Viewed and Captured at the Electron Microscopy Facility at Miami University on a Zeiss Supra 35 FEG-VP Scanning Electron Microscope Operating at 5 Kv

MIAMI UNIVERSITY

The Graduate School

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation

Jennifer Marie Hatchel

Candidate for the Degree:

Doctor of Philosophy

______Dr. Mitchell Balish, Director

______Dr. Eileen Bridge, Reader

______Dr. Kelly Abshire, Reader

______Dr. Xiao-Wen Cheng

______Dr. Richard Moore, Graduate School Representative

ABSTRACT

STRUCTURE AND FUNCTION OF THE ELECTRON-DENSE CORE IN MYCOPLASMA PNEUMONIAE AND ITS RELATIVES By Jennifer Hatchel

Among the mycoplasmas, the human pathogen Mycoplasma pneumoniae is the best- characterized at the cellular level. It has a polar structure, the attachment organelle, which mediates adherence to host cells, is the leading end during gliding motility, and plays a role during cell division. Within the attachment organelle is a detergent-insoluble electron-dense core composed of several proteins, some of which have been identified through the study of cytadherence deficient mutants of M. pneumoniae. Mutants lacking proteins in the electron-dense core are avirulent, suggesting that the core is essential for the proper formation of the attachment organelle, which in turn is essential for virulence. We used scanning electron microscopy (SEM) and time-lapse microcinematography to test the relationship between ultrastructure and gliding motility in M. pneumoniae and some of its close phylogenetic relatives, which vary in ultrastructure, gliding characteristics, host range, and pathogenic potential. Our results show that Mycoplasma amphoriforme, a novel species found in the respiratory tract that is possibly pathogenic to humans, is motile and shares morphological characteristics with its closest relatives, M. pneumoniae and the avian pathogen, Mycoplasma gallisepticum. Using SEM and time- lapse microcinematography, we find that the morphology of seven species of the M. pneumoniae cluster correlates with phylogeny rather than with gliding motility characteristics. We also find that in most species the electron-dense cores have fibers and filaments that remain attached to the base of the core after detergent treatment, but disappear after treatment with DNase, suggesting that they are DNA. It has been hypothesized that the electron-dense core plays a role during cell division, which might utilize protein-DNA interactions between the core and the chromosome. Using fluorescence in situ hybridization (FISH) coupled to immunofluorescence, we attempted to further investigate specific interactions between the oriC region of the chromosome and the electron-dense core in M. pneumoniae. The data suggest that a variable region of the chromosome associates with the base of the electron-dense core, although the FISH protocol still needs optimization. Overall, my work suggests that gliding motility has a major role in the partitioning of the chromosomes in cell division and a minor role in pathogenicity. STRUCTURE AND FUNCTION OF THE ELECTRON-DENSE CORE IN MYCOPLASMA PNEUMONIAE AND ITS RELATIVES

A DISSERTATION

Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology

Jennifer Marie Hatchel Miami University Oxford, Ohio 2009

Dissertation Director: Dr. Mitchell Balish Table of Contents Page INTRODUCTION………………………………………………… 1 A. Significance of studying M. pneumoniae and its relatives……... 1 B. Phylogeny and cell morphology of M. pneumoniae and relatives. 3 C. Attachment organelle proteins…………………………………... 7 D. Gliding motility and division …………………………………… 12 E. Cell division requires the duplication and migration of the attachment organelle…………………………………………….. 14 F. Hypotheses ………………………………………………………. 15

Chapter 1: Ultrastructure and gliding motility of Mycoplasma amphoriforme, a possible human respiratory pathogen……….. 16 Abstract……………………………………………………………. 17 Introduction………………………………………………………... 18 Materials and Methods…………………………………………….. 20 Results……………………………………………………………... 22 Discussion…………………………………………………………. 39

Chapter 2: Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives…………………………………………………………… 44 Abstract……………………………………………………………. 45 Introduction………………………………………………………... 46 Materials and Methods…………………………………………….. 49 Results……………………………………………………………... 51 Discussion…………………………………………………………. 66

Chapter 3: Fluorescence in situ hybridization, as a possible method for characterizing the interaction between the electron-dense core and the chromosome in Mycoplasma pneumoniae...... 72 Abstract……………………………………………………………. 73 Introduction………………………………………………………... 74 Materials and Methods…………………………………………….. 78 Results……………………………………………………………… 80 Discussion…………………………………………………………. 85 SUMMARY AND CONCLUDING REMARKS……………….. 90 REFERENCES…………………………………………………… 100

iii

List of Tables Page Table 1 Dimensions of the cytoskeleton-like structure of M. 29 amphoriforme and M. pneumoniae

Table 2 Gliding motility parameters. (Chapter 1) 36

Table 3 Gliding motility parameters. (Chapter 2) 54

Table 4 Whole cell dimensions (in nm) ± SD, mean of 30 cells. 57

Table 5 Electron-dense core dimensions (in nm) ± SD, mean of 65 30 cells.

iv List of figures Page Figure 1 Phylogenetic tree of the Mollicutes and some walled 4 relatives based on 16S rRNA sequences

Figure 2 Schematic drawing of the adhesins and the electron- 9 dense core in M. pneumoniae

Figure 3 Scanning electron micrographs of mycoplasma cells 23 grown attached to coverslips.

Figure 4 Scanning electron micrographs of presumptively 25 dividing M. amphoriforme cells grown attached to coverslips.

Figure 5 Scanning electron micrographs of mycoplasma 27 cytoskeletal structures.

Figure 6 Consecutive phase-contrast images of M. amphoriforme 31 gliding motility at 5-s intervals at 37°C.

Figure 7 Measurement of M. amphoriforme gliding speed by 34 time-lapse microcinematographic analysis.

Figure 8 Distribution of mycoplasma velocities about the mean. 37

Figure 9 Phylogenetic tree based on 16S rRNA sequences of 52 mycoplasmas within the M. pneumoniae cluster.

v Figure 10 Scanning electron micrographs of mycoplasma cells 55 grown on glass coverslips.

Figure 11 Scanning electron micrographs of mycoplasma cells 59 grown on glass coverslips.

Figure 12 Scanning electron micrographs of mycoplasma 61 electron-dense cores.

Figure 13 Schematic of a typical M. amphoriforme cell. 69

Figure 14 Schematic representation of expected results in a M. 81 pneumoniae cell after FISH with a Cy3-labeled probe and immunofluorescence against the attachment organelle protein, HMW1.

Figure 15 FISH using probes against the 16S rRNA gene and the 83 oriC sequences in M. pneumoniae.

Figure 16 Comparison of FISH in Caulobacter crescentus and 87 Mycoplasma pneumoniae.

vi Acknowledgments The first person I would like to thank is my advisor, Dr. Mitchell Balish. Thank you for your guidance, patience, and support over the last five years. I have learned so much during my time here at Miami, and I am grateful to have had the opportunity to work in your lab. My committee members, Dr. Kelly Abshire, Dr. Eileen Bridge, Dr. Xiao-Wen Cheng, and Dr. Richard Moore have been extremely helpful during this process as well. Thank you for all of your suggestions, for always challenging me to be a better scientist, and for making me think critically about my experiments. Thank you to my lab mates, Dominika and Ryan. I cannot thank you enough for being who you are because it has shaped who I have become. Thank you for being there when I needed someone to talk to, especially at the end. Rachel, even though we didn’t work together in the lab for long, I want to thank you for being my friend. Thanks for encouraging me to be a better person. I wish you well on your journey to completing your degree. Thanks also to Jason, Jason, Jackie, Jianli, Karthik, and Rachael, for making my time here more enjoyable. Thank you to Barb and Darlene for making it easier to work here. I appreciate all the help when ordering items or when the copy machine didn’t like me. Thanks to the rest of the department for being supportive and willing to help. I am glad to have known all of you. To my friends, both here in Ohio and back home in Tennessee, thanks for the support and encouragement. Sara, Matt, Paige, Amanda, Cameron, Lynne, Becky, Dennis, and Frank, thank you for not only being my neighbors, but also for being people I could count on in times of need. I will miss living in our building. I also want to thank Dr. Becky Balish, Annika, and Ian for all that you have done for me. Thanks also to Tommie, Maggie, and Connor for entertaining Alexis while I was at school. I couldn’t have finished without you. THANKS!! Last but not least, I want to thank my family. Those of you in Tennessee, thank you for understanding my decision to go to school in Ohio even though it meant that sometimes I had to be in the lab over holidays instead of being there with you. Thank you for believing in me. Finally, Mark and Alexis, you saw me at the highest of highs and lowest of lows. Thank you for having patience with me and encouraging me through it. I love you!!

vii INTRODUCTION The bacterial genus Mycoplasma contains over 100 known species. Mycoplasmas lack a cell wall, and their genomes have experienced reductive evolution (sizes range between 580 -1350 kb), making them the genetically least complex organisms capable of self- replication in a laboratory setting. In nature, the absence of many biosynthetic pathways leads these organisms to depend on a host for nutrients; thus, they are always associated with a vertebrate host. Several Mycoplasma species are significant pathogens of humans or animals, making them an important subject for study. Mycoplasma pneumoniae, a human respiratory pathogen that causes tracheobronchitis and other diseases, (Waites and Talkington, 2004) is the best characterized mycoplasma and has close relatives that are also present, and sometimes pathogenic, in humans, agriculturally significant animals, and wildlife. These bacteria have a distinct polar organelle called the attachment or terminal organelle (Kirchhoff et al., 1984; Balish and Krause, 2002). This unusual structure, which is not found in model bacteria like Escherichia coli, functions as a hub of activities related to cytadherence, gliding motility, and cell division. However, details of the mechanism underlying the operation of this organelle are lacking. A. Significance of studying M. pneumoniae and its relatives M. pneumoniae was first isolated in 1944 from the sputum of a patient with primary atypical pneumonia (Eaton et al., 1944). The typical infection is slow to develop and includes symptoms of a sore throat, hoarseness, fever, and cough (Atkinson et al., 2008). Treatment for a M. pneumoniae infection is commonly a macrolide antibiotic, such as azithromycin, but tetracyclines and fluoroquinolones are effective as well (Waites and Talkington, 2004; Atkinson et al., 2008). While resistance to tetracycline and fluoroquinolones has never been shown in M. pneumoniae, macrolide-resistance can occur (Atkinson et al., 2008). With the resistance to the common therapies on the rise, it is important to understand the mechanisms underlying M. pneumoniae pathogenesis to develop new means of treatment when antibiotics are no longer effective. The closest relatives of M. pneumoniae include other significant pathogens. The human urogenital pathogen, Mycoplasma genitalium (Jensen, 2004) and the avian pathogen, Mycoplasma gallisepticum (Levisohn and Kleven, 2000) are capable of

1 causing disease by damaging ciliated epithelial cells and eliciting a host immune response that results in the release of cytokines (Lam, 2004; Ekiel et al., 2009). These three organisms have also all been shown to cause conjunctivitis (Salzman et al., 1992; Björnelius et al., 2004; Dhondt et al., 2005). Antigenic variation of membrane proteins has also been shown in M. genitalium and M. gallisepticum, but not formally in M. pneumoniae (Atkinson et al., 2008; Kleven, 2008; Ekiel et al., 2009). In contrast to M. pneumoniae, M. genitalium infections are typically in the urogenital tract (Jensen, 2004), and M. gallisepticum infections are in the respiratory tract and conjunctiva of avian species (Levisohn and Kleven, 2000). Another close relative of M. pneumoniae, Mycoplasma amphoriforme, was identified as a possible opportunistic pathogen associated with immunosuppressed patients suffering from chronic bronchitis (Webster et al., 2003; Pitcher et al., 2005). It is more closely related to M. gallisepticum than to M. pneumoniae, and its closest relative, Mycoplasma testudinis, is a putative commensal isolated from the cloaca of a healthy tortoise (Hill, 1985). While it has not been confirmed to be a pathogen, M. amphoriforme appears to pose a risk to those suffering from chronic respiratory illnesses and is difficult to treat with antibiotics due to resistance to most common therapies (Webster et al., 2003). Currently, efforts are underway to identify this organism in clinical samples from patients with respiratory complications in order to gauge the significance of M. amphoriforme as a pathogen (Balish and Waites, unpublished). The difficulty in treating M. amphoriforme infections underscores the importance of understanding the mechanisms of its pathogenicity. Damage to host cells by M. pneumoniae is caused by the production of peroxide, a by-product of glycerol metabolism (Low et al., 1968; Miles et al., 1991; Hames et al., 2009), and superoxide anions that prevent host cells from being able to break down the peroxide (Low, 1971; Almagor et al., 1984). Recently, a protein with sequence homology to the S1 subunit of pertussis toxin was identified in M. pneumoniae and was named community-acquired respiratory distress syndrome toxin (CARDS TX), although its role in disease has yet to be established (Kannan and Baseman, 2006). These factors, in addition to cytokines released as a response to the infection, conspire to cause cell damage, including a loss of cilia, increased vacuolation, and a general decrease in cellular

2 function that ultimately leads to exfoliation of the cells (Atkinson et al., 2008). This damage may be responsible for the persistent, hacking cough associated with a typical M. pneumoniae infection (Waites et al., 2007). Although M. pneumoniae is typically thought of as an extracellular pathogen, studies of other mycoplasmas, including Mycoplasma penetrans, isolated from the urine of patients with human immunodeficiency virus (HIV) infections, and Mycoplasma fermentans, another human mycoplasma, have shown an ability to penetrate and infect the host epithelial cells (Waites and Talkington, 2004). Having the ability to survive within host cells would protect the organism from the host immune responses and also antibiotic therapies, which may explain the persistence of M. pneumoniae, although the extent to which this particular organism does this is still unknown (Baseman et al., 1995; Rottem, 2002). B. Phylogeny and cell morphology of M. pneumoniae and relatives Mycoplasmas are evolutionarily related to gram-positive bacteria even though they lack a cell wall. In 1989, the small subunit of the 16S rRNA was used to develop a classification system of over 50 mycoplasma species and their walled relatives (Weisburg et al., 1989). This group divided the class Mollicutes into five groups: the pneumoniae group, the hominis group, the spiroplasma group, the anaeroplasma group, and a fifth group that contained only a single organism, Asteroleplasma anaerobium (Weisburg et al., 1989). These five groups were also divided into fifteen clusters (Fig. 1), some containing multiple species while others contained as few as two species. Eight species, including M. pneumoniae, M. genitalium, M. gallisepticum, Mycoplasma imitans, M. amphoriforme, M. testudinis, Mycoplasma pirum, and Mycoplasma alvi, comprise the M. pneumoniae phylogenetic cluster within the pneumoniae group based on 16S rRNA sequences (Johansson and Pettersson, 2002). M. pneumoniae, M. genitalium, M. amphoriforme, M. gallisepticum, and M. testudinis are described above. M. imitans, a close relative of M. gallisepticum, (Abdul-Wahab et al. 1996) is pathogenic to ducks, geese, and partridges. M. alvi (Pettersson et al., 1996) was isolated from a cow and is considered to be a commensal. M. pirum (Montagnier et al., 1990) is most often associated with cell culture contamination but is also found in humans, where its pathogenicity has not been established (Tham et al., 1994).

3 Fig. 1. Phylogenetic tree of the Mollicutes and some walled relatives based on 16S rRNA sequences. The M. pneumoniae cluster is highlighted. Adapted from Johansson and Pettersson, 2002.

