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Mechanosensitive Channel Protein (Mscl) in Mycoplasma Bovis

Permanent Link: http://ufdc.ufl.edu/UFE0043842/00001

Material Information

Title: Mechanosensitive Channel Protein (Mscl) in Mycoplasma Bovis
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Chow, Cheng Fen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: mycoplasma
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The class Mollicutes are composed of the smallest free-living microorganisms. Mollicutes have a minimal genome, arose by reductive evolution, and lack cell walls. Without a cell wall, Mollicutes are generally more susceptible than other bacteria to environmental stressors. Mechanosensitive channel (MscL) protein forms membrane pores that can open and close during osmotic changes to regulate osmotic pressure changes between the microbial cell and its environment. Using in silico analysis of available complete genome sequences of Mollicutes, genes were identified that contained the MscL domain in the genera Acholeplasma, Mycoplasma, and Candidatus Phytoplasma but not in the genera Ureaplasma or Spiroplasma. These species can colonize host sites with wide osmotic ranges, and some of the species are known to persist in the environment. The objectives of this study were (1) to characterize known MscL proteins among the Mollicutes, (2) to determine the phylogenetic and evolutionary relationship of the MscL protein, and (3) to determine the evolutionary force of the mscL gene in different isolates of Mycoplasma bovis and the closely related Mycoplasma agalactiae. Based on multiple sequence alignment, different Mollicutes species had a variation in the length of the protein. Most commonly, the variation in length was associated with the amino- and carboxyl-terminal portions of the protein; however, the mscL domain also varied to some degree. With the exception of M. bovis and M. agalactiae, the genomic context of mscL was usually species-dependent and only a few species shared any common genes near the mscL locus. Based on Bayesian and maximum likelihood (ML) analysis, Mycoplasma and Phytoplasma species formed two separate clades, suggesting that the mscL gene may have been acquired from two distinct common ancestors. The most likely common ancestors for Mycoplasma sp. derived from animal and human hosts were Gram-positive Lactobacillus. However, there are some discrepancies between the results on the common ancestor for Phytoplasma sp. Bayesian analysis showed that the Phytoplasma sp. shared a common ancestor with Clostridium, a Gram-positive bacterium. In contrast, ML analysis could not conclude the common ancestor for Mollicutes. Interestingly, from the fast minimal tree and supported by the BlastP search it showed the MscL from the Gram-negative Campylobacter had the highest homology to MscLs from Phytoplasma species. Within an individual or closely related species, little variation in the mscL gene was observed. ClustalW alignment of different isolates of M. bovis and the closely related M. agalactiae showed nearly identical homologous nucleic acid sequences. The ratio of non-synonymous and synonymous mutation frequency was calculated at each nucleic acid, and the results showed that most of the sites within the mscL gene experienced purifying selection. Future studies will be needed to determine if the MscL protein confers resistance to osmotic shock and its potential role in environmental persistence.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cheng Fen Chow.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Brown, Mary B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043842:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043842/00001

Material Information

Title: Mechanosensitive Channel Protein (Mscl) in Mycoplasma Bovis
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Chow, Cheng Fen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: mycoplasma
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The class Mollicutes are composed of the smallest free-living microorganisms. Mollicutes have a minimal genome, arose by reductive evolution, and lack cell walls. Without a cell wall, Mollicutes are generally more susceptible than other bacteria to environmental stressors. Mechanosensitive channel (MscL) protein forms membrane pores that can open and close during osmotic changes to regulate osmotic pressure changes between the microbial cell and its environment. Using in silico analysis of available complete genome sequences of Mollicutes, genes were identified that contained the MscL domain in the genera Acholeplasma, Mycoplasma, and Candidatus Phytoplasma but not in the genera Ureaplasma or Spiroplasma. These species can colonize host sites with wide osmotic ranges, and some of the species are known to persist in the environment. The objectives of this study were (1) to characterize known MscL proteins among the Mollicutes, (2) to determine the phylogenetic and evolutionary relationship of the MscL protein, and (3) to determine the evolutionary force of the mscL gene in different isolates of Mycoplasma bovis and the closely related Mycoplasma agalactiae. Based on multiple sequence alignment, different Mollicutes species had a variation in the length of the protein. Most commonly, the variation in length was associated with the amino- and carboxyl-terminal portions of the protein; however, the mscL domain also varied to some degree. With the exception of M. bovis and M. agalactiae, the genomic context of mscL was usually species-dependent and only a few species shared any common genes near the mscL locus. Based on Bayesian and maximum likelihood (ML) analysis, Mycoplasma and Phytoplasma species formed two separate clades, suggesting that the mscL gene may have been acquired from two distinct common ancestors. The most likely common ancestors for Mycoplasma sp. derived from animal and human hosts were Gram-positive Lactobacillus. However, there are some discrepancies between the results on the common ancestor for Phytoplasma sp. Bayesian analysis showed that the Phytoplasma sp. shared a common ancestor with Clostridium, a Gram-positive bacterium. In contrast, ML analysis could not conclude the common ancestor for Mollicutes. Interestingly, from the fast minimal tree and supported by the BlastP search it showed the MscL from the Gram-negative Campylobacter had the highest homology to MscLs from Phytoplasma species. Within an individual or closely related species, little variation in the mscL gene was observed. ClustalW alignment of different isolates of M. bovis and the closely related M. agalactiae showed nearly identical homologous nucleic acid sequences. The ratio of non-synonymous and synonymous mutation frequency was calculated at each nucleic acid, and the results showed that most of the sites within the mscL gene experienced purifying selection. Future studies will be needed to determine if the MscL protein confers resistance to osmotic shock and its potential role in environmental persistence.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cheng Fen Chow.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Brown, Mary B.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043842:00001


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1 MECHANOSENSITIVE CHANNEL PROTEIN (MSCL) IN MYCOPLASMA BOVIS By CHENG FEN CHOW A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Cheng Fen Chow

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3 To my f amily and Eric

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4 ACKNOWLEDGMENTS I would like to thank my thesis advisor, Dr. Mary Brown, for giving me the opportunity to work in her lab, for her expertise, guidance, and most importantly, her patience. I also want to express my gratitude and appreciation to the other committee members, Dr. Michael Fields and Dr. Joel Brendemuhl for their support and help. In addition, I want to thank Dr. James Wellehan and Dr. Tom Walzek for all the helpful advice, expertise and support. I am very grateful for Linda and Alex for coming to the rescue whenever I have encountered laboratory technical difficulties. Lastly, the most special thanks go to my family for their unconditional love and support. I would not be who I am today without them.

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5 TABLE OF CONTENTS page ACKN OWLEDGMENTS ....................................................................................................... 4 LIST OF TABLES .................................................................................................................. 7 LIST OF FIGURE S ............................................................................................................... 8 ABSTRACT ......................................................................................................................... 10 CHAPTER 1 INTRODUCTION.......................................................................................................... 12 Overview of Mollicutes ................................................................................................. 12 Classi fication and Phylogeny ...................................................................................... 14 Mycoplasma bovis ........................................................................................................ 14 Large Conduc tance Mechanosensitive Channels (MscL) ......................................... 18 Evolutionary Forces on Proteins ................................................................................. 20 Gene Transfer in Mollicutes ......................................................................................... 21 Goals of Study .............................................................................................................. 23 2 MATERIAL AND METHODS ....................................................................................... 27 In Silico Analysis .......................................................................................................... 27 Phylogeny ..................................................................................................................... 28 Evolutionary Forces ..................................................................................................... 31 3 RESULTS ..................................................................................................................... 33 Homology of MscL Sequence Based on BlastP Search ............................................ 33 Characterization of the MscL Protein .......................................................................... 33 Genomic Context of mscL Gene ................................................................................. 35 Phylogeny ..................................................................................................................... 35 Evolutionary Forces Acting on Isolates of M. bovis and M. agalactiae ..................... 38 4 CONCLUSIONS AND FUTURE DIRECTIONS ......................................................... 84 The MscL Protein in Mollicutes ................................................................................... 84 Variations in the MscL Protein ..................................................................................... 86 Genomic Context and Implications for Gene Transfer ............................................... 88 Phylogenetic and Evolutionary Relationships ............................................................ 89 Selective Pressures on MscL in M. bovis and M. agalactiae Isolates ...................... 94 Future Directions .......................................................................................................... 96 LIST OF REFERENCES .................................................................................................... 98

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6 BIOGRAPHICAL SKETCH ............................................................................................... 106

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7 LIST OF TABLES Table page 3 -1 BlastP result of MscL protein using Mycoplasma bovis PG45 as the reference query ........................................................................................................ 40 3 -2 BlastP result of MscL protein using Mycoplasma penetrans HF 2 as the reference query ........................................................................................................ 41 3 -3 BlastP result of MscL using Candidatus Phytoplasma asteris Aster Yell ows strain as a reference query ..................................................................................... 42 3 -4 Descriptive characteristics of MscL in Mollicutes .................................................. 43 3 -5 Description of the transmembrane domain (TM), cytoplasmic domain (CD) and extracellular domain (ED) in Mollicutes .......................................................... 44 3 -6 MscL protein of these Mollicutes species do not have a signal peptide. .............. 45 3 -7 List of MscL -containing bacteria sharing a high homologous sequence with Mollicutes ................................................................................................................ 46

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8 LIST OF FIGURES Figure page 1 -1 Classification of Mollicutes ...................................................................................... 24 1 -2 Phylogenetic tree of Mol licutes with genome sequencing projects ...................... 25 1 -3 Monomeric structure of MscL Transmembrane (TM) domain 1 and 2 are c onnected by a periplasmic loop ............................................................................ 26 3 -1 ClustalW alignment of MscL protein. ...................................................................... 47 3 -2 Color scheme guide for the identification of each gene function. ......................... 49 3 -3 Genomic context of mscL in Acholeplasma laidlawii ............................................. 50 3 -4 Genomic context of mscL in Phytoplasma asteris Aster Yellows strain ............... 51 3 -5 Genomic context of mscL in Phy toplasma asteris Onion Yellows strain. ............. 52 3 -6 Genomic context of mscL in Phytoplasma australiense ........................................ 53 3 -7 Genomic context of mscL in Phytoplasma mali ..................................................... 54 3 -8 Genomic context of mscL in Mycoplasma. agalactiae PG2. ................................. 55 3 -9 Genomic context of mscL in Mycoplasma alligatoris A21JP2. ............................. 56 3 -10 Genomic context of mscL in Mycoplasma anatis 1340 ......................................... 57 3 -11 Genomic context of mscL in Mycoplasma. arthritidis 158L31 ............................. 58 3 -12 Genomic context of mscL in Mycoplasma bovis PG45 ......................................... 59 3 -13 Genomic context of mscL in Mycoplasma columbium SF7. ................................. 60 3 -14 Genomic context of mscL in Mycoplasma crocodyli PM145 ................................. 61 3 -15 Genomic context of mscL in Mycoplasma fermentans JER .................................. 62 3 -16 Genomic context of mscL in Mycoplasma gallisepticum str. R (low) .................... 63 3 -17 Genomic context of mscL in Mycoplasma hominis PG21 ..................................... 64 3 -18 Genomic context of mscL in Mycoplasma mobile 163K ........................................ 65 3 -19 Genomic context of mscL in Mycoplasma penetrans HF 2 .................................. 66

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9 3 -20 Genomic context of mscL in Mycoplasma synoviae 53 ........................................ 67 3 -21 Similarities in the genomic context of the mscL gene between Mycoplasma bovis PG45 (a), Mycoplasma agalactiae P G2 (b), and Mycoplasma fermentans JER (c). ................................................................................................. 68 3 -22 Similarities in the genomic context of the mscL gene between Mycoplasma alligatoris A21JP2 (a) and Mycoplasma crocodyli MP145 (b) .............................. 69 3 -23 A fast minimum tree of Mycoplasma bovis PG45 .................................................. 70 3 -2 4 A fast minimum tree of Mycoplasma penetrans HF 2 ........................................... 71 3 -25 A fast minimum tree of Phytoplasma asteris Aster Yellows strain ........................ 72 3 -26 MAFFT alignment of 18 Mollicutes species and 34 bacteria sharing a high h omologous MscL protein sequence ...................................................................... 73 3 -27 Maximum likelihood tree. ........................................................................................ 79 3 -28 Bayesisan tree. ........................................................................................................ 80 3 -29 ClustalW alignment of mscL nucleic acid sequence of Mycoplasma bovis isolates ..................................................................................................................... 81 3 -30 Positive and purifying selection at each amino acid of Mycoplasma bovis .......... 83

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MECHAN OSENSITIVE CHANNEL PROTEIN (MSCL ) IN MYCOPLASMA BOVIS By Cheng Fen Chow December 2011 Chair: Mary Brown Major: Animal Molecular and Cellular Biology M ollicutes are the smallest free-living microorganisms have a minimal genome, and lack cell walls making them more susceptible than other bacteria to environmental stressors. Mechanosensitive channel (MscL) proteins regulate osmotic pressure changes between the microbial cell and its environment. Using in silico analysis of available complete genome sequences of the class Mollicutes, we identified the MscL domain in the genera Acholeplasma, Mycoplasma, and Candidatus Phytoplasma but not in the genera Ureaplasma or Spiroplasma. The objectives of this study were (1) to characterize known MscL proteins among the Mollicutes (2) to determine the phylogenetic and evolutionary relationship of the MscL protein, and (3) to determine the evolutionary force of the m scL gene in different isolates of Mycoplasma bovis and the closely related Mycoplasma agalactiae. The MscL proteins v aried primarily in the length of the aminoand carboxyl terminal portions of the protein. The genomic context of the mscL gene locus was usually species -dependent Based on Bayesian and maximum likelihood (ML) analysis, Mycoplasma and Phytoplasma species formed two separate clades, suggesting that the mscL gene may have been acquired from two distinct common ancestors. The most

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11 likely common ancestors for Mycoplasma sp. derived from animal and human hosts were Gram -positive Lac t obacillus sp. while the Phyt oplasma sp. shared a common ancestor with Clostridium a G ram -positive bacterium In contrast, ML analysis could not conclude the common ancestor for Mollicutes T he fast minimal tree and BlastP search results suggested that the MscL from the Gram -negative Campylobacter had the highest homology with Phytoplasma species. Within an individual or closely related species, little variation in the mscL gene was observed. The ratio of non-synonymous and synonymous mutation frequency at each nucleic acid showed that most of the sites experienced purifying selection. Future studies will be needed to determine if the MscL protein confers resistance to osmotic shock and its potential role in environmental persistence.

