen, 2006), other lines of evidence implicate the adhesins in gliding. The unrelated adhesin Gli349 of Mycoplasma mobile, whose tip structure is distinct from that of M. pneumoniae (Nakane & Miyata, 2007), is proposed to undergo conformational changes, driven by ATP hydrolysis by an associated protein, that allow for successive substrate attachment-and- release cycles, propelling cells forward (Miyata, 2010). Although these proteins lack homologs in M. pneumoniae, antibodies against P1 inhibit motility prior to causing detachment from the surface (Seto et al., 2005a), implicating P1 in a similar process in M. pneumoniae. Likewise, defects in the surface-exposed C-terminal region of P30 are associated with reduced motility. M. pneumoniae mutant II-3, which has a frameshift mutation in MPN453, the P30-encoding gene, produces no detectable P30 protein fragments, is non- adherent, and has reduced levels of attachment organelle protein P65 (Romero-Arroyo et al., 1999; Jordan et al., 2001; Chang et al., 2011). In the slowly gliding strain II-3R, a

52 pair of frameshift mutations results in production of a variant P30 in which a region of 17 amino acids is altered (Hasselbring et al., 2005). Similarly, gliding speed is significantly diminished in mutant II-7 (Hasselbring et al., 2005), a spontaneous non-motile mutant expressing a truncated P30 in which 48 amino acids are deleted from the surface-exposed domain (Dallo et al., 1996). Successive truncation of the C-terminus of P30 results in strains with increasingly smaller amounts of P30 and increasingly reduced ability to adhere and glide (Chang et al., 2011). The Mycoplasma genitalium ortholog of P30, P32, is compositionally distinct from P30 in its C-terminal region but fully complements mutant II-3 (Relich & Balish, 2011), suggesting that the overall structure of the C- terminal region is similar in both proteins. These data suggest that P30 affects gliding speed in ways that relate to its composition and/or stability. To test the role of P30 quantity in gliding motility, we generated several M. pneumoniae strains in which a C-terminally 6X-His-tagged P30 (P30His) is under the control of the M. pneumoniae ldh promoter (Halbedel et al., 2007) in place of the native promoter. These strains were found to have reduced levels of P30 as compared with wild- type M. pneumoniae cells, and also produced different amounts of P30 as compared with each other. These strains enabled us to analyze the impact of P30 levels on gliding speed and other P30-dependent characteristics, including cell morphology.

53

Materials and Methods

Propagation of bacterial strains. Mycoplasma pneumoniae was grown in SP-4 broth (Tully et al., 1979) in tissue culture flasks; for transformants, 18 µg ml-1 gentamicin (Sigma-Aldrich, St. Louis, MO) was included. For assays that required plating of M. pneumoniae strains, SP-4 with or without 18 µg ml-1 gentamicin plus 1% (wt/vol) Noble agar (Becton, Dickinson and Company, Franklin Lakes, NJ) was used. In all cases, mycoplasmas were incubated in ambient air at 37°C. Escherichia coli strains harboring -1 modified pMT85 plasmids were grown in LB medium containing 100 µg ml ampicillin.

PCR and mutagenesis. The polymerase chain reaction (PCR) was used to amplify a 170-bp region immediately upstream of the M. pneumoniae ldh translational start codon using the primers LDHBamHI (5’GGATCCTGTTAATTATACTTTTGCC-3’) and LDHClaI (5’-CATCGATAATTTATTCAAGCTTGCC-3’). The following thermal cycler reaction conditions were used with Taq polymerase (New England BioLabs, Ipswich, MA) and the EasyStart Micro50 kit (Molecular BioProducts, San Diego, CA): 95°C for 3 min for initial template denaturation; 35 cycles of 95°C for 1 min, 54°C for 30 s, and 72°C for 1 min; and a final 5-min incubation at 72°C. Five-µl aliquots of reactions were resolved on a 2% (wt/vol) agarose gel for confirmation of amplicons. A total of 13 separate reactions were pooled, precipitated with ethanol, and air-dried. The amplicon pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0 and stored at - 20°C until further use. Site-directed mutagenesis was used to modify a previously constructed plasmid containing a P30His construct of M. pneumoniae wild-type strain M129 (pOO21; in press) in order to accommodate the insertion of the ldh promoter region in place of the native promoter that lies upstream of the gene encoding protein P21 (Hahn et al., 1998). Briefly, the primers Mut21-ClaI-Fwd (5’-GAAGAAACCCATCGATAGAGAAGAG TTAAATATTG-3’) and Mut21-ClaI-Rev (5’CAATATTTAACTCTTCTCTATC GATGGGTTTCTTC-3’) were used to engineer a ClaI restriction site immediately upstream of the P21 translational start site by using the QuikChange® II Site Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA) according to the

54 manufacturer’s instructions. Because the original construct within pOO21 was engineered to contain a BamHI restriction site upstream of the native promoter, double digestion with BamHI and ClaI of mutagenized pOO21 allowed us to remove the native promoter region and ligate the ldh promoter amplicon in its place. The resulting plasmid, pOO34, was cloned in Escherichia coli DH5α under selection with 100 µg ml-1 ampicillin on Luria- Bertani agar. Clones were screened for the presence of the ldh promoter by restriction analysis of purified plasmids from E. coli transformants using BamHI and EcoRI. To assess sequence validity, plasmids containing an ~170-bp DNA fragment were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) followed by capillary electrophoresis on an Applied Biosystems 3130xl genetic analyzer (Applied Biosystems, Foster City, CA) at the Miami University Center for Bioinformatics and Functional Genomics.

Transformation of M. pneumoniae II-3. The Tn4001-containing plasmid pMT85 (Zimmermann & Herrmann, 2005) was used to introduce the insert into competent M. pneumoniae II-3. The pOO34 insert was excised by restriction digestion with BamHI and EcoRI. Following digestion of pOO34, the insert was ligated into complementary sites within Tn4001 of pMT85. The recircularized plasmid was next introduced into competent M. pneumoniae II-3 cells via electroporation according to a standard protocol (Hedreyda et al., 1993). Following pulsing of the cell-plasmid mixture, cells were transferred to SP- 4 broth, incubated for 1 h at 37°C, diluted 10-fold, and plated in 100-µl volumes on SP-4 + 1% (wt/vol) Noble agar with and without gentamicin. Plates were incubated at 37°C with ambient air in a humidified chamber. Multiple gentamicin-resistant colonies were picked to control for transposon insertion-site positional effects. The transformants were designated M. pneumoniae 36-series. Colony subcultures were grown in 250-µl aliquots of SP-4 broth containing gentamicin until the pH indicator was yellow (approximately 7 d) after which time the broth was passed through a 0.22-µm syringe filter and re-plated on SP-4 + 1% (wt/vol) Noble agar containing gentamicin using 100-µl aliquots of 10-fold dilutions. This filter cloning procedure was carried out a total of three times to assure the acquisition of genetically homogeneous transformants. SP-4 stocks were made from the third filter-cloned broth subcultures and frozen at -80°C until further use. For generation

55 of M. pneumoniae 24-A, the original pOO21 insert, containing the P30His-encoding gene under control of its native promoter, was subcloned into pMT85 and subsequently introduced into M. pneumoniae II-3. Transformants were isolated using the protocol described above for M. pneumoniae 36-series transformants.