4 5 Bacteria of the genus Mycoplasma are pleomorphic, a trait often attributed to the lack of a cell wall (Balish, 2006), and exhibit morphologies ranging from rod-shaped (Mycoplasma insons; May et al., 2007) to coccus-shaped (Mycoplasma hyopneumoniae; Mare and Switzer, 1965) to flask-shaped (M. pneumoniae; Bredt, 1968). The flask shape of M. pneumoniae is conferred by the presence of a polarized structure, the terminal organelle or attachment organelle, which confers both cytadherence and gliding motility (Krause, 1996; Balish, 2006). The attachment organelle is unlike any other structure used for adherence in other forms of life in that it is an extension of the cell itself rather than an appendage that is attached to the cell. Other members of the M. pneumoniae cluster, M. genitalium, M. imitans, M. gallisepticum, and M. pirum have been observed to have a similar attachment organelle structure (Abu-Zahr and Butler, 1978; Del Giudice et al., 1985; Bradbury et al., 1993; Tully et al., 1993). However, prior to the current studies, the details of their morphological similarities were unclear. Structures resembling the M. pneumoniae attachment organelle are not present in all mycoplasmas, but they have been observed in several other species of the genus Mycoplasma. Cells of Mycoplasma mobile, a distantly related species of the M. sualvi cluster (Fig. 1), have a “head-like structure,” which is functionally an attachment organelle, even though the attachment in this organism is mediated by the base of the structure (the “neck region”) rather than the tip (Kirchhoff and Rosengarten, 1984; Kirchhoff et al., 1984). Other motile mycoplasmas also have attachment organelle-like structures. M. penetrans, a member of the M. muris cluster (Fig. 1), which was isolated from humans infected with HIV, also has an attachment organelle, but its internal structure does not closely resemble that of M. pneumoniae (Lo et al., 1992; Jurkovic and Balish, unpublished). Thin-section micrographs of M. pneumoniae reveal a cytoskeleton- like electron-dense structure within the attachment organelle (Biberfeld and Biberfeld, 1970). This electron-dense core is insoluble in the detergent Triton X–100 (TX) (Meng and Pfister, 1980; Gobel et al., 1981). Cytadherence mutants defective in attachment organelle biogenesis have allowed for the identification of many of the proteins found in the attachment organelle (Krause and Balish, 2004). More recently, electron cryotomography has been used to obtain high-resolution images of the M. pneumoniae electron-dense core (Henderson and Jensen, 2006; Seybert et al., 2006). These images

6 detail the structure of the core, which can be divided into three regions: a terminal button, a rod made up of two parallel subunits, and a bowl-shaped base (Henderson and Jensen, 2006). While M. mobile and M. penetrans have an external morphology that is similar to yet distinct from that of M. pneumoniae, these species lack a structure strictly resembling the electron-dense core of M. pneumoniae (Kirchhoff and Rosengarten, 1984; Miyata, 2005). In M. mobile, this structure resembles the bell and tentacles of a “jellyfish” and, similar to the electron-dense core of M. pneumoniae is found within the membrane and remains after the cells are treated with TX (Nakane and Miyata, 2007). In M. penetrans, the internal structure is not divided into distinct regions (Jurkovic and Balish, unpublished). No homologs of the M. pneumoniae attachment organelle proteins are present in M. mobile (Jaffe et al., 2004) or M. penetrans (Sasaki et al., 2002). Therefore the proteins found in the cytoskeletal structures of these species are completely different from those found in M. pneumoniae, suggesting that whereas the overall mechanisms for adherence and motility might be similar, the machinery in each of these distantly related organisms is distinct. The widespread nature of attachment organelles among distantly related mycoplasmas raises intriguing evolutionary and mechanistic questions concerning both the origins and workings of this novel structure. Comparing attachment organelle- containing species is of great potential value in identifying characteristics that have been inherited from their common ancestor, as well as characteristics that have evolved independently. This is accomplished by looking at characteristics found in the majority of related species in the cluster. The features found in most species were probably also found in the common ancestor, while those that are different, most likely diverged. Understanding attachment organelles in this way will contribute to a deeper understanding of mycoplasma pathogenesis. C. Attachment organelle proteins Transmembrane adhesins mediate cytadherence. The known adhesins of M. pneumoniae are proteins P1 (Feldner et al., 1982; Hu et al., 1982; Krause et al., 1982) and P30 (Baseman et al., 1987; Seto and Miyata, 2003). P1 and accessory proteins B and C cluster on the attachment organelle surface but are also found at other locations on the

7 cell in smaller amounts (Baseman et al., 1982; Feldner et al., 1982; Seto et al., 2001). P30, however, is found only at the attachment organelle tip (Fig. 2) (Baseman et al., 1982; Seto and Miyata, 2003). The adhesins function in adherence to host cells in vivo and to glass or plastic surfaces in vitro. It is thought that the adhesins are receptors for sialic acid found on the host cell surface or, in vitro, on sialoproteins that have been deposited on the glass or plastic from serum in the media (Nakane and Miyata, 2007). Rabbit antibodies against these proteins block adherence to these surfaces (Feldner et al., 1982; Hu et al., 1982; Morrison-Plummer et al., 1986; Razin and Jacobs, 1992; Svenstrup et al., 2002). P1 is considered to be the primary adhesin based on numerous lines of evidence. Addition of antibodies against P1 to a population of attached and motile cells results in decreased motility and ultimately a release from the substrate (Seto et al., 2005a). Mutants that fail to localize P1 to the attachment organelle are avirulent due to their inability to attach to host surfaces (Baseman et al., 1982). P1 is essential for M. pneumoniae virulence (Baseman et al., 1982), and related species also have homologs of this adhesin. One homolog of P1 is found in M. genitalium, MgPa (Hu et al., 1987; Inamine et al., 1989) and two are present in M. gallisepticum, GapA (Goh et al., 1998) and CrmB (Papazisi et al., 2003), although only GapA has been well-characterized. In these species, antibodies against their respective adhesins result in loss of adherence, indicating a similar role for these homologs (Goh et al., 1998; Svenstrup et al., 2002). Additionally, P1 homologs have been found in M. amphoriforme, M. testudinis, M. alvi, and M. pirum (Balish, unpublished; Tham et al., 1994), suggesting that P1 is a common feature of the species in the M. pneumoniae cluster. P1 has not been identified in any other organism. A second transmembrane adhesin protein, P30 in M. pneumoniae, localizes to the tip of the attachment organelle (Fig. 2) (Dallo et al., 1996). This protein also has homologs in the other species of the M. pneumoniae cluster (Reddy et al., 1995; Hnatow et al., 1998; Boguslavsky et al., 2000). Like P1, antibodies to P30 in M. pneumoniae or its homologs in the other species interfere with attachment to surfaces (Morrison- Plummer et al., 1986). P30 also plays a role in gliding motility, as mutants missing this protein are unable to glide (Romero-Arroyo et al., 1999) while a mutant which is missing

8 Fig. 2. Schematic drawing of the adhesins and the electron-dense core in M. pneumoniae. Purple and black arrows indicate surface proteins. Red arrows point to the components of the electron-dense core (terminal button, rod, and base). The green arrow represents HMW1 interacting with HMW2. Adapted from Balish, 2006a.

10 some of the C-terminal repeats of P30 is capable of gliding at a reduced speed (Hasselbring et al., 2005). Interestingly, a revertant that has a frameshift mutation that corrects the reading frame in all but 17 residues, produces an altered P30 protein is cytadherent, but gliding speed is not fully complemented (Hasselbring et al, 2005). These data point toward an important role for P30 in gliding motility. Electron-dense core of Mycoplasma pneumoniae In addition to the adhesins, other essential proteins are found in the electron-dense core of the M. pneumoniae attachment organelle (Fig. 2). The terminal button is proposed to contain the proteins HMW3 (Stevens and Krause, 1992) and P65 (Balish and Krause, 2005), which is surface-exposed (Proft et al., 1995), and has a close association with other proteins in the electron-dense core (Jordan et al., 2001; Seto et al., 2001). The rod is proposed to be made up predominantly of protein HMW2 (Balish et al., 2003) and is composed of two parallel subunits (Hegermann et al., 2002; Henderson and Jensen, 2006). The proteins P24, P28, and P41 are associated with the base of the electron-dense core (Krause et al., 1997; Jordan et al., 2001; Kenri et al., 2004). HMW1, like P65, is surface-exposed (Seto et al., 2001; Seto and Miyata, 2003), yet plays an important role in the stabilization of HMW2 (Balish et al., 2001). P200 is proposed to play a role in gliding motility and colonization (Jordan et al., 2007). While the specific function of each of these proteins is unknown, the overall role that they play in the formation of the attachment organelle is essential. Most of these proteins play a role in stabilizing or localizing other proteins during the assembly of the electron-dense core, thus ensuring the proper formation of the attachment organelle (Krause and Balish, 2004). The core proteins are also responsible for localizing the adhesins to the attachment organelle, thus allowing for cytadherence. Without a core, the attachment organelle does not form properly which results in a failure of the adhesins to localize to the tip, and without the adhesins the cell cannot adhere to host cells making it avirulent (Balish and Krause, 2002; Seto and Miyata, 2003). Pathogenicity of these organisms strongly relies on their ability to adhere to their host (Sobeslavsky et al., 1968).

11 D. Gliding motility and division Gliding motility in mycoplasmas is different from motility seen in other bacteria due to a lack of flagella or pili on their surfaces (Kirchhoff et al., 1984; Kirchhoff, 1992). A small but significant number of species of Mycoplasma are motile and movement is in the direction of the attachment organelle (Bredt, 1979). The best-characterized species with regards to gliding motility is M. mobile, because of its fast speed of 2.0-4.5 µm/s (Uenoyama et al., 2005). For mycoplasmas, gliding is dependent on adherence via adhesins found on the attachment organelle, which leads the cell in a continuous unidirectional pattern. The genomes lack genes required for the other mechanisms of gliding motility, suggesting that the mycoplasma mechanisms for gliding are unique to these organisms (Miyata, 2005). Gliding motility of mycoplasmas was first observed for Mycoplasma pulmonis (Andrewes and Welch, 1946). Gliding motility was first observed for M. pneumoniae in vitro in 1968 (Bredt, 1968). In M. pneumoniae, the average speed is 0.3-0.4 µm/s in the direction of the attachment organelle, excluding the time spent in rest periods (Bredt, 1968). The ability to move is thought to aid in pathogenicity by allowing the cells to spread out during an infection (Jordan et al., 2007). Previous reports have shown that, among the members of the M. pneumoniae cluster, M. pneumoniae, M. genitalium, and M. gallisepticum exhibit gliding motility on surfaces (Bredt, 1968; Kirchhoff et al., 1984; Kirchhoff, 1992), whereas M. alvi is non-adherent and non-motile (Bredt, 1979). While motility is observed in these species, the exact mechanism behind this feature is unknown. Direct evidence for the location of the motor being within the attachment organelle comes from the observation that M. pneumoniae mutants missing P41, though capable of gliding, often exhibit detachment of the attachment organelle from the cell body. These detached organelles, but not the cell body, continue to glide (Hasselbring and Krause, 2007a). M. mobile has different gliding characteristics than mycoplasmas of the M. pneumoniae cluster. M. mobile cells exhibit no rest periods, and their gliding speed averages between 2.0-4.5 µm/s (Rosengarten and Kirchhoff, 1987; Miyata et al., 2002). Motility has been studied most extensively in this species because of its high speed and the ease with which it can be observed. Rapid-freeze and freeze-fracture rotary-shadow electron microscopy was used to identify the motility machinery of M. mobile (Miyata

12 and Peterson, 2004). Three large proteins were found to localize at the “neck” region and each has a different function (Miyata, 2005). Gli123 functions in the positioning of the Gli349 and Gli521 proteins as evidenced by their inability to localize properly in a mutant missing Gli123 (Uenoyama and Miyata, 2005). Gliding-deficient mutants and antibodies were used to elucidate the functions of Gli349 and Gli521 (Miyata et al., 2000; Kusumoto et al., 2004; Seto et al., 2005b). Gli349 was determined to be an adhesin based on its role in hemadsorption and glass binding, analogous to M. pneumoniae P1, with which it has no homology (Uenoyama et al., 2004). The postulated function of Gli521 is to contribute to the force needed for movement as antibodies against this protein stopped movement but not attachment in M. mobile cells (Seto et al., 2005b). There is a connection between the Gli proteins and the cytoskeletal structure. Mutants missing the Gli proteins had “jellyfish” structures that were unorganized when compared to those of wild-type (Nakane and Miyata, 2007). M. mobile has protruding “spike” structures 50 nm in length that localize at its neck and are found in different conformational states that may provide evidence that these structures bind and release the glass in order to move the cell (Miyata and Peterson, 2004; Miyata, 2005). The identification of these conformations led to the development of a model of motility for M. mobile. Miyata proposes that there are five phases to binding: 1) initial binding; 2) tight binding; 3) stroke; 4) release; and 5) return (Miyata, 2005). Numerous lines of evidence suggest that the spikes are composed of Gli349: 1) the distribution of the spikes corresponds to the gliding proteins; 2) the spikes are absent in a Gli349 mutant; and 3) the dimensions of the spike match the predicted volume of one Gli349 monomer (Miyata, 2005; Adan-Kubo et al., 2006). There is controversy over the mechanisms responsible for M. pneumoniae gliding motility, and two opposing hypotheses have emerged. One of the hypotheses focuses on the binding and release of the adhesins in a manner similar to that of the bind-and-release mechanism proposed for M. mobile. This hypothesis is supported in M. pneumoniae as both the adhesins, P1 (Seto et al., 2005a) and P30 (Hasselbring et al., 2005), have been shown to play a role in motility. The second hypothesis focuses more on the electron- dense core. It states that the cells “inchworm” along as a result of conformational changes in proteins in the electron-dense core that lengthen and shorten the rod, thus allowing for

13 movement (Henderson and Jensen, 2006). It has been suggested that these conformational changes are due to flexible parts of the electron-dense core and allow for dissociation, displacement, and association of “legs” on the cell surface with the solid surface which would propel the cell forward (Seybert et al., 2006; Henderson and Jensen, 2006; Miyata, 2008). The conformational changes suggested by this hypothesis would result in morphological differences among species with different gliding speeds. These morphological differences would be observed as a variation in the length of the core itself or as a large variation in the standard deviations of the rod length measurement that would correlate with motility speed. The current study addresses these hypotheses. E. Cell division requires the duplication and migration of the attachment organelle. Dividing cells have been seen by scanning electron microscopy in multiple mycoplasma species (Bredt, 1968; Seto et al., 2001; Hasselbring et al., 2006). It has been shown that during M. pneumoniae cell division the attachment organelle duplicates itself (Seto et al., 2001) and that the new attachment organelle remains attached to the surface while the old attachment organelle pulls the cell away (Hasselbring et al., 2006a), allowing for migration of the attachment organelle to the opposite pole before cytokinesis occurs. Duplication of the attachment organelle also is accompanied by an increase in the amount of genetic material within the cell, and the amount of DNA also increases as the distance between the two attachment organelles increases (Seto et al., 2001). Furthermore, newly synthesized DNA is enriched in a subcellular fraction of M. gallisepticum cells that is enriched for attachment organelles (Maniloff and Quinlan, 1974). These data suggest a possible interaction between the chromosome and the attachment organelle during cell division, which is probably mediated through the electron-dense core. However, very few proteins involved in DNA-pole linkages in other bacteria are present in mycoplasmas, leaving the components involved in this interaction unknown. The absence of homologs of partitioning machinery found in model bacteria also suggests that mycoplasmas use a novel mechanism for segregating their chromosomes. It is conceivable that such a mechanism could be driven by interactions between the chromosome and the migrating attachment organelle during division.