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12 CHAPTER 1 INTRODUCTION Overview of Mollicutes Mollicutes (commonly referred to as mycoplasmas) represent the simplest and smallest free-living microorganisms and have arisen by reductive evolution (de Crecy Lagard et al. 2007, Sirand-Pugnet, Citti et al 2007, Sirand-Pugnet Lartigue et al 2007,). For simplicity, unless italicized, the term mycoplasma will be used to refer to Mollicutes in general. As a consequence of reductive evolution, mycoplasmas have small genome si zes ranging from 580 to 2200 kb an d abe rrant stop codon usage (Barr et al. 2004). Instead of a cell wall, mycoplasmas are surrounded by a single cell membrane that contains cholesterol as a crucial component. As a result, they have pleiomorphic shapes (Casewell et al. 2008). For the most part, these microbes colonize mucosal surfaces of the respiratory and urogenital tracts and do not persist in the environment. Mycoplasmas have evolved from Gram -positive bacteria and are most closely related to Bacillus, Clostridium and Streptococcus. Two branches [the AAP (Archebacteria and Phytoplasma) and the SEM ( Spiroplasma, Mesoplasma, Mycoplasma/Ureaplasma) branches] of low G C content minimal genome bacteria occurred ~650 million years ago (Kunisawa 2002, Maniloff 2002) via genome reduction. Mycop lasmas have a low G+C content and, as a result, mycoplasmas have altered the codon usage by using the stop codon UGA rather than UGG to preferentially code for tryptophan (Iriarte et al 2011). As a result of degenerative evolution, mycoplasma have fewer rRNA operons, tRNA genes and simplified metabolic pathways (Cordova et al. 2002) Because of their limited metabolic pathways, mycoplasmas lack

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13 the ability to synthesize many amino acids, nucleotides and fatty acids. Mycoplasmas therefore have fastidious growth requirements and are dependent on their hosts for nutrients ( de Crecy -Lagard et al. 2007) Mollicutes can be isolated from a wide range of hosts and can act as commensal flora or as either opportunistic or frank pathogens. Mycoplasmas wi th pathogenic potential can cause diseases in humans, animals, insects and plants (Brown et al. 2010) In humans and animals, mycoplasmas colonize mucosal surfaces, such as the respiratory, gastrointestinal and urogenital tract. Human pathogens of signif ic ance include Mycoplasma genitalium Mycoplasma hominis and Mycoplasma pneumonia, Ureplasma parvum and U urealyticum These bacteria can lead to diseases with high morbidity and low mortality (Barr et al. 2004). In animals, mycoplasmas can cause debilitating diseases and infections in livestock, consequently, leading to economical losses creating a huge impact on agricultural industries (Hanzlicek et al. 2011). Major clinical manifestations include but are not limited to pneumonia, otitis media, mastitis, urogenital disease, arthritis and meningitis (House et al. 2011) Candidatus Phytoplasma s p. and Spiroplasma s p. are plant pathogens that are often transmitted by insect vectors. The bacteria reside in the phloem, which is a complex tissue in the vascular system of higher plants that functions to translocate sugars and other dissolved nutrients within the plant or to store nutrients (Kube et al. 2008). The mechanosensitive channel ( MscL ) protein that is the focus of this thesis is found in diverse Mollicut es species representing all genera with sequenced genomes except Spiroplasma. The microbes with the mscL gene are associated with avian, fish, human, plant, reptilian, ruminant, and swine hosts.

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14 Classification and Phylogeny The general classification of my coplasmas is shown in Figure 1 1 Mycoplasmas are members of the phylum Tenericutes There are four orders within the class of Mollicutes : Mycoplasmatales Entomoplasmatales Acholeplasmatales and Anaeroplasmatales (Brown et al. 2010). The sequenced genom es available for this study were found in all orders except Anaeroplasmatales. The evolutionary relatedness of Mollicutes is most commonly assessed by 16S rRNA gene sequences, which is a highly conserved housekeeping gene in most bacteria (Wolf et al. 2004) Further, whole genome comparisons and multiple gene analyses ha ve confirm ed the validity of the 16S rRNA as a standard (Carridge III 2004) A phylogenetic tree inferred from the 16S rRNA sequence for the fully sequenced mycoplasma has constructed genomes and was constructed using the MolliGen website (http://cbib1.cbib.u-bordeaux2.fr/molligen3b/SPECIES/phylo.php), a dedicated database that provides annotated genome sequence data for 38 mycoplas m a species (Figure 1-2). Based on the 16S rRNA phylogenet ic tree, the genus Mycoplasma is further divided into four main groups: Spiroplasma, Hominis, Pneumoniae, and Phytoplasma. In the latest addition of Bergys Manual of Determinative Bacteriology (Brown et al. 2010), the Hominis and Pneumoniae groups have be en further separated into clusters (not shown) and a hemotropic cluster has been added. Mycoplasma bovis In the U.S., M bovis is an important pathogen of ruminants (House et al. 2011; Manusell et al. 2011) and can result in significant economic loss (Whit e et al 2010). This pathogen has been discussed in detail in a recent Consensus Statement of the American College of Veterinary Internal Medicine (ACVIM) (Manusell et al. 2011).

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15 Earnings from dairy industries are reduced due to poor milk quality and decreased milk production from M. bovis -infected animals. In the U.S., about $108 million per year of dairy revenues are lost due to M. bovis infections (Nicholas et al. 2008). The beef industry shares similar losses caused by M. bovis infections. Weight loss and low carcass value from infected animals can lead to a $32 million per year loss to the industry ( Nicholas et al. 2008). Clinical presentations associated with M. bovis are mastitis, chronic pneumonia, keratoconjunctivitis, art hritis, and tenosynovitis (Caswell et al. 2007, Maunsell et al. 2011). In adult dairy cattle, M. bovis is primarily associated with mastitis. Mycoplasmal mastitis usually involves multiple quarters at the same time, and presumptive diagnosis is based on abnormal milk color (brown to tan), decline in milk production, and elevated somatic cells in milk. Definitive diagnosis requires culture and/or polymerase chain reaction (PCR). While many animals are subclinical, some infected animals will show clinical sig ns of fever, loss of appetite, and arthritis. (Kirk et al. 2004, Nicholas et al. 2008). Arthritis is a sequelae of M. bovis induced infections like mastitis and pneumonia (Nicholas et al. 2008). Arthritis is developed when M. bovis infects joints, primaril y the stifle, hock, shoulder and elbows. Joints of infected animals will swell and cause pain, which can lead to lameness (Caswell et al. 2007). Mycoplasma bovis has detrimental effects on calves by causing pneumonia, otitis media, and arthritis (Nicholas et al. 2008, Maunsell et al. 2011). Calves with pneumonia may have non-specific signs of fever, abnormal breathing sounds depression, nasal discharge, coughing, loss of appetite, and weight loss ( Nicholas Ayling 2003, Nicholas et al. 2008). Mycoplasma bovis is often isolated from the lungs of pneumonic calves

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16 (Nicholas et al. 2008). Otitis media is another frequent clinical presentation caused by M. bovis Infected dairy and beef calves often showed signs of unilateral or bilateral ear droop, fever, head tilt, head shaking and scratching ( Manusell 2007, Walz et al. 1996). In cases, otitis media can progress to otitis interna with clinical signs of ataxia, recumbency, horizontal nystagmus, head tilt, and death. Mycoplasma bovis is highly contagious and is widely spread among ruminants, especially within the bovine population ( Nicholas et al. 2008). Transmission ( H ouse et al. 2011, Nicholas et al. 2003, Maunsell et al. 2011) of the bacteria is commonly through inhalation of respiratory secretions from an infected animal and from ingestion of contaminated milk. Mechanical transmission involves use of contaminated equipment (Fox et al. 2005). Transmission of M. bovis from an infected calf to a nai ve calf only takes 24 hours, which shows the bacteria can be rapidly transmitted. In addition, stress from handling and transportation can increase shedding of M. bovis in nasal secretion from infected calves (Caswell et al. 2007). Disease control and prevention includes good hygiene protocols and a good surveillance program. Disinfect ion of bedding, stalls, and equipment can minimize the spread of the bacteria. Treatment for M. bovis is difficult because it is often refractory to antibiotics Further, because there is no cell wall, antibiotics like penicillin that target the cell wall are ineffective (Caswell et al. 2007). However, antibiotics may be of use in treat ing secondary bacterial infections (Nicholas et al. 2003). There ar e mixed data on the effectiveness of the antibiotics in treatments of M. bovis In a study conducted by Ayling et al. 2000, most Europe an and North American strains of M. bovis were still responsive in vitro to antibiotics with the exception of newer strains that did show

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17 antibiotic resistance. In contrast, a nimals with M. bovis clinical disease failed to respond to treatments even though the isolates were sensitive in vitro (Haines et al. 2001). Taken together, these two studies suggest that the ineffecti veness of antibiotic treatments is not a result of the acquisition of resistance determinants by M. bovis. Vaccination has met with limited success. Currently, bacterin and autogenous vaccines are approved and available in the U.S However, vaccines are not effective in the prevention of M. bovis from colonizing the upper respiratory trac t and do not prevent lung lesions ( Maunsell et al. 2001, Soehnlen et al. 2011) In some instances, increased disease severity is seen in vaccinated animals (Nicholas et al. 2002). Although generally most mycoplasmas are thought not to persist outside the host, some m ycoplasmas, including M. bovis can persist for long periods under different pH and temperature conditions and in various environments (Nagatomo et al. 2001). Mycoplasma bovis can survive up to 8 months in a sand pile and its concentration is depende nt on temperature and precipitation (JusticeAllen 2010). In vivo, m ycoplasma can colonize diverse sites within the host (Nagatomo et al. 2001). One potential mechanism by which mycoplasmas can persist in different host sites as well as in the environment outside the host is by controlling osmositc pressure changes. Mechanosensitive channel proteins are common in prokaryotes and function to control osm otic pressure changes but have not been described in mycoplasmas. A very recent study (Aug 2011) published since initiation of this thesis showed that inhibition of MscL of Phytoplasma asteris Onion Yellows strain resulted in decreased growth in planta (Oshima et al. 2011), supporting the concept that mscL may be important for survival. Based on genome sequence data of the M. bovis F1 strain, our laboratory found that a

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18 gene (MYBF 029) encoding MscL was present. A standard BLAST search on the MolliGen search engine revealed the presence of MscL in the genomes of several other mycoplasmal and phytoplasmal pathogens, including the M. bovis PG45 type strain. Large Conductance Mechanosensitive Channels (MscL) Mechanosensitive (MS) channels are wide spread amo ng living organisms, i ncluding eukaryotes and eubacteria ( Kung et al. 2010, Perozo 2006).Mechanosensitive channels w ere first found in Escherichia coli using patch clamp analysis (Perozo 2006, Pivetti et al. 2003). The channel can transduce mechanical forc es into electrochemical signals to allow cells to respond accordingly. Embedded in the membrane, MS channels can detect and minimize mechanical tensions or forces to maintain the physical integrity of bacterial cells. By opening and clos ing the pore, the M S channel can regulate membrane tensions created by osmotic and turgur pressure imbalance to prevent cell lysis (Yefimov et al. 2008). Acting as safety valves, MS channels give microbes the ability to survive in extreme environments like hot springs, basic or acidic waters, saline water and volcanic vents at the bottom of the sea floor at temperatures over 100 C (Martinac et al. 2003). There are two families of MS channel proteins: MscL and MscS (Pivetti et al. 2003). Both families of proteins exist as hom omeric complexes that span the bilipid membrane and form physical pore structures. These pores open and close to provide protection from osmotic damage. When the external osmotic pressure decreases (and especially during rapid shifts), the epitopes on the Msc protein act as proton sensors, and the channels are activated to open and release osmolytes and water (Batiza et al. 2002, Ewis & Lu 2005, Jeon & Voth 2008, Kloda et al. 2008 Nomura et al. 2007, Yefimov et al. 2008). During increases in external osmot ic pressures, the MS channel

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19 responds to changes in the membrane caused by alteration in lipoprotein tension (Elmore & Dougherty 2003, Meyer et al. 2006, Moe & Blount 2005, Nomura et al. 2007, Powl et al. 2005). Indeed, the presence of anionic phospholipids is critical for both the rate and extent of flux in MscL (Powl et al. 2008). Detailed analyses have demonstrated conserved and key domains for effective function and mechanosensitivity (Chiang et al. 2005, Iscal et al. 2007, Jeon & Voth 2008, Tsai et al. 2005). Studies of Msc L function and responses to electrochemical and osmotic gating have been important in providing a better understanding of the biophysical properties of membranes and their response to osmotic stress. Although MscL was first isolated in E. coli the crystal structure (http://www.rcsb.org/pdb/explore.do?structureId=2OAR ) was first determined for Mycobacterium tuberculosis (Jeon et al. 2008). The mscL gene is conserved acr oss various species of bacteria; for example, E. coli and M. tubercul osis share 37% homologous sequences (Chang et al. 1998). MscL has a homope n tameric structure that consist s of five identical subunits (Okaley et al. 1999). Each subunit is composed of two transmembrane alpha-helices denoted TM1 and TM2, N and C terminal helices and a periplasmic loop (Kung et al. 2010) (Figure 1-3) Both N and C -terminal helices are in the cytoplasm. TM1 helices line the pore with hydrophilic resides making the inner c hannel polar. TM1 helices are packed together to keep the pore closed when the channel is at rest. TM2 helices are connected to TM1 helices by periplasmic loops in the extracellular space (Chen et al. 2008, Chang et al 1998, Kloda et al. 2008, Kung et al. 2010, Okaley et al. 1999). The full mechanism of how mscL opens is still not fully understood. How ever, it has been suggested that tension exerted onto the cell

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20 membrane will cause the cytoplasmic ends of TM1 to move a part from each other and TM2 becomes wedged between them forming a pore (Ch i ang et al. 2005). The function of M scL is to allow solutes to cross through the membrane to relieve pressure generated by osmotic downshock. To balance osmotic differences, MscL will open and close to regulate solute and water in order to maintain a homeostatic status between the cell and its environment. For example, when the environment of the bacteria changes from a high osmolarity to a low osmolarity it will cause a rapid influx of water into the cell, thus increasing membrane tension, which can cause the cell to burst. This is called hypoosmotic shock. Hypoosmotic shock can increase the turgor pressure withi n a cell. To balance pressure, M scL will open to release solutes from within the cell to minimize additional influx of water (Booth et al 2007) Once the channel is open, it is permeable to solutes ranging from 400 to 500 Da and the solutes often include both disaccharides and amino acids. Evolutionary Forces on Proteins The genomic content of prokaryotes experiences both gene loss and gene acquisition from unknown sources at a substantial rate (Lerat et al. 2005). This phenomenon might be the result of selective evolutionary forces acting upon the bacteria. Macroevolutionary forces focuses on the gene transfer between distant organisms. In contrast, microev olutionary forces explain the selection forces acting on the genomic sequences of an organism (Novichkov et al. 2009). To examine the evolutionary forces acting upon a protein, the ratio ( ) of non-synonymous (Ka) and synonymous (Ks) i s calculated at each amino acid site (Doron-Faigenboim et al. 2005). Non -synonymous change occurs when there is a change in the nucleotide sequence,

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21 resulting in a change in the amino acid present in the encoded protein. In contr ast, a synonymous mutation in the nucleotide sequence does not alter the amino acid in the encoded protein (Doron-Faigenboim et al. 2005, Massingham et al. 2004). The ratio determines the level of polymorphism within a species and the rate of divergence between species by estimating the purifying and positive selection (Biswas et al. 2006, Doron-Faigenboim et al. 2005). Positive selection occurs when the changes in the nucleotides of a specific gene results in changes in the protein, and this diversity is thought to increase the overall fitness of the organism (May et al. 2009). Purifying selection occurs when the changes result in structural changes that are detrimental, for example an altered active site. If this occurs, then the gene (and the mutation) would be removed (Biswas et al. 2006). The ratio is calculated using likelihood ratio test (LRT) by comparing a null model that assumes no positive selection to a model that does assume positive selection (Doron-Faigenboim et al. 2005). The whole sequenc e is used to calculate the ratio allowing for a more accurate estimation of evolutionary forces acting upon the gene/protein. If the ratio is > 1, it indicates positive selection. However, if is < 1, it shows purifying selection (Doron-Faigenboim et al. 2005). Since synonymous mutation does not affect protein sequence, it is assumed to be neutral selection and ultimately becomes fixed (Massingham et al. 2004). If there are more nonsynonymous mutations, it is suggestive of adaptive evolution (Massingh am et al. 2004). Gene Transfer in Mollicutes Genes can be deleted, duplicated or inserted within a bacterial genome. Acquisition of genes can be achieved through two different mechanisms: duplication/modification or lateral transfer (Hao & Golding 2004). L ateral gene transfer