Hemadsorption assay. To assess cytadherence capability of transformants, cells were plated to SP-4 agar and incubated in a humidified chamber at 37°C for approximately 7 d. Hemadsorption (HA) was carried out as previously described (Sobeslavsky et al., 1968) using sheep blood in Alsever’s solution, and an additional wash in 150 mM NaCl,

3.2 mM NaH2PO4, 13.6 mM Na2HPO4, pH 7.2 (PBS) was also performed. Images were captured using a Leica DM IRB inverted phase-contrast microscope (Leica Microsystems, Wetzlar, Germany) and equipped with a SPOT charge-coupled device camera and the accompanying imaging software (Diagnostic Instrument, Inc., Sterling Heights, MI).

Immunolocalization of P30His: To detect P30His in the M. pneumoniae 36-series transformants, a modification of the protocol by Jordan et al. (2001) was used. The modifications included use of 200-µl aliquots of cells, incubation of cells with 2 µg ml-1 anti-6X-His primary antibody (Immunology Consultants Laboratories, Newberg, OR), and incubation of cells with a 1:100 dilution of a FITC-conjugated secondary antibody (KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Coverslips were mounted on glass microscope slides using VECTASHIELD (Vector Laboratories, Burlingame, CA) before being examined with a Leica DM IRB inverted microscope (Leica Microsystems, Weilburg, Germany). Images were manipulated with SPOT v4.1.1 software and Adobe Photoshop (Adobe, San Jose, CA) to minimize background fluorescence while maintaining the integrity of cellular P30His foci.

Immunoblotting. M. pneumoniae 36-series transformants and mutant II-3 were grown in 50 ml SP-4 with or without gentamicin, respectively, until mid-logarithmic phase of growth, then centrifuged at 17,400 x g at 4°C. Cell pellets were washed 3 times with PBS and resuspended in 150 mM NaCl, 20 mM Tris HCl, pH 7.5 by trituration with a 25-

56 gauge needle and a 1-ml syringe. Protein quantification was carried out using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL). For detection of the proteins HMW2 and HMW3, 5 µg of protein was resolved on a 5% (vol/vol) sodium dodecyl sulfate-polyacrylamide gel (Laemmli, 1970). For detection of HMW1, P1, P30, P65, and P200, 5 µg of protein was resolved on a 12% gel. For estimation of relative P30His concentration in 36-series transformants, samples of M. pneumoniae 24-A protein of 0.5, 1, 2, 3, 4 and 5 µg were run alongside 5-µg protein aliquots of each of the 36-series transformants and electrophoresed through 12% gels. Proteins were transferred to a nitrocellulose membrane for antibody detection (Towbin et al., 1979). The following antiserum dilutions were used for protein detection: 1:10,000 for HMW1 (Stevens & Krause, 1991) and HMW3 (Stevens & Krause, 1992) antisera; 1:1,000 for anti-P1 (Baseman et al., 1982), anti-P65 (Proft et al., 1995), and anti-6X-His antisera (Immunology Laboratory Consultants, Inc., Newberg, OR); 1:2,000 for HMW2 (Krause et al., 1997) and P200 (Proft et al., 1996) antisera; and 1:250 for P30 antiserum (Romero-Arroyo et al., 1999). Primary antibodies were detected with an alkaline phosphatase-conjugated goat anti-rabbit antibody (Promega, Madison, WI).

Glycerol induction assays. M. pneumoniae 36-series transformants were cultured in SP- 4 broth containing gentamicin with or without 0.01%, 0.1%, and 1% (vol/vol) glycerol in 24-well tissue-culture plates. Following incubation of plates at 37°C for up to 48 h, plates were examined with an inverted compound microscope to assess adherence of cells. As a control, the transformant M. pneumoniae 24-A, which produces P30His and is phenotypically indistinguishable from wild-type cells, was included. For detection of

P30His, 5 µg protein from lysates of M. pneumoniae transformant 36-D grown in SP-4 broth in the absence or presence of 1% added glycerol was subjected to immunoblotting with anti-6X-His as described above.

Motility analysis. To assess gliding properties of transformants, phase-contrast microcinematography was performed as previously described (Relich et al., 2009) with the following modifications: to prepare 50-µl motility stocks, adherent monolayers were

57 scraped into 1 ml SP-4 broth containing gentamicin, and images were taken at varying intervals. Analysis of motility movies was done using SPOT v4.1.1 imaging software.

Scanning electron microscopy. SP-4 stocks of M. pneumoniae 36-series transformants and mutant II-3 were inoculated into a 24-well tissue culture plate containing 18-mm diameter sterile glass coverslips and SP-4 broth with or without 18 µg ml-1 gentamicin, respectively. Plates were incubated until mid-logarithmic phase growth (when the pH indicator was orange), at which time the broth was aspirated from each well and cells were fixed with 1.5% glutaraldehyde, 1% formaldehyde, 0.1 M sodium cacodylate, pH 7.2, for 30 min at room temperature. Subsequently, coverslips were dehydrated with increasing concentrations of ethanol, critical-point dried, mounted, and gold sputter- coated as previously described (Hatchel et al., 2006). Micrographs were captured with a Zeiss Supra 35 FEG-VP scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) at the Miami University Center for Advanced Microscopy and Imaging.

58

Results

Native ldh promoter activity is sufficient for P30 gene expression. In their experiments to characterize ldh promoter activity in M. pneumoniae, Halbedel and colleagues (2007) found that 1% glycerol significantly increased ldh expression and lactate dehydrogenase protein levels. This observation suggested that the ldh promoter, extending 53 bases upstream of the translation start site (Halbedel et al., 2007), could be induced with exogenous glycerol. In an effort to obtain a construct in which production of P30 in M. pneumoniae could be modulated, we placed its coding gene, MPN453, under the control of the ldh promoter. We amplified the 170-bp region directly upstream of the translational start codon of M. pneumoniae wild-type strain M129 ldh, which was subsequently ligated in place of the native promoter region in a construct for expression of P30His, including the P21-encoding gene MPN454, which has no impact on attachment organelle function (Relich & Balish, 2011). For construct delivery into competent M. pneumoniae II-3 cells, we used the Tn4001-containing vector pMT85 (Zimmermann & Herrmann, 2005). Following transformation of M. pneumoniae II-3 and purification of clones, we incubated cells of several transformants in SP-4 broth containing 18 µg ml-1 gentamicin with 0.01%, 0.1%, 1%, or no glycerol in 24-well tissue-culture plates. Following incubation of plates at 37°C, adherent cells and microcolonies were observed in all wells (data not shown). P30His levels were unaffected by glycerol as determined by immunoblot with a monoclonal 6X-His antibody (Fig. 10), indicating that the ldh promoter is not suitable for modulation of P30 expression using glycerol in this manner.