14 F. Hypotheses By comparing the characteristics of multiple species, we hope to accomplish the following: 1) to understand mechanisms underlying pathogenicity and virulence that relate to the attachment organelle; 2) to identify the mechanisms behind attachment organelle function by comparing species that have similar attachment organelles with a range of distinct properties; and 3) to understand how and why attachment organelle structure and function has diverged so much among these related species. The goal of chapter one was to characterize M. amphoriforme, a novel, possibly pathogenic organism, with respect to its closest relatives, M. pneumoniae and M. gallisepticum, by testing whether it exhibits similarities or differences in cell morphology and gliding motility. Our null hypothesis was that its morphology is similar to its relatives and that it exhibits gliding motility. We tested this hypothesis using scanning electron microscopy (SEM) and time-lapse microcinematography. The goal of chapter two was to characterize the eight species in the M. pneumoniae cluster by testing their similarities in morphology and gliding motility. Our hypothesis was that characteristics of morphology correlated with gliding motility in these species, addressing the issue of the mechanism of gliding motility in M. pneumoniae and its relatives. We tested this hypothesis using SEM and time-lapse microcinematography. Finally, the goal of chapter three was to characterize the interaction between the electron-dense core and the chromosome. Our hypothesis was that the origin of replication (oriC) or a region near it interacts specifically with the electron-dense core. We tested this hypothesis using fluorescence in situ hybridization (FISH), which has been successful in identifying such interactions in other bacteria (DuTeau et al., 1998; Jensen and Shapiro, 1999).

CHAPTER 1

Ultrastructure and gliding motility of Mycoplasma amphoriforme, a possible human respiratory pathogen

Jennifer M. Hatchel, Rebecca S. Balish, Matthew L. Duley, and Mitchell F. Balish

Microbiology. 2006. 52: 2181-2189.

16 Abstract Despite their small size and reduced genomes, many mycoplasma cells have complex structures involved in virulence. Mycoplasma pneumoniae has served as a model for the study of virulence factors of a variety of mycoplasma species that cause disease in humans and animals. These cells feature an attachment organelle, which mediates cytadherence and gliding motility and is required for virulence. An essential component of the architecture of the attachment organelle is an internal detergent-insoluble structure, the electron-dense core. Little information is known regarding its underlying mechanisms. Mycoplasma amphoriforme, a close relative of both M. pneumoniae and the avian pathogen Mycoplasma gallisepticum, is a recently discovered organism associated with chronic bronchitis in immunosuppressed individuals. In this work, we describe both the ultrastructure of M. amphoriforme str. A39T as visualized by scanning electron microscopy and the gliding motility characteristics of this organism on glass. Though externally resembling M. gallisepticum, M. amphoriforme cells were found to have a Triton X-100-insoluble structure similar to the M. pneumoniae electron-dense core but with different dimensions. M. amphoriforme also exhibited gliding motility using time- lapse microcinematography; its movement was slower than that of either M. pneumoniae or M. gallisepticum.

17 Introduction Within the Mycoplasma genus of the bacterial class Mollicutes, the pneumoniae group (Johansson & Pettersson, 2002) consists of numerous species, some commensal and some pathogenic, that live in association with vertebrates, including humans. Mycoplasma amphoriforme is a recently discovered member of the M. pneumoniae cluster of the pneumoniae group (Webster et al., 2003); its closest relatives include the avian respiratory pathogen Mycoplasma gallisepticum (Levisohn & Kleven, 2000), the human respiratory pathogen Mycoplasma pneumoniae (Waites & Talkington, 2004), and the human urogenital tract pathogen Mycoplasma genitalium (Jensen, 2004). Analysis of 16S ribosomal RNA sequence indicates a closer relationship with M. gallisepticum than with the others. M. amphoriforme strain A39T was identified as a frequent inhabitant of the respiratory tracts of immunodeficient patients with chronic bronchitis, but not of immunocompetent patients (Webster et al., 2003); it was cultured independently from such patients multiple times (Pitcher et al., 2005). The details of the clinical case presented (Webster et al., 2003) suggest that M. amphoriforme is an opportunist that is likely pathogenic, especially in immunodeficient individuals. The species of the M. pneumoniae cluster (Johansson & Pettersson, 2002) have numerous distinctive morphological features in common, including a flask-shaped appearance with a prosthecal polar structure usually called the attachment organelle in M. pneumoniae literature and the terminal bleb in M. gallisepticum literature (Kirchhoff et al., 1984; Balish & Krause, 2002). Best studied in M. pneumoniae, the attachment organelle is the primary site at which the mycoplasma cell attaches (cytadheres) to the host cell using localized adhesin proteins (Feldner et al., 1982; Hu et al., 1982; Baseman et al., 1987; Krause, 1996). The attachment organelle contains an electron-dense core (Biberfeld & Biberfeld, 1970) which is insoluble in the nonionic detergent Triton X-100 (TX) (Meng & Pfister, 1980; Gobel et al., 1981) and regarded as cytoskeletal. M. pneumoniae, M. genitalium, and M. gallisepticum also exhibit gliding motility on surfaces (Kirchhoff, 1992); cells invariably glide in the direction of the attachment organelle (Bredt, 1968). Although the mechanism of gliding is not known, loss of virulence due to mutations in the attachment organelle adhesin protein P30 is associated with reduced speed in M. pneumoniae (Hasselbring et al., 2005), suggesting that motility

18 is a virulence-associated trait. Duplication of the attachment organelle is also linked with the cell division process (Bredt, 1968; Seto et al., 2001). Cytadherence and virulence of M. pneumoniae depend upon a specific set of proteins associated with the attachment organelle, including those required for the presence of the electron-dense core (Balish and Krause, 2002; Seto and Miyata, 2003). Although ultrastructural aspects of the M. genitalium attachment organelle and the M. gallisepticum terminal bleb are less well-studied, loss of virulence in both species is associated with disruptions to homologues of these M. pneumoniae proteins (Mernaugh et al., 1993; Dhandayuthapani et al., 1999; Papazisi et al., 2002; Mudahi-Orenstein et al., 2003; May et al., 2006). However, despite the work that has been done in M. pneumoniae, the relationship between the physical features of the attachment organelle and the processes of cytadherence, gliding motility, and attachment organelle duplication and cell division remains obscure. Since M. amphoriforme is a possible human pathogen whose closest relatives include gliding mycoplasmas that have attachment organelles and associated cytoskeletal structures, we wanted to determine whether M. amphoriforme str. A39T shared these virulence-associated features. We have used scanning electron microscopy (SEM) to investigate the morphological features of M. amphoriforme, identifying a probable homolog of the cytoskeleton-like electron-dense core of M. pneumoniae that is distinct from the cytoskeletal structures of M. gallisepticum, despite the external appearance of the cell resembling that of M. gallisepticum much more closely than that of M. pneumoniae. We have also characterized the gliding motility properties of M. amphoriforme. The data indicate that M. amphoriforme is characterized by a novel combination of morphological features of both M. pneumoniae and M. gallisepticum.

19 Materials and Methods Growth and culture conditions. M. amphoriforme strain A39T (generously provided by J. Jensen, Staatens Serum Institut, Copenhagen, Denmark) was grown in plastic tissue culture flasks from frozen stocks for 4-6 days at 37 °C in SP-4 broth (Tully et al., 1979) to mid-log phase (phenol red indicator was orange). Cells adhered to the plastic surface of the flask. For motility stocks, cells were prepared according to Hasselbring et al. (2005), with minor modifications. Cells were grown to mid-log phase in 10 ml SP-4 broth. The supernatant was removed and the cells were scraped into 1 ml of fresh SP-4 broth. This suspension was then dispensed into 50-µl aliquots and stored at -80 °C. M. pneumoniae strain M129 and M. gallisepticum strain Rlow (generously provided by S. Geary, University of Connecticut, Storrs, CT, USA) were also cultured at 37 °C in SP-4 broth, with mid-log phase achieved after 2-3 days; motility stocks were prepared as for M. amphoriforme. Time-lapse microcinematographic analysis. Thirty-five-microliter samples of motility stock were inoculated into 765 µl SP-4 broth supplemented with 3 % gelatin in individual chambers of 4-well chamber slides (Nalge Nunc Intl.). The suspension was passed 7× through a 25-gauge needle and incubated 3 h at the appropriate temperature. Cells attached to the slide were visualized using a Leica DM IRB inverted microscope equipped with a 100× objective. The sample was held at the appropriate temperature (see text) using a heating chamber. Phase-contrast images were captured at fixed intervals using a SPOT charge-coupled device camera and accompanying software (Diagnostic Instruments, Inc.). Twenty-seven consecutive images were merged in different color channels using Adobe Photoshop CS version 8.0, enabling visualization of cell movement. The initial image is magenta, the final image is yellow, and the merged image of all 27 frames is cyan. Immotile cells appear black, whereas motile cells are in color (see Fig. 7). The distance traveled by each cell corresponds to the length of the cyan line from the tip of the magenta cell to the tip of the yellow cell as measured using the SPOT software. The speed of each cell was computed by dividing the distance traveled by the duration of movement. Cells moving between fewer than 10 of the 27 frames were counted as motile but their speeds were not included in calculations.

20 Scanning electron microscopy (SEM). Stocks were inoculated into 24-well plates containing 1 ml SP-4 broth and glass coverslips coated with 0.01 % poly-L-lysine. Cells were grown 1-3 days before processing. For analysis of TX-insoluble structures, coverslips were incubated 30 min at 37 °C in a solution containing 2 % TX in Tris-NaCl buffer (TN; 20 mM Tris-HCl, pH 7.5/150 mM NaCl) (Stevens & Krause, 1991). For analysis of both whole cells and TX-insoluble structures, coverslips were rinsed in TN, 4 × 5 min, and then fixed 30 min in 1 % glutaraldehyde / 2 % formaldehyde / 0.1 M sodium cacodylate, pH 7.2. Coverslips were subsequently rinsed with 0.1 M sodium cacodylate, 4 × 10 min, and dehydrated through a series of ethanol washes from 25 % to 100 %. After dehydration, the coverslips were critical point-dried and sputter-coated with 15 nm of gold. Images were viewed and captured at the Electron Microscopy Facility at Miami University on a Zeiss Supra 35 FEG-VP scanning electron microscope operating at 5 kV. Dimensions of the TX-insoluble structures were measured using SPOT software.

21 Results The cellular morphology of M. amphoriforme is similar to that of M. gallisepticum but distinct from M. pneumoniae. Thin sections of M. amphoriforme observed by transmission electron microscopy suggest a flask-shaped morphology (Webster et al., 2003; Pitcher et al., 2005) consistent with related mycoplasma species of the pneumoniae group (Kirchhoff et al., 1984; del Giudice et al., 1985; Bradbury et al., 1993). SEM of whole M. amphoriforme cells (Fig. 3a) confirmed that like their relatives in the pneumoniae group, they are pleomorphic, with a flask shape. These images also revealed that in many cells the polar extension terminates in a single knob-like structure whose shape is like that of the terminal bleb of M. gallisepticum (Fig. 3b) but distinct from that of the M. pneumoniae attachment organelle (Fig. 3c). M. pneumoniae cells also consistently have a trailing filament (Fig. 3c); this was observed only very rarely in M. gallisepticum and M. amphoriforme (data not shown). Some M. amphoriforme cells appeared to be larger with at least two extensions with knobs at opposite poles (Fig. 4a), closely resembling similar forms reported in M. gallisepticum (Morowitz & Maniloff, 1966). Others had three polar structures (Fig. 4b), and some cells appeared to have a pair of extensions in close proximity at one pole, occasionally with a third extension at the opposite pole (Fig. 4c). M. amphoriforme has TX-insoluble structures similar to those of M. pneumoniae but distinct from M. gallisepticum. Glass-adherent cells of all three species were extracted in 2 % TX. As observed by SEM, TX-insoluble structures of M. amphoriforme that remained adherent to the glass surface following extraction generally resembled those of M. pneumoniae (Fig. 5). These M. amphoriforme structures had elements resembling the terminal button, rod, base, and fibres, though each element was larger, more consistently observed, and better defined in M. amphoriforme than in M. pneumoniae (Fig. 5a). Fibers extending from the base were found in all M. amphoriforme specimens and exhibited considerable variability in orientation. Base-associated fibers were only rarely preserved in M. pneumoniae (Fig. 5b). The width of the rod-like portion of the M. amphoriforme TX-insoluble structure was 88 ± 12 nm (Table 1), nearly double the width of the M. pneumoniae rod. The total length of the rod and the approximately spherical, terminal button-like structure was 254 ±20 nm in M. amphoriforme (Table 1),

22 Fig. 3. Scanning electron micrographs of mycoplasma cells grown attached to coverslips. Images of cells are aligned with attachment organelle-like structure at the top. Panel (a), M. amphoriforme; panel (b), M. amphoriforme cells with a single polar structure; panel (c), M. gallisepticum; panel (d), M. pneumoniae. Scale bar for panel (a), 1 µm; scale bar for panels (b), (c), and (d), 250 nm.

24 Fig. 4. Scanning electron micrographs of presumptively dividing M. amphoriforme cells grown attached to coverslips. Panel (a), cells with attachment organelle-like structures at opposite poles; panel (b), cells with three protrusions roughly equidistant; panel (c), cells with adjacent protrusions. Scale bar, 250 nm.

26 Fig. 5. Scanning electron micrographs of mycoplasma cytoskeletal structures. M. amphoriforme [panel (a)], M. pneumoniae [panel (b)], and M. gallisepticum [panel (c)] cells grown attached to coverslips and extracted with 2 % TX (see methods). T, terminal button; R, rod; B, base; F, fibrous extensions. Arrows indicate longitudinal clefts; carets indicate individual fibres. Scale bars, panels (a) and (b), 250 nm; panel (c), 1 µm.

28 TABLE 1. Dimensions of the cytoskeleton-like structure of M. amphoriforme* and M. pneumoniae*. M. amphoriforme M. pneumoniae Terminal button width 88 ± 12† 44 ± 10 Terminal button length 73 ± 12 ND Rod width 82 ± 15 45 ± 8 Rod length 181 ± 17 ND Base width 164 ± 16 64 ± 14 Base length 90 ± 16 56 ± 14 (Terminal button + rod) length 254 ± 20 225 ± 20 Total length‡ 343 ± 23 282 ± 25

ND, not determined due to inconsistent resolution of the boundary between the terminal button and the rod. * Measurements (in nm) taken from 29 representative structures for M. amphoriforme and 34 representative structures for M. pneumoniae (see Fig. 3). † Mean ± SD. ‡ Excluding the fibers.

29 slightly longer than we measured for M. pneumoniae. The base in the M. amphoriforme structure was irregular but roughly rectangular, 164 ± 16 nm wide and 90 ± 16 nm long (Table 1), substantially larger than that of M. pneumoniae (Fig. 5). Despite the close phylogenetic relationship with M. amphoriforme and M. pneumoniae, the TX-insoluble fraction of M. gallisepticum was entirely dissimilar, with filamentous or fibrous masses lacking obvious organisation and occupying the volume of the entire cell (Fig. 5c). In M. pneumoniae, the two parallel components of the rod have been observed distinctly only in an hmw3- mutant (Willby & Krause, 2002) and in cross-section (Hegermann et al., 2002). In contrast, images of the TX-insoluble structures of M. amphoriforme clearly revealed that some rods were approximately doubled in width with a longitudinal cleft, suggestive of a double rod extending from a single base (Fig. 5a, images 4-6). Furthermore, in some cases, the rods were partly (Fig. 5a, image 7) or completely (image 8) separated, though still apparently extending from a single base, conceivably corresponding to cells with two extensions at a single pole (see Fig. 4c). In M. pneumoniae this cleft was clearly observed only rarely (Fig. 5b, image 7). M. amphoriforme cells exhibit slow gliding motility. Since SEM images indicated that M. amphoriforme has a combination of ultrastructural features of M. pneumoniae and M. gallisepticum, both of which exhibit gliding motility (Bredt, 1979), we investigated gliding motility in M. amphoriforme. Consecutive phase-contrast images of individual fields revealed that individual M. amphoriforme cells glided (Fig. 6). Although the polar protrusion (see Fig. 3) was difficult to see clearly at this resolution, it appeared that cells were moving in the direction of a tapered pole, consistent with this structure (Fig. 6, arrowhead). Cells moved in paths that were clockwise, counterclockwise, and approximately straight, and also changed directions (data not shown). Snapshots of living cells attached to glass under previously established optimal conditions for motility (Hasselbring et al., 2005) were captured at regular intervals; the paths taken by individual cells were characterised with respect to velocity and analysed as a population. We used a merged image of 27 consecutive frames captured at intervals adjusted to ensure that consecutive images of a given cell were overlapping. The false- colored merged image was overlaid with false-colored images of the first and last frames in order to indicate unambiguously the starting and stopping points for each cell;

30 Fig. 6. Consecutive phase-contrast images of M. amphoriforme gliding motility at 5-s intervals at 37°C. Three representative cells are indicated by arrows orientated horizontally, vertically, and diagonally. In the first frame (0 s), the carets point to the tapered ends of the indicated cells. In the last frame (40 s), the asterisks indicate the positions of each of the indicated cells at 0 s. Scale bar, 1 µm.