PAGE 22

22 (LGT) can occur between distantly related species often involving d eletion or insertion of genes in larger genomic units thus, creating variation in genome sizes and bacterial pathogenicity (Hao & Golding 2004) When bacteria are in close proximity to each other there is an increased opportunity for LGT to occur ; it has been suggested that this facilitates the ability to occupy a new niche or reside in a novel environment (Hao & Golding 2004). Horizontal gene transfer (HGT) occurs am ong closely related species and contributes to the evolutionary diversity of genomes Possible ways for HGT to occur include transformation, transduction, conjugation and membrane fusion (Teachman et al. 2002). Evidence for transformation and transduction is lacking in mycoplasmas; however, mycoplasmal genomes contain i nsertion s equence (IS) elements, transposases, and i ntegrative c onjugative e lements (Calcutt et al 2002, Dybvig et al 2007; Sirand-Pugnet, Citti et al 2007; Thomas et al 2005), suggesting the potential for HGT and possibly LGT via conjugation. T he evolutionary constraints on minimal genomes have led to the speculation that most of the reduction has been due to gene loss, and that HGT and LGT ev ents were limited in these microorganisms However, it is now recognized that these events may be more common than previously thought (Arnold et al. 2008, Dagan & Martin 2007, Dagan et al 2008). Mycoplasmas that lack phylogentic relatedness but that colon ize common hosts can share similar genes suggesting horizontal gene exchange may occur within the host For example, HGT has been demonstrated between M. bovis and M. agalactiae (Marenda et al 2005, Thomas et al 2005), M. agalactiae and the Mycoplasma m ycoides cluster (Sirand -Pugnet, Lartigue et al 2007), and yellowing

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23 Phytoplasma s p. and Spiroplasma kunkelii (Bai et al 2006). Recently it has been suggested that the heat shock protein, GroEL, in M. penetrans was acquired from Helicobacter pylori by LGT ( Clark & Tillier 2010) Finally, under experimental conditions, HGT was demonstrated in M. pulmonis presumably via conjugation or cell fusion. Goals of Study In silico analysis of the genome sequences of a clinical field isolate, M. bovis F1 strai n and a proprietary vaccine strain in our laboratory identified the presence of the mscL gene. Subsequently, the complete genome sequence for M. bovis PG45 (Wise et al. 2010), M bovis strain Hubei -1 (Li et al. 2011) as well as the closely related M. agalactiae type strain PG2 and field strain 5632 genomes (Nouvel et al. 2010) were released. This provided the impetus to study the evolutionary forces acting upon the mscL gene and its encoded protein in the Class Mollicutes in general and in the ruminant pathogens M. bovis and M. agalactiae specifically The goals of this project were (1) t o characterize known MscL proteins among the members of the Class Mollicutes for which genome sequences were available; (2) t o determine phylogenetic and evolutionary relationship among the MscL proteins in the Class Mollicutes ; and (2) t o determine the degree of diversifying (positive) and purifying selection of the mscL gene in different strains of M. bovis and the closely related M. agalactiae.

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24 Figure 1-1 Classification of Moll i cutes.

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25 Figure 12. Phylogenetic tree of Mollicutes with genome sequencing projects. The tree was constructed using 16S rRNA sequences. In this figure, the Moll i cutes are divided into four clusters: Spiroplasma, H ominis, Pneumoniae and Phytoplasma. Hosts are noted by green (plant), blue (animal) and red (human) circles. High lights indicate species containing the MscL protein (yellow). M. anatis 1340 and M. columbinum SF7 are newly identified MscLcontaining species, therefore, they are not included in this tree. (Source: http://cbib1.cbib.ubordeaux2.fr/molligen3b/SPECIES/phylo.php Last accessed October, 2011)

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26 Figure 1-3. Monomeric structure of MscL Transmembrane (TM) domain 1 and 2 are connected by a periplasmic loop. Both TM domains span th e single membrane layer of the m ycoplasma cell.

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27 CHAPTER 2 MATERIAL AND METHODS In Silico A nalysis Initial BlastP search from the National Center for Biotechnology Information (NCBI, http://blast.ncbi.nlm.nih.gov/) was performed using the M scL protein sequence of the M. bovis PG45 as the reference. Each BlastP search result was given an expectation value as a measure of statistical significance (Sayers et al 2011). The expectation value, or E-value, i s a parameter represent ing the number of hits expected to occur by chance in a search (Hall, 2011). The other Mycoplasma species that contain the mscL gene were identified from the BlastP search. An additional two references, M. penetrans HF -2 and P. aster yellow s, were also used in the BlastP search. A total of 18 Mycoplasma species were found and used in this research. The gene was present in Acholepl asma laidlawii Mycoplasma agalactiae PG2 M. alligatoris A21JP2 M. anatis 1340, M. arthritidis 158L3-1 M. bovis PG45 M. columbinum SF7 M. crocodyli MP145 M. fermentans JER M. gallisepticum str. R(low) M. hominis PG21 M. mobile 163K M. penetrans H F -2 M. synoviae 53, Candidatus Phytoplasma asteris Aster Yellow strain C. P. asteris Onion Yellow strain C. P. australiense, and C. P. mali The amino acid length and the molecular weight of the M scL protein for each Mycoplasma species were obtained from MolliGen (http://cbib1.cbib.ubordeaux2.fr/molligen3b/), a web database providing annotated genome sequences data for 38 Mycoplasma species. ClustalW is a multiple sequence alignment program. It aligns input protein or nucleic acid sequences The program color -codes each amino acid by its chemical property allowing a clear view of the similarities and differences in the alignment. All of

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28 the 18 Mycoplasma sequences in FASTA format were aligned using ClustalW program from the European Bioinfor matics Institute (EPI) server (http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). The genomic context of each Mycoplasma was examined by looking at the genes that are up and downstream from the mscL gene. The whole genome of each Mycoplasma species was obtained from the NCBI genome database to locate the mscL gene. The gene or the locus tag was used to search for the location of the mscL gene. Once the mscL gene was located, genes that are in the vicinity were identified. The genome contexts of 18 Mycoplasma species were analyzed to identify similarities and differences between the species. The M scL protein sequences of each Mycoplasma species were entered into Philius prediction server from the Yeast Resource Center (http://www.yeastrc.org/philius/pages/philius/runPhilius.jsp;jsessionid=D1236CF9D9372 7253D6A9B6F6D5B00A6). The server predicted the location of transmembrane helix region, extracellular domain, cytoplasmic region and presenc e of signal peptide. In addition, the server also provided the length of each region. Phylogeny Based on the in silico results, the mscL gene was identified in 18 species within the class Mollicutes. The gene was present in Acholeplasma laidlawii Mycoplasma agalactiae PG2 M. alligatoris A21JP2, M. anatis 1340, M. arthritidis 158L31 M. bovis PG45 M. columbinum SF7 M. crocodyli MP145 M. fermentans JER M. gallisepticum str. R (low) M. hominis PG21, M. mobile 163K M. penetrans HF 2 M. synoviae 53, Candidatus Phytoplasma asteris Aster Yellow strain C. P. asteris Onion Yellow strain C. P. australiense, and C. P. mali AT. The host range represented by these Mollicutes

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29 species include plant, animal and human pathogens. In some cases, the mscL gene sequence was available for multiple strains of a given species. When this occurred, only one representative strain was used unless otherwise noted. Because Mollic utes use UGA as a codon for tryptophan rather than as a stop codon ( Iriarte et al 2011), a BLASTP rather than a BLASTN search was performed for each Mollicutes species Each Mycoplasma was used as a reference for the BlastP search. Only the top 5 non-Mycoplasma bacteria from the search result with an E -value <106 were chosen. A total of 33 MscL protein sequences from different bacteria species were used for the Bayesian and maximum likelihood analysis. In selected studies, Mycobacterium tuberculosis w as chosen as an outgroup because its M scL protein has been crystallized (Chang et al 1998; PDB 2OAR http://www.rcsb.org/pdb/explore.do?structureId=2OAR ). Several multiple alignment programs are available. The MAFFT program was chosen for the phylogenetic studies because of its accuracy and speed. MAFFT is one of the fastest methods used to align nucleic acid or protein sequences (Katoh et al. 2005). MAFFT will compare all the sequences then perform progressive alignment and finally refine the alignment (Katoh et al 2010). Sequences of the M scL proteins were obtained from BLASTP and aligned in MAFFT format using the MAFFT server by Computational Biology Research Center ( http://mafft.cbrc.jp/alignment/server/ ). Two different approaches were used to construct the phylogenetic trees. First, sequences were run on MrBayes, a Bayesian program that uses the Metropolis Coupled Monte Carl Markov Cha in (MCMC) method to approximate the posterior probability distribution of trees (Altekar et al 2002, Hall 2011). Bayesian analysis

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30 employs several evolutionary models to estimate branch lengths. Each branch length is a numeric number representing the amount of genetic changes between the ancestor and its descendant (Hall 2011). The evolutionary models can be manually selected or the program will select the best -fitted model based on the given data (i.e. protein sequence alignment) to construct a phylogenet ic tree. Bayesian analysis uses posterior probability as a measure of confidence level for each branching pattern. The MAFFT aligned M scL protein sequences were converted into Phylip 3.2 format using a biosequence conversion tool called the Readseq from the European Bioinformatics Institute (EBI). In the converted file, specific in-text codes that specify the evolution model, generation time, number of taxon and the length of the sequence were included. The program, Mr.Bayes was downloaded from its developers website (http://mrbayes.sourceforge.net/). To run the program, the input data (the alignment file) needed to be in the same folder as the program itself. In addition, t he progr am was a command-line interface and therefore required the input of specific commands. After opening of the program, execute filename.txt was typed in the prompt. Once the program was done acknowledging the data, the screen showed the list of taxons in the input data. Then, t he command line mcmc was entered and the program started the analysis of the data. The phylogenetic tree, the output, was viewed using a graphical viewing program called the Figtree ( http://tree.bio.ed.ac.uk/software/figtree/). Maximum likelihood analys is was the second method used to infer the evolutionary tree. ML finds the tree that maximizes the probability of the genetic data given the tree. Similar to Bayesian analysis, ML also uses the evolutionary models to

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31 estimate the branch length. However, ML uses bootstrap values to measure its confidence measurement for the branching pattern. The same MAFFT protein sequence alignments used for the Bayesian analysis were also used for the maximum likelihood analysis. The file was converted to phylip/phylip 4.0 format using Readseq. The input data was submitted to an online maximum likelihood analysis web server, RaxML BlackBox ( http://phylobench.vital it.ch/raxml -bb/ ). The output of the phylogenetic tree w as viewed using the Figtree program Evolutionary Forces The nucleic acid sequences of the mscL gene from four different clinical isolates of M. bovis (M. bovis PG 45 M. bovis F1 strain M. bovis P strain and M. bovis hubei ) and 1 M. agalactiae PG2 strain were used. The F1 was sequenced in our lab and the P strain is a proprietary strain from a company. M. bovis hubei and M. agalactiae PG2 were obtained from the NCBI genome database. All the sequences were submitted as FASTA files and analyzed using Selecton v2.4 software suite (DoronFaigenboim et al 2005 Stern et al 2007). The software computes the non-synonymous (aminoacid altering, Ka) to synonymous (silent, Ks) substitutions ratio. This Ka/Ks ratio is used to estimate both positive and purif ying selection at each amino-acid site. The default model used by the server, M8, employs the maximum likelihood method (ML) to determine the Ka/Ks ratio by comparing between two hypotheses: a null model that assumes no positive selection and an alternativ e model that assumes positive selection does occur (DoronFaigenboim et al 2005). The model cant calculate the p-value; therefore, a confidence interval was calculated instead to demonstrate the reliability of

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32 the Ka/Ks ratio. The output file was in a color -coding scheme to show positive selection, purifying selection and neutral selection.

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33 CHAPTER 3 RESULT S Homology of MscL Sequence Based on BlastP Search BlastP search results using Mycoplasma bovis PG45 MscL protein as the reference query showed significant homologous protein sequences with other Mycoplasma species (Table 31). The low E -values indicated a highly homologous sequence of MscL that was shared between M. bovis PG45 and M. agalactiae PG2. In addition, the BlastP revealed M. bovis PG45 and M. fermentans JER had a similar MscL sequence as well. In contrast, M. penetrans HF -2 did not share high homologous sequences with any Mycoplasma species (Table 32). When the MscL sequence of M. penetrans HF -2 was used as t he reference query, its sequence was more similar to non-Mycoplasma bacteria than to members of the Mollicutes When Phytoplasma asteris Aster Yellows was used as the reference query, a highly homologous MscL protein sequence was seen with other Phytoplasm a species especially to P. asteris Onion Yellows (Table 3-3) However, the MscL of Mycoplasma sp. did not share high levels of homology with the MscL of Phytoplasma species. Interestingly, the Evalues for several gram -negative bacteria ( Campylobacteria, Bacteroides and Yersinina) indicate they shared similar MscL protein with the Phytoplasma species. Characterization of the MscL Protein The ClustalW alignment (Figure 31) of all the amino acids of the MscL proteins from Mycoplasma and Phytoplasma species allowed for the identification of conserved sequences across all the species. From the alignment, each amino acid across all the species were indicated by an asterisk (*) at bottom of the alignment, and a colon (:) w as used to represent highly simi lar sequences The color -coded scheme represented the

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34 chemical properties of the amino acids. This allowed for an easy discernment of the similarities and differences within the alignment. Therefore, at a glance, the conserved and variant regions among all the Mollicutes species could be identified. The MscL protein sequence of the 18 Mollicutes species varied in amino acid length from 131 to 220 residues (Table 3-4). M. alligatoris A21JP2 had the shortest MscL protein sequence whereas M. gallisepticum str R (low) had the longest. From the ClustalW alignment, it is evident that the MscL protein sequence was conserved across the Mollicutes species (Figure 3-1). Not unexpectedly the transmembrane domains to the MscL protein were the most similar across speci es. However, the MscL protein did show some interspecies variation, most notably at the aminoand carboxy terminus and, to a lessor extent, the extracellular loop. Upon further examination, the alignment showed the MscL protein sequences for M. bovis PG4 5 and M. agalactiae PG2 were almost identical, hence the very low E -value from the BlastP search. Mycoplasma alligatoris A21JP2 and M. crocodyli MP145 also shared highly similar MscL protein sequences with each other. From the alignment, the MscL sequence of M. bovis PG45 and M. agalactiae PG2 shared some similarities with M. fermentans JER. These similarities were supported by the low E values from the BlastP search. Furthermore, the MscL sequence of P. asteris Aster Yellows and P. asteris Onion Yellows we re found fairly conserved, but there were unexpected differences in the length of the protein given these are two strains of the same species. The transmembrane (TM) and cytoplasmaic domains were identified to examine the functionality of the conserved se quences and the variants in the MscL protein of the Mycoplasma species. Most of the MscL sequences consisted of two TM domains with