However, we noted that the amount of P30His produced in the transformant we selected, 36-D, was distinctly lower than in transformant 24-A, a derivative of mutant II-3 in which P30His was produced under the control of its native promoter (Relich & Balish, 2011). We further noted that some transformants seemed to produce more unattached microcolonies than others, suggesting that, despite being adherent, P30His levels could differ among transformants. Transformants were grown on SP-4 agar containing gentamicin and were then assayed for cytadherence capability using the hemadsorption assay. All M. pneumoniae 36-series transformants assayed were cytadherent, which was

59 indicated by the presence of sheep erythrocytes attached to the surface of the colonies (Fig. 11). In addition, indirect immunofluorescence microscopy confirmed the presence of P30His exclusively at the tips of the attachment organelles of transformants, represented by transformant M. pneumoniae 36-C (Fig. 12).

Levels of P30 differ in M. pneumoniae 36-series transformants. To determine relative

P30His levels among the adherent M. pneumoniae 36-series transformants, we immunoblotted lysates from multiple transformants and compared them to M. pneumoniae 24-A. This transformant produces P30His in place of P30 at a level somewhat lower than that of P30 in wild-type M. pneumoniae (Fig. 13B), yet it glides at nearly the same speed as wild-type cells (Relich & Balish, 2011), indicating that the slight reduction in the amount of P30 has little impact on gliding speed. Different transformants exhibited varying amounts of 6X-His signal, corresponding to varying low amounts of P30 production in these transformants. Among the M. pneumoniae transformants producing

P30His under the control of the ldh promoter, 36-C and 36-I produced the most P30His, approximately 50% and 15% of 24-A, respectively (Fig. 13A). M. pneumoniae 36-D and 36-G produced the least, as both were approximately 10% of 24-A (Fig. 13A). The reduction in P30His levels was confirmed by immunoblotting with a polyclonal P30 antiserum (Fig. 13B). To verify that the introduced constructs were not mutated within the M. pneumoniae II-3 cells, we PCR-amplified and sequenced the ldh-P30His operon constructs from these four transformants. Analysis of sequences from each of the four transformants indicated that the variability in P30His levels was not due to acquisition of mutations.

Levels of other attachment organelle proteins are unaffected in the transformants. To test whether the phenotypes observed in the M. pneumoniae 36-series transformants were the result of deleterious transposon insertions, we performed immunoblots for the proteins P65 (Fig. 13C), HMW1, HMW2, HMW3, P1, and P200 (data not shown), all of which are implicated in M. pneumoniae attachment organelle-associated processes. One very poorly adherent transformant did not produce P1, suggesting that we were able to detect second site mutations in attachment organelle genes; it was not further considered.

60

Among the remaining transformants, all of these proteins were present at wild-type levels. Of particular significance is that P65 is present at somewhat reduced steady-state levels in M. pneumoniae mutant II-3, indicating a direct requirement for P30 in its stabilization (Jordan et al., 2001). These data indicate that P65 is stabilized by even low levels of P30, and, importantly, that the phenotypes of these strains, described below, are independent of P65 levels.

Gliding speed is greatly diminished in M. pneumoniae cells producing low levels of P30. Spontaneous mutations in the P30-encoding gene resulting in either amino acid substitutions or deletions negatively impact gliding motility in ways that are not fully understood (Hasselbring et al., 2005; Chang et al., 2011), possibly implicating the amount of P30 in gliding characteristics. Transformant M. pneumoniae 24-A, encoding a C-terminally 6X-His-tagged P30 under transcriptional control of its native promoter, glided at approximately wild-type speed despite producing less P30 than wild-type M. pneumoniae (Relich & Balish, 2011), indicating that neither the 6X-His tag nor the lower amount of P30 produced by this transformant affect P30 function in gliding. In contrast, gliding motility analysis of M. pneumoniae 36-C, 36-D, 36-G, and 36-I indicated significant decreases in gliding speed. 36-C, which produced the most P30His among the transformants, approximately one-half that of 24-A (Fig. 13A), glided at an average speed of 86 nm s-1 ± 42 nm s-1, which is three times slower than both the wild-type and 24-A (Hatchel et al., 2006; Relich & Balish, 2011). These data indicate that when P30 concentration dwindles to a point somewhere below that found in 24-A, decreasing amounts of P30 are associated with decreased speed. Gliding motility analysis of 36-D, 36-G, and 36-I was complicated by the fact that very few cells were motile and those that did move were so slow that accurate analysis of speed was precluded, but in all cases they glided at approximately 1/100 to 1/30 the speed of wild-type cells.

Cellular morphology is altered by decreased gliding speed. In addition to loss of cytadherence and gliding properties by the P30 null mutant II-3, cellular morphology is also altered. Rather than the typical spindle-like appearance of the wild-type M. pneumoniae strain M129, those of II-3 exhibit profound pleomorphy (Romero-Arroyo et

61 al., 1999). They typically have multiple tip structures, reinforcing a link between gliding and normal cell division. Also, whereas gliding-competent cells are capable of forming adherent microcolonies from which individual cells emerge and eventually glide away, II-3 cells largely remain closely associated, growing in suspended microcolonies.

Scanning electron microscopy indicated transformants producing successively less P30His exhibited successively more aberrant cellular morphology, approaching that of mutant II-

3 (Fig. 14). Transformant 36-C, which produced the most P30His of the four transformants analyzed and glided the fastest among them, was indistinguishable from wild-type.

Transformant 36-I, which produced P30His at levels between those of 36-C and 36-D/36- G appeared very similar to wild-type as well, but with a higher degree of pleomorphy.

Finally, transformants 36-D and 36-G, which produced the least P30His, appeared noticeably different from wild-type cells. Also, fewer cells of these transformants were attached to the surfaces of the coverslips, and in broth, these two transformants produced mostly suspended microcolonies (data not shown). Although the morphologies of even the slowest transformants were more like wild-type cells than like mutant II-3 cells, these data suggest that attachment organelle-mediated motility contributes to the acquisition of a normal, extended shape by M. pneumoniae cells.

62

Fig. 10. Immunoblot analysis of P30His in the transformant M. pneumoniae 36-D grown in the presence of 1% added glycerol (+) or no added glycerol (-). M. pneumoniae 24-A and mutant II-3 were included as positive and negative controls, respectively.

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Fig. 11. Hemadsorption analysis of wild-type M. pneumoniae strain M129, P30 null mutant II-3, and the 36-series transformants. Strains are indicated in the upper left of each panel. WT, wild-type M. pneumoniae strain M129. Scale bar, 25 µm.

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Fig. 12. Immunolocalization of P30His in transformant M. pneumoniae 36-C, a representative for all other M. pneumoniae 36-series transformants. The fluorescent signal is the bright focus adjacent to the cell body, visualized by phase-contrast microscopy as dark. Bar: 2 µm.

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Fig. 13. Immunoblot analysis of various attachment organelle proteins in the M. pneumoniae 36-series transformants. (a), anti-6X-His; (b), anti-P30; and (c), anti-P65.