32 as a result, immotile cells were black in the final image, whereas motile ones appeared in color (see Fig. 7). Finally, the length of the path between the tip of the starting image and the final image was measured. As a positive control, motility of M. pneumoniae at 37 °C was measured at intervals of 1 s at an average speed of 336 nm/s ± 59 (Table 2), with 51 % of cells gliding at a given time, consistent with previous results (Radestock and Bredt, 1977; Hasselbring et al., 2005; Seto et al., 2005a). As measured at 37 °C using 2-s intervals (Table 2), M. gallisepticum str. Rlow glided with an average speed of 131 nm/s ± 38, faster than reported previously for M. gallisepticum (Bredt, 1979), with 64 % gliding at a time. Multiple fields of gliding M. amphoriforme cells were captured at 5-s intervals and analysed in this manner (Fig. 7). At 37 °C, M. amphoriforme cells glided at 49 nm/s ± 19, with 53 % of cells moving at any instant (Table 2). At room temperature M. amphoriforme cells attached poorly and moved more slowly; among those cells that did attach to the glass surface, a substantially smaller fraction was motile (data not shown). The large standard deviation in the speed of M. pneumoniae was distinct from that reported by previous workers (Hasselbring et al., 2005) (Table 2). However, for each species, gliding rates were unimodal, with a substantial proportion of cells gliding up to two-fold faster than the average speed (Fig. 8). The relatively low standard deviation of mean velocities from field to field for each species (Table 2) indicates that the range of cell velocities is present in each field, as opposed to conditions varying during analysis of each field.

33 Fig. 7. Measurement of M. amphoriforme gliding speed by time-lapse microcinematographic analysis. Phase-contrast images of cells attached to glass slides held at 37 °C were captured at 5-s intervals. Twenty-seven consecutive images were merged and the resulting track was measured (see methods). Panel (a), image at 0 s; panel (b), image at 130 s; panel (c), merged image of all 27 frames between 0 s and 130 s; panel (d), false-colored overlay of panels (a) (magenta), (b) (yellow), and (c) (cyan). See methods for details. For each cell, the distance was measured from the leading end of the magenta image to that of the yellow image along the cyan path, the number of frames in which the cell had moved was determined, and the speed was calculated. Scale bar, 1 µm.

35 TABLE 2. Gliding motility parameters.

Mean Total # % cells Mean Range of speed of Species # motile moving/ speed speeds field cells* cells frame† (nm/s)‡ (nm/s) (nm/s)§ M. amphoriforme 1034 621 53 49 ± 19 15 – 133 48 ± 4 M. gallisepticum 421 278 64 131 ± 38 50 – 286 131 ± 3 M. pneumoniae 149 80 51 336 ± 59 197 – 538 335 ± 8

* Total number of motile cells in all fields. † Because different populations of cells are motile in different frames, the total number of motile cells over an observation period is greater than the total number of motile cells in any one frame. ‡ Mean speed of motile cells ± SD. § Mean of mean speeds of motile cells in each field counted ± SD.

36 Fig. 8. Distribution of mycoplasma velocities about the mean. Gliding speeds of the individual motile cells for which data are shown in Table 2 were grouped into bins of 0.2 × the mean, designated x, for each species. The percentage of cells with speeds in a given range out of the total population of motile cells was plotted against the speed as a fraction of the mean speed for each species. White bars, M. amphoriforme (x = 49); black bars, M. gallisepticum (x = 131); grey bars, M. pneumoniae (x = 336).

38 Discussion Mycoplasma virulence is multifactorial, with contributions from activities associated with adherence (Balish & Krause, 2002), competition with host cells for nutrients (Razin et al., 1998), production of harmful molecules like peroxide (Tryon & Baseman, 1992), phase variation of surface molecules (Yogev et al., 2002), and motility (Miyata, 2005), in addition to immunopathological factors (Simecka, 2005). The molecular bases for these traits vary among mycoplasma species and are largely not well understood. In the human pathogen M. pneumoniae, molecules and cellular structures associated with cytadherence have been identified, including adhesins and cytadherence accessory proteins, all of which appear to be involved in formation and function of a polar protrusion, the attachment organelle (Krause & Balish, 2004), which is also instrumental in gliding motility. M. gallisepticum also has such a structure, albeit with a somewhat different appearance, and possesses related molecules, as do other species of the M. pneumoniae cluster (Balish & Krause, 2005), suggesting that this cluster of related species uses a common set of virulence factors. However, these factors have been well-studied only in M. pneumoniae to date, and our understanding of them is limited. Elucidating correlations between attachment organelle-associated characteristics and virulence- associated properties of species other than M. pneumoniae is essential for understanding the common molecular underpinnings of virulence in species of the M. pneumoniae cluster. At the same time, differences identified among these species will underscore specific adaptations of each, casting crucial light on the disease process itself. M. amphoriforme, a likely human pathogen (Webster et al., 2003; Pitcher et al., 2005), is a particularly relevant choice for such studies. Our results indicate that in contrast to the notion that all of these related mycoplasma species cause disease in essentially the same way at the cellular level, M. amphoriforme has a novel combination of the virulence-associated features of its relatives. M. amphoriforme cells resembled those of M. gallisepticum, not M. pneumoniae, in overall morphology, including the absence of a trailing filament and the presence of a short polar protrusion terminating in a widened knob. In striking contrast, M. amphoriforme cells had discrete TX-insoluble structures with significant resemblance to the M. pneumoniae electron-dense core, which is essential for the architecture and

39 functions of the attachment organelle in cytadherence and gliding motility (Balish & Krause, 2002). A similar TX-insoluble structure was not distinguishable in M. gallisepticum cells treated in a like manner amidst a remarkably complex mass of material (Fig. 5); extraction with concentrations of TX up to 10% did not substantially alter the appearance of the fraction (data not shown). These results do not discount the possibility of such an element being present within the M. gallisepticum TX-insoluble fraction, but they demonstrate that a range of ultrastructural complexity not previously anticipated is present among the species of the M. pneumoniae cluster. Although similarities to related species make it likely that the polar structure of M. amphoriforme is likely to be involved in infection, its role remains to be determined. We propose that as in M. gallisepticum, the polar protrusions observed in M. amphoriforme (Figs. 3, 4) function as sites of attachment, though this remains to be demonstrated formally; however, the contribution to this structure by TX-insoluble cell components is likely to be more similar to M. pneumoniae than to M. gallisepticum. Even so, the M. amphoriforme TX-insoluble structure is nearly twice as wide as and slightly longer than that of M. pneumoniae, with a much more prominent terminal button and base (Fig. 3); this difference in ultrastructure might point toward differences in properties associated with attachment organelle function, though it is not yet possible to propose the specific relationship between structural and functional differences. Like M. pneumoniae, M. amphoriforme has a set of large TX-insoluble proteins (data not shown); future studies will address the identities, locations, and cellular roles of these proteins. Two observations concerning the ultrastructure of the M. amphoriforme TX- insoluble structure relate to proposed roles of the electron-dense core in attachment organelle duplication in M. pneumoniae. First, the M. amphoriforme TX-insoluble structure commonly revealed a cleft within the rod portion (Fig. 5), its visualization perhaps facilitated by the greater width of the rod as compared with M. pneumoniae. This cleft might delimit the two parallel elements of the M. pneumoniae core (Hegermann et al., 2002); it also might represent a feature unique to M. amphoriforme or, at any rate, one that has not been described in M. pneumoniae. Second, it was possible to see occasional M. amphoriforme images in which a base had two separate and apparently single rods emanating from it (Fig. 5A, images 7 and 8). A possible interpretation of

40 these two observations is that the electron-dense core in both M. pneumoniae and M. amphoriforme consists of a paired rod whose two parallel elements separate longitudinally during attachment organelle duplication, as has been proposed (Krause & Balish, 2004). Alternatively, the new rod might be synthesised de novo on the same base as the original rod, with division occurring at the base. It is unclear why identical treatment of M. pneumoniae and M. amphoriforme results in differential preservation of these presumably homologous structures; perhaps the interaction between the base and the rod is stronger in M. amphoriforme, or this structure might be differentially TX- insoluble in the two species. Regardless, because duplication of attachment organelles and migration to the opposite pole in stages is postulated as the means of M. pneumoniae cell division (Bredt, 1968; Seto et al., 2001), images of relatively large M. amphoriforme cells with two or three protrusions (Fig. 4) reinforce the idea that duplication of the organelle and cell division are linked. In addition to cytadherence and cell division, a third aspect of mycoplasma physiology involving the attachment organelle is gliding motility. In M. pneumoniae as well as in Mycoplasma pulmonis and Mycoplasma mobile, which also exhibit polar protrusions despite lacking homologs of attachment organelle components, the polar structure leads the cell during gliding motility (Miyata, 2005). A handful of novel proteins has been directly implicated in gliding in these species, with an ATP-dependent attach-and-release mechanism suggested (Hasselbring et al., 2005; Seto et al., 2005a, 2005b; Uenoyama & Miyata, 2005). Despite the fact that the polar structure of M. amphoriforme is only marginally resolvable by light microscopy, time-lapse microcinematographic images of glass-adherent M. amphoriforme cells suggested that as in the other species, gliding occurs in the direction of a tapered pole (Fig. 6). However, the average speed was some 7-fold slower than that of M. pneumoniae and nearly 3-fold slower than that of M. gallisepticum (Table 2). Neither the molecular basis for gliding motility nor its physiological role is clear, but its conservation in species with different characteristics is further evidence that it is important. In conclusion, M. amphoriforme, a recently-discovered likely respiratory pathogen of the immunosuppressed, has virulence-associated ultrastructural features in common with the related species M. pneumoniae and M. gallisepticum. However, these

41 features are present in a unique combination, the overall morphology resembling the latter and the internal structure resembling the former. The dimensions of the M. amphoriforme TX-insoluble structure, which is a probable component of an attachment organelle, differ distinctly from those of the comparable structure in M. pneumoniae. M. amphoriforme also exhibits gliding motility, with a speed that is slower than either of the other species. The presence of distinct varieties and combinations of established virulence characteristics from other species underscores the fact that an understanding of M. amphoriforme-associated disease must derive from direct characterization of this organism, not just from application of information concerning its relatives.

42 ACKNOWLEDGEMENTS This work was supported by startup funds from Miami University. We are grateful to Jorgen Jensen and Steve Geary for supplying mycoplasma strains, to Xiufeng Wan for help with statistical analysis, to Duncan Krause and members of his lab and of the Balish lab for helpful discussions, and to Duncan Krause, Luis Actis, and Anne Hooke for help with the manuscript.

CHAPTER 2

Attachment organelle ultrastructure correlates with phylogeny, not gliding motility properties, in Mycoplasma pneumoniae relatives

Jennifer M. Hatchel and Mitchell F. Balish

Microbiology. 2008. 154: 286-295.

44 Abstract The Mycoplasma pneumoniae cluster is a clade of eight described species which all exhibit cellular polarity. Their polar attachment organelle is a hub of cellular activities including cytadherence and gliding motility, and its duplication in the species M. pneumoniae is coordinated with cell division and DNA replication. The attachment organelle houses a detergent-insoluble, electron-dense core whose presence is required for normal function. Although mutant analysis has led to the identification of attachment organelle proteins, the mechanistic basis for the activities of the attachment organelle remains poorly understood, with gliding motility attributed alternatively to the core or to the adhesins. In this study we investigate attachment organelle-associated phenotypes, including gliding motility characteristics and ultrastructural details, in seven species of the M. pneumoniae cluster under identical conditions, allowing direct comparison. We identified gliding ability in three species in which it has not previously been reported, Mycoplasma imitans, Mycoplasma pirum, and Mycoplasma testudinis. Across species, ultrastructural features of attachment organelles and their cores do not correlate with gliding speed, and morphological features of cores are inconsistent with predictions about how these structures are involved in the gliding process, disfavoring a prominent, direct role for the electron-dense core in gliding. Additionally, we found M. pneumoniae to be an outlier in terms of cell structure with respect to its close relatives, suggesting that it has acquired a special set of adaptations during its evolution.

45 Introduction The bacterial genus Mycoplasma includes over 100 species. These organisms lack cell walls and in nature are always associated with vertebrate hosts. Several mycoplasma species exhibit a polarized cell morphology, exemplified by Mycoplasma pneumoniae, a significant causative agent of assorted respiratory and non-respiratory human disease conditions (Waites and Talkington, 2004). This surface-adherent organism exhibits the pleomorphy typical of its genus, but is characterized by a prosthecal terminal organelle or attachment organelle at one pole (Balish, 2006b). This structure is the site at which adhesins are clustered, enabling the cell to interact productively with host cells (cytadhere). The attachment organelle is the leading end of the cell during gliding motility (Balish and Krause, 2006) and the location of the gliding motor (Hasselbring and Krause, 2007a, b). Gliding appears to be important for spreading from the initial infection site (Jordan et al., 2007). Duplication of the M. pneumoniae attachment organelle at a site adjacent to the existing one accompanies initiation of DNA replication (Seto et al., 2001). The cytoplasm within the M. pneumoniae attachment organelle lacks DNA, is clear of large particles, and contains a Triton X-100 (TX)-insoluble structure, the electron-dense core (Biberfeld and Biberfeld, 1970), which appears to be connected to an ill-defined network of cytoskeletal filaments present throughout the cell body (Meng and Pfister, 1980; Göbel et al., 1981). Proper assembly of the core is essential for formation of a functional attachment organelle (Krause and Balish, 2004), making the core essential for survival in vivo. The core consists of several subdomains, including a distal terminal button which is probably in direct contact with the attachment organelle tip, a perpendicularly striated double rod, and a proximal complex that includes a bowl-shaped component at the base (Henderson and Jensen, 2006). Although studies of M. pneumoniae attachment organelle mutants have led to the identification of many attachment organelle proteins and to the outline of an assembly pathway (Krause and Balish, 2004), how each protein contributes to the structure of the core remains poorly understood. Mycoplasma gliding is poorly understood compared to other prokaryotic forms of motility. The M. pneumoniae motility mechanism has been addressed through characterization of mutants with differences in colony spreading phenotypes, resulting in

46 the identification of a moderate number of genes of both known and unknown function (Hasselbring et al., 2006b). In Mycoplasma mobile, a distant relative whose adherence, motility, and terminal organelle components are unrelated to those of M. pneumoniae (Miyata, 2005), gliding is proposed to involve the cyclical binding and release by the adhesin Gli349 (Uenoyama et al., 2004) of carbohydrate moieties present on a wide variety of host cell proteins, including those deposited on the surface of the microscope slide from serum in vitro (Nagai and Miyata, 2006). A similar binding-and-release process might occur in M. pneumoniae, albeit through the use of an unrelated set of adhesins. In support of this hypothesis, the adhesins P30 (Hasselbring et al., 2005) and P1 (Seto et al., 2005a) have both been implicated in gliding motility of M. pneumoniae. Alternative hypotheses have focused on the electron-dense core as the major component of the M. pneumoniae motor, citing differences in fine features of individual M. pneumoniae cores (Henderson and Jensen, 2006) or postulating that the core is capable of twisting within the attachment organelle (Hegermann et al., 2002). Core-centered models require that the morphology of the core would change during motility, such as by shortening or twisting, and one might further predict that organisms with different motile properties would exhibit corresponding differences in the morphology of the core. The ability to distinguish among these models is predicated on an understanding of the relationship between motile properties and attachment organelle features. One approach to the basis for mycoplasma motility is the characterization of relatives of M. pneumoniae that have a similar motility mechanism but differing motility characteristics, thereby constituting a series of “naturally-occurring mutants.” Eight species are currently assigned to the M. pneumoniae cluster based on 16S rRNA sequence similarity (Johansson and Pettersson, 2002) and the presence of an attachment organelle that contains a core (Balish and Krause, 2005). Gliding has been described for some of these species, including M. pneumoniae, Mycoplasma genitalium, M. gallisepticum, and M. amphoriforme, while Mycoplasma alvi has been reported to be non-motile (Bredt, 1979; Hatchel et al., 2006). Mean speeds reported in this cluster range from 50 nm/s for M. amphoriforme to >300 nm/s for M. pneumoniae (Hatchel et al., 2006). Homologs of the M. pneumoniae attachment organelle proteins required for cytadherence and motility are restricted to the M. pneumoniae cluster (Tham et al., 1994;

47 Dhandayuthapani et al., 1999; Papazisi et al., 2002), making it likely that a common fundamental mechanism involving this set of proteins underlies attachment organelle- mediated functions in each of these organisms. For most of these species, detailed analysis of the dimensions of the electron-dense cores has not been performed. Correlating molecular and structural aspects of attachment organelle components with cytadherence and motility-related properties in each species will reveal which components are likely participants in the motility process, directing future research on the mechanistic aspects of mycoplasma gliding motility. We have therefore examined each species of the M. pneumoniae cluster with respect to gliding motility and, for motile species, the morphological features of attachment organelles and cores. Time-lapse microcinematographic analysis revealed gliding in all species of the M. pneumoniae cluster except M. alvi. However, scanning electron microscopy (SEM) revealed substantial differences in cell and attachment organelle dimensions among all species which, while largely associated with phylogenetic relatedness, correlated poorly with gliding motility characteristics, not supporting a direct role for the core in motility. Additionally, SEM revealed evidence of DNA association with the proximal end of the core in most species.