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35 the exception of Phytoplasma astris Aster Yellows which had three TM domains (Table 3 -5). The lengths of the cytoplasmic regions varied between species; however, the most pronounced differences were observed in Phytoplasma species where the length ranged from 15 to 51 amino acids in length. In contrast, the TM domains for both Mycoplasma and Phytoplasma species were uniform in length. In addition, the MscL protein from all the Mycoplasma and Phytoplasma did not contain a signal peptide (Table 3-6). Genomic Context of mscL Gene To examine the genomic context, genes from upand downstream of mscL were id entified from genome sequences (Figure 32 to Figure 3-21). Mycoplasma bovis PG45 and M. agalactiae PG2 had an identical genomic context. This was not surprising because these two species are closely related based on whole genome sequence comparisons (Bashiruddin et al 2005, Sirand-Pugnet et al 2007). Additionally, the genomic context of M. fermentans JER shared a few common genes to that of M. bovis PG45 and M. agalactiae PG2 (Figure 320). Mycoplasma alligatoris A21JP2 and M. crocodyli MP145 also shared an identical genomic context with each other but different from other Mycoplasmas (Figure 321). Again, this was expected because of the high homologous MscL protein sequence they share. The same degree of similarities between the genomic contexts of P. a steris Aster Yellows and P. asteris Onion Yellows was not observed (Figure 3-3 and 3-4). No other significant similarities in genomic context were observed among the remaining mscL genes. Phylogeny Three fast minimum trees were constructed based on the BlastP results. The fast minimum tree of M. bovis PG45 showed it had a close evolutionary relationship with

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36 other Mycoplasma species (Figure 3-22). The clustering of M. bovis PG45 with M. agalactiae PG 2 and M. alligatoris A21JP2 with M. crocodyli MP145 were not a surprise due to high conservation of the MscL protein sequences between these two groups. However, in the tree, other non-Mycoplasma bacteria were also found. The two bacteria, the Gram -negativ e Flavobacteria and Gram -positive Streptococcus shared an evolutionary relationship with M. arthritidis 158L31 suggesting that they might have acquired the mscL gene from a common ancestor. The fast minimum tree of M. penetrans HF -2 was reflective of the BlastP search (Figure 3-23). The Mycoplasma penetrans HF 2 and Gram -positive bacterium, Exiguovabacterium sp. AT1b was branched off from the same node suggestive of sharing a common ancestor. Together M. penetrans HF -2 and Exiguovab acterium sp. AT1b formed a cluster with another Gram -positive bacterium, Clostridium perfringens D. str. JG51721. From the fast minimum tree, one can infer that M. penetrans HF -2 had acquired its mscL from a Gram -positive ancestor. The Phytoplasma asteris Aster Yellows and P. asteris Onion Yellows clustered together on the fast minimum tree (Figure 324) The tree revealed P. australiense was the species that is most closely related to the Gram -negative bacteria, Campylobacter, Yersinia, Aeromonas, and Bac teroides and shared a common ancestor. The MAFFT alignment of MscL protein sequences of 18 species from the class of Mollicutes and 34 non-Mycoplasma species was performed (Figure 3-25). The MAFFT alignment was used as an input file for the Bayesian and ma ximum likelihood analysis. The maximum likelihood (ML) tree showed Mycoplasma and Phytoplasma species

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37 formed an individual and separate cluster supported by strong bootstrap values (Figure 3 -26). The P. asteris Aster Yellows and P. asteris Onion Yellow clu stered and together with P. mali branched off from a common ancestor The ML tree showed all the Phytoplasma species were clustered together and diverged with Odoribacter, a gram negative bacteria from a common ancestor. However, the divergence was not ful ly supported due to a low bootstrap value; therefore, it must be discarded under this analysis. The Mycoplasma species also formed an individual clade and surprisingly, M. penetrans HF -2 was found within the cluster. Some of the bootstrap values for the b ranching patterns within the Mycoplasma clade were not supportive; therefore the topology of the clade was not conclusive. Nonetheless, the Mycoplasma clade was still valid. The identification of the common ancestor for Mycoplasma species was unsuccessful due to lack of confidence level for the majority branching patterns of the ML tree. The Bayesian tree revealed the same individual clustering of the Phytoplasma and Mycoplasma species (Figure 327). Each clade was formed with a strong confidence. The Phytoplasma species clustered together supported with strong posterior probabilities. However, the individual branching pattern for the Phytoplasma was different from the ML tree. By this analysis, P. asteris Aster Yellows and P. asteris Onion Yellows did not share a common ancestor with P. mali. Instead, they and P. australiense formed a cluster and shared a common ancestor with P. mali

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38 The Bayesian tree showed the Phytoplasma species and the Gram -positive bacterium Clostridium were closely related and diverged from a common ancestor. As for the Mycoplasma species, the clade formation and its topology were formed with confidence, but the topology was different from the ML tree. The only similarities between the two trees were the two clusterings of M. bovis PG45 with M. agalactiae PG2 a nd M alligatoris A21JP2 with M. crocodyli MP145 Also, both trees agreed on the evolution of M. mobile 163K and the rest of the Mycoplasma species, which had diverged at the same time. Therefore, they shared a common ancestor. However, the earliest or the oldest Mycoplasma was M. penetrans H F -2 that shared a common ancestor with the species that gave rise to the rest of the Mycoplasma species. In addition, the Mycoplasma clade was found most related to Lactobacillus, a Gr am positive bacterium. Evolutionary Forces Acting on Isolates of M. bovis and M. agalactiae The mscL nucleic acid sequences of different isolates of M. bovis PG45 and M. agalactiae PG2 were aligned using ClustalW (Figure 328). The alignment showed the nucleic acid sequence of mscL was highly homologous among all the i solates. The Selecton web server was used to detect the evolutionary force at each amino acid site. The amino acid sequences of MscL were inferred f rom mscL gene sequences of the M. bovis PG45 isolates and M. agal actiae PG2. The result showed that most of the amino acids experienced either neutral or purifying selection (Figure 3-29). The color -coded scheme permits for identification of the selective pressure at each site and the degree of the pressure. The majority of the amino acid sites experienced purifying selection, where only two sites at amino acid positions 54 and 99 that had undergone positive selection. The amino acid at the position 54 was a serine and at position 99, a histidine.

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39 Serine residue resided in the extracellular domain of the MscL protein. In contrast, histidine was a part of the cytoplasmaic domain of the MscL sequence.

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40 Table 3-1. BlastP result of MscL protein using Mycoplas ma bovis PG45 as the reference query Accession Description Host E value YP_004056093.1 M bovis PG45 Bovine 1.00E 93 YP_001256696.1 M agalactiae PG2 Bovine 9.00E 84 YP_003922812.1 M fermentans JER Human 6.00E 60 YP_002000100.1 M arthritidis 158L3 1 Rodent (rat) 5.00E 47 ZP_08730294.1 M a columbinum SF7 Avian (pigeon) 4.00E 46 YP_003559891.1 M crocodyli MP145 Reptile (crocodile) 3.00E 35 YP_003302602.1 M hominis PG21 Human 4.00E 32 ZP_08703671.1 M anatis 1340 Avian (duck) 1.00E 27 YP_016316.1 M mobile 163K Fish 6.00E 27 ZP_06610634.1 M alligatoris A21JP2 Reptile (alligator) 5.00E 26 NP_853324.2 M. gallisepticum str. R (low) Avian (galliforme) 5.00E 24 ADC31198.1 M gallisepticum str. F Avian (galliforme) 5.00E 24 YP_278558.1 M synoviae 53 Avian (galliforme) 7.00E 22

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41 Table 3-2. BlastP result of MscL protein using Mycoplasma penetrans HF -2 as the reference query Accession Description E value NP_757716.1 M penetrans HF 2 9.00E 152 EGF82475.1 Batrachochytrium dendrobatidis JAM81 6.00E 20 ZP_02952541.1 Clostridium perfringens D str. JGS1721 8.00E 20 YP_002885666.1 Exiguobacterium sp AT1b 5.00E 19 YP_003151058.1 Cryptobacterium curtum DSM 15641 5.00E 19 YP_003777428.1 Herbaspirillum seropedicae SmR1 6.00E 19 YP_003754811.1 Hyphomicrobium denitrificans ATCC 51888 1.00E 18

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42 Table 3-3. BlastP result of MscL using Candidatus Phytoplasma asteris Aster Yellows strain as a reference query. Accession Description E value YP_456223.1 C P asteris Aster Yellows 4.00E 98 NP_950310.1 C P asteris Onion Yellows 2.00E 90 YP_001798791.1 C P australiense 3.00E 41 YP_002004364.1 C P mali 5.00E 26 ZP_03610112.1 Campylobacter rectus RM3267 3.00E 25 YP_004161041.1 Bacteroides helcogenes 2.00E 22 NP_671312.1 Yersinia pestis KIM 10 2.00E 22

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43 Table 3 -4. Descriptive characteristics of MscL in Mollicutes Locus name Species name MscL aa length Molecular weight MALL_0252 M. alligatoris A21JP2 131 14 728 MCRO_0239 M. crocodyli MP145 132 14996 PAa_0113 P. australiense 134 15471 GIG_02848 M. anatis 1340 135 14896 MCSF7_01896 M. columbinum SF7 135 14486 MFE_03250 M. fermentans JER 139 15366 ATP_00331 P. mali 139 16339 MBOVPG45_0250 M. bovis PG45 140 15233 MAG_5570 M. agalactiae PG2 140 15233 AYWB_027 P. asteris Aster Y ellows 145 16509 MMOB6190 M. mobile 163k 151 16844 MARTH_orf609 M. arthritidis 158L3 1 152 16647 PAM_058 P. asteris Onion Yellows 172 19762 MHO_0650 M. homonis PG12 172 19128 MS53_0435 M. synoviae 53 173 19488 ACL_0213 A. laidl a wii 178 20010 MYPE3270 M. penetrans HF 2 215 24623 MGA_0284 M. gallisepticum str. R(low) 220 24196

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44 Table 3-5. Description of the transmembrane domain (TM ), cytoplasmic domain (CD) and extracellular domain (ED) in Mollicutes Species AA Length # TM CD 1 TM helix 1 ED TM helix 2 CD 2 TM helix 3 ED1 M. alligatoris A21JP2 131 2 1 15 16 38 39 68 69 93 94 131 M. crocodyli MP145 132 2 1 16 17 39 40 70 71 93 94 132 M. anatis 1340 135 2 1 17 18 39 40 72 73 95 96 135 M. columbinum SF7 135 2 1 17 18 39 44 70 71 93 94 135 M. fermentans JER 139 2 1 15 16 38 39 70 71 93 94 139 M. bovis PG45 140 2 1 17 18 39 40 69 70 93 94 140 M. agalactiae PG2 140 2 1 17 18 39 40 68 69 93 94 140 M. mobile 163K 151 2 1 28 29 50 51 84 85 109 110 151 M. arthritidis 158L3 1 152 2 1 27 28 50 51 82 83 105 106 152 M. synoviae 53 167 2 1 15 16 38 39 70 71 93 94 167 M. homonis PG21 172 2 1 23 24 49 50 92 93 114 115 172 M. penetrans HF 2 215 2 1 31 32 53 54 101 102 124 125 215 M. gallisepticum str. R (low) 223 2 1 30 3153 5486 87109 110223 P australiense 134 2 1 15 16 38 39 75 76 94 95 134 P mali 139 2 1 21 22 41 42 86 87 106 107 139 P asteris Aster Yellows 145 3 1 20 21 41 42 46 47 66 67 78 79 103 104 145 P asteris Onion Yellows 172 2 1 51 52 79 80 111 112 134 134 172 A laidlawii 178 2 1 26 27 46 47 104 105 127 128 178

PAGE 45

45 Table 3-6. MscL protein of these Mollicutes species do not have a signal peptide. Species Amino Acid Length Signal Peptide M. alligatoris A21JP2 131 No M. crocodyli MP145 132 No M. anatis 1340 135 No M. columbinum SF7 135 No M. fermentans JER 139 No M. bovis PG45 140 No M. agalactiae PG2 140 No M. mobile 163K 151 No M. arthritidis 158L3 1 152 No M. synoviae 53 167 No M. homonis PG21 172 No M. penetrans HF 2 215 No M. gallisepticum strain R (low) 223 No P australiense 134 No P mali 139 No P asteris Aster Yellows 145 No P asteris Onion Yellows 172 No A laidlawii 178 No

PAGE 46

46 Table 3-7. List of MscL -containing bacteria sharing a high homologous sequence with Mollicutes Accession Species name NP215500 Mycobacterium tuberculosis H37Rv CAMRE0001 Campylobacter rectus RM3267 NZACYV Vibrio mimicus VM573 B5650910 Aeromonas veronii B565 Y4019 Yersinia pestis KIM 10 CTU38180 Cronobacter turicensis z3032 ECL04667 Enterobacter cloacae subsp. cloacae ATCC 13047 HMPREF9086 Enterobacter hormaechei ATCC 49162 KPN03691 Klebsiella pneumoniae subsp. pneumoniae MGH 78578 ODOSP1394 Odoribacter splanchnicus DSM 20712 HMPREF9412 Paenibacillus sp. HGF5 HMPREF0660 Porphyromonas gingivalis TDC60 BACHE1450 Bacteroides helcogenes P 36 108 POREN0001 Porphyromonas endodontalis ATCC 35406 CLOCEL1815 Clostridium cellulovorans 743B EAT1B1294 Exiguobacterium sp. AT1b HMPREF0890 Lactobacillus gasseri 202 4 HMPREF0401 Fusobacterium sp. 11_3_2 SPIBUDDY2105 Spirochaeta sp. Buddy HMPREF9488 Coprobacillus sp. 29_1 ANACOL03286 Anaerotruncus colihominis DSM 17241 NZAAXX Flavobacteria bacterium BAL38 CAPGI0001 Capnocytophaga gingivalis ATCC 33624 SDD27957 Streptococcus dysgalactiae subsp. dysgalactiae ATCC 27957 HMPREF8578 Streptococcus sanguinis ATCC 49296 FLUTA3374 Fluviicola taffensis DSM 16823 PLABR1065 Planctomyces brasiliensis DSM 5305 ATU0528 Agrobacterium tumefaciens str. C58 HSERO4050 Herbaspirillum seropedicae SmR1 HDEN0670 Hyphomicrobium denitrificans ATCC 51888 TMATH1405 Thermoanaerobacter mathranii subsp. mathranii str. A3 HMP0721 Pseudoramibacter alactolyticus ATCC 23263 BATDEDRAFT Batrachochytrium dendrobatidis JAM81

PAGE 47

47 Figure 3-1. ClustalW alignment of MscL protein. Sequences of MscL were obtained from M. homonis PG12 M. arthritidis, M. agalactiae PG2 M. bovis PG45 M. fermentans JER M. columbium SF7 M. crocodyli MP145 M. alligatoris A21JP2 M. synoviae 53 M. gallisepticum str. R (low), M. anatis 1340, M. mobile 163k P asteris Onion Yellows strain, P. asteris Aster Yellows strain P. australiense, P. mali, A laidlawii, and M. penetrans HF -2. Amino acids are colored coded according to its chemical properties. Small and hydrophobic incl. aromatic tyrosine are in red. Acidic amino acids are in blue. Basic amino acids and histidine are in magenta. Amino acids wit h hydroxyl, sulfhydryl, amine function group and glycine are in green. Other unusual amino acid, imino acids are in grey. A (asterisk) indicates positions have a single, fully conserved residue. A : (colon) indicates conservation between groups of strong ly similar properties. A (period) indicates conservation between groups of weakly similar properties.