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Fig. 14. Scanning electron microscopy of M. pneumoniae 36-series transformants expressing different low levels of P30. Strains are indicated in the upper left of each panel. WT, wild-type M. pneumoniae strain M129. Scale bar, 1 µm.

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72

Discussion

Although a tetracycline-inducible gene expression system for Mycoplasma agalactiae has been described (Breton et al., 2010), at present, exogenous proteins can only be produced constitutively in M. pneumoniae. The inducible nature of ldh in M. pneumoniae (Halbedel et al., 2007) offered the potential for glycerol-mediated regulation of gene expression, leading us to test whether the ldh promoter would function in that manner. However, although the ldh promoter served to enable production of P30His, we could not recapitulate glycerol induction, suggesting that elements within ldh itself also contribute to inducibility. Nonetheless, we were able to use the ldh promoter to obtain significant underexpression of the P30His-encoding gene as compared with the native promoter. Interestingly, different transformants produced different amounts of protein, enabling us to use these transformants to study the effects of various reduced levels of P30 on M. pneumoniae gliding motility. Conceivably, the differences in protein production in different transformants might result from random insertion of Tn4001 at different chromosomal loci (Mahairas & Minion, 1989), but this remains to be tested. At any rate, analyzing multiple strains resulting from transformation with a relatively weak promoter might be a generally useful tool for studying stoichiometry-oriented questions in M. pneumoniae. In M. pneumoniae, the mechanism for this gliding is unknown but might be driven by conformational changes in adhesins by analogy with the unrelated structure in M. mobile (Adan-Kubo et al., 2006; Nakane & Miyata, 2007). An essential component of the attachment organelle that is required for adherence and gliding motility in M. pneumoniae is the transmembrane protein P30. Loss or mutation of this protein deleteriously affects cytadherence, gliding motility, morphology, and stabilization of attachment organelle protein P65. The exact role of P30 in gliding motility is not known, but it has been speculated that P30 is needed to stabilize the major M. pneumoniae adhesin, P1, which might be directly involved in motility (Hasselbring et al., 2005; Seto et al., 2005a). It is conceivable that P30 directly transduces the force needed for cell propulsion in a manner analogous to M. mobile Gli349, which directly interacts with surfaces and uses a force generated and transmitted by other proteins to thrust the cell

73 forward (Fig. 2; Uenoyama et al., 2004; Miyata, 2010). If so, then reduction in the number of P30 molecules would result in fewer substrate binding-and-release cycles, slowing the movement of the cell. A second possibility is that the cell surface-exposed C- terminal region of P30 interacts with P1 in a manner analogous to M. mobile Gli521 (Seto et al., 2005b; Miyata, 2010) in its interaction with Gli349. In this model, P30 would transmit the propulsive force generated by unknown proteins to P1, which in turn would move the cell forward. If P30 concentration were reduced, fewer P1 propulsive events would occur, leading to slower cell movement.

Efforts to overproduce P30 by expressing the P30His-encoding gene in wild-type M. pneumoniae cells resulted in only a very small amount of extra P30, with no increase in gliding speed (data not shown), suggesting that there is an upper limit to the amount of P30 that M. pneumoniae will produce. Moreover, the absence of a major difference in gliding speed between wild-type cells and those of transformant 24-A (Relich & Balish, 2011), suggests that there is also an upper limit to gliding speed. Interestingly, with the exception of Mycoplasma testudinis, no species in the M. pneumoniae cluster glides faster than M. pneumoniae (Hatchel & Balish, 2008). It will be interesting to determine whether M. testudinis, whose speed is an order of magnitude greater than M. pneumoniae, employs an alternative gliding mechanism; assuming so, it may be that wild-type M. pneumoniae exhibits the maximum gliding speed afforded by the attachment organelle components it shares with its relatives. Our results here provide further evidence for P30 playing important roles in gliding motility. In particular, analysis of transformants producing different, reduced amounts of P30 points to a positive correlation between the amount of P30 produced and gliding speed that is independent of the P30-dependent protein P65. Six other species of the M. pneumoniae cluster are motile, with mean gliding speeds varying over a 60-fold range (Hatchel & Balish, 2008). Reduction of M. pneumoniae gliding speed to approximately that of M. genitalium due to a mutation in the attachment organelle protein P200 prevents colonization of, but not adherence to, epithelial cells (Jordan et al., 2007), suggesting that the gliding speed in each species is calibrated to other as yet unidentified features of the organism. Experiments aimed at determining the roles of both the

74 composition and the amount of P30 in controlling gliding speed across these species are underway.

75

Acknowledgements

This work was supported by Public Health Service grant R15 AI073994 from the National Institutes of Health and by funds granted by the Miami University Doctoral- Undergraduate Opportunities in Scholarship program. We thank Dr. Duncan Krause (University of Georgia) for the generous gift of rabbit polyclonal P30 antiserum and Dr. Luis Actis (Miami University) for his gift of the FITC-conjugated secondary antibody. We also thank the Miami University Center for Bioinformatics and Functional Genomics and the Miami University Center for Advanced Microscopy and Imaging for use of their facilities and equipment.

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CHAPTER 3

Novel Cellular Organization in a Gliding Mycoplasma, Mycoplasma insons

Ryan F. Relich, Aaron J. Friedberg, and Mitchell F. Balish Journal of Bacteriology. 2009. 191:5312-5314.

77

Summary

Mycoplasmas that are known to exhibit gliding motility possess a differentiated tip structure. This polar organelle mediates cytadherence and gliding motor activity and contains a cytoskeleton-like component that provides structural support. Here, we describe gliding motility and a unique cytoskeleton in Mycoplasma insons, which lacks any obviously differentiated tip structure.

78

Introduction

Bacterial cell polarity underlies a number of critical processes, including the appropriate clustering of chemotactic receptors (Lybarger et al., 2001), chromosome segregation (Hazan & Ben-Yahuda, 2006), and placement of specialized polar structures (Goley, et al., 2007). The bacterial cytoskeleton is an important component of the mechanism underlying the generation of polarity. The bacterial actin, MreB, is crucial for the morphology of elongated bacterial cells, directing peptidoglycan synthesis along the long axis of the cell (den Blaauwen et al., 2008). In most well-studied bacterial systems, cell shape is dictated by the interaction between the cytoskeleton and the cell wall biosynthetic machinery. In contrast, bacteria of the class Mollicutes lack cell walls. Despite the absence of such a structure, many species maintain cell polarity and distinctive shapes. In Mycoplasma pneumoniae, one cell pole is organized as a terminal organelle. This tip structure, also called the attachment organelle, is readily distinguished from the more pleomorphic cell body by virtue of its slender, elongated appearance and regular dimensions. Adhesins are concentrated at the terminal organelle, which also contains a motor activity for gliding motility (Balish, 2006). Essential for the formation of the M. pneumoniae attachment organelle is a Triton X-100 (TX)-insoluble cytoskeletal structure, the electron-dense core (Biberfeld & Biberfeld, 1970), which contains novel proteins found only in M. pneumoniae and its close relatives (Balish and Krause, 2006). The distantly related species Mycoplasma mobile has a terminal organelle that also functions in gliding motility, with adhesin and cytoskeleton molecules that are completely unrelated to those of M. pneumoniae present at its cell-proximal end (Balish, 2006; Nakane et al., 2007). Interestingly, MreB is absent from both species (Himmelreich et al., 1996; Jaffe et al., 2004). The difference in composition between the terminal organelles of M. pneumoniae and M. mobile suggests that the two have evolved similar structures independently. Thus, mycoplasmas are innovators of cytoskeletons, and the variation among species provides a rich opportunity to learn a great deal about the generation of cell polarity.