48 Materials and Methods Growth and culture conditions. The strains used in this study were: M. pneumoniae strain M129; M. genitalium strain G37; M. gallisepticum strain Rlow; M. imitans strain 4229; M. amphoriforme strain A39; M. testudinis strain 01008; M. pirum strain 70-159; and M. alvi strain Ilsley. Cells were grown in plastic tissue-culture flasks from frozen stocks for 2-10 d at 37°C (30°C for M. testudinis) in SP-4 broth (Tully et al., 1979) to mid-exponential phase (phenol red indicator was orange). M. alvi was grown in a modified SP-4 formulation containing a 1:1 mixture of fetal bovine and porcine sera (HyClone, Logan, UT) and grown until slightly turbid. Motility stocks were prepared as previously described (Hatchel et al., 2006). For M. testudinis and M. pirum, cells were passaged 3-5 times in SP-4 broth to enrich for plastic-attached subpopulations prior to further use and analysis. Phylogenetic tree. The 16S rRNA sequence for each species was entered into BioEdit Sequence Alignment Editor v. 7.0.5.2 (Hall, 1999) for trimming and neighbor-joining alignment by CLUSTAL X v. 1.8 (Thompson et al., 1997). The phylogenetic tree was generated from these data by NJplot (Perrière and Gouy, 1996). SEM. SEM was performed as previously described (Hatchel et al., 2006) with minor modifications. Briefly, stocks were grown in 24-well plates containing glass coverslips for 3 h - 1 d before processing. For analysis of TX-insoluble structures, coverslips were incubated 30 min at 37°C in a solution containing 2% TX in TN buffer (20 mM Tris HCl, pH 7.5, 150 mM NaCl). For DNase treatment, cells were grown 1-4 d on coverslips before processing. TX extraction was followed by a 30 min incubation with 5 U bovine pancreas DNase I (Sigma-Aldrich, St. Louis, MO) in 500 µl 1X DNase reaction buffer

(10 mM Tris-HCl, pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2) at room temperature. For RNase A treatment, TX-extracted coverslips were incubated 30 min in 500 µl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM ethylenediaminetetraacetic acid) containing 100 µg RNase A (Qiagen Inc., Valencia, CA). For whole cells, coverslips were fixed 30 min in fixative A (1.5% glutaraldehyde/1% formaldehyde/0.1 M sodium cacodylate, pH 7.2; M. pneumoniae, M. genitalium, M. gallisepticum, M. imitans, and M. pirum) or fixative B (1% glutaraldehyde/2% formaldehyde/0.1 M sodium cacodylate, pH 7.2; M. amphoriforme and M. testudinis). For visualization of TX-insoluble material, fixative A

49 was used for all species. Dehydration, critical point drying, and gold coating were performed as previously described (Hatchel et al., 2006). Images were viewed and captured on a Zeiss (Carl Zeiss, Inc., Thornwood, NY) Supra 35 FEG-VP scanning electron microscope operating at 5 kV. Dimensions of whole cells, TX-insoluble structures, and structures following DNase treatment were measured individually using SPOT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Time-lapse microcinematographic analysis. Motility sequences were captured as previously described (Hatchel et al., 2006). Briefly, cells were inoculated into chamber slides containing SP-4 + 3% gelatin. After 3 h incubation at 37°C, cells attached to the slide were visualized using a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany). The sample was held at 37°C in a heated chamber. Phase-contrast images were taken at appropriate time intervals and later merged into false-color channels using Adobe (San Jose, CA) Photoshop CS version 8.0. The speed of each cell was computed by measuring the distance traveled and dividing it by the duration of movement. Cells moving in fewer than 10 of 27 frames were considered to be motile, but were not included in the calculation of mean speed.

50 Results Gliding motility. Of the eight species found in the M. pneumoniae cluster based on 16S rRNA sequences (Fig. 9; Pitcher et al., 2005), only M. alvi did not attach to glass and exhibit gliding motility, as previously reported (Bredt, 1979); this species was therefore not considered for further investigation. For M. pirum and M. testudinis, only a small minority of the population derived from the initial stocks attached to glass. In these cases, all further studies were performed on plastic-attached subclones. Efforts to enrich for a similar population of M. alvi cells were unsuccessful. Gliding characteristics, which were previously determined by our method for M. pneumoniae, M. amphoriforme, and M. gallisepticum, were assayed for the remaining species (Hatchel et al., 2006). Mean speeds ranged from 28 nm/s for M. pirum to 2,971 nm/s for M. testudinis (Table 3). M. testudinis was the fastest, though with the lowest percentage of motile cells. Though cells glided in the direction of the attachment organelle, large-scale cellular movement appeared generally random for most species. However, ~80% of M. genitalium and M. testudinis cells moved in nearly circular patterns (not shown). There was no relationship between phylogeny and gliding speed. Attachment organelle and cell morphology. To test whether gliding characteristics correlated with ultrastructural features of the attachment organelle, we analyzed both whole cells and TX-insoluble electron-dense cores in each of the seven adherent, motile species by SEM. Attachment organelles were divided into substructures for common reference: the distal moiety was termed the knob, and the proximal portion, the shaft (Fig. 10H). Dimensions of individual attachment organelles and cells (Table 4) were measured. M. gallisepticum (Fig. 10A), M. imitans (Fig. 10B), M. amphoriforme (Fig. 10C), and M. testudinis (Fig. 10D) exhibited short, wide attachment organelles, with mean lengths from 120-150 nm, and widths from 130-150 nm (Table 4). The knob was distinct from the shaft in M. gallisepticum and M. amphoriforme, sometimes in M. testudinis, but not in M. imitans. The M. pirum attachment organelle, which also featured a prominent knob, was long, at 250 nm, and moderately wide, at 100 nm (Table 4), and uniquely exhibited 3-4 parallel striations perpendicular to the long axis (Fig. 10E). M. genitalium (Fig. 10F) and M. pneumoniae (Fig. 10G) had narrow shafts, at only 80 nm in width. The M. genitalium structure, at 170 nm, was substantially shorter than that of M. pneumoniae, which at 290

51 Fig. 9. Phylogenetic tree based on 16S rRNA sequences of mycoplasmas within the M. pneumoniae cluster. Scale bar, 0.1 substitutions per site.

53 TABLE 3. Gliding motility parameters % cells Mycoplasma species Mean speed Range moving/ (nm/s) ± SD (nm/s) Frame M. pneumoniae str. M129 a 336 ± 59 197 - 538 51 M. genitalium str. G37 111 ± 22 62 - 172 72 a M. gallisepticum str. Rlow 131 ± 38 50 - 286 64 M. imitans str. 4229 107 ± 31 41 - 194 65 M. testudinis str. 01008 2,971 ± 570 2,184 – 4,742 37 M. amphoriforme str. A39 a 49 ± 19 15 - 133 53 M. pirum str. 70-159 28 ± 8 12 - 58 45 a Data from Hatchel et al. (2006).

54 Fig. 10. Scanning electron micrographs of mycoplasma cells grown on glass coverslips. Three representative cells are shown for each species. Images are aligned so that the attachment organelle is at the top. (A) M. gallisepticum; (B) M. imitans; (C) M. amphoriforme; (D) M. testudinis; (E) M. pirum; (F) M. genitalium; (G) M. pneumoniae; (H) schematic of mycoplasma cell. Open and black arrows indicate a ridge-like feature at the base of the attachment organelle shaft in M. gallisepticum and M. amphoriforme respectively; white arrows indicate striations on the M. pirum attachment organelle that are perpendicular to the long axis; black arrowheads indicate the curvature of M. testudinis and M. genitalium attachment organelles. Scale bar, 250 nm.

56 TABLE 4. Whole cell dimensions (in nm) ± SD, mean of 30 cells

Attachment Whole cell Cell body organelle Knob length Length width length width Length width M. pneumoniae 1,500 ± 420 520 ± 90 220 ± 30 290 ± 40 80 ± 10 ND a ND M. genitalium 590 ± 50 420 ± 50 280 ± 50 170 ± 20 80 ± 10 80 ± 10 80 ± 10 M. gallisepticum 830 ± 120 710 ± 110 330 ± 70 120 ± 20 150 ± 20 100 ± 20 150 ± 20 M. imitans 620 ± 50 500 ± 50 350 ± 30 120 ± 10 140 ± 20 ND ND M. amphoriforme 770 ± 70 620 ± 70 360 ± 60 150 ± 20 150 ± 20 90 ± 10 140 ± 20 M. testudinis 680 ± 100 560 ± 100 280 ± 20 120 ± 10 130 ± 20 ND ND M. pirum 720 ± 110 480 ± 90 230 ± 20 250 ± 30 100 ± 10 100 ± 20 100 ± 10

a ND, not determined because knob and shaft were not uniformly distinguishable.

57 nm was the longest of the cluster, consistent with previously noted differences (Lind et al., 1984). Knobs were indistinct in M. pneumoniae attachment organelles (Fig. 10G). The two species that exhibited circular gliding paths had curved attachment organelles. For M. testudinis, bent attachment organelles were occasional (Fig. 10D), whereas M. genitalium attachment organelles were all curved 10°-30° from the long axis of the cell (Fig. 10F), and the knob, prominent in this species, was off-center in the direction of this curvature. Measurements of cell bodies revealed a positive correlation between cell width and attachment organelle width (Table 4). M. pneumoniae and M. pirum cells were distinctly narrow in comparison to the others (Figs. 10E, G; Table 4). In contrast, cell length appeared unrelated to other features (Table 4). M. pneumoniae cells were unique in the prominence of the trailing filament found at the pole opposite the attachment organelle (Fig. 10G). This structure, whose length varied greatly, drove the mean length of the M. pneumoniae cell to 1500 nm (Table 4). M. gallisepticum and M. amphoriforme cell bodies often had a ridge-like feature in the vicinity of the base of the attachment organelle shaft (Figs. 10A, C). Cell division We observed SEM fields of cells from each of the seven adherent species of this cluster (Fig. 11A-G) to characterize cells that appeared to be in the process of dividing. Either the appearance of two attachment organelles at the same pole on a single cell or two attachment organelles pointing in different directions was taken to indicate that cell division was in progress. All species except M. imitans (Fig. 11B) were observed to have cells exhibiting one or both of these phenotypes. Dividing M. pirum cells were only found to have attachment organelles at opposite poles (Fig. 11E), and in M. gallisepticum, adjacently paired attachment organelles were not observed (Fig. 11A), suggesting that in these species the new attachment organelle might not form immediately adjacent to the old one as in M. pneumoniae. Triton X-100-insoluble structures Substructures of the electron-dense core of the attachment organelle, namely the terminal button, the rod, the base, and a tuft of fibrous material (Fig. 12H; Hatchel et al., 2006),

58 Fig. 11. Scanning electron micrographs of mycoplasma cells grown on glass coverslips. Each image is a representative field of cells. (A) M. gallisepticum; (B) M. imitans; (C) M. amphoriforme; (D) M. testudinis; (E) M. pirum; (F) M. genitalium; (G) M. pneumoniae. Arrows indicate cells that have two attachment organelles pointing at opposite poles; arrowheads indicate cells with two attachment organelles not yet at opposite poles on a single cell. Scale bar, 1 µm.

60 Fig. 12. Scanning electron micrographs of mycoplasma electron-dense cores. Under TX only, cells were grown on glass coverslips and extracted with 2% TX for 30 min at 37°C (see Materials and Methods). Under TX + DNase, cells were grown on glass coverslips, extracted with TX as above, and then treated with DNase I for 30 min at room temperature (see Materials and Methods). Two representative cores are shown for each species with and without DNase treatment, with terminal buttons aligned at the tops of images. (A) M. gallisepticum; (B) M. imitans; (C) M. amphoriforme; (D) M. testudinis; (E) M. pirum; (F) M. genitalium; (G) M. pneumoniae; (H) schematic of the mycoplasma electron-dense core substructures; (I), M. gallisepticum treated with TX and then RNase A. Arrows point to notches in M. gallisepticum and M. imitans bases. Scale bar, 250 nm.

62 have been described. Although we previously had difficulty resolving structures in M. gallisepticum (Hatchel et al., 2006), the use of a fresh preparation of TX resulted in consistent observation of core-like structures in all seven species (Fig. 12). Rod length was conserved across species over a narrow range from 130-170 nm; inclusion of the terminal button raised the values to 180-240 nm (Table 5). However, rod width varied substantially among species, from 30 to 80 nm (Table 5), and the variation correlated well with phylogenetic relatedness. The length of the M. imitans rod (Table 5) is underestimated on account of being variably obscured by the unusual base (see below). The slight difference between the current results and those previously reported for rod width in M. amphoriforme might owe to the use of a different fixative preparation (Hatchel et al., 2006). Terminal button length and width varied somewhat across species, and although the variation appeared unrelated to gliding speed, width appeared to have a phylogenetic component, with M. pneumoniae and M. genitalium exhibiting the narrowest terminal buttons (Table 5). The M. pneumoniae terminal button was frequently difficult to distinguish from the rod (Fig. 12G), as previously described (Hatchel et al., 2006). The M. genitalium rod exhibited a pronounced lateral curvature, and the terminal button was consistently offset in the direction of this curvature (Fig. 12F). Curvature of the electron-dense core was also visible in ~25% of M. testudinis cells, though in this species the curvature was more abrupt and located close to the terminal button (Fig. 12D). Except in M. pneumoniae, the base of the electron-dense core was obscured by irregular material. Treatment of adherent TX-insoluble fractions with DNase I resulted in highly consistent loss of this material, exposing the base of the core (Fig. 12). Incubation in buffer alone had no effect, nor did treatment with RNase A (Fig. 12I). Pronase caused loss of all visible structures (data not shown). In M. genitalium (Fig. 12F) and M. amphoriforme (Fig. 12C) the DNase-sensitive material was predominantly filamentous, with some clumpier regions; in M. gallisepticum (Fig. 12A), M. imitans (Fig. 12B), and M. testudinis (Fig. 12D) this mass was mostly nodular, with occasional filaments; and in M. pirum (Fig. 12E) the material had a smooth appearance with some short filaments extending from it. These filaments are consistent with the appearance of loops of DNA that have been locally denuded of protein (Cunha et al., 2001). Taken together, these data

63 are consistent with the identification of the core-associated material as DNA, rendered condensed by treatment with detergent and salt (Cunha et al., 2001), although whether the interaction between the DNA and the core is physiologically relevant is unclear. Treatment with DNase had no significant effects on measurements of other core substructures, except for occasional lengthening of the rod due to its being less obscured. The dimensions of the exposed bases were highly variable across species (Table 5; Fig. 12). The base of M. pneumoniae was by far the smallest in both length and width. At the other extreme was M. imitans, which had a very large and highly irregularly shaped base (Fig. 12B). The M. gallisepticum and M. imitans bases consistently had a notch-like feature of irregular orientation (Figs. 12A, B). No relationship between base dimensions and gliding speed or phylogeny was apparent across species. In fact, although attachment organelle morphology generally correlated well with phylogeny, there was no relationship detected between gliding speed and any element of attachment organelle morphology.