PAGE 48

48 Figure 3-1 C ontinued

PAGE 49

49 Figure 3-2. Color scheme guide for the identification of each gene function.

PAGE 50

50 Figure 3-3. Genomic context of mscL in Ach oleplasma laidlawii The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in A. laidlawii are listed in the table.

PAGE 51

51 Figure 3-4. Genomic context of mscL in Phytoplasma asteris Aster Yellows strain. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in P. asteris Aster Yellows strain are listed in the table.

PAGE 52

52 Figure 3-5. Genomic context of mscL in Phytoplasma asteris Onion Yellows strain. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in P. asteris Onion Yellows strain are listed in the table.

PAGE 53

53 Figure 3-6. Genomic context of mscL in Phytoplasma australiense. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in P. australiense are listed in the table.

PAGE 54

54 Figure 3-7. Genomic context of mscL in Phytoplasma mali The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in P. mali are listed in the t able.

PAGE 55

55 Figure 3-8. Genomic context of mscL in Mycoplasma. agalactiae PG2. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. agalactiae PG2 are listed in the table.

PAGE 56

56 Figure 3-9. Genomic context of mscL in Mycoplasma alligatoris A21JP2. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. alligatoris A21JP2 are listed in the table.

PAGE 57

57 Figure 3-10. Genomic context of mscL in Mycoplasma anatis 1340. The mscL gene i s denoted by the yellow arrow. Genes upstream and downstream from mscL in M. anatis 1340 are listed in the table.

PAGE 58

58 Figure 3-11. Genomic context of mscL in Mycoplasma. arthritidis 158L3-1. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. arthritidis 158L3-1are listed in the table.

PAGE 59

59 Figure 3-12. Genomic context of mscL in Mycoplasma bovis PG45 The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. bovis PG45 are listed in the table.

PAGE 60

60 Figure 3-13. Genomic context of mscL in Mycoplasma columbium SF7 The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. columbium SF7 are listed in the table.

PAGE 61

61 Figure 3-14. Genomic context of mscL in My coplasma crocodyli PM145. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. crocodyli MP145 are listed in the table.

PAGE 62

62 Figure 3-15. Genomic context of mscL in Mycoplasma fermentans JER. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. fermentans JER are listed in the table.

PAGE 63

63 Figure 3-16. Genomic context of mscL in Mycoplasma gallisepticum str. R (low). The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. gallisepticum str. R (low) are listed in the table.

PAGE 64

64 Figure 3-17. Genomic context of mscL in Mycoplasma hominis PG21. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. h ominis PG21 are listed in the table.

PAGE 65

65 Figure 3-18. Genomic context of mscL in Mycoplasma mobile 163K. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. mobile 163K are listed in the table.

PAGE 66

66 Figure 3-19. Genomi c context of mscL in Mycoplasma penetrans HF -2. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. penetrans HF -2 are listed in the table.

PAGE 67

67 Figure 3-20. Genomic context of mscL in Mycoplasma synoviae 53. The mscL gene is denoted by the yellow arrow. Genes upstream and downstream from mscL in M. synoviae 53 are listed in the table.

PAGE 68

68 Figure 3-21. Similarities in the genomic context of the mscL gene between Mycoplasma bovis PG45 (a), Mycoplasma agalactiae PG2 (b), and Mycoplasma fermentans JER (c). Genes that are shared by M. bovis PG45 M. agalactiae PG2 and M. fermentans JER are listed in the table.

PAGE 69

69 Figure 3-22. Similarities in the genomic context of the mscL gene between Mycoplasma alligatoris A21JP2 ( a ) and Mycoplasma crocodyli MP145 (b) Genes that are shared by M. alligatoris A21JP2 and M. crocodyli MP145 are listed in the table.

PAGE 70

70 Figure 3-23. A fast minimum tree of Mycoplasma bovis PG45.

PAGE 71

71 Figure 3-4. A fast minimum tree of Mycoplasma penetrans HF -2

PAGE 72

72 Figure 3-25. A fast minimum tree of P hytoplasma asteris Aster Yellows strain.

PAGE 73

73 NP215500 ----------------------------------------MLKGFKEFLARGNIVDLAV CAMRE0001 -------------------------------------MSFVKEFKEFAMRGNVIDMAV NZACYV -------------------------------------MSLLKEFKAFASRGNVIDMAV Sputw3181 -------------------------------------MSLIKEFKAFASRGNVIDMAV B5650910 -------------------------------------MSLIQEFKAFAARGNVIDMAV y4019 -----------------------------------MLAMSFMKEFREFAMRGNVVDLAV CTU38180 -------------------------------------MSFFKEFREFAMRGNVVDLAV ECL04667 -------------------------------------MSFVKEFREFAMRGNVVDLAV HMPREF9086 -------------------------------------MSFIKEFREFAMRGNVVDLAV KPN03691 -------------------------------------MSFLKEFREFAMRGNVVDLAV Odosp1394 -------------------------------------MSFLKEFKTFAMRGNVVDMAV HMPREF9412 -------------------------------------MNLLKEFKTFALKGNVLDLAI HMPREF0660 -------------------------------------MSKLIQEFKEFAVKGNAVDMAV Bache1450 -----------------------------------MGKTSFLQDFKSFAMKGNVIDMAV POREN0001 -------------------------------------MSKLVSEFKEFAMRGNVMDMAV Clocel1815 ----------------------------------------MLKEFKEFAMKGNVVDLAV EAT1b1294 ----------------------------------------MWKEFKKFAMRGNVIDLAV HMPREF0890 ----------------------------------------MIKEFKEFISRGNMMDLAV HMPREF0401 -------------------------------------MKKLLEEFKAFVMRGNVVDMAV SpiBuddy2105 ---------------------------------MAGKSKMLGEFKTFITRGNVMDLAV HMPREF9488 -------------------------------------MKKLISEFKEFAVKGNVIDMAV ANACOL03286 -------------------------------MNVKEKGSGFISEFKQFIARGNVMDMAV NZAAXX -------------------------------------MGMISEFKDFIAKGNAMDLAV CAPGI0001 -------------------------------------MGFLKEFKEFA VKGNVVDLAI SDD27957 ----------------------------------------MIKELKAFLFRGNVIDLAV HMPREF8578 -------------------------------------MKMLKDLKEFLLRGNVVDLAV Fluta3374 ----------------------------------------MLKEFKDFISRGNVVDLAV Plabr1065 ----------------------------------------MLREFRNFIARGNVMDLAV Atu0528 ----------------------------------------MLNEFKTFIARGNVMDLAV Hsero4050 -------------------------------------MSMLKDFRAFAIKGNVVDLAV Hden0670 ----------------------------------------MLEEFKKFVLRGNVVDLAV Tmath1405 ----------------------------------------MLKGFKEFIMRGNVLDLAV ACL0213 -------------MG-----------KQIKISKEQRKKGFLNGFKNFIMRGNVIDMAV HMP0721 ---------------------------------MKKTKGFFSEFK EFLSRGNVMDLAI PA0113 -------------------------------------MTFFQGFKNFITRGNVINLAV PAM058 MLFLYFDTITKIITKLIFNFLLKRSFEMIENYNFTSKSLQFAKGFKAFIARGNVINLAV AYWB027 ---------------------------MIENYNFTSKSLEFAKGFKAFIARGNVINLAV ATP00331 -----------------------------MNINKILNNNFTKGFKKFINKGNVLNLSI BATDEDRAFT MSEPQQSRNQSVGDKL-----------KGGAFKSVKAVGNVFDDFKAFLNKGNVVDLAV MHO_0650 -------------MT-----------SKELEEKKHYIKKSYIDAKKIVSRGNMFMLAI MAG5570 ------------------------------------MFKKSVNDAWASVKRGNMLLLAI MBOVPG45 ------------------------------------MFKKSVSDAWTSVKRGNMLLLAI MFE03250 ------------------------------------MFKKSCKDAWIVVKRGNMFMLAI MCSF7_01896 ------------------------------------MFKKAGKEAWGVVKRGNMFMLAI MARTHORF609 -------------MT-----------QKELNDKKKVFKNAYKDAKKSITKGNMFMLAI MCRO0239 ------------------------------------MIRKSFKQAKETLKKGNIFMLAV MALL0252 ------------------------------------MITKSMKQAKDALKKGNIFMLAV GIG_02848 ------------------------------------MFKKAVKSAKAHIQRGNMFMLAI MMOB6190 -------------MA-----------IKKPFKKDTIPAKSYDEAKSALKRGNILMLAV MS53_0435 -------------------------------------MKKSWQDAKANIKRNNFLLLAV MGA0284 ---------MLNMKD----------LFNKDKQVSLSKNAMKNATQVVKRGNIFMLAI MYPE3270 ------------MKKE-----------SKKMKIKNPLKGQSWKEFKKLVSSRGNLIDLAV :.* ::: Figure 3-26. MAFFT alignment of 18 Mollicutes species and 34 bacteria sharing a high homologous MscL protein sequence.

PAGE 74

74 NP215500 AVVIGTAFTALVTKFTDSIITPLI-----NR IGVNAQSDVGIL---------RIGIGGG CAMRE0001 GVVIGGAFGKIVSSLVGDVIMPVV-----GV LTGGVN --FTDL---------KFTLKEA NZACYV GIIIGAAFGKIVSSFVADIIMPPI-----GI ILGGVN --FSDL---------SFVLLAA Sputw3181 GIIIGAAFGKIVSSFVADVIMPPI-----GI ILGGVN --FSDL---------SIVLQAA B5650910 GIIIGAAFGKIVSSFVGDVIMPPI-----GL ILGGVD --FSDL---------AVTLKAA y4019 GVIIGAAFGRIVSSLVADIIMPPL----GL LLGGVD --FKQF---------HFVLRAA CTU38180 GVIIGAAFGKIVSSLVADIIMPPL-----GL LIGGID --FKQF---------ALTLRPA ECL04667 GVIIGAAFGKIVSSLVADIIMPPL-----GL LIGGID --FKQF---------AFTLREA HMPREF9086 GVIIGAAFGKIVSSLVADIIMPPL-----GL LIGGID --FKQF--------AFTLREA KPN03691 GVIIGAAFGKIVSSLVADIIMPPL-----GL LIGGID --FKQF---------AVTLRDA Odosp1394 GIIIGGAFGKIVSSLVSDIIMPPI-----GL LIGGVK --FESL---------KIVLKHA HMPREF9412 GVIIGAAFGKIVSSLVSDIIMPVI-----GL LLGGVD --LSGL---------KATIGDA HMPREF0660 GVIIGGAFGKIVSSIVDDIIMPPI-----GW LIGGVN --FSDL---------KWTLPAV Bache1450 GVIIGGAFGKIVSSIVADVIMPPI-----GL LVGGVN --FTDL---------KWVMKPA POREN0001 GIIIGGAFGKIVSSLVADVIMPPI-----TL LTSGSN --IEDL---------KWVLREA Clocel1815 GVIIGGAFGKIVSSLVNDVIMPLF -----GI ILGGIN --FTSL---------TLNIRGT EAT1b1294 AVVLGAAFTAIVNSLVNDIFMPLL-----GI IIGGID --FSSL---------KASILGV HMPREF0890 GVIIGAAFTAIVNSLVKDLINPLI-----GL FIGKID --LSNL---------KFTVGEA HMPREF0401 GVIIATAFGKIVTSLVNDIFMPII-----GV LIGNMN --FSDL---------QIKLGTP SpiBuddy2105 GIIIGSAFTAIINSLVKDILMPFI-----GL ILGGVS --FIDL---------KIVITEA HMPREF9488 GVIIGSAFGKIVSSLVNDIIMPVV-----TL MTGATD --FSRL---------SIVLKEP ANACOL03286 GVIVGGAFKAIADSLTADIIMPII-----GI FVKENS --FSDL---------SVSIGAA NZAAXX GVIIGASFGAIVNSLVSDVITPAL-----LNPALKAAQ --VEDL---------AGLKTDG CAPGI0001 GVIIGGAFGAIVSSLVADVITPLL-----LTPAFKATG --AENL---------QDLVWNSDD27957 AVIIGSAFGAIVTSFVNDIITPLI-----LNPALKAAN --VENI---------TQLTWNHMPREF8578 GVIIASAFGAIVTSFVNDIITPLL -----LNPALEAAK --VQNI---------AELAWNFluta3374 AVIIGAAFGAIVTSLVADIITPLI-----LQPVIEKAG --VANL---------AEVSWNPlabr1065 AVILGGAFSAIVSSLVKDIITPGL-----LNPVMKAAQ --VEKL---------EGLVWNAtu0528 GVIIGAAFSKIVDSVVNDLIMPIV-----GA IFGGFD --FSNY--------FLPLSSN Hsero4050 GVIIGGAFGKIVSSLVEDIIMPIV-----GK IFGGLD --FANY---------YLPLNGQ Hden0670 GVVIGVAFGAIVSSLVADLIMPII-----GA VTGGLD --FSNY---------YLPLSDK Tmath1405 AVIIGAAFNKVVNSLVVDVLTPLI-----GA IFGAPD --FSAL---------KLG ---ACL0213 GVIVGGAFGKIVTSLVNDIILPPI-----GV LLGGVE --FRDL---------QALIHQK HMP0721 GLIIGSAFTAIVTSLNNDIISPLL-----G --LFGGVD --FSNL---------MVKIGGN PA0113 AVVIGQLFSKIVSSLVADIMMPPF-----SL LFNETKGLQGL---------KWCIKED PAM058 AVVIGQLFAKIVSSLVADIIMPLF-----SL LFNYTGALKDL---------KLEIKAN AYWB027 AVVIGQLFAKIVSSLVADIIMPLF-----SL LFNYTGALKDL---------KFTIKAN ATP00331 AFVISQLFSKIVNSLSSDIIMPFM-----NL LFSSDKNDFSDL---------RFRITSN BATDEDRAFT GLVMGAAFTAVVTSLVGDLITPLI-----GL ATQSN--LENM---------FYVLRCP MHO_0650 GLLLGASFGALVSSLANDVIMSAI-----TK AVGMKN -LDAWVVWPGIHATKETG--MAG5570 GVLIGASFNAVISSLANDVIMAAI-----AS LFNVQA --VSEL---------KAG ---MBOVPG45 GVLIGASFNAVISSLANDVIMAAI-----AS LFNVSA --VSEL---------KAG ---MFE03250 GLLLGTAFNAVVSSLANDVIMAAI-----AK AFNVDE --VKDL---------KAG ---MCSF7_01896 GLLLGASFGAVVTSLANDVIMAAI-----AS LFKLDD --VKDL---------KSG ---MARTHORF609 AVLIGAAFGAVVSSLANDVIMAAI-----AK IWNASS --VEDL---------KVAG--MCRO0239 AFILGVVFNAVVSSLANDVIMSAI-----AS KLNFAD --LAQM---------KYN ---MALL0252 AFLIGVVFNAVVTSLANDVIMSAI-----AK HLGFEE --LAKM---------QHN ---GIG_02848 GLLLGTVFGAVVSSLANDIIMSYI-----STNILKYNN --LDDY---------IVS ---MMOB6190 GLLLGTVFGALVASFANDILLGAIGIGINALGININN --FS DL---------S FQ--MS53_0435 AFLLGAVTNAMISSFANDVVLSYI-----AN AFGIKN --LQEW---------KLEN--MGA0284 GLLLGTSFNAVIASLANDVIIAAI-----AK LYNVQD --LQKW---------QVQ ---MYPE3270 ALIIGTAFTAIVTSLVNDIIMPLI-----GA AAGKS--LSEL---------VWYLDIN ..::. : .. .:. Figure 3-26 Continued