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Many mycoplasmas, including Mycoplasma insons, found in the respiratory tract and blood of healthy green iguanas, lack a distinct tip structure (May et al., 2007). M. insons shares with its two closest relatives, Mycoplasma cavipharyngis and Mycoplasma fastidiosum, a slender rod shape. A series of regularly spaced, electron-dense striations perpendicular to the long axis is visible in thin sections by transmission electron microscopy, and scanning electron microscopy reveals a somewhat twisted appearance along the long axis (May et al., 2007). To understand the underlying cellular organization, we investigated the ultrastructure and behavior of M. insons cells attached to glass.

80

Materials and Methods

Cellular morphology analysis: For whole-cell analysis, cells were grown for 24 h on glass coverslips in SP-4 broth at 30°C (Hatchel et al., 2006; May et al., 2007; Tully et al., 1979). For cytoskeleton analysis, coverslips with attached cells were incubated in Triton X-100 (TX) in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl for 30 min at 37°C and then fixed and further processed as described above for whole cells. Imaging was performed on a Zeiss Supra 35 FEG-VP scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany).

Motility analysis. Stocks of M. insons were prepared by growing cells in SP-4 broth to mid-exponential phase (phenol red indicator was orange; approximately 24 h) at 30°C. After incubation, cultures were centrifuged for 20 min at 17,400 x g, resuspended in 1 ml of fresh SP-4 broth and frozen at -80°C in 50-µl aliquots. Subsequently, motility stocks were suspended in SP-4 broth plus 3% (wt/vol) gelatin, syringe-triturated seven times, passed through 0.45 µm syringe filters into chamber slides (Thermo Fisher Scientific, Rochester, NY), and incubated for 3 h at various temperatures before phase-contrast imaging with a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany). A heated chamber was used to maintain temperatures above ambient during analysis. Sequential frames were merged and analyzed as previously described (Hatchel et al., 2006).

81

Results

Cellular and subcellular features. The morphologies of whole cells and TX-insoluble material of M. insons were assessed by scanning electron microscopy. M. insons cells appeared as nonbranching rods lacking distinguishable tip structures, as previously described (May et al., 2007) (Fig. 15A and B). After treatment of cells with A22, an MreB depolymerizing agent (Gitai et al., 2005), cells remained rod shaped (not shown), suggesting an absence of MreB. The majority of cells lay flat on the coverslip; however, 8 to 10% of cells were attached at only one end, suggesting polar distribution of adhesins (Fig. 15C). Treatment with 3.2% TX revealed loose bundles of 5 to 8 roughly parallel filaments ~30 nm in width (Fig. 17A and B). Extraction with only 1.2% TX revealed the presence of filaments that were partially obscured by remnants of the cell membrane (Fig. 17C), reminiscent of the twisted appearance previously described (May et al., 2007). Filament bundles from 3.2%-TX-extracted cells were an average length of 1.18 ± 0.28 µm with a range of 0.65 to 1.85 µm (n = 140), whereas whole cells had an average length of 0.78 ± 0.28 µm with a range of 0.30 to 2.09 µm (n = 180). The slightly larger average in filament bundle length might indicate that the cytoskeleton is under longitudinal compression in intact cells. No clear evidence of polarity of these cells was observed in these analyses, although further study is necessary to understand whether they have a role in the polarization of the adherence machinery. Structures corresponding to the electron- dense bands observed by transmission electron microscopy (May et al., 2007) were not observed.

Gliding motility analysis. At 24°C, very few M. insons cells adhered to the glass surface. At 30°C, cells were capable of attaching and glided at an average speed of 25 ± 7 (mean ± standard deviation) nm s-1 (n = 28). At 37°C, M. insons cells glided at an average speed of 30 ± 14 nm s-1 (n = 45). These speeds were similar to the average speed of Mycoplasma pirum cells (28 nm s-1; Hatchel et al., 2008) making these two the slowest gliding mycoplasmas characterized. Approximately 7% of cells glided at either temperature. This filed and others were observed for a 2-h period. During this time, individual motile cells stopped and started moving and occasionally pivoted. However,

82 no cell ever changed the polarity of its movement over this extended period of observation. Figure 16 shows eight sequential phase-contrast of M. insons cells incubated at 37°C taken approximately 5 s apart. Three cells within the field shown glided; their paths and directions are indicated in the bottom right panel. The cell indicated with the diagonal arrow shows an example of pivoting over the course of the first three frames. These observations constitute the first report of gliding motility in a mycoplasma lacking a differentiated tip structure. The unidirectionality exhibited by these cells, together with the observation of unipolar attachment to the substrate in a portion of cells similar to the portion of motile cells, suggested an underlying polarity.

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Fig. 15. Scanning electron micrographs of whole cells of M. insons attached to a glass coverslip. (A) Low-magnification view. (B) Single cell lying flat on the coverslip. (C) Single cell attached by one pole to the coverslip. Bars: panel A, 2 µm; panel C, 0.5 µm; magnification of panel B is the same as for panel C.

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Fig. 16. Consecutive phase-contrast images of M. insons in a chamber slide at ~5-s intervals at 37°C. Three representative cells are indicated by arrows. In the first frame (0 s), the carets point to the leading end of gliding cells. In the last frame, the paths of the gliding cells are represented, with arrows indicating the direction of gliding. Bar: 1 µm.

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Fig. 17. Scanning electron micrographs of M. insons attached to glass coverslips and extracted with TX. (A) Low-magnification view of cells extracted with 3.2% TX. (B) High-magnification view of three separate cells extracted with 3.2% TX. (C) High- magnification view of three separate cells extracted with 1.2% TX. Bars: panel A, 2 µm; panel C, 200 nm; magnification of panel B is the same as for panel C.

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89

Discussion

This report constitutes the first description of motility in a mycoplasma lacking an obviously differentiated tip structure and with an undifferentiated cytoskeleton extending throughout the entire cell. The internal organization of M. insons is entirely distinct from that of any previously described mycoplasma, representing a novel paradigm for mycoplasma cell polarity. The results suggest that the evolution of polarity in mycoplasmas has taken multiple independent pathways.

90

Acknowledgments

This work was supported by National Institutes of Health grant AI073994. We thank D. Brown and M. May (University of Florida) for many helpful discussions during the preparation of this research and Y. Brun (Indiana University) for his generous gift of A22.

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APPENDIX A

Transformation of Mycoplasma insons with a P30GFP Construct in an Attempt to Define Polarity.

