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Investigating Amrz-Mediated Activation of Pseudomonas Aeruginosa Twitching

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Investigating AmrZ-mediated activation of Pseudomonas aeruginosa twitching motility and alginate production DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Binjie Xu Graduate Program in Microbiology The Ohio State University 2015 Dissertation Committee: Daniel J. Wozniak, Ph.D., Advisor Irina Artsimovitch, Ph.D. Robert S. Munson, Jr., Ph.D. John S. Gunn, Ph.D. Copyright by Binjie Xu 2015 Abstract The Gram negative bacterium Pseudomonas aeruginosa is ubiquitous in the natural environment and responsible for various human infections. Successful P. aeruginosa infections require many virulence factors such as flagella, type IV pilus and alginate, whose expression is under tight control by transcription factors such as AmrZ. AmrZ activates type IV pilus-mediated twitching motility and alginate production while repressing the flagella machinery. However, molecular mechanisms of AmrZ-mediated activation of twitching motility and the alginate biosynthesis operon promoter (algD promoter) remain elusive and are the emphases of this study. Specifically, DNA microarray analyses were performed in order to identify AmrZ- dependent gene(s) that is required for twitching motility. The AmrZ regulon contains 112 genes, many of which are involved in virulence. However, this regulon does not contain a gene that is known to be necessary for twitching motility. In addition, we used multiple biochemical methods to show that AmrZ tetramerizes via its C-terminal domain, the loss of which results in diminished DNA binding and reduced efficiency during AmrZ- ii mediated activation and repression. Demonstration of AmrZ tetramerization also assists in understanding the activation mechanism of the algD promoter. This complex promoter contains a number of transcription factor binding sites with many in the far upstream region. It is unclear how those transcription factors from far upstream contribute to transcription activation. This study identified four AmrZ binding sites within this promoter and showed that each is required for algD transcription activation. We also illustrated phase-dependent activation of this promoter, suggesting interactions between upstream and downstream factors. Furthermore, in vitro fluorescence resonance energy transfer experiments indicate the formation of trans DNA-AmrZ complexes, suggesting that two DNA molecules are held together by AmrZ, likely via AmrZ oligomerization. These results thus provide new support for the DNA looping model of the algD promoter. Taken together, the work described in this document has significantly enhanced our knowledge of AmrZ-mediated regulation, full understanding of which may provide insights into discovering new means of interfering this regulation and successful control of P. aeruginosa infections. iii Dedication This document is dedicated to my family and friends for their love, care and support iv Acknowledgments First and foremost, I would like to express my greatest gratitude to my Ph.D. advisor Dr. Daniel J. Wozniak, who has been a phenomenal scientific mentor inside the lab as well as a precious friend off work. Besides Dr. Wozniak, all current and previous “Woz Lab” members have fostered my growth and maturity as a scientist and as a person. They have offered substantial assistance to promote my professional development. I would like to specially thank Mohini Bhattacharya, Sheri Dellos-Nolan, Dr. Dominique H. Limoli and Preston J. Hill for helping with numerous presentation practices and paper editing. I was also lucky to work with the undergraduate student Amyla Tan and the rotation student Qian Shen. Both of them are smart, self-motivated and hard-working, and should take the credit of multiple experiments. I would also like to appreciate the guidance of my dissertation committee throughout graduate school. Bob, Irina and John have offered numerous insightful advices, all of which are instrumental during the completion of my dissertation projects. I would like to thank Dr. Jesse Kwiek for his helpful career advices. All members in the Center for v Microbial Interface Biology have created a unique work environment that is packed with expertise and fun. Many experiments were conducted in collaboration with scientists from other disciplines. We thank Dr. Richard Fishel and Randal Soukup for gel filtration and FRET experiments, and appreciate the collaboration of Dr. Vicki H. Wysocki and Yue Ju for mass spectrometry experiments. Laboratories of Dr. Fishel and Dr. Wysocki are both located at OSU. Whole genome sequencing was conducted and analyzed by Benjamin Kelly from the Peter White laboratory at Center for Microbial Pathogenesis in Nationwide Children’s Hospital, Columbus, OH. We want to thank Genomics Shared Resource of OSU Comprehensive Cancer Center for performing DNA microarray experiments. We would also like to thank Dr. Thomas Hollis (Wake Forest School of Medicine, Winston-Salem, NC) for sharing recombinant AmrZ and its variants, and express our gratitude to Dr. Laura Jennings in the Matthew Parsek laboratory (University of Washington, Seattle, WA) for measuring c-di- GMP concentrations for us. We also thank Dr. Matthew Wolfgang (University of North Carolina, Chapel Hill, NC), Drs. Reuben Ramphal/Jeevan Jyot (University of Florida, Gainesville), and Dr. Joe J. Harrison (University of Calgary, Calgary, Alberta, Canada) for sharing strains and plasmids. We acknowledge Dr. Gayan Senavirathne for valuable scientific discussions, and Dr. Nrusingh Mohapatra for assistance with the β-galactosidase assay. vi Vita July 2005 ........................................................Leping No.1 High School, Jiangxi, China 2009................................................................B.S. Life Sciences, University of Science and Technology of China, Hefei, China 2009 to present ..............................................Graduate Assistant, Department of Microbiology, The Ohio State University, Columbus, OH, USA Publications 1. Jones, C. J., Newsom, D., Kelly, B., Irie, Y., Jennings, L. K., Xu, B., … Wozniak, D. J. (2014). ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di- GMP synthesis and biofilm development by Pseudomonas aeruginosa. PLoS Pathogens, 10(3), e1003984. http://doi.org/10.1371/journal.ppat.1003984 vii 2. Pryor, E. E., Waligora, E. A., Xu, B., Dellos-Nolan, S., Wozniak, D. J., & Hollis, T. (2012). The transcription factor AmrZ utilizes multiple DNA binding modes to recognize activator and repressor sequences of Pseudomonas aeruginosa virulence genes. PLoS Pathogens, 8(4), e1002648. http://doi.org/10.1371/journal.ppat.1002648 3. Xu, B., Ju, Y., Soukup, R. J., Ramsey, D. M., Fishel, R., Wysocki, V. H., & Wozniak, D. J. (2015). The Pseudomonas aeruginosa AmrZ C-terminal domain mediates tetramerization and is required for its activator and repressor functions. Environmental Microbiology Reports. http://doi.org/10.1111/1758-2229.12354 4. Xu, B., & Wozniak, D. J. (2015). Development of a novel method for analyzing Pseudomonas aeruginosa twitching motility and its application to define the AmrZ regulon. PLoS One, 10(8). http://doi.org/10.1371/journal.pone.0136426 Fields of Study Major Field: Microbiology viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1. Introduction ....................................................................................................... 1 Pseudomonads and Pseudomonas ................................................................................... 1 Pseudomonas aeruginosa and its clinical significance ................................................... 3 Pseudomonas aeruginosa pathogenesis in acute and chronic infections ........................ 5 Virulence factors of Pseudomonas aeruginosa ............................................................... 9 Alginate production and its regulation ........................................................................ 9 ix Flagella and FleQ....................................................................................................... 11 Type IV pilus and twitching motility ........................................................................ 13 Biofilm and its significance ....................................................................................... 16 Control by transcription factors ..................................................................................... 19 The versatile transcription factor AmrZ ........................................................................ 23 Key methods used in this study ....................................................................................
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