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Sortase Enzymes and Their Integral Role in the Development of Streptomyces Coelicolor

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Sortase Enzymes and Their Integral Role in the Development of Streptomyces Coelicolor Sortase enzymes and their integral role in the development of Streptomyces coelicolor Sortase enzymes and their integral role in the development of Streptomyces coelicolor Andrew Duong A Thesis Submitted to the School of Graduate Studies In Partial Fulfillment of the Requirements of the Degree of Master of Science McMaster University Copyright by Andrew Duong, December, 2014 Master of Science (2014) McMaster University (Biology) Hamilton, Ontario TITLE: Sortase enzymes and their integral role in the development of Streptomyces coelicolor AUTHOR: Andrew Duong, B.Sc. (H) (McMaster University) SUPERVISOR: Dr. Marie A. Elliot NUMBER OF PAGES: VII, 77 Abstract Sortase enzymes are cell wall-associated transpeptidases that facilitate the attachment of proteins to the peptidoglycan. Exclusive to Gram positive bacteria, sortase enzymes contribute to many processes, including virulence and pilus attachment, but their role in Streptomyces coelicolor biology remained elusive. Previous work suggested that the sortases anchored a subset of a group of hydrophobic proteins known as the long chaplins. The chaplins are important in aerial hyphae development, where they are secreted from the cells and coat the emerging aerial hyphae to reduce the surface tension at the air-aqueous interface. Two sortases (SrtE1 and SrtE2) were predicted to anchor these long chaplins to the cell wall of S. coelicolor. Deletion of both sortases or long chaplins revealed that although the long chaplins were dispensable for wild type-like aerial hyphae formation, the sortase mutant had a severe defect in growth. These two sortases were found to be nearly redundant, as deletion of individual enzymes led to only a modest change in phenotype. In vitro analysis of sortase cleavage activity showed that both sortases recognized the unique LAXTG pentapeptide sequence found in the long chaplins, and 11 other putative substrate proteins. Transcriptional analysis revealed that a number of genes typically expressed during aerial hyphae development were not expressed in a sortase deletion mutant. This suggests that the sortases have a role in transcriptional regulation, a phenomenon that has not been described previously. Current work is focused on addressing the mechanism(s) by which sortases affect transcriptional regulation, with a specific focus on the role of the proteins that they anchor to the cell wall (sortase substrates) in aerial growth. I Acknowledgements First, and foremost, I’d like to thank Dr. Marie A. Elliot for all of her efforts in supporting my academic career. She’s been a supportive and kind mentor that has encouraged me to perform my best and is someone who I greatly admire for her thirst for knowledge and compassion for others. I’d like to thank my committee members, Dr. Turlough Finan and Dr. Lori Burrows for their honest and helpful feedback for these past few years. The input that I’ve received has helped both my research and my evolution as a critical person. I’d also like to thank Dr. Alba Guarné for introducing me to the bench, which sparked my interests in primary research. I would also like to extend special thanks for Dr. Juliet Daniel for her continuous support and guidance. These professors have taught me that mentorship is too often an ignored, but invaluable component of life; of which, the successes of millions are dependent. I have been lucky to work with some fantastic people during my time in the lab. All have had an impact, but in particular, I’d like to thank: Dr. David Capstick for his friendship and support, while being an encyclopedia of science and pop culture; Dr. Chan Gao for being that weird, cat-loving, big sister I never had; Dr. Henry Haiser for the intellectual conversations about science in pubs; Dr. Julia Swiercz for being my annoying ‘lab little sister’ with a huge heart; Dr. Hindra and Dr. Mary Yousef for being two of the nicest people I have ever worked with, and for teaching me the value of family; Dr. Emma Sherwood for her optimism and wonderful lab quirks that I never get tired of discovering; Matthew Moody for never being afraid to share his weirdness and whose integrity I greatly admire; Danielle Sexton for being one of the most caring people I’ve ever met, and despite that, also being my lab twin (CATCATCAT); Rachel Young for being so kind, brilliant, selfless, and badass at the same time. Beyond school, I’d like to thank my family, and especially my sister Anita, for reminding me of life beyond work. I also want to thank the people who have become like family over these past few years: Catherine Nguyen for being so wonderful, uncool, and generous with her kindness. Her friendship is one that I will always cherish for the impact she has had on me; Nolan D’Souza for being a confidant, a brother, and whose tenacity is something of legends; and to Sanjan George for reminding me to remember the important things in life. I’d also like to extend my gratitude towards the Kwan family for their kindness these last few years. Special thanks goes to Michelle J. Kwan, who has contributed so much of the positivity in my life, and without whom, I would not have made it to this point. Her patience, compassion, and encouragement have done so much in providing everlasting support through this journey. II Table of Contents Abstract ............................................................................................................................ I Acknowledgements ........................................................................................................ II List of Figures ................................................................................................................. V List of Tables .................................................................................................................. VI List of Abbreviations .................................................................................................... VII Chapter 1: Introduction .................................................................................................. 1 1.1 The Actinomycetes ............................................................................................ 1 1.2 Streptomyces coelicolor ................................................................................... 1 1.3 The developmental life cycle of S. coelicolor ................................................. 2 1.4 Regulation of development ............................................................................... 2 1.4.1 Vegetative growth .......................................................................................... 3 1.4.2 Aerial hyphae formation ................................................................................. 3 1.4.3 Sporulation ..................................................................................................... 3 1.5 Surfactants in development .............................................................................. 4 1.5.1 SapB .............................................................................................................. 4 1.5.2 Chaplins ......................................................................................................... 5 1.6 Sortases .............................................................................................................. 6 1.6.1 Sortase A ....................................................................................................... 7 1.6.2 Sortase B ....................................................................................................... 7 1.6.3 Sortase C ....................................................................................................... 8 1.6.4 Sortase D ....................................................................................................... 9 1.6.5 Sortases in the actinomycetes: SrtE and SrtF ............................................... 9 1.7 Sortases and the chaplins ................................................................................ 9 1.8 Aims of this thesis ........................................................................................... 10 1.9 Figures ............................................................................................................ 11 Chapter 2: Materials and methods .............................................................................. 14 2.1 Bacterial strains and plasmids .......................................................................... 14 2.2 Bacterial culturing ............................................................................................. 14 2.3 Bacterial and molecular genetic techniques .................................................. 15 2.3.1 Plasmid isolation from Escherichia coli ....................................................... 15 2.3.2 Agarose gel electrophoresis ........................................................................ 15 2.3.3 PCR amplification ......................................................................................... 15 2.3.4 Purification of PCR products ......................................................................... 16 2.3.5 Purification of DNA fragments from agarose gels ......................................... 16 2.3.6 Restriction digest of DNA .............................................................................. 16 2.3.7 Phosphorylation and dephosphorylation of DNA .......................................... 16 2.3.8 DNA ligation .................................................................................................
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