To date, the vast majority of motile Mycoplasma species that have been characterized possess a distinct polar tip structure on the forward end of the cell. In several of the well- characterized species, including Mycoplasma mobile and Mycoplasma pneumoniae, the gliding machinery appears to be localized to this tip structure. For M. mobile, gliding occurs as a result of energy from ATP hydrolysis being channeled through a series of proteins including a leg-like protein, Gli349, which changes conformation, advancing the cells across a surface (Miyata, 2010). The molecular mechanisms responsible for gliding in the human respiratory tract pathogen M. pneumoniae and its close relatives remain obscure; however, the tip structure, known as the attachment organelle in these organisms was experimentally demonstrated to house the machinery used to generate gliding motility (Hasselbring et al., 2007). In both of these cases, the ultrastructural attributes are distinct, which suggests multiple ways by which gliding motility has evolved in these organisms. Unlike the aforementioned species, Mycoplasma insons lacks any type of distinguishable tip structure, yet it glides unidirectionally at a very slow speed (Relich et al., 2009). Our investigations have uncovered a novel cytoskeleton within this organism, which we hypothesize is associated with gliding motility. However, without genome sequence data, it is difficult to investigate the underpinnings of both polarity and motility generation in this organism. Despite the scarcity of information about the cell biology of M. insons, we attempted to localize the site of motility generation in this organism by use of genetic complementation with an M. pneumoniae P30 adhesin-green fluorescent protein (P30GFP) construct. Because P30 both localizes to the attachment organelle tip and is required for gliding motility in M. pneumoniae, this construct was an appealing test subject for use in our M. insons studies. Our hope was that we could introduce this construct into M. insons cells and get expression of it. After acquisition of transformants

92 expressing P30GFP, we then focused our experiments to determine if P30GFP was trafficked to the motility-generating apparatus, and if so, what might P30GFP tell us about how M. insons motility is generated in relation to M. pneumoniae or other motile species. Mycoplasmas were grown in SP-4 broth (Tully et al., 1977) in plastic tissue culture flasks in an ambient air atmosphere. Cultures of wild-type and transformant strains of M. insons type strain I17P1T were incubated at 30°C; cultures of M. pneumoniae strain pne8G cultures were incubated at 37°C. For selection of M. insons transformants and M. pneumoniae strain pne8G, 10 µg ml-1 tetracycline or 18 µg ml-1 gentamicin, respectively, was included in all media. For plating of mycoplasmas, SP-4 containing 1% Noble agar and the appropriate antibiotic was used. Escherichia coli strains harboring pTF20 constructs were grown in Luria-Bertani medium containing 10 µg ml-1 tetracycline.

In order to transform the P30GFP-encoding construct from the plasmid pKV244 (Jordan et al., unpublished) into M. insons, the suicide vector pTF20 (Dybvig et al., 2008) was used. However, pTF20 was first modified to accommodate this insert by introduction of a BglII restriction site downstream of the EcoRI restriction site. To do so, site-directed mutagenesis was done utilizing the primers a70g_c71a (5’ CAATTGACGCGGCCGCAGATCTAGCCGGTCGAC 3’) and a70g_c71a-rev (5’ GTCGACCGGCTAGATCTGCGGCCGCGTCAATTG 3’) and the QuikChange® II Site Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA) according to the manufacturer’s instructions. Next, the insert was subcloned into the BglII and EcoRI sites of pTF20. The resulting plasmid, pOO39, was propagated in Escherichia coli strain TOP10. For transformation of M. insons strain I17P1T (May et al., 2007), electroporation was used according to the protocol of Hedreyda et al. (1993). Electroporated cells were diluted and plated to SP-4 agar containing tetracycline. Multiple tetracycline-resistant colonies were filter-cloned as previously described (chapters 1 and 2). Five separate transformants, designated M. insons 39-A through E, were grown in 10 ml selective SP-4 and aliquots were frozen at -80°C until further use.

To detect the P30GFP-encoding genetic construct in the M. insons transformants, PCR was utilized to amplify a 450-bp fragment from within the P30-encoding gene,

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MPN453. The primers MPN_453-region FWD (5’ AGGACGTGGTGGTTGCATTC 3’) and MPN_453-region REV (5’ CGCCAAGAACAGTTAGCGGA 3’) were used along with the following reaction conditions: 95°C for 3 minutes for initial template denaturation; 30 cycles of 95°C for 30 s, 57°C for 60 s, 72°C for 45 s; and a final 5-min incubation at 72°C to complete extension. After PCR, 10-µl aliquots of each reaction were electrophoresed on a 2% (w/v) agarose gel and bands were detected using an ultraviolet light transilluminator. Cell pellets of relevant strains were prepared from 25-ml mid-logarithmic phase SP-4 cultures, with the appropriate antibiotic selection, by centrifugation at 17, 400 x g for 20 min at 4°C. Cells were washed three times with 150 mM NaCl, 3.2 mM NaH2PO4,

13.6 mM Na2HPO4, pH 7.2 (PBS) and resuspended in 150 mM NaCl, 20 mM Tris HCl, pH 7.5. Protein concentrations were determined by the bicinchoninic acid method (Thermo Fisher Scientific, Rockford, IL). Five-µg protein samples from each strain were separated on a 12% (vol/vol) sodium dodecyl sulfate-polyacrylamide gel (Laemmli, 1970) and transferred to a nitrocellulose membrane (Towbin et al., 1979) for detection of

P30GFP. After blocking of the membrane in 5% (w/v) skim milk for 2 h, a 1:250 dilution of polyclonal anti-M. pneumoniae P30 antiserum (Romero-Arroyo et al., 1999) was used to probe the blot. Next, a monoclonal goat anti-rabbit IgG (Fc) alkaline phosphatase- conjugate (Promega, San Luis Obispo, CA) secondary antibody was used. Between antibody incubation steps, the membrane was washed 5 times for 5 min each with 50 mM

Tris base / 150 mM NaCl / 0.05% (v/v) Tween 20, pH 7.5. P30GFP bands were detected with a solution of 5-bromo-4-chloro-3’-indolyphosphate p-toluidine and nitro-blue tetrazolium chloride.