64 TABLE 5. Electron-dense core dimensions (in nm) ± SD, mean of 30 cells Terminal button + Terminal button Rod rod length Base length Width Length Width length width M. pneumoniae ND a 60 ± 10 ND 50 ± 10 240 ± 20 70 ± 10 70 ± 10 M. genitalium 50 ± 10 60 ± 20 160 ± 20 50 ± 10 220 ± 20 110 ± 20 120 ± 20 M. gallisepticum 90 ± 10 90 ± 20 140 ± 30 30 ± 0 230 ± 30 120 ± 40 140 ± 40 M. imitans 60 ± 20 60 ± 20 130 ± 60 40 ± 10 180 ± 70 240 ± 60 160 ± 50 M. amphoriforme 70 ± 10 90 ± 20 160 ± 20 60 ± 10 220 ± 20 90 ± 20 140 ± 30 M. testudinis 80 ± 10 80 ± 10 170 ± 20 80 ± 10 240 ± 20 150 ± 40 120 ± 20 M. pirum 90 ± 10 90 ± 10 130 ± 10 80 ± 10 210 ± 10 100 ± 20 110 ± 10 a ND, not determined because rod and terminal button were difficult to distinguish.

65 Discussion Gliding motility and ultrastructure In the present study, all species of the M. pneumoniae cluster except M. alvi exhibited gliding motility, raising the total number of motile mycoplasma species to nine with the addition of M. imitans, M. testudinis, and M. pirum. These seven organisms exhibited a wide range of speeds and proportions of cells moving at a given time. M. testudinis populations had a low frequency of motile cells, but interestingly, the motile cells glided as rapidly as M. mobile, whose gliding speed was previously unchallenged among mycoplasmas (Miyata, 2005). Its ultrastructure and phylogenetic position suggest that M. testudinis has a mechanistically similar motor to that of its close relatives, but that it and M. mobile have evolved, perhaps under similar unidentified pressures, to glide rapidly. Alternatively, despite its close relatedness to M. pneumoniae and its similar appearance to its relative, M. testudinis might have acquired a different mechanism for motility, perhaps through horizontal gene transfer. However, the absence of a relationship between the motor mechanism and gliding speed is observed in Mycoplasma pulmonis, which appears to have the same gliding machinery as M. mobile (Seto et al., 2005b), despite its mean speed being much slower (Bredt and Radestock, 1977). Aside from the curvature of the rod in relation to large-scale near-circular movement in M. genitalium and M. testudinis, no measured dimension of the attachment organelle appeared to correlate with gliding characteristics. Curved paths were previously indicated for M. genitalium (Pich et al., 2006). The curvature of these structures is quite different in either species, with the rod of the M. genitalium core exhibiting a shallow curvature throughout (Fig. 12F), and that of the M. testudinis core, when curved, being sharply bent at a single location (Fig. 12D). Importantly, this curvature is distinct from the bend observed in M. pneumoniae cores in cells that are not attached to the substrate (Henderson and Jensen, 2006). We do not observe this bend under our conditions in M. pneumoniae or any other species, perhaps because when attached to the substrate the attachment organelle is under tension. In no case did we observe cores that were twisted, as predicted by one model (Hegermann et al., 2002). Although the M. pneumoniae core is clearly suggested to have a broad and a narrow side (Regula et al., 2001; Hegermann et al., 2002; Henderson and

66 Jensen, 2006), there was little variation in rod width within any species. The inchworm model, an alternative model for core function during gliding (Henderson and Jensen, 2006), invokes repeated contraction and expansion of the core along its length, which might have been borne out by substantial differences in rod length of cells within a single species. However, except for M. imitans, whose rod was difficult to measure because of obfuscation by the large, irregular base, distributions of the length of the terminal button plus the rod were not dramatic. It is possible that the degree of contraction is too small to be measured by our techniques, but also possible that contraction does not occur. Also, observations of gliding cells did not suggest any quantized movement, though the step size could be smaller, or the steps faster, than our ability to detect. The consistency in the dimensions of cores within each species leads us to propose that the electron-dense core, though an important determinant in attachment organelle biogenesis, is not especially dynamic, and not directly involved in motility. Cell division Whether or not the electron-dense core has direct involvement in gliding motility, it might well play a direct role in some other process. Its roles in normal attachment organelle formation and adhesin localization in M. pneumoniae are well-documented (Krause and Balish, 2004), though other core-lacking mycoplasmas like M. mobile (Shimizu and Miyata, 2002) form functional attachment organelles. The special relationship between attachment organelle formation and DNA replication in M. pneumoniae might highlight a role for the electron-dense core in coordination between the two events. An increase in the amount of cellular DNA is accompanied by appearance of a second attachment organelle adjacent to the first one in M. pneumoniae, and the distance between the two organelles increases with continually increasing DNA levels (Seto et al., 2001). Furthermore, time-lapse microcinematography of dividing M. pneumoniae cells reveals that a new attachment organelle generally appears adjacent to the old one, and the new one anchors the cell in place while the old one glides away from it (Hasselbring et al., 2006a), resulting in the two structures being present at opposite poles. Finally, in M. gallisepticum, newly synthesized DNA is specifically enriched in a biochemical fraction itself enriched for attachment organelles (Maniloff and Quinlan, 1974). Although it could be artifactual, the physical interaction observed in this study

67 between the cellular DNA and the electron-dense core is intriguing and will be a subject of future studies. Evolution Clearly, the electron-dense core and the attachment organelle, as well as gliding motility, were present in the last common ancestor of the M. pneumoniae cluster; these features were presumably conferred by acquisition of the set of cytadherence-associated genes present in M. pneumoniae and its relatives from an unidentified source. The considerable lengths of the M. pneumoniae and M. pirum structures appear to have arisen independently, since the M. genitalium attachment organelle is not much longer than that of M. amphoriforme. Curvature in M. genitalium and M. testudinis also appears to have developed independently, as discussed above. Thus, the progenitor of these species most likely harbored a short, wide attachment organelle reminiscent of M. gallisepticum and M. amphoriforme. As the knob is markedly reduced only in M. pneumoniae, it was probably prominent in the common ancestor. Additionally, the trailing filament appears to be unique to M. pneumoniae and therefore unlikely to have been present in the parent species. Finally, the presence of faster gliding only in M. pneumoniae and especially M. testudinis suggests that slower motility was probably ancestral. M. pneumoniae appears to be the most derived member of its cluster with respect to attachment organelle and other ultrastructural phenotypes. Its speed is not close to that of any of its relatives, and the reduction in the size of the distal elements of the structure is likely an adaptation to an unknown pressure. Numerous proteins involved in gliding are apparently unique to the subclade containing M. pneumoniae and M. genitalium (Hasselbring et al., 2006b), and it is conceivable that some of these proteins are involved in processes special to one or both of these organisms. Thus, extrapolation of M. pneumoniae attachment organelle-related data to other species ought to be applied cautiously. In contrast, M. amphoriforme (Fig. 13) might be the most primitive, with its slow speed and average dimensions; as such, it might constitute a more general model for attachment organelle function in species of the M. pneumoniae cluster.

68 Fig. 13. Schematic of a typical M. amphoriforme cell. External features are in black; internal features are in gray. Dimensions are based on data from Tables 4 and 5. Scale bar, 50 nm.

70 Acknowledgments This work was supported by funds from Miami University. SEM was performed at the Miami University Electron Microscopy Facility. We thank the following for strains: L. Duffy and K. Waites, University of Alabama-Birmingham (M. genitalium); S. Kleven, University of Georgia (M. imitans); M. Brown, University of Florida (M. testudinis); M. Davidson, Mollicutes Culture Collection, Purdue University (M. alvi, M. pirum). Thanks also to R. Balish and E. Bridge for suggestions about the manuscript.

CHAPTER 3

Fluorescence in situ hybridization as a method for characterizing the interaction between the electron-dense core and the chromosome in Mycoplasma pneumoniae

Jennifer M. Hatchel and Mitchell F. Balish

72 Abstract Mycoplasma pneumoniae cells are polarized, with an attachment organelle containing an electron-dense core at one pole. The electron-dense core is associated with the chromosome. During cell division, a nascent attachment organelle appears next to the existing attachment organelle as the amount of genetic material is increasing, followed by migration of one structure to the opposite pole prior to cytokinesis. This organization suggests a role for the physical linkage between the DNA and the electron-dense core in segregation of chromosomes. In this study we examine the interaction between the electron-dense core and the chromosome using fluorescence in situ hybridization (FISH) in Mycoplasma pneumoniae, assessing the feasibility of using this technique in this organism. We find that the DNA probes against the 16S rRNA gene sequence and the oriC region bind specifically in M. pneumoniae, while the negative control does not. However, the focus for the 16S rRNA probe is very diffuse, whereas the focus for the oriC probe is more compact. The localization of the oriC probe is inconsistent with a permanent interaction between the oriC locus and the electron-dense core. Further optimization of the protocol is necessary.

73 Introduction Cell division, which includes faithful replication and segregation of DNA, is a necessity for all living cells. Readiness of the cell, the ability to duplicate and partition the chromosome, and efficiency of the division machinery are three important factors that influence when division is most likely to occur (Balish and Krause, 2005). First, the cells must be in an environment that is conducive to division, including the availability of nutrients. The size of the cell and the integrity of the DNA also play important roles in determining whether the cell is ready to divide (Balish and Krause, 2005). Second, the chromosome must be replicated, beginning at the origin of replication (oriC) (Mott and Berger, 2007). Once the chromosome is duplicated, each copy must be partitioned to opposite poles of the cell, thus ensuring that each daughter cell gets a complete copy of the chromosome. Finally, once the partitioning is complete, cytokinesis, or division of the cytoplasm, occurs resulting in two daughter cells. Bacterial cell division has been extensively studied in model organisms, including Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, and while many aspects of the process have been uncovered, much remains unknown. In these bacteria, coordination between the cell membrane, the cell wall, and in the case of gram-negatives such as E. coli the outer membrane, is essential in properly dividing cells (Balish and Krause, 2005). In model bacteria, the process of partitioning the chromosomes starts as early as DNA replication due to the location of the replication machinery itself (Lemon and Grossman, 1998, 2000; Haeusser and Levin, 2008). In these systems, replication occurs in the middle of the dividing cell, and as new DNA is synthesized, it is condensed and pushed toward the poles, which ultimately separates the two chromosomes (Haeusser and Levin, 2008). The Mollicutes, unlike these model bacteria, lack cell walls, suggesting that the details of their cell division process may differ substantially from model bacteria (Balish and Krause, 2005). Mycoplasmas, which belong to the class Mollicutes, are associated with vertebrate hosts, and have experienced reductive evolution that makes their genome the smallest among organisms capable of self-replication in a laboratory setting. Mycoplasma pneumoniae is the causative agent of atypical (walking) pneumonia, tracheobronchitis, and other diseases (Waites and Talkington, 2004) and has been associated with asthma

74 (Esposito et al., 2000; Waites and Talkington, 2004; Atkinson et al., 2008). Its closest relatives include the human urogenital pathogen, Mycoplasma genitalium (Jensen, 2004) and the avian pathogen, Mycoplasma gallisepticum (Levisohn and Kleven, 2000). All these species have a polar attachment organelle that mediates attachment to host cells in vivo or glass and plastic in vitro (Kirchhoff et al., 1984; Balish and Krause, 2002). The attachment organelle also plays a role in gliding motility (Bredt, 1968) and cell division (Seto et al., 2001). Cells with multiple attachment organelles have been observed using electron microscopy, and it is assumed that these cells are undergoing cell division (Bredt, 1968; Seto et al., 2001). In M. pneumoniae, cell division requires the duplication and migration of the attachment organelle, which happens in concert with replication and partitioning of the chromosome (Seto et al., 2001). In dividing cells, a nascent attachment organelle forms next to the existing one, and then migrates to the opposite pole before cytokinesis (Seto et al., 2001; Hasselbring et al., 2006a). The cotranscription of genes for attachment organelle proteins P30, HMW1, and HMW3 (Waldo et al., 1999; Krause and Balish, 2001), and HolA (Kim et al., 1997; Song et al., 2001), a protein involved in loading DNA polymerase onto the DNA (Balish and Krause, 2005), further supports a link between attachment organelle assembly and initiation of DNA replication in M. pneumoniae. Fluorescent microscopic analysis supports a correlation between the amount of DNA and the number of P1 foci, which represents the number of attachment organelles (Seto et al., 2001). The chromosome was visualized by staining the cells with 4’,6- diamidino-2-phenylindole (DAPI), and antibodies against P1 were used to identify the attachment organelle. Cells with one P1 focus have the smallest amount of DNA; cells with two foci at opposite poles, suggesting that migration has been completed, have the most DNA; and cells with two foci that had not completely migrated have intermediate amounts of DNA. Additionally, the attachment organelle houses a cytoskeleton-like electron-dense core (Biberfeld and Biberfeld, 1970), which appears to have a physical interaction with the chromosome, at least in M. pneumoniae relatives (Quinlan and Maniloff, 1973; Hatchel and Balish, 2008), suggesting the involvement of the attachment organelle during partitioning events in M. pneumoniae.

75 The interaction between the attachment organelle and the chromosome is not unique as interactions between the DNA and the poles are observed in other bacteria and these interactions are necessary for partitioning events. In C. crescentus, partitioning is mediated by ParB (Toro et al., 2008), which binds to the parS sites near the origin of replication and spreads along the DNA to form a nucleoprotein (Sullivan et al., 2009). ParA, a Walker-box ATPase, interacts with the ParB-parS complex to partition the chromosomes to the opposite poles and to position the chromosome in the proper orientation (Sullivan et al., 2009). In B. subtilis, homologs of ParA and ParB are called Soj and Spo0J, respectively (Ireton et al., 1994). Both proteins are necessary for positioning of the origins, but it is Spo0J that is essential for chromosome segregation (Sullivan et al., 2009). Interestingly, in E. coli, homologs of these proteins are used only for plasmids, not for the chromosome, indicating other mechanisms for segregation of the chromosome (Ben-Yehuda et al., 2003). M. pneumoniae encodes a homolog of ParA/Soj, but not a homolog of ParB/Spo0J, and its function is still unknown (Balish and Krause, 2005). It is conceivable that in M. pneumoniae, as the nascent attachment organelle migrates to the opposite pole, it pulls the chromosome along with it, thus driving partitioning in the absence of a ParB/Spo0J homolog. The mechanism driving cell division in this organism has fundamental differences from those seen in model bacteria. By studying cell division in M. pneumoniae, it may be possible to learn how a complex protein structure within the attachment organelle is involved in chromosome partitioning and also how the complex events are coordinated in a minimal organism. The possible interactions between the electron-dense core and the chromosome during cell division include: 1) the core binds to a specific region of the chromosome at all times; 2) the core has specific interactions with variable regions; or 3) the core has non-specific interactions with the chromosome. We propose that the interaction between the base of the electron-dense core and the chromosome occurs at a specific location. We hypothesized that the oriC region of the chromosome interacts with the base of the electron-dense core in M. pneumoniae because of the link between attachment organelle biogenesis and DNA replication. We used FISH coupled to immunofluorescence to localize regions of the chromosome with respect to the attachment organelle. If our hypothesis is correct, we

76 expect to see the oriC probe localizing at a small or fixed distance from HMW1, which represents the attachment organelle. If our hypothesis is not correct, then we expect to see the oriC probe localizing at a large or variable distance from HMW1.