PAGE 75

75 NP215500 Q----------------------TIDLNVLLSAAINFFLIAFAVYFLVVLP--YNTL -CAMRE0001 V ----------GD------TAAVTVNYGSFIQTMVDFTIIAFCIFCVVKA--INSL -NZACYV Q----------GD------APAVVIAYGKFIQTVVDFTIIAFAIFMGLKA--INSL -Sputw3181 Q----------GD------APSVVIAYGKFIQTVIDFTIIAFAIFMGLKA--INTL -B5650910 E----------GT------TPAVVIAYGKFIQTIIDFLIISFAIFMGLKA--INTL -y4019 E----------GT------IPAVVMNYGTFIQSIFDFVIVALAIFSAVKL--MNKL -CTU38180 V----------GD------TPAVIMHYGVFIQNVFDFIIVAFAIFLAIKV--INRL -ECL04667 Q----------GD------IPAVVMHYGVFIQNVFDFVIVAFAIFMAIK L --INKL -HMPREF9086 Q----------GD------IPAVVMHYGVFIQNVFDFVIVAFAIFMAIKL--INRL -KPN03691 Q----------GD------VPAVVMHYGVFIQNVFDFIIVAFAIFMAIKL--MNKL -Odosp1394 H----------TDAVTGKVTEAVSINYGNFINTALDFLIIAFSIFLFVKL--INSM -HMPREF9412 -----------------------TLTYGVFLQTVVDFLIVSFSIFMFIRT--LNRF -HMPREF0660 E----------IPGVTA-PAPATINYGNFLQTLLDFIIIAFCVFMMVKG--INKL -Bache1450 E------------IIDGEEVAAVTLNYGNFMQATFDFLIIAFSIFLFIRL--LTKL -POREN0001 V----------IEGGEIT RPEVAMTVGTFLQAVLDFFIIAFVIFMLIKG--MNKL -Clocel1815 -----------------------VVNYGTFIQNIVDFIIISFTIFVVIKV--INKL -EAT1b1294 -----------------------DVLYGNFIQQIVSFFLIAIALFLIVKV--INRL -HMPREF0890 -----------------------TFKYGSFLNAVINFLIIALVVFFL IKL --VNKM -HMPREF0401 V----------EG------VEQAAIRYGAFIQEVINFLIIAFCIFVFIKV--VISL -SpiBuddy2105 T----------AE------TAEVAIMYGNFIQKVVDFLIIAFVVFMIVRT--INRL -HMPREF9488 V----------DG------SQGISLMYGSFLQNVIDFLIIALVIFLMLKCIVKISSL -ANACOL03286 -----------------------TITYGNFIQAVLNFLIMAFVVFCMVKG--INRF -NZAAXX -----------------------GILYGKFLAAVISFLVIAFVIFLLVKA--MNSM -CAPGI0001 -----------------------GVAYGKFLAAVINFLCVAFVLFMLVKG--INKF -SDD27957 -----------------------GVKYGNFLGAVINFLIIGTSLFFVVKA--AEKA -HMPREF8578 -----------------------GVTYGKFLSAVINFLVVGTVLFFVIKG--IEKA -Fluta3374 -----------------------GVLYGKFLAAVINFFVVAFCIFMIVKL--MNRA -Plabr1065 -----------------------GVRYGSFLAAVINFLIIAFVIFLLVRI--VSNV -Atu0528 VN ---------ATSLAAAREQGAVFAYGNFLTVLINFLILAWIIFLMVKG--VNKL -Hsero4050 AY ---------GLPLAEARKAGAVFAYGNFLTILINFLILAFIIFQMVRA--INKA -Hden0670 VHT--------GVAYADAKKEGAVLGYGAFITALLNFLIIAAVLFMVIQ A --MNRL -Tmath1405 -----------------------PIAIGNFLNAVVNFIIVSAAIYFFIVAP--MNAI -ACL0213 PVLDD-NGAQVVIDGIAQFNNVYIRYGNLIQIILEFLIIAFSIYLVLYI--FIKR -HMP0721 -------------------ANSPVIKYGNFITAVINFLITGLVLFVIIKI--VSHA -PA0113 ---------------------VYMNYGIFLQNILEFVIFSFAIYLILTI--FSRT -PAM058 ---------------------TYLGYGNFLQTILEFLLLSFIIYTILTL--ISWK -AYWB027 ---------------------TYLCYGNFLQTILEFLLLSFIIYTILTL--MSWK -ATP00331 --------------------SNI YIYYGKFLKTVFEFLIMSFFIYIILII--VFRQ -BATDEDRAFT KNQTNCRDLLYGTIGDANKAGVVTWNYGRFVQMVINFVIIALIVFFIVKA--YSAA -MHO_0650 -----------------------GIFIGKFLGALIQFIIVSTFIFIGLMIVFTIKNS -MAG5570 -----------------------PVLIGKFLAALISFIIVALIIFLF LILYFLIKNA -MBOVPG45 ----------------------SVLIGKFLAALISFIIVTIIIFLFLIIYFLIRNA -MFE03250 -----------------------PILIGKFLAALISFLIVATVIFILLVVVFLVKNA -MCSF7_01896 -----------------------PVFIGKFLAALIAFLIVATIIFVALYLVFIIKLS -MARTHORF609 -----------------------TIKIGKFLAALIQFVIVATVVFFTLLIVFSIKNA -MCRO0239 -----------------------GILYGKFLATLINFIVVSLFLFLMLSFYFLIVNF -MALL0252 -----------------------GILYGKFLAAVINFFVVAVFLFFMLTGYFLLVNF -GIG_02848 ----------------------GMKVGKFLGVLLNFVIVTLFIFAVLVVYYLFYNM -MMOB6190 -----------------------TIRYGNFISALLTFIIVSLFIFFALFIYFVIRNI -MS53_0435 -----------------------GILIGKFLGTVIQFAIVMVLLFFVLFLFFVIKNN -MGA0284 -----------------------GIFIGKFFAALLSFVIINVILVSALFVSYYIIEV -MYPE3270 -------NQPHFLPNGDINPQAIIIKYGNFLQVIINFFIIALTMFSVVKAYLSIRKSYK :. : : Figure 3-26 Continued

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76 NP215500 -------------------R ---------------KKGEVE--QPG ------------CAMRE0001 -------------------K ---------------KPKVEE--PKAAE--P API--NZACYV -------------------K ---------------RKEEEE--APKAP--P AP---Sputw3181 -----------------K ---------------RKE EE--APKAP--P TP---B5650910 -------------------K ---------------KKQ EE--EAAAP--A GP---y4019 -------------------R ---------------REKAEE--EPATP--P AP---CTU38180 -------------------H ---------------QKK -------PKEA--A GP---ECL04667 -------------------N ---------------RKK EE----PAAA--P AP---HMPREF9086 -------------------N ---------------RKK EE--PAAAP--P AP---KPN03691 -------------------N ---------------RKK EE--APAAP--P AP---Odosp1394 -------------------K ---------------RKE ET--KPTPP--P AP---HMPREF9412 -------------------K ---------------RKE EI--KAEEP--P AP---HMPREF0660 -------------------S ---------------KKK EE--EPVAPAPDPEP---Bache1450 ------------------T ---------------EKKKDE--KQATPVSPAP---POREN0001 -------------------R ---------------KAK PE--EPATP--A EP---Clocel1815 -------------------N ---------------KDRQKK--EEVKE--A KK---EAT1b1294 -------------------Q ---------------REK EV--EEAAI--P TP---HMPREF0890 -------------------M ---------------PKK EV--EEDD----P TP---HMPREF0401 -------------------Q ---------------KKK EE--KPAPA--P EP---SpiBuddy2105 -------------------RERLDA----------KKREEE--KAKAAATPAPAPVT HMPREF9488 -------------------R ---------------QKEEKE--DMREE--K GP---ANACOL03286 -------------------H ---------------RKA EK--APPMP--P AP---NZAAXX -----------------------------------KKKQEP--APAAP--S GP---CAPGI0001 -----------------------------------KKKEEP--APEAP--A GP---SDD27957 -------------------M ---------------PKKQEE--EVVEV--A AP---HMPREF8578 -------------------QNL -------------RKKEEV--VEEAP--A AP---Fluta3374 -------------------MSLR------------KKKEEE--TPAAP--A EP---Plabr1065 -------------------QKQF------------DDEPEP--EKKDP----AP---Atu0528 -------------------RDSV------------DRKKIE--EKPDA--A PP---Hsero4050 ------------------RDLA------------SKHEEA--APAAP--A PT---Hden0670 -------------------T ---------------RKEK-----AVEKP--A EP---Tmath1405 -------------------R ---------------QRKAKE--KEQTP--P EP---ACL0213 -------------------KEQEEKFIAEEKARLEKLEAEK--NPPKP --A PK---HMP0721 -------------------ANKL------------PLIGDD--EVAEPTTKTCP--Y PA0113 -------------------KLE -------------KPKSEL--KLILE----SL---PAM058 -------------------NPLQ------------KDKVDK--NMLLL--QQSL---AYWB027 -------------------NPLQ------------KEKTDK--NMLLL--QQSL---ATP00331 -------------------NLN -------------SEKVNI--ESLT-----------BATDEDRAFT -------------------FCRKTDVIVKDCVYCCKEIPLD--ATRCPFCT SPVQIP MHO_0650 -------------------ILYA ------------KAKKNPIEEEIKEE--P IP---MAG5570 -------------------VEAR------------KAKKNP--PVVVV--A AP---MBOVPG45 -------------------IEHR------------KAKRNP--PVAVV--A TP---MFE03250 -------------------IETR------------KFKKNP--PV AEE--P KP---MCSF7_01896 -------------------LQAR------------KERLNP--TPAPE--V VP---MARTHORF609 -------------------IEYA------------KAKKMPI --EPEAE--P TP---MCRO0239 -----------------------------------KNRKKV-EPVKEA--P KP---MALL0252 -----------------------------------KNRKKV--VVKEA--P KP---GIG_02848 -------------------KKAK------------EEKAKA--AEAPA--PKVP---MMOB6190 -------------------RLDN------------LKKKYPERYVVAA--P KP---MS53_0435 -------------------YVEH------------KNRKNP--PQPVV----KPL--MGA0284 -------------------RKAI------------KAKQLL--AEGVNPTTTKPIEIMYPE3270 NKFYGFTYEEYFAFIKEGKKRKEIKLLAIERDLKLKEKEEK--EKAEK----EK---Figure 3-26 C ontinued

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77 NP215500 DTQVVLLTEIR-----DLLAQT ---------NGD -------------------------CAMRE0001 PADVALLTEIR-----DLLKNK -------------------------------------NZACYV TKDQELLSEIR-----DLLKAQ ---------QDK -------------------------Sputw3181 TKEEELLSEIR-----DLLKAQ ---------QEK -------------------------B5650910 TKDQELLTEIR-----DLLKSQ ---------QER -------------------------y4019 TTEEILLAEIR-----DLLKAQ ---------HTK -------------------------CTU38180 SKEQVLLTEIR-----DLLKEQ ---------NNQ RP----------------------ECL04667 TKEEVLLTEIR-----DLLKEQ ---------NNR --V ----------------------HMPREF9086 TKEEVLLTEIR-----DLLKEQ ---------NNR --V ----------------------KPN03691 SKEEVLLSEIR-----DLLKEQ ---------NNR --S ----------------------Odosp1394 SKEEQLLTEIR-----DLLKGK ---------NS--------------------------HMPREF9412 SKEEVLLAEIR-----DLLKQQ ---------NQASEK----------------------HMPREF0660 TNEEKLLSEIR-----DLLKNK -------------------------------------Bache1450 SKEEVLLTEIR-----DILKEK ---------K ---------------------------POREN0001 SDEVKLLGEIK-----ELLKKQ ---------AEK -------------------------Clocel1815 SEDIVLLEEIR-----NLLKEN ---------RRV ------------------------EAT1b1294 TKEEQLLTEIR-----DLLKDR ---------SL--------------------------HMPREF0890 TNEELYLRQIR-----DLLQEK ---------NK--------------------------HMPREF0401 TKEEVLLTEIR-----DALNKI ---------ADK -------------------------SpiBuddy2105 SADVVLLTEIR-----DLLKKK -------------------------------------HMPREF9488 TTEELLIEIR-----DLLKEK -------------------------------------ANACOL03286 SNEEKLLIEIR-----DLLKEK -------------------------------------NZAAXX TQEQLLAEIR-----DLLKK-------------------------------------CAPGI0001 TQEELLTEIR-----DLLKKN ---------N ---------------------------SDD27957 TQEELLTEIR-----DLLANK -------------------------------------HMPREF8578 TELEVLQEIK-----ALLEKK -------------------------------------Fluta3374 SNEEKLLMEIR-----DLLKNK -------------------------------------Plabr1065 STEARLLTQIR-----DLLKQQ ---------TEVGKPADDQA -----------------Atu0528 PEDVKLLTEIR-----DLLKTR -------------------------------------Hsero4050 PEDVLLLREIR-----DSLKKQ ---------G ---------------------------Hden0670 SAELKVLTDIR-----DLLARK -------------------------------------Tmath1405 SEEVKLLREIL-----EVLKEK -------------------------------------ACL0213 PEDIQLLIEIR-----DLLKKN ---------EK--------------------------HMP0721 CKSEIDVTATRCPHCTSQLGEH ---------TAA -------------------------PA0113 QKEITLLQEIK-----NNLQQN ---------NNKK------------------------PAM058 DKEIALLEEIK-----DILKSN ---------K --------------------------AYWB027 NKEIALLEEIK-----NILKSN ---------K ---------------------------ATP00331 KEQIILIKEIK-----EILKKK ---------N ---------------------------BATDEDRAFT HSDSDILTKNN-----DDVAVQ ---------LPT -------------------------MHO_0650 TTEELILNELK-----KLNENL ---------LETKNSGNKKDLKNDLK-----------MAG5570 TTDELILQELK-----KLNENI ---------EELRRSQLK-------------------MBOVPG45 TTDELILQELK-----KLNENI ---------EELRRNQLK-------------------MFE03250 TTEELILAEIK-----KLN ERL ---------EKLEQPKK--------------------MCSF7_01896 TKEELILAELK-----KISASL ---------EQKK------------------------MARTHORF609 TNEELILAELK-----KLNSQI ---------ENLNRNQN--------------------MCRO0239 TTEELILEELR-----KLNEKL ---------NAKK------------------------MALL0252 STDELILEELK-----KLNEKL ---------ESRK------------------------GIG_02848 TTEELILAELK-----ELNQNM ---------KK--------------------------MMOB6190 STDELILEELR-----TLNRAK ---------RL--------------------------MS53_0435 TVDELILKELQ-----QLNSNI ---------TKNQLSQDMQTSLSALETSARKTSSRSAS MGA0284 NKDEKIIELLE--YNNMLLQQQIEIFGKAHNMKVEILSKKFSDSVISVPFDINKPEPQS MYPE3270 NSVESILKDIR-----TIMEEN ---------AKLLKENNDISRKTTKTLR ---------: Figure 3-26 Continued