In an attempt to visualize P30GFP expression in M. insons 39-series transformants, fluorescence microscopy was used. Briefly, motility stocks of M. insons 39-series transformants were prepared as previously described (Relich et al., 2009). Motility stocks were next suspended in 750 µl SP-4 broth containing tetracycline and 3% (wt/vol) gelatin, triturated 7 times with a 25-g needle, and passed into wells of 4-well borosilicate chamber slides (Thermo Fisher Scientific, Rochester, NY). Inoculated chamber slides were incubated for 3 h at 30°C and then viewed using epifluorescence illumination with a

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Leica DM IRB inverted microscope (Leica Microsystems, Weilburg, Germany) equipped with a chamber maintained at 30°C. The Tn4001TF2-containing plasmid pTF20 has previously been used to transform M. insons wild-type strain I17P1T by means of electroporation (May, unpublished). To that end, we hypothesized that this plasmid would be a useful vehicle for transferring a

P30GFP-encoding construct into M. insons in an attempt to understand polarity and gliding motility in this organism. To do so, we first introduced the restriction enzyme recognition site BglII downstream of the native EcoRI site within Tn4001TF2 of pTF20 by means of site directed mutagenesis. Next, the P30GFP-encoding construct from pKV244 was ligated into these sites resulting in the plasmid pOO39. This plasmid was next transformed into M. insons strain I17P1T by means of electroporation. Three rounds of filter-cloning was performed on five different tetracycline-resistant colonies acquire genetically homogenous stocks, which were further characterized to identify both transformation success and P30GFP production. Following filter-cloning, cells of M. insons 39-series transformants were analyzed by PCR to determine if the P30GFP-encoding construct was successfully transposed into their chromosomes. To do so, PCR primers were engineered to amplify a 450-bp fragment located within the P30 gene (MPN453). Figure A1 demonstrates that this fragment was detected in 4 of the 5 M. insons 39-series transformants as well as the positive control template from M. pneumoniae wild-type strain M129, but not from the negative water control or wild-type M. insons. We next analyzed cell lysates and whole cells grown in glass chambers slides for evidence of P30GFP production. For the former, whole-cell lysates were probed with a polyclonal anti-P30 antiserum (Romero-Arroyo et al., 1999), which detected the protein fusion from the positive control, M. pneumoniae pne8G, but not in the M. insons 39- series transformants (Fig. A2). Subsequently, epifluorescence microscopy was used in an attempt to visualize P30GFP in M. insons 39-series transformants, but fluorescent foci were not seen in any fields.

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Fig. A1. Detection of MPN453 fragment in Mycoplasma insons 39-series transformants using PCR. A band approximately 450 bp in size, which corresponds to the expected size of the MPN453 fragment, was detected in the positive control and the M. insons transformants 39-A, 39-C, 39-D, and 39-E. L, 100 bp DNA ladder; lanes 1 and 2, positive control (wild-type Mycoplasma pneumoniae M129 DNA) and negative control (no DNA template), respectively; lane 3, wild-type Mycoplasma insons I17P1T template; lanes 4 through 8, Mycoplasma insons transformants 39-A, 39-B, 39-C, 39-D, and 39-E, respectively. 10-µl aliquots of each reaction were loaded into their respective wells.

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L 1 2 3 4 5 6 7 8 L

450 bp

450 bp

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Fig. A2. Absence of P30 expression in the Mycoplasma insons 39-series transformants. M. pneumoniae P30 was not detected in any of the M. insons 39-series transformants. Lanes 1 and 2, positive-control (M. pneumoniae pne8G protein) and negative-control (M. pneumoniae II-3); lane 3, wild-type M. insons I17P1T, lanes 4 through 8, Mycoplasma insons transformants 39-A, 39-B, 39-C, 39-D, and 39-E, respectively. 5-µg aliquots of protein from each were blotted.

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99

CONCLUDING REMARKS and FUTURE DIRECTIONS

The paucity of information concerning the causal mechanisms of gliding motility and the genesis of cellular morphology in Mycoplasma species provided an impetus for us to investigate M. pneumoniae and M. insons. Analysis of these species reveals differences concerning ultrastructure, and for M. pneumoniae, the transmembrane adhesin P30 was implicated in motility. We believe that the role of P30 in M. pneumoniae gliding is analogous to components of the gliding machinery of M. mobile. More specifically, we speculate that P30 is the functional analog of either Gli349 or Gli521 (Fig. 2; Miyata, 2010). With regard to M. insons, we propose that it too possesses either transmembrane adhesins or extracellular proteins that drive motility, and that the cytoskeletal filaments of this organism facilitate gliding in lieu of an attachment organelle. Current evidence suggests that Mycoplasma species have distinct motility-related proteins; homologs of mycoplasma motility proteins do not exist within walled bacteria. Extensive studies have demonstrated significant differences between cellular ultrastructure in these organisms, suggesting that motility has evolved differently among the members of this genus (Balish & Krause, 2005). Of the motile Mycoplasma species, most possess an apical membrane extension, called the attachment organelle, which is indispensible for adhesion to host cells and gliding motility (Razin & Jacobs, 1992; Rottem, 2003; Balish & Krause, 2005). The attachment organelles of the species comprising the M. pneumoniae phylogenetic cluster, as well as the features associated with their attachment organelles, are distinct, yet the components of this prostheca are conserved among these species (Hatchel et al., 2008). For example, the attachment organelle of the human respiratory tract pathogen M. pneumoniae is straight, but the attachment organelle of its closest genetic relative, the human genital tract pathogen M. genitalium, is curved (Fig. 3). The motility parameters of these two organisms are also distinct; the gliding speed of M. genitalium is slower than M. pneumoniae and the cells of the former organism move predominately in circles (Hatchel et al., 2008). Similar differences also exist between the other species comprising the M. pneumoniae phylogenetic cluster. The most extreme example of these is the gliding properties of the tortoise commensal M. testudinis. The average gliding speed for these cells is

100 approximately 3,000 nm s-1 and many cells possess curved attachment organelles (Hatchel & Balish, 2008).

The M. pneumoniae P30 ortholog, P32, from M. genitalium does not confer species- specific phenotypes, but rather complements wild-type M. pneumoniae phenotypes. We performed experiments aimed at identifying the contribution of the attachment organelle protein P30 in the processes of gliding motility and morphogenesis through the use of orthologous gene replacement in the M. pneumoniae P30 null mutant II-3. Data generated from these experiments validates the necessity for P30 in M. pneumoniae gliding motility, and interestingly, indicates that P32 is a suitable surrogate for P30. Also, we have determined that the differences in morphology and gliding motility between M. pneumoniae and M. genitalium are not associated with P30 / P32. The reasons for these observations are unclear, but we propose that, despite inherent differences between these proteins, the relatively high sequence similarity among the transmembrane domains of these two proteins allows for the substitution of P30 with P32 in M. pneumoniae, the notion of which is supported by the work of Chang et al. (2011). Another possibility is the conservation of several proline-rich repeat regions within the C-terminus of both P30 and P32. However, further investigations are needed to evaluate these possibilities. Future experiments that could prove to be of value include transformation of P30 orthologs from other species within the M. pneumoniae phylogenetic cluster into M. pneumoniae II-3 in order to determine if these more highly divergent genes confer species-specific attributes from their originating strains. Also, modifications to P30s, including truncations and amino acid substitutions, by site-directed mutagenesis might prove useful for identifying how sequence specificity affects P30 function. Such experiments are underway currently using the P30-encoding ortholog from M. testudinis, the fastest gliding species within this cluster.