77 Materials and Methods Cell cultures. M. pneumoniae M129 cells were cultured as previously described (Tully, 1983). Labeling the FISH probes. Probes for the 16S rRNA sequence and the oriC sequence were synthesized by PCR with primers designed against sequences in the genome just outside of the gene of interest. Primer sequences were: A: 5’-AGAGTTTGATCCTGGCTCAGGA-3’; B: 5’- GTAGGGATACCTTGTTACGACT-3’; C: 5’-TTCTTTATTAGTATACGCGC-3’; and D: 5’-TAATCAAAACTTTCATGCCC-3’ (A and B, 16S; C and D, oriC). The DNA template was M. pneumoniae genomic DNA that was isolated using a phenol/chloroform protocol. Samples were denatured at 94°C for 5 min. Subsequent rounds of amplification with the 16S primers (A and B) consisted of 45 s denaturation at 94°C, 1 min annealing at 59°C, and 2 min extension at 72°C. Subsequent rounds of amplification with the oriC primers (C and D) consisted of 45 s denaturation at 94°C, 1 min annealing at 43°C, and 2 min extension at 72°C. After 35 rounds of amplification, the samples were held at 72°C for an additional 7 min. The negative control was plasmid pTF20 (generously provided by M. May and D. Brown, University of Florida), which was linearized using EcoRI (Promega, San Luis Obispo, CA). The DNA was run on a 1% agarose gel containing 10 µg/ml ethidium bromide. The appropriate size bands were removed from the gel and purified using a Qiagen Gel Extraction kit (Qiagen, Valencia, CA) or Gene Clean (MP Biomedicals, Solon, OH). The DNA was quantified using a NanoDrop spectrophotometer and subsequently labeled covalently with the Cy3 fluorophore as directed by the manufacturer of the Mirus Label-IT Kit (Mirus Corporation, Madison, WI). The probe solution was stored in the dark at -20°C. Fluorescence in situ hybridization (FISH) and immunofluorescence. FISH was modified from Jensen and Shapiro (1999). Briefly, M. pneumoniae was grown overnight in SP4 medium on glass coverslips and fixed with 1.5% glutaraldehyde/1% formaldehyde/0.1 M sodium cacodylate, pH 7.2 for 15 min at room temperature (RT) and 45 min at 4°C. The coverslips were washed twice in wash buffer (PBS-0.05% Tween-20) at RT for 5 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT, and washed again in wash buffer twice for 5 min at RT. Next, the

78 coverslips were washed twice in 2X SSCT (10X SSCT is 1.5 M NaCl/150 mM NaCitrate/0.1% Tween-20, pH 7.0) for 5 min at RT and incubated in 2X SSCT + 50% formamide at 37°C for 30-60 min. Then 50 µl of hybridization solution (3X SSCT/50% formamide/10% dextran sulfate/4-8 ng/µl of each probe) was dropped onto a piece of parafilm on the bottom of a plastic petri dish. The coverslips were inverted onto a drop of hybridization solution (one coverslip/50 µl). The DNA was denatured by floating the petri dish in a water bath set at 94°C for 2 min, and the probes were hybridized overnight at 42°C. The coverslips were then washed twice in 2X SSCT + 50% formamide for 30 min at 37°C, one time in 2X SSCT + 25% formamide for 10 min at 37°C, and three times in 2X SSCT for 10 min at 37°C. Next, the coverslips were placed in immunofluorescence (IF) block (PBS/5% powdered milk/0.02% azide/0.05% Tween-20, pH 7.2) and incubated overnight at 4°C. A primary rabbit antibody against the attachment organelle protein HMW1 (Stevens and Krause, 1991) was diluted 1/100 in diluent (IF block diluted 1/2.5 in PBS) and coverslips were inverted onto a 100-µl drop of antibody solution in a humid chamber for 1 h at RT. The coverslips were then washed four times in wash buffer for 5 min each at RT. They were then incubated for 1 h at RT with the secondary antibody, a Cy2-conjugated donkey-anti-rabbit diluted 1/100, (Jackson Immunological Laboratories, West Grove, PA) followed by four more washes in wash buffer for 5 min each at RT. The DNA was stained with 1 µg/ml 4’,6-diamidino-2-phenylindole (DAPI) for 30 min at RT. The coverslips were then rinsed briefly in water before being mounted on a glass microscope slide using 3-5 µl of Vectashield (Vector Laboratories, Burlingame, California) mounting media. The coverslips were sealed with clear fingernail polish and were viewed on a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany). Phase- contrast images were captured at fixed intervals using a SPOT charge-coupled device camera and accompanying software (Diagnostic Instruments, Inc.). Individual images were merged in different color channels using Adobe Photoshop CS version 8.0.

79 RESULTS PCR primers were designed using the 16S rRNA and the origin of replication sequences in M. pneumoniae. The PCR products for each set of primers were purified, labeled with a Cy3 fluorophore (red), and used to probe the chromosome. The probe against the oriC region was approximately 0.8 kb; the probe against the 16S region was approximately 1.5 kb. These probes are not expected to hybridize with DNA in other bacteria due to a lack of sequence homology. As a negative control, a probe against a mycoplasma transformation vector, pTF20 (Dybvig, unpublished), which is not present in wild-type M. pneumoniae cells, was used as was a cell with no probe. Indirect immunofluorescence was used to identify the attachment organelle using Cy2-labeled (green) antibodies against the protein, HMW1. DAPI (blue) was used to visualize the entire chromosome, which is approximately 816 kb. A schematic representation of the expected FISH results is shown in Fig. 14. Probes specific to sequences within the M. pneumoniae chromosome bound to the chromosome in permeabilized M. pneumoniae cells (Fig. 15). The probes for the 16S rRNA region and the oriC were hybridized to M. pneumoniae cells independently, since both were labeled with the Cy3 fluorophore (Fig. 15, Cy3/DAPI column). The 16S rRNA probe and the oriC probe fluoresced in the Cy3 channel and localized within the chromosome, as visualized by an overlap of red and blue (Fig. 15, Cy3/DAPI; merge columns). HMW1 localized to the attachment organelle (Fig. 15, DAPI/HMW1 column). The red spots from the oriC probe were more compact than those arising from the 16S probe but varied considerably in their distances from the green foci. The red spots from the 16S probe were much more diffuse than the green anti-HMW1 signal, making localization difficult to assess. The probe against the pTF20 plasmid (Fig. 15) did not fluoresce, indicating that the binding of the probes was specific. Controls with no probe gave results similar to those with the negative control (data not shown).

80 Fig. 14. Schematic representation of expected results in a M. pneumoniae cell after FISH with a Cy3-labeled probe and immunofluorescence against the attachment organelle protein, HMW1. A) control cell; B) oriC probe; C) 16S rRNA probe; D) pTF20 probe. In all cells, blue represents the chromosome, green represents the anti- HMW1 antibody, and red represents the Cy3-labeled probe.

81 A B

C D

82 Fig. 15. FISH using probes against the 16S rRNA gene and the oriC sequences in M. pneumoniae. In each image green represents the attachment organelle, blue represents the entire chromosome, and red represents the probe. Phase contrast images are shown on the far left. Scale bar = 1 µm.

84 Discussion Unlike model bacterial cells, some mycoplasmas, including M. pneumoniae, must undergo division in the context of the presence of a polar structure, the attachment organelle, which duplicates and migrates to the opposite pole before cytokinesis (Seto et al., 2001; Hasselbring et al., 2006a). This process is temporally linked to an increase in the DNA (Seto et al., 2001). Once the nascent organelle is formed, it remains attached to the surface, and the old attachment organelle regains motility, pulling the dividing cell away from the nascent organelle and positioning itself at the opposite pole in the process (Hasselbring et al., 2006a). In the presumably analogous species M. gallisepticum, attachment organelles are enriched for newly synthesized DNA (Maniloff and Quinlan, 1974). These data strongly suggest that the electron-dense core coordinates events between the attachment organelle and replication of the chromosome for cell division. In order for this to occur, the electron-dense core must interact with the chromosome in some manner. Recently, we have provided evidence that DNase-sensitive material, presumably the chromosome, is attached to the base of the electron-dense core in species of the M. pneumoniae group (Hatchel and Balish, 2008). In model bacteria, C. crescentus and B. subtilis, the partitioning machinery, ParB and Spo0J respectively, bind sites found near the origin of replication on the chromosome (Ireton et al., 1994; Toro et al., 2008). As the chromosome is duplicated, the partitioning sites become available for binding the machinery that will segregate them to the opposite poles (Lemon and Grossman, 1998, 2000; Haeusser and Levin, 2008). Our hypothesis is that a specific sequence, possibly the origin of replication, is itself or is located near the region of the chromosome responsible for the interaction between the M. pneumoniae chromosome and the electron-dense core, which may serve as the partitioning machinery. In this study, we used FISH to address this hypothesis. Our work focused on the interaction between the electron-dense core and the chromosome. Using fluorescence in situ hybridization, we attempted to identify whether the region of the chromosome associated with the core was a conserved region at or near the oriC. The experiment was to bind a Cy3-labeled DNA probe to the oriC region of the chromosome, while also labeling HMW1, an attachment organelle protein with the Cy2- labeled secondary antibodies that recognize the primary rabbit antibody (Stevens and

85 Krause, 1991). By measuring the distance between the oriC focus and HMW1 in multiple cells, we would be able to say that the oriC region is always a set distance away from the attachment organelle or that the distance varied from cell to cell. The former would indicate that the region associated with the core was conserved because the distance was always the same, while the latter would indicate that the sequence was variable. The signal from the oriC probe resulted in foci that were less diffuse than those from the 16S probe (Fig. 15, Cy3/DAPI column). The distances between the oriC probe and HMW1 were variable, which suggests that the interaction between the electron-dense core and the chromosome is at a variable region and not at the oriC. This refutes our hypothesis. In contrast to the foci from the oriC probe, the foci for the 16S probe were routinely rather diffuse (Fig. 15, Cy3/DAPI column). The diffuse nature of the spot could be a result of any of a number of things: a) the resolving power of the microscope; b) the size of the probes; c) some aspect of the FISH protocol; or d) the probe binding to ribosomes instead of DNA. Even though mycoplasmas are small bacteria, 1-2 microns in size, the resolution of the light microscope is most likely not the problem. Immunofluorescence studies have shown that the resolving power of similar microscopes is sufficient to distinguish proteins at the distal and proximal ends of the attachment organelle of M. pneumoniae, which is only about 250 nm in size (Kenri et al., 2004). The size of the probes should not make a difference, as larger probes have been shown to produce compact spots in other bacteria (Fig. 16) (DuTeau et al., 1998; Jensen and Shapiro, 1999). In the future, various aspects of the protocol should be tested. A key step that may play a role in the compactness of the focus is the fixation protocol. Fixation of nucleoids without inducing artifacts is empirical, and probably differs considerably across the bacterial kingdom (Eltsov and Zuber, 2006). Perhaps the fixation technique followed in this protocol produces artifacts that interfere with the hybridization of the probe. Experimenting with fixation techniques may lead to a more optimal protocol. Another possibility is that the hybridization protocol is not optimal for mycoplasmas. This step requires an overnight incubation at 42°C, which may be too long for these small bacteria. The incubation procedure followed in this protocol may have optimized binding of the probes to ribosomes in close proximity to the chromosome, ultimately leading to a diffuse

86 Fig. 16. Comparison of FISH in Caulobacter crescentus and Mycoplasma pneumoniae. Left panel (modified from Jensen and Shapiro, 1999); red: Cy3-labeled origin probe, green: Fluor-X-labeled terminus probe. Right panel; red: Cy3-labeled origin probe, green: Cy2-labeled secondary antibody to HMW1, attachment organelle protein. Scale bar = 2 µm.

88 focus. Because this probe was made against the ribosomal RNA gene sequence, it is possible that this signal results not from binding to the chromosome, but rather to ribosomes throughout the cytoplasm. In the future, to guard against this interaction, RNase could be included in the FISH protocol, or probes against regions not containing ribosomal sequences should be used. The specificity of the probe could lead to binding at multiple sites, although we did not see this with our negative control. This could be tested by using the same probe in a Southern blot and looking for multiple bands. A significant factor to consider is that our hypothesis is incorrect, and the electron-dense core is capable of associating specifically or non-specifically with multiple regions of the chromosome as division occurs. This is supported by the observation that the oriC region is a variable distance away from the attachment organelle. As the attachment organelle migrates to the opposite pole, the electron-dense core could be simultaneously interacting with regions of the chromosome, which may vary depending on the timing of division events. It is conceivable that the replication machinery is located near the base of the electron-dense core and the DNA gets threaded through it during replication. The other possibility is that the replication machinery is mobile and can actually move along the chromosome. As replication occurs, the nascent strand of DNA may be threaded through the base of the electron-dense core, and this association segregates the chromosomes. This process may represent a novel mechanism for chromosome segregation, one that uses a complex structure rather than individual proteins working together to partition the DNA. Our work contributes to the understanding of cell division in M. pneumoniae through the development of a new technique used to study the interaction between the electron-dense core and the attachment organelle. While the protocol is not completely optimized, this study lays the groundwork for future investigations in this manner. Our work is also significant because it further supports evidence that the attachment organelle has roles in processes other than gliding motility and pathogenesis.

89 SUMMARY AND CONCLUDING REMARKS The attachment organelle and pathogenicity In the current study, structural differences were found between the attachment organelle of M. pneumoniae and species related to it. M. pneumoniae has a straight and narrow attachment organelle, while its closest relative, M. genitalium has a curved and narrow attachment organelle. In contrast, the attachment organelles of related species, M. gallisepticum, M. imitans, M. amphoriforme, and M. testudinis are short and wide, yet have unique features. Our results show substantial variation in attachment organelle morphology and motile properties, even between closely related species. In this way, related species are like naturally occurring mutants that may hold the key to identifying the mechanisms behind the attachment organelle functions in members of the M. pneumoniae phylogenetic group (Hatchel and Balish, 2008). Pathogenicity in M. pneumoniae relies on the ability to adhere to host cells (cytadherence), which is mediated by the formation of a complete and functional attachment organelle; both the adhesins, and the structural proteins that make up the electron-dense core within the attachment organelle are essential to this process (Krause and Balish, 2004). Cytadherence mutants of M. pneumoniae which lack some of these proteins are incapable of making a functional electron-dense core or attachment organelle, leaving them avirulent, indicating an essential role for this structure in pathogenicity (Krause and Balish, 2004). Mutants of M. pneumoniae have allowed for the identification of several of the proteins involved (Krause and Balish, 2004), and the use of electron cryotomography has allowed visualization of how these proteins come together to make the electron-dense core (Henderson and Jensen, 2006; Seybert et al., 2006). Future studies are needed to determine the exact location of each protein within the core, as well as any novel proteins that may be found in species other than M. pneumoniae. The data from my work suggests that in species that are closely related to M. pneumoniae, the formation of the electron-dense core happens in a manner similar to that of M. pneumoniae, however the proteins involved may be different. Identifying individual proteins in the core will help to determine the exact proteins that are essential for formation of the core, which makes them essential for pathogenicity.