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78 NP215500 -------SPGRHGGRGTPSPTDGPRASTESQ CAMRE0001 ------------------------------NZACYV ------------------------------Sputw3181 ------------------------------B5650910 ------------------------------y4019 ------------------------------CTU38180 ------------------------------ECL04667 ------------------------------HMPREF9086 ------------------------------KPN03691 ------------------------------Odosp1394 ------------------------------HMPREF9412 ------------------------------HMPREF0660 ------------------------------Bache1450 ------------------------------POREN0001 ------------------------------Clocel1815 ------------------------------EAT1b1294 ------------------------------HMPREF0890 ------------------------------HMPREF0401 ------------------------------SpiBuddy2105 ------------------------------HMPREF9488 ------------------------------ANACOL03286 ------------------------------NZAAXX ------------------------------CAPGI0001 ------------------------------SDD27957 ------------------------------HMPREF8578 ------------------------------Fluta3374 ------------------------------Plabr1065 ------------------------------Atu0528 ------------------------------Hsero4050 ------------------------------Hden0670 ------------------------------Tmath1405 ------------------------------ACL0213 ------------------------------HMP0721 ------------------------------PA0113 ------------------------------PAM058 ------------------------------AYWB027 ------------------------------ATP00331 ------------------------------BATDEDRAFT -------G ----------------SNYRR-MHO_0650 ------------------------------MAG5570 ------------------------------MBOVPG45 ------------------------------MFE03250 ------------------------------MCSF7_01896 ------------------------------MARTHORF609 ------------------------------MCRO0239 ------------------------------MALL0252 ------------------------------GIG_02848 ------------------------------MMOB6190 ------------------------------MS53_0435 SKKLNKK-----------------------MGA0284 VEAANLEKMLRPNAQSAVSASNWSGTEGLIG MYPE3270 ------------------------------Figure 3-26 C ontinued

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79 Figure 3-27. Maximum likelihood tree. The Phytoplasma are clustered together (blue); the Mycoplasma form another independent cluster (green). Bootstrap value (black) at each node is the confidence level for the branching pattern.

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80 Figure 3-28. Bayesisan tree. The Phytoplasma are clustered together (blue); the Mycoplasma form another independent cluster (green). Posterior probability (black) at each node is the confidence level for the branching pattern.

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81 MBOVPG45 ATGTTCAAAAAATCGGTAAGTGATGCATGAACATCAGTTAAACGTGGTAATATGCTACTA 60 MYBP ATGTTCAAAAAATCGGTAAGTGATGCATGAACATCAGTTAAACGTGGTAATATGCTACTA 60 MYBF ATGTTCAAAAAATCGGTAAGTGATGCATGAACATCAGTTAAACGTGGTAATATGCTACTA 60 MMOB ATGTTCAAAAAATCGGTAAGTGATGCATGAACATCAGTTAAACGTGGTAATATGCTACTA 60 MAG5570 ATGTTCAAAAAATCAGTAAACGATGCATGAGCATCAGTTAAACGTGGCAATATGCTACTA 60 **************.****. *********.**************** ************ MBOVPG45 TTAGCTATTGGTGTTTTAATTGGTGCCTCATTTAATGCAGTTATTAGTTCATTAGCAAAC 120 MYBP TTAGCTATTGGTGTTTTAATTGGTGCCTCATTTAATGCAGTTATTAGTTCATTAGCAAAC 120 MYBF TTAGCTATTGGTGTTTTAATTGGTGCCTCATTTAATGCAGTTATTAGTTCATTAGCAAAC 120 MMOB TTAGCTATTGGTGTTTTAATTGGTGCCTCATTTAATGCAGTTATTAGTTCATTAGCAAAC 120 MAG5570 TTAGCAATTGGTGTTTTAATTGGTGCTTCATTCAATGCAGTTATTAGCTCATTAGCAAAC 120 *****:******************** ***** ************** ************ MBOVPG45 GATGTTATAATGGCTGCAATAGCTTCTTTATTCAATGTTTCTGCAGTTTCCGAGTTAAAA 180 MYBP GATGTTATAATGGCTGCAATAGCTTCTTTATTCAATGTTTCTGCAGTTTCCGAGTTAAAA 180 MYBF GATGTTATAATGGCTGCAATAGCTTCTTTATTCAATGTTTCTGCAGTTTCCGAGTTAAAA 180 MMOB GATGTTATAATGGCTGCAATAGCTTCTTTATTCAATGTTTCTGCAGTTTCCGAGTTAAAA 180 MAG5570 GATGTGATAATGGCTGCGATAGCTTCTTT ATTCAATGTACAAGCAGTTTCTGAACTAAAA 180 ***** ***********.********************: .:******** **. ***** MBOVPG45 GCTGGCTCTGTTTTAATTGGTAAATTCTTAGCTGCTTTAATTTCATTCATTATTGTTACT 240 MYBP GCTGGCTCTGTTTTAATTGGTAAATTCTTAGCTGCTTTAATTTCATTCATTATTGTTACT 240 MYBF GCTGGCTCTGTTTTAATTGGTAAATTCTTAGCTGCTTTAATTTCATTCATTATTGTTACT 240 MMOB GCTGGCTCTGTTTTAATTGGTAAATTCTTAGCTGCTTTAATTTCATTCATTATTGTTACT 240 MAG5570 GCTGGCCCTGTTTTAATTGGGAAGTTCTTAGCAGCATTAATTTCATTTATCATTGTTGCT 240 ****** ************* **.********:**:*********** ** ******.** MBOVPG45 ATTATAATTTTTCTATTCTTAATTATTTATTTCTTAATTAGAAATGCTATAGAGCATAGA 300 MYBP ATTATAATTTTTCTATTCTTAATTATTTATTTCTTAATTAGAAATGCTATAGAGCATAGA 300 MYBF ATTATAATTTTTCTATTCTTAATTATTTATTTCTTAATTAGAAATGCTATAGAGCATAGA 300 MMOB ATTATAATTTTTCTATTCTTAATTATTTATTTCTTAATTAGAAATGCTATAGAGCATAGA 300 MAG5570 CTTATTATTTTCCTTTTCTTAATCCTTTATTTCTTGATTAAGAATGCAGTTGAAGCAAGA 300 .****:***** **:******** .**********.****..*****:.*:**. .:*** MBOVPG45 AAAGCAAAAAGAAATCCTCCAGTTGCAGTGGTTGCCACTCCTACAACTGATGAATTAATA 360 MYBP AAAGCAAAAAGAAATCCTCCAGTTGCAGTGGTTGCCACTCCTACAACTGATGAATTAATA 360 MYBF AAAGCAAAAAGAAATCCTCCAGTTGCAGTGGTTGCTACTCCTACAACTGATGAATTAATA 360 MMOB AAAGCAAAAAGAAATCCTCCAGTTGCAGTGGTTGCTACTCCTACAACTG ATGAATTAATA 360 MAG5570 AAGGCTAAGAAGAATCCACCTGTTGTGGTTGTTGCTGCTCCAACAACAGATGAATTAATA 360 **.**:**.*..*****:**:**** .** ***** .****:*****:************ MBOVPG45 CTTCAAGAGCTTAAAAAACTTAATGAAAACATTGAAGAATTAAGAAGAAATCAATTAAAG 420 MYBP CTTCAAGAGCTTAAAAAACTTAATGAAAACATTGAAGAATTAAGAAGAAATCAATTAAAG 420 MYBF CTTCAAGAGCTTAAAAAACTTAATGAAAACATTGAAGAATTAAGAAGAAATCAATTAAAG 420 MMOB CTTCAAGAGCTTAAAAAACTTAATGAAAACATTGAAGAATTAAGAAGAAATCAATTAAAG 420 MAG5570 CTCCAAGAACTTAAGAAACTTAATGAAAACATTGAAGAATTAAGAAGAAGTCAATTAAAG 420 ** *****.*****.**********************************.********** MBOVPG45 TAA 423 MYBP TAA 423 MYBF TAA 423 MMOB TAA 423 MAG5570 TAG 423 **. Figure 3 -29. ClustalW alignment of mscL nucleic acid sequence of Mycoplasma bovis i solates. Amino acids are colored coded according to its chemical properties. Small and hydrophobic incl. aromatic tyrosine are in red. Acidic amino acids are in blue. Basic amino acids and histidine are in magenta. Amino acids with hydroxyl, sulfhydryl, amine function group and glycine are in green. Other unusual amino acid imino acids are in grey. A (asterisk) indicates positions have a sing le, fully conserved residue. A : (colon) indicates conservation between groups of strongly similar properties. A

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82 (period) indicates conservation between groups of weakly similar properties.

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83 Figure 3-30. Positive and purifying selection at each amino acid of Mycoplasma bovis. Shades of yellow (color 1 and 2) indicates >1. Shades from white to magenta (3 -9) indicates <1. Most of the amino acids experienced some degree of purifying se lection. There were only two amino acid sites that underwent positive selection.

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84 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS The MscL Protein in Mollicutes The mscL gene was identified in Mollicutes species that infect a wide range of hosts including animal s, birds fish, plants and insect vectors, reptiles and humans. Within the Mollicutes, mscL was identified by in silico analysi s of the available genomes in the family Mycoplasmataceae genus Mycoplasma but was not found in the genus Ureaplasma. The mscL gene also was not identified in the available genomes in the family Spiroplasmatacea genus Spiroplasma nor in the members of the closely related Mycoplasma mycoides cluster. MscL was found in the available genomes of the Acholeplasmataceae. The remaining families of the Mol licutes did not have genome sequences available for in silico analysis. For reference purposes, we c hose t he MscL protein sequence from M. bovis PG45. Sequences with greater similarities to the query sequence will have the lowest Evalue, with E -6 generally accepted as a cutoff point Based on the E -value and the ClustalW alignment, M. bovis PG45 and M. agalactiae PG2 had nearly identical MscL protein sequences Both of these species are bovine pathogens with somewhat similar clinical presentation. T herefore, it was not a surprise that they shared a highly homologous MscL protein sequence. In fact, these two species are known to be quite similar (Thomas et al. 2005) What was surprising was the absence of mscL in the M. m ycoides cluster, since th ese pathogens cause similar disease patterns, share evolutionary roots, and also occupy ruminant hosts. Mycoplasma fermentans JER, which colonizes humans, also shared a similar MscL protein sequence with M. bovis PG45 and M. agalactiae PG2

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85 The two reptile pathogens, M. alligatoris A21JP2 and M. crocodyli MP145 shared a nearly identical MscL protein sequenc e as well, but it had a lower homology with the MscL protein sequence found in the bovine pathogens. Interestingly, the genomic context in which the mscL gene was found was identical for M. bovis PG45 and M. agalactiae PG2 ; M. alligatoris A21JP2 and M. crocodyli MP145 also had identical genomic contexts with each other (see discussion below). One MscL protein that did not share a high ly homologous sequence with MscLs for other Mycoplasma species was identified in the putative human pathogen M. penetrans HF -2 Instead, t he outlier M. penetrans HF -2 had an MscL protein sequence that showed the high est degree of homology with non-Mollicutes species. The BlastP result showed the MscL sequence from M. penetrans HF -2 was more similar to Gram positive bacteria and fungi like Batrachochytrium Clostridium, and Exiguobacterium. M ycoplasma penetrans HF 2 is different from most Mycoplasma species in that it is an intracellular pathogen (Sasaki et al. 2002) and therefore has a different host environment than a mucosal surface. However, M. penetrans HF 2 and Batrachochytrium have very different hosts and thus the similar ity in MscL sequence would not be predicted to be a result of the host environment. Batrachochytrium dendr obatidis causes chytridiomycosis, an infectious disease in amphibians (Johnson et al. 2003). Therefore, this finding was very intriguing and suggests that factors other than the host are at play and contributing to the similarity of the MscL sequence between these two organisms. MscL was not detected in Phytoplasma sp. when M. bovis PG45 was used as the query in the BlastP search. However, the Molligen search using mscL and

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86 mechanosensitive channel as queries allowed the identification of the mscL gene in Phytoplasma sp because the genus Phytoplasma is in a different order from the genus Mycoplasma, it is not surprising that the MscL protein is quite different (Bertaccini et al. 2007 Christensen et al. 2005, Firrao et al. 2007, Gasparich et al. 2010, Sugio et al. 2011, Weintraub et al. 2006) In fact, the host niche for Phytoplasma sp. is the phloem of plants, so these are essentially intracellular pathogens. In addition, these noncultivable pathogens frequently colonize insect vectors, and thes e infected insects play a role in disease transmission (Bertaccini et al. 2007 Christensen et al. 2005, Firrao et al. 2007, Gasparich et al. 2010, Sugio et al. 2011, Weintraub et al. 2006). Although their homology to the MscL proteins of other Mollicutes w as low the MscL proteins identified in Phytoplasma sp. shared a highly homologous MscL sequence with each other. The sequence homology for the MscL protein of P. asteris Onion Yellows and P. asteris Aster Yellows was very high, as would be predicted from strains of the same species. By BlastP analysis, the Phytoplasma species and Gram negative bacteria like Campylobacter Bacteroides and Yersinia shared similar MscL protein sequences. This fin d ing suggests that the mscL gene may have been acquired by transfer of Gram -negative bacteria rather than as a result of the accepted G ram positive evolutionary pathway for Mollicutes Variations in the MscL P rotein The length of the MscL protein sequence varied greatly among Mycoplasma species rang ing from 13 1 to 220 amino acids. Mycoplasma alligatoris A2JP2 had the shortest protein, with only 131 amino acids in contrast to M. gallisepticum str. R (low ), which consisted of 220 amino acids. Variations in the amino acid length can be

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87 significant with respect to overall MscL protein function and may even provide evolutionary information on how the mscL gene was acquired. A s would be expected, the transmembrane (TM) domains showed very little variation among the different MscL proteins. Since these two domains form the critical pore structure, this was not unexpected. The predicted TM domai ns within the MscL protein sequence showed MscL crosses the membrane multiple times. Most multipass TM proteins consist of alpha-helices that span the membrane and do not have a signal peptide (Chou et al 1999, UniProt Consortium 2011). Annotation results showed that most of the MscL proteins from Mollicutes contained two TM domains. However, P. asteris Aster Yellows strain had three TM domains. The TM domains were similar in length and conserved in sequences among the Mycoplasma and Phytoplasma species, demonstrating the functional part of the protein. The Mollicutes have only a single membrane; therefore, it makes sense that TM domains have uniform lengths. The TM1 domain lines the inside of the channel and is surrounded by the TM2 domain (Perozo 2006). Therefore, TM1 domains can converge or diverge to open or close the MscL channel (Kung et al. 2010). The ClustalW alignment demonstrated that the majority of the amino acid variations were at the aminoand carboxyl terminals which form the two cytoplasmic domains. The role of the cytoplasmic domains is st ill not well understood in other bacteria and has not been addressed at all for Mollicutes. However, deletion of the C terminal domain of the E. coli MscL had no major impact on the function of the protein (Oakley et al. 1999).