The amount of P30 produced by M. pneumoniae affects gliding motility speed. We hypothesized that P30 governs gliding speed as a function of its concentration. To test this, we attempted to control the amount of P30 by developing an inducible promoter system that used the glycerol-sensitive M. pneumoniae ldh promoter to drive P30

101 expression. Although we were not capable of controlling P30 we were able to acquire transformants that produced varying low levels of P30, demonstrable by immunoblotting. Subsequent analysis of transformants by phase contrast microcinematography revealed that these transformants glided at significantly slower speeds. In fact, most transformants could not be analyzed because they glided so slowly. Scanning electron microscopy revealed a correlation between the amount of P30 and cellular morphology; as P30 concentration decreased, cellular morphology becomes more aberrant. Also, we confirmed the stabilization of several other attachment organelle proteins in these transformants, including the protein P65, which requires P30 for stability. The data generated from these experiments provide valuable insight into the calibration of gliding motility speed for M. pneumoniae. It is interesting to speculate that perhaps the same relationships between P30 concentration and gliding speed exists within the other members of the M. pneumoniae phylogenetic cluster. Also, it is intriguing to consider how modifications of P30 might affect the functionality of this protein. Such modifications might include manipulation of the proline-rich C-terminal repeat region or generation of P30 ortholog chimeras.

The role of P30 in gliding motility in M. pneumoniae phylogenetic cluster members. Because P30 is known to be necessary for the adherence and gliding motility of M. pneumoniae, it provided an attractive target for the study of attachment organelle proteins with regard to their contribution to mycoplasma motility and morphology. Our data suggest not only that M. genitalium P32 is a suitable surrogate for P30, but also that it controls gliding speed in a concentration-dependent manner. However, we are still uncertain about how these proteins function in accomplishing these feats. To that end, we speculate that P30 and its orthologs function by analogy to the unrelated gliding protein Gli349 from the fish pathogen M. mobile. Purification and analysis of Gli349 demonstrates that this protein exists in multiple conformations, which are perhaps recapitulated in vivo. The model of M. mobile gliding invokes the use of this extracellular protein as a leg that consumes energy from ATP hydrolysis to mediate successive substrate attachment-and-release cycles. The net effect of which essentially pulls the cells forward (Fig. 2; Miyata, 2010). We propose that for M. pneumoniae and its

102 relatives, P30 acts in much the same was as Gli349. However, examination and purification of P30 remains to be done, as well as identification of possible binding partners of this adhesin. A second possibility is that P30 functions in a manner that is analogous to the M. mobile protein Gli521, which transduces the energy from the ATPase to the Gli349 leg protein. Since P30 has been demonstrated to facilitate stabilization of the major M. pnuemoniae adhesin P1, it stands to reason that perhaps P30 does not directly cause movement of cells, but rather it transfers the propulsive force to P1 or other as yet unidentified proteins that directly effect movement.

The generation of polarity and gliding motility in M. insons. The rod-shaped species Mycoplasma insons is unusual not only because is it one of the few Mycoplasma species that is regularly rod-shaped, but because it is also capable of gliding motility in the absence of any distinguishable polar features. We adapted techniques commonly used to study M. pneumoniae and many other mycoplasmas for the examination of M. insons. Detergent extraction of whole cells followed by scanning electron microscopy revealed a network of detergent-insoluble cytoskeletal filaments that lay along the cellular long axis. The composition and use of these filaments are unknown, but we speculate that they are key for maintenance of cellular morphology in this organism. Treatment of live cells with the compound A22 indicated that the bacillary shape of M. insons is not due to the presence of MreB, a well-characterized cytoskeletal protein of many rod-shaped bacteria including E. coli. We were not able, however, to identify structures used to generate polarity in this organism, so further examination of M. insons is justified, especially after the acquisition of genome sequence data. Microcinematography was used to provide a comprehensive assessment of M. insons gliding motility, which indicated that cells attach and glide optimally at temperatures characteristic of its host species. With regard to its movement, we hypothesize that the cytoskeletal filaments, either in conjunction with transmembrane adhesins or alone, drive motility. We speculate that motility-associated proteins, either extracellular or transmembrane, mediate movement of this organism, perhaps utilizing the cytoskeletal filaments in this process. Because this species does not glide any faster at

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37°C than it does at 30°C, its optimal growth temperature, it is unlikely that a Brownian motor drives movement. To further our understanding of this organism, the genome of the type strain is being sequenced.

The role of gliding motility in the mycoplasma life cycle. The idea that Mycoplasma species use gliding motility within host animals to colonize, disseminate, and avoid phagocytosis is not new. However, evidence to support this notion is lacking. While it may be possible that motility serves as a means for translocation within hosts, we speculate that the motile forces generated by cells are mainly used for cell division. Evidence for this is provided by analysis of M. genitalium mutants lacking the bacterial tubulin homolog FtsZ. Lluch-Senar et al. (2010), demonstrated that the gliding forces alone were sufficient to allow for cytokinesis. In addition, the P30 null mutant M. pneumoniae II-3 is highly pleomorphic and often grows in clumps, indicating a possible need for gliding motility to correct aberrant cell division. During mycoplasma cytokinesis, the attachment organelle is first duplicated and migrates to the opposite cell pole, most likely under the influence of cell gliding, as proposed by Hasselbring et al. (2006). In this model of M. pneumoniae cell division, the new attachment organelle remains stationary and is pulled to the opposite cell pole by gliding motility forces generated by the initial attachment organelle. Eventually, the gliding forces pull the daughter cells apart. It is conceivable in nature that the gliding phenomenon seen in the laboratory may not necessarily occur, as it is likely that accessory adhesins present all over the mycoplasma cell surface anchor cells rather tightly to the surface of the host epithelium. To successfully duplicate in this environment, gliding motility forces may be needed to overcome adherence forces, and rather than moving considerable distances, cells may only move far enough to ensure that cytokinesis is complete. We propose that in all motile Mycoplasma species, gliding is used for cell division rather than for translocation. We have learned from our analyses and previous observations that the ultrastructural details of Mycoplasma species are largely distinct among the members of different phylogenetic clusters, and by examination of a novel species, we have discovered yet another example of distinct ultrastructural components.

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We believe that, despite dissimilar ultrastructural components of many Mycoplasma species, the means by which species glide require either transmembrane adhesins (e.g., P30 or P1) or extracellular proteins (e.g., Gli349) that directly contact the substrate to pull cells along. These proteins either directly generate the energy required for moving or utilize energy channeled through associated proteins.

The applicability of this research to other facets of mycoplasma cell biology. We have reported for the first time the successful use of orthologous gene replacement in mycoplasmas for studying the function of a protein crucial for motility. It may be possible to utilize this approach to parse out the role of other attachment organelle or cellular proteins important in mycoplasma motility and morphogenesis. However, orthologous gene replacement is currently limited to use in circumstances in which a mutant for the protein of interest exists in the organism being studied, as approaches for the targeted generation of null mutants in M. pneumoniae are lacking. It is also interesting to speculate how the process of gliding motility in M. pneumoniae and potentially other species might be affected by overproduction of P30 and its orthologs. Eventually, it may be possible to obtain a functional inducible promoter system to modulate P30 expression so that an entire array of P30 concentrations can be obtained in transformants. Data generated from analysis of these cells could pinpoint the exact concentration of P30 or other motility related proteins necessary to cause differences in gliding parameters. Such techniques may also be amenable for use in other species to characterize as yet unknown processes.

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