90 The information we learned about the morphology and gliding motility of M. pneumoniae and its relatives gets us one step closer to understanding how these organisms, especially M. pneumoniae, use the attachment organelle and how it contributes to virulence. The features of attachment organelle morphology do not correlate with adherence ability or gliding speed, suggesting that the specific morphology of the attachment organelle is not responsible for virulence properties. Instead, the affinity for host cells and gliding speed appear to be independent of this characteristic. Gliding motility is proposed to aid M. pneumoniae in spreading during an infection as evidence shows that a P200 mutant is unable to colonize human bronchial epithelial cells (Jordan et al., 2007). Nonetheless, understanding differences in motility properties across species can contribute to the understanding of how they cause disease. Even though we found that morphology correlates with phylogeny, each species still has characteristics that make it distinct from even its closest relatives (Fig. 10). These data suggest that the morphology of the attachment organelle may not be important at all or that the benefits of a particular shape have yet to be understood. There may also be a correlation between gliding motility speed and pathogenicity, with the mycoplasmas that have slower speeds being less pathogenic than those that are capable of fast gliding, with the possible exception of M. testudinis, a fast mycoplasma isolated from a healthy tortoise whose pathogenicity is not well established. We speculate that the relationship between gliding speed and pathogenicity is based on the ability of the organism to adhere to host cells. In addition to the attachment organelle-associated adhesins, other receptors on the mycoplasma surface interact with molecules such as fibronectin, mucin, and surfactant proteins on host cells (Kannan et al., 2005). In vivo, we envision that these interactions act like brakes, inhibiting the motility that we study in vitro. Therefore, the gliding speeds of the mycoplasmas in vivo might be similar; the difference in speeds we observe in vitro is due to the absence of these opposing forces. Similar motility-derived forces across species in vivo would be used to carry out force-requiring functions, including cell division and colonization. Perhaps the differential attachment organelle-independent interactions with host cells is related to pathogenicity, with more pathogenic species having stronger interactions, ultimately

91 reflected by faster gliding in vitro. Future experiments can address this issue by measuring these opposing forces and comparing them between mycoplasmas. Attachment organelle function Gliding motility In addition to mediating cytadherence, the attachment organelle also functions as the leading end during gliding motility (Balish and Krause, 2006) and houses the gliding motor (Hasselbring and Krause, 2007a, b). In M. pneumoniae, it is proposed that the ability to glide on host cells helps the organism spread during an infection (Jordan et al., 2007). Two hypotheses have been proposed to explain gliding motility in mycoplasmas, one that implicates the adhesins as principal component of the motor and another that implicates the electron-dense core. In M. mobile, a distant relative of M. pneumoniae that has unrelated structures for cytadherence and motility (Miyata, 2005), gliding is proposed to be mediated through its adhesin, Gli349, which has the ability to bind and release its substrate in a manner that moves the organism along (Uenoyama et al., 2004). A similar bind-and-release pattern is proposed for the adhesins of M. pneumoniae, P1 and P30, which localize to the attachment organelle and have been shown to play a role in gliding motility (Hasselbring et al., 2005; Seto et al., 2005a). The second hypothesis is based on small structural differences seen in M. pneumoniae cores (Henderson and Jensen, 2006) or the idea that the core itself can undergo morphological changes, such as a change in length, during the gliding process (Hegermann et al., 2002; Henderson and Jensen, 2006). Our approach toward addressing this question was to examine morphologies of members of the M. pneumoniae cluster and compare them with gliding motility speeds to see if there was a correlation between the two that would support the second of the proposed hypotheses. In order to do this, we treated whole cells with TX and analyzed the insoluble electron-dense cores by SEM, which allows the surfaces of these structures to be visualized. Prior to this study, the electron-dense cores of these species, with the exception of M. pneumoniae, had been observed only in thin section micrographs. Transmission electron microscopy studies (TEM) of individual M. pneumoniae cores have been performed (Regula et al., 2001), but these cores were from cells that were fixed while in suspension, not in an adherent state. The advantage of using SEM to look

92 at conformational differences in the electron-dense core is that the cells were attached to a substrate prior to fixation and were engaged in the interactions favoring gliding. Looking at cores under these conditions, tells us more about the conformational changes that result from gliding motility because the cells are under tension when they are fixed. Cells in suspension may exhibit conformational changes that are a result of the release of tension rather than those resulting from motility. The images that were obtained allowed for comparisons between the components of the cores in all adherent species in the M. pneumoniae cluster. We identified an electron-dense core within species in the M. pneumoniae cluster that had not previously been shown to have a core in this manner. Recently, a similar technique was used in M. mobile, which was found to have a cytoskeleton-like structure completely unrelated to the electron-dense cores of our study (Nakane and Miyata, 2007). This structure is complex, lacks homologs of the M. pneumoniae core proteins, and has a jellyfish-like structure, rather than the terminal button, rod, and base structure seen in M. pneumoniae. Whether or not the cytoskeletal structures of M. mobile and M. pneumoniae are fundamental components of the mechanism of gliding motility, these structures represent bacterial cytoskeletons that are unlike those found in model bacteria. Individual measurements of each core component were taken and compared to identify differences that would indicate conformational changes that are occurring as the cells are gliding on the surface. If the conformational changes result in a shortening or lengthening of the core, we would expect to see that rod length is noticeably variable in a population of cells. This difference would also correlate with different speeds in the different species. We might expect to see longer rods in the faster species because they would be able to take larger steps as the cell moved forward, while the slower species would be expected to have noticeably shorter rods. Another possibility is that the differences in the conformations would result in a larger range of step sizes, which would manifest themselves as differences in the standard deviations for rod length. Although we did not see differences in these structures that would support the hypothesis that the core is the motor for motility, it is possible that differences are present but not easy to recognize using SEM. A potentially significant disadvantage of this technique is that the addition of a gold coat to the cores may not be even throughout a single sample or among

93 different samples making measurements of the core less accurate. These inaccuracies in measurements may obscure conformational changes that are found in the core as a result of motility, causing a misinterpretation of the results that led to us rejecting our hypothesis. Perhaps the conformational changes are too small to be measured using still images of SEM or the size of the “inch-worm” steps is too small to detect. Even though similarities exist among the attachment organelles and the electron-dense cores of these species, something still unknown is controlling speed, and raises the questions of how maintaining different speeds is beneficial to each species and how speed is controlled. Using time-lapse microcinematography, we were able to visualize gliding motility for the first time in M. amphoriforme and later on in other species. Other techniques have been used to observe and quantitate mycoplasma motility, including similar observations of still images with measurements based on merged images (Pich et al., 2006) or measurements based on changes in the x and y coordinates of a cell (Hasselbring et al., 2005) and live images of motility captured using a video camera attached to the microscope (Miyata et al., 2000). The advantages to our approach to time-lapse microcinematography are that: 1) the intervals between images can be optimized for each species; 2) the images can be merged to observe the path of motile cells; 3) the incubation temperature can be changed as needed to optimize the conditions; and 4) we were able to calculate speeds of individual cells based on the distance traveled over a defined period of time, while also taking into account resting periods seen in these species. Phase-contrast images were captured and analyzed to determine gliding speed in M. amphoriforme, along with M. pneumoniae and M. gallisepticum as positive controls (Fig.6, Fig. 7, Table 2). The gliding speed of M. amphoriforme was determined to be 48 ± 4 nm/s, which was approximately 3 times slower than M. gallisepticum and 7 times slower than M. pneumoniae (Radestock and Bredt, 1977; Hasselbring et al., 2005; Seto et al., 2005a). We also examined gliding motility in M. pirum, M. testudinis, and M. imitans, species of the M. pneumoniae cluster with no previously reported motility data. Comparing all of the species in the M. pneumoniae cluster, we found that speed varied anywhere between 28 nm/s for M. pirum and ~3000 nm/s for M. testudinis (Table 3). Speed did not correlate with phylogeny as did the dimensions of the attachment organelle and the electron-dense cores. In fact, M. amphoriforme and M. testudinis are each others

94 closest relatives, yet the difference between their gliding speeds is almost 60-fold. Based on this data, we concluded that the attachment organelle features could not be correlated with motility and therefore could not support the hypothesis that the electron-dense core undergoes conformational changes that result in gliding in these species. Cell division The attachment organelle is proposed to play a role in cell division in addition to its role in gliding motility. Using scanning electron microscopy (SEM), we examined whole cells of M. amphoriforme and found that some cells of M. amphoriforme were found to have multiple attachment organelles, some being at opposite poles, while others were at the same pole (Fig. 4), which suggested that these cells were in the process of dividing. Similar morphologies have also been seen in M. pneumoniae (Seto et al., 2001). The presence of cells with two attachment organelles at one pole and one attachment organelle at the opposite pole suggests that multiple rounds of division are occurring simultaneously, and that additional rounds of division can start before the previous one has finished. As a result attachment organelle biogenesis occurs early in cell division and migration of one of the attachment organelles must occur before cytokinesis. This finding is significant because it supports a correlation between the attachment organelle and the events of cell division. We also examined fields of whole cells of the remaining species in the M. pneumoniae cluster (Fig. 11) and found other examples of cells with multiple attachment organelles. We found that two morphologies were common: 1) having two attachment organelles on opposite poles or 2) having two attachment organelles at the same pole of an individual cell. Depending on the species, with the exception of M. imitans, one or both of these morphologies were observed (Fig. 11). This result is significant because it supports the idea that this mechanism of cell division is consistent within this group of related species. Our observations that in the M. pneumoniae group, the electron-dense core is linked to the chromosome, suggests a role for the attachment organelle in separating duplicated chromosomes during cell division. The bacterial chromosome is also associated with the poles in well-characterized species such as Caulobacter crescentus and Bacillus subtilis, and this association is a component of the partitioning process (Toro et al., 2008). In C. crescentus and B. subtilis, the partitioning events are mediated

95 through ParB (Toro et al., 2008) and its homolog Spo0J (Cervin et al., 1998), respectively. Mycoplasmas lack evidence of homologs of ParB/Spo0J for partitioning of the chromosome (Balish and Krause, 2005). The absence of these proteins together with the physical association between the chromosome and the electron-dense core suggests that the proteins of the electron-dense core may function to partition the chromosomes to the opposite poles. During division of M. pneumoniae, a new attachment organelle forms next to the old one and then migrates to the opposite pole before cytokinesis and the split into two daughter cells (Seto et al., 2001; Hasselbring et al., 2006a). The cotranscription of genes for attachment organelle proteins P30, HMW1, and HMW3 (Waldo et al., 1999; Krause and Balish, 2001), and HolA (Kim et al., 1997; Song et al., 2001), a protein involved in loading DNA polymerase onto the DNA (Balish and Krause, 2005), further supports a link between attachment organelle assembly and initiation of DNA replication in M. pneumoniae. It is possible that the electron-dense core pulls the bound chromosome to the opposite pole as it migrates there. The models of cell division propose that the attachment organelle duplicates and migrates to the opposite pole during cell division (Seto et al., 2001; Hasselbring et al., 2006). Gliding motility plays a role in the cell division process, as the nascent attachment organelle remains attached to the surface while the existing attachment organelle glides away (Hasselbring et al, 2006). It is the movement of the existing attachment organelle that is proposed to allow for migration of the nascent organelle to the opposite pole and that gliding ultimately pulls the two daughter cells apart (Hasselbring et al., 2006). This ultimately raises the question of why the mycoplasmas move. It is conceivable that the primary role of gliding motility is to partition the chromosomes and pull the daughter cells apart during cell division, rather than for movement of the cells and the spreading of infection. Mutants that are missing proteins in the electron-dense core have problems completing the division cycle and are observed as filamentous masses using SEM (Krause and Balish, 2001), supporting this idea. Although future studies are needed to test whether the migration of the electron- dense core is the mechanism for partitioning of the chromosome in these species, our work focused on the physical interaction between the electron-dense core and the chromosome. Techniques for looking at this interaction are currently limited and those

96 that are available rely on the proper preservation of the DNA during fixation. The small size of a mycoplasma cell, approximately 1-2 microns, increases the difficulty of these investigations. Fluorescence in situ hybridization (FISH) is a technique that has been used to identify this type of relationship in other bacteria (Jensen and Shapiro, 1999; Kadoya et al., 2002), but no reports of this technique are present in mycoplasma literature. Using FISH, we attempted to identify whether the region of the M. pneumoniae chromosome associated with the core was a conserved region at or near the oriC. The experiment was to bind a fluorescently labeled DNA probe to the oriC region of the chromosome, while also fluorescently labeling HMW1, an attachment organelle protein (Stevens and Krause, 1991). By measuring the distance between the two foci (oriC and HMW1), we would be able to test whether the oriC region is always a specific distance away from the attachment organelle or whether the distance varied from cell to cell. The former would indicate that the region associated with the core was conserved because the distance was always the same, while the latter would indicate that the sequence was variable. The foci produced by the oriC DNA probe were variable distances away from HMW1 (Fig. 15), suggesting that the interaction between the core and the chromosome is not permanently localized to the oriC region. This data does not support our hypothesis and suggests that the electron-dense core is capable of non- specific binding to the chromosome. It is conceivable that the replication machinery is near the base of the electron-dense core and remains stationary while the chromosome is mobile or the replication machinery may be mobile and capable of moving along the chromosome as it duplicates. Future studies are needed to investigate this possibility. The foci produced by the 16S DNA probe was extremely diffuse (Fig. 15), making it difficult to measure the distance between the spots. It is possible that these spots are diffuse because the 16S probe is binding to ribosomes, rather than the DNA, and the addition of RNase to the protocol might result in more compact foci. Future experiments should focus on optimizing conditions for this technique, as well as identifying probes that are not against ribosomal sequences. Under proper conditions, FISH could be very useful for localizing areas of the chromosome in relation to the attachment organelle. Other techniques such as cloning or Southern blotting could also be used to identify the sequence associated with the core. Knowing how the DNA interacts

97 with the attachment organelle this relationship will help in the understanding of cell division in these organisms. Hopefully, our work with FISH will provide a starting point for others in using this technique with mycoplasmas. Attachment organelle evolution in M. pneumoniae and relatives The attachment organelle, the electron-dense core, and gliding motility appear to be traits inherited from a common ancestor of the M. pneumoniae cluster. Using SEM, we compared seven of the eight species in the M. pneumoniae cluster, the exception being M. alvi, a species that we found to be non-adherent, which agrees with previous reports (Bredt, 1968). External morphology of these species correlated with phylogeny in that the closely related species were more likely to share similarities than those that were more distantly related (Table 4). When looking at the electron-dense cores in these species, once again dimensions correlated with phylogeny (Table 5). The correlation between attachment organelle dimensions and phylogeny is helpful in identifying the features that were most likely found in the last common ancestor for this cluster. Short, knobby attachment organelles were common among the species. Specific features such as a trailing filament in M. pneumoniae and a curved attachment organelle in M. genitalium and M. testudinis are rare among the related species and most likely represent characteristics that have diverged independently from those of the common ancestor. Looking at these features in more detail allows us to have a better understanding of the ancestor, which in turn allows us to see how evolution occurred in these species. This is valuable for understanding attachment organelle functions in general, which may lead to a better understanding of pathogenicity, gliding motility, and cell division. Prior to this study, M. pneumoniae was thought of as the model mycoplasma, mainly because structurally, it is the best-characterized. The work in this study is significant because it suggests that M. amphoriforme would be a “better” choice for a model mycoplasma (Fig. 13). It has average attachment organelle dimensions and a reduced motility speed that could represent an ancestral state for these species. This work suggests the importance of comparative analysis between Mycoplasma species when trying to identify the mechanisms behind attachment organelle functions, as well as any other mycoplasma traits. Recently the genomes of M. amphoriforme, M. testudinis, M. alvi, and M. pirum have been sequenced (Wilson and Balish, unpublished). Combining

98 the morphology and motility data from this study with the genomic information from the sequence analysis will further the understanding of attachment organelle functions in the species of the M. pneumoniae cluster.

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