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88 Genomic Context and Implica tions for Gene Transfer A better understanding of the genomic context permits analysis of the function of a specific gene, but also can provide information on the interactions between the target gene and its neighboring genes. For example, in Mycoplasma genitalium genes that were in close vicinity to each other encoded proteins that had similar functions or interacted in similar pathways (Huynen et al 2000). Furthermore, prokaryotic genes that were spatially associated with each other also had more prote in interactions (Huynen et al. 2000). Therefore, comparative genome analysis allows for the prediction of functional interactions between proteins (Huynen et al 2000). In addition, the presence of elements associated with gene mobility such as i nsertion s equence elements, transposases, and i ntegrative c onjugative e lements (Calcutt et al 2002, Dybvig et al. 2007; Sirand-Pugnet, Citti et al 2007; Thomas et al 2005) near specific genes may suggest the potential for horizontal and/or lateral gene transfer via conjugation. The genomic context of the mscL gene was for the most part, unique and species -dependent. The closely related M. bovis PG45 and M. agalactiae PG2 had the mscL gene in an identical genomic context with each other with some similarity observed in M. fermentans JER. This is consistent with the BlastP result and clustalW alignment that showed all three species had low E -values and highly homologous protein sequences, suggesting that the gene may have been acquired by a common ancestor and/or a common mechanism. M. bovis PG45, M. agalactiae PG2, and M. fermentans JER had an identical genomic context upstream of mscL. However, a different suite of genes was found downstream of mscL in M. fermentans JER Interestingly, MFE03310 encoded a putative transposase, suggestive of a gene transfer event. The length and the protein sequence of MscL from Mycoplasma alligatoris

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89 A21JP2 and M. crocodyli MP145 were identical; therefore, it was not unexpected to see th at their genomic contexts like M. bovis PG45 and M. agalactiae PG2 were also the same. Phylogenetic and Evolutionary R elationships The variations at the aminoand carboxyl terminals of the MscL protein of Mollicutes species inspired us to further examine the evolutionary relationship of the MscL protein among Mollicutes species and with other non-Mollicutes bacteria. The fast minimum evolution (ME) method was applied to the BlastP search results to construct phylogenetic trees. The ME method used the sum of the total estimated branch length (S) a nd the tree with the smallest S value was designated the most preferred tree (Takahashi et al 2000). Mycoplasma bovis PG45 clustered with other Mycoplasma species in the fast minimum tree, suggests that that have acquired the mscL gene from a common ancestor, likely a Gram -positive Clostridium or Gram -negative Flavobacterium Similar clusters from the fast minimum tree were also observed in the 16S rRNA tree generated by MolliGen. The close clustering of M. bovis PG45, M. agalactiae PG2 and M. fermentans JER was supported by the E values, 16S rRNA tree and by our ME tree as well. In addition, M. alligatoris A21JP2 and M. crocodyli MP145 were again found clustering together. Due to high homology of the MscL protein sequence, it was expected that these speci es would be evolutionary closely associated with each other. The outlier M. penetrans HF -2 clustered with Gram -positive Clostridium and Exiguobacterium species in the fast minimum tree. Because it is an intracellular pathogen, M. penetrans HF -2 might have experienced a selective pressure by the host that influenced the MscL sequence. Nonetheless, the results supported that in the genus Mycoplasma, the mscL gene was most likely acquired from Gram -positive

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90 bacteria, which is consistent with the evolutionary p attern of the standard 16S rRNA tree. Phytoplasma species showed a close evolutionary relationship among themselves. In addition, the fast minimum tree showed Phytoplasma species shared an evolutionary relationship with Gram -negative bacteria like Campylobacter, Yersinia, Bacteroides and Aeromonas. This finding was surprising since Mollicutes have evolved from Gram -positive bacteria. However, the MscL protein is widely found in both Gram negative and Gram -positive bacteria. Therefore, it is possible for Phy toplasma species to obtain its MscL from Gram -negative bacteria, especially since these species are common in soil and plants. The optimal tree generated from any analysis might not be the true tree because its topology and branching patterns are influenced by the number of nucleotides and sequences compared (Takahashi et al 2000). When the ME method that was used to generate the fast minimum tree described above is applied to a small number of sequences, the resulting tree tends to be greater or equal to the true tree (Takahashi et al 2000). Therefore, for a smaller number of sequences, the fast minimum tree is used. Maximum -likelihood (ML) and Bayesian analyses are preferred for large numbers of sequences. We analyzed 54 sequences; therefore we also used ML and Bayesian as well as fast minimum tree methods (Takahashi et al 2000). The ML analysis is widely used to create reliable molecular phylogenies (Guindon et al 2003). The ML method has a higher accuracy in constructing phylogenetic trees than other evolutionary analyses like neighbor joining and maximum parsimony (Ogden et al 2006). The ML method uses a statistical parameter to produce a distribution creating the most probable evolutionary tree from a given dataset. Bootstrap values are

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91 usually given in percentages and used in ML as estimates for the reliability of evolutionary trees (Hall 2011). Bootstrap is a statistical re-sampling technique by replacing characters (e.g., a single nucleotide across all taxa) of a given dataset (Cummings et al 2003, Hall 2011). After 100 to 2,000 replications of re-sampling, the generated bootstrap values are then used to construct a new tree (Cummings et al 2003, Hall 2011) The new tree is then compared to the original tree that was estimated using the same parameter and method (Hall 2011). The higher the bootstrap value, the more confidence there is in the topology of the tree (Hall 2011). From the ML tree, all of the Msc L -containing Mycoplasma species formed a cluster. Based on the E value and the fast minimal evolution tree, we found that M. penetrans HF -2 did not have a highly homologous MscL protein sequence with other Mycoplasma species and clustered instead with other bacteria. However, unlike the fast minimum tree, the ML tree clustered M. penetrans HF -2 with the rest of the Mycoplasma species. A supportive value below 50 is deemed non-supportive, and that specific branching pattern should be discarded. The clusteri ng of M. penetrans HF 2 with the other Mycoplasma species had a supportive value of 58, so it was within the minimal criterion for acceptance. Within the cluster of all the Mycoplasma species, some of the branching patterns were poorly supported based on the criterion of a supportive value below 50. However, two sets of branching [ M. agalactiae PG2 and M. bovis PG45 ] and [ M. crocodyli MP145 and M. alligatoris i A21JP2] were highly supported with values of 97 and 88. This demonstrated that M. bovis PG45 and M. agalactiae PG2 contain minimal evolution changes in their MscL protein sequence and the same applies to M. alligatoris and M.

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92 crocodyli This is further supported by BlastP, ClustalW, and the similarity in genomic context. The Phytoplasma species also f ormed an individual cluster with high bootstrap values of 57 to 99. Because most clades were not highly supported, identification of common ancestors for Mycoplasma and Phytoplasma species was not possible with certainty. The Bayesian method is an alternative method with the ability to use complex evolutionary models and can accommodate uncertainty in phylogeny. In addition, it is used to estimate large phylogenies (Huelsenbeck et al 2002). The Bayesian method has the same level of performance as the ML ( Ogden et al 2006). However, the Bayesian model differs from ML in that it uses the data and a substitution model to generate the most likely tree whereas the ML method finds the most likely tree that fits the dataset (Hall 2011). The Bayesian analysis wil l choose a tree as a starting point and its likelihood is accessed. The topology and the branch length will be changed, forming a new tree and its likelihood will be determined. This whole process is termed generation and will continue until there is no si gnificant likelihood changes among the trees (Hall 2011). Once that is achieved, a consensus tree will be constructed. Because the Bayesian analysis uses prior probabilities in determining the most likely tree, it is used as a measurement of confidence for the topology or formation of clades (Hall 2011, Huelsenbeck et al 2001). The tree generated using the Bayesian method showed similar clustering patterns in the Mollicutes as did the ML tree. The Mycoplasma species clustered together with posterior pro babilities of 0.74 to 1, which demonstrated a high level of confidence. Phytoplasma species were also clustered together and supported by high posterior

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93 probabilities. In addition, the Bayesian tree suggested that the Mycoplasma species have acquired the mscL gene from the same ancestor as Lactobacillus a Gram -positive bacterium. The Phytoplasma species and the Gram -positive bacterium Clostridium have acquired the mscL gene from a common ancestor. These results support the established concept that Mollicu tes are the descendents of Gram -positive bacteria like Bacillus, Clostridium and Streptococcus (Kunisawa 2002,Maniloff 2002). Therefore, it is not surprising that the Bayesian analysis of the MscL protein also showed similar results. The goal was to use various methods to examine the evolutionary relationship of the MscL protein from all the Mycoplasma and Phytoplasma species and their relation with other bacteria. Each evolutionary analysis offered a separate tree for the evolution of the MscL protein. There is no single perfect method that can generate the true tree; therefore, best methods are employed to make the optimal tree. There were some discrepancies among the fast minimum, ML and Bayesian trees. The fast minimum tree offers a rough evolutionary relationship because of its usage of a simple statistical model to calculate the evolutionary distance between species. The most commonly used methods are the Bayesian and ML analyses because of their well -suited statistical models. The ML method uses bootst rap value as a measure of uncertainty and instead of using an evolutionary model, it is based on re-sampling of the original data (Huelsenbeck et al 2002). In contrast, the Bayesian method uses a specific evolutionary model that is best fitted to the data to measure the uncertainty (Huelsenbeck et al 2002). Due to the differences in calculating uncertainties, it explains the discrepancies between these two analyses. Furthermore, parsimony (ME) and ML

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94 methods tend to provide a lower confidence level than t he Bayesian analysis, which was evident in our results (Huelsenbeck et al 2002). Both ML and Bayesian trees offered a different topology of the branching patterns for the Mycoplasma and Phytoplasma species. The branching patterns within each cluster vari ed by methods; however, the ML tree offered a topology that was more similar to the 16S rRNA tree generated by MolliGen. Both trees showed M. bovis PG45 clustering with M. agalactiae PG2 and M. alligatoris A21JP2 clustering with M. crocodyli MP145. In addition, both trees revealed that M. fermentans JER had a close evolutionary relationship with M. bovis PG45 and M. agalactiae PG2; all three were descended from a common ancestor. Each evolution analysis has its own pitfalls and can only offer an optimal tree conditional to the data and other parameters of that specific analysis. As a result, there was no definitive answer on the evolution of the MscL protein from the class of Mollicutes; therefore, more studies are needed to elucidate its phylogeny. This will be possible as additional species are sequenced. Selective P ressures on MscL in M. bovis and M. agalactiae I solates The ClustalW alignment revealed the conserved sites shared among the M ollicutes species. Conserved sites may be structurally important and are often the active sites for protein-protein interactions (Doron-Faigenboim et al 2005, Stern et al 2007). Conserved sites usually are considered the result of purifying selection, resulting from synonomous nucleic acid substitutions that do not al ter the amino acid. Sites that undergo nucleic acid substitutions resulting in a different amino acid are considered to be positive or diversifying selection, and are thought to provide a survival advantage to the microbe (DoronFaigenboim et al 2005, Hal l 2011, Stern et al 2007). Diversifying

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95 selection generally does not occur in active sites or structural components of a protein. Thus, we would predict that the MscL protein would have minimal diversifying selection and would maintain highly conserved residues. All the evidence showed that M. bovis PG45 and M. agalactiae PG2 shared a nearly identical MscL protein sequence. Therefore, we used sequences from M. bovis PG45, M. agalactiae PG2, and three additional strains of M. bovis for further analysis The ClustalW alignment of the amino acids from the four M. bovis isolates revealed that these were identical sequences; the nucleic acid sequences differed by only one nucleotide and this was a point mutation that resulted in a synonymous change. The amino ac id and nucleic acid sequences from M. a galactiae we re quite similar to M. bovis with two notable exceptions. The amino acids of the M. bovis and M. agalactiae isolates were examined to determine the evolutionary force at each amino acid site. In order to do so, the ratio of synonymous and non-synonymous ( ) was calculated for each amino acid site. This ratio is used as an indicator of selective pressure at the protein level (Yang et al 2000). The calculates the number of synonymous and non-synonymous substitutions per each amino acid site (Yang et al 2000). If >1, it is indicative of positive selection. In contrast, when < 1, it means purifying selection. However, when = 1, it is suggestive of a neutral mutation (Yang et al 2000). The web server S electon (http://selecton.tau.ac.il ). Selecton was used to determine the selective pressure. It employs an M8 model, which detects positive sites using likelihood-ratio test by comparing a null model that does not allow >1 to another model that does (Yang et al 2000, Yang et al 2005).

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96 The result revealed that most of the amino acid sites experienced some degree of purifying selection. However, there were two sites at positions 54 and 99 in the sequence that had undergone positive selection. This selection was only seen in M. agalactiae. The amino acid position 54 was a serine (S) in M bovis and a glutamine (Q) in M. agalactiae. The residue is found in the extracellular domain. Both S and Q are polar amino acids with uncharged side chains; however the serine residue can be glycosylated. The amino acid at position 99 was histidine (H) in M bovis and alanine (A) in M. agalactiae. Residue 99 is found in the cytoplasmic domain 2. This difference is significant in that H is hydrophilic and basic whereas alanine is hydrophobic and uncharged. The impact, if any, of these changes on the overall function of the MscL protein (Martinac et al. 2003) remains to be determined. It is interesting that the MscL protein had significant variation among species, but it appears that within a species the protein is highly conserved. Future D irections There are a number of questions that remain to be answered with respect to the mscL gene and its encoded protein. The role of MscL in the ability of M. bovis to persist in the environment is of interest as it may have direct relevance to disease transmission and potential therapeutic applications. Experiments could be designed to test the hypothesis that MscL increases the ability of M. bovis to survive osmotic shock and also to persist in the environment. A specific mechanosensitive channel inhibitor is available and was recently shown to inhibit the growt h of Phytoplasmas (Oshima et al. 2011) Therefore, it would be important to see if this is also true for M. bovis. If so, then the MscL protein could be a potential vaccine target. Finally, understanding the functional role, if any, of the CD and ED loops would be of interest.

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97 From an evolutionary standpoint, it would be important to do a more intensive survey of Mollicutes species, not just those with full genome sequences available. However, the challenge would be to develop a rapid screening system to identify species with the mscL gene and then to actually be able to find, amplify, and sequence the gene. A more easily addressed question would be to examine multiple strains of species known to have the mscL gene and determine if those pr oteins, like the MscL of M. bovis are highly conserved. Pathogens like M. gallisepticum and M. synoviae would be good candidates as there are large collections of clinical isolates available.

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106 BIOGRAPHICAL SKETCH Cheng-Fen Chow was born in Taiwan and moved to the United States when she was 13 years old. She graduated from University of Central Florida in 2005 with a Bachelor of Sciences in Micro Molecular Biology and from Washington State University in 2008 with a Bachelor of Sciences degree in Biology. She received her Masters of Science in Animal Molecular Cell Biology from the University of Florida in 2011.