CHARACTERIZATION OF STRINGENT RESPONSE REGULATORS (p)ppGpp, AND DksA IN Xanthomonas citri subsp. citri By YANAN ZHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Yanan Zhang To my family ACKNOWLEDGMENTS First, I would like to express my thanks to my supervisor Dr. Nian Wang, a Professor in Microbiology and Cell Science Department of University of Florida. Four years ago, he accepted me as a PhD student in his lab and opened the gate for me to science research. His diligence and enthusiasm for science is very impressive. He always encourages me to be familiar with the literature, tackle challenging questions, think deeper, try new methods and learn how to grow into an independent scientist. Besides, I really appreciate that he gives me so much freedom so that I can carry out my research more independently following my interest. In addition, I want to give my thanks to my committee members: Drs. Tony Romeo, Julie Maupin-Furlow, Jeffrey Jones, and Frank White. Their guidance, suggestions, scientific thought as well as encouraging words help me to be a better researcher. My thanks also go to the former and current lab members in Dr. Nian Wang’s lab for creating an accommodating and collaborative research environment and offering so much support in many aspects. I would like to list all the lab mates who help me with my research as follows: Xiaofeng Zhou, Nadia Riera Faraone, Maxuel Andrade, Yunzeng Zhang, Shuming Wang, Jin Xu, Doron Teper, Sheo Shankar Pandey, Donghuan Lee, Shuo Duan, Camila Ribeiro, Yuanchun Wang, Yixiao Huang, Erica Carter, Jinyun Li, Hongge Jia, Fernanda Nogales da Costa Vasconcelos, Xiaoen Huang, Tirtha Lamichhane, Ali Parsaeimehr, Zhiqian Pang, Hang Su, and Wenxiu Ma. I cannot say enough thanks to Dr. Doron Teper who taught me how to do some basic experiments one on one when I entered to the lab. I am so glad that he became one of my best friends now. Dr. Monika Oli from Microbiology and Cell Science Department also gave me much confidence to overcome my language barrier. 4 Most importantly, I owe a lot to my family. All of this would be impossible without their unconditional love. I want to say thanks to my father, my mother, my sister, my brother in law and my lovely nephew. At last, I want to say that all my effort is worthwhile and the last four years are memorable! I believe that I have built up some skills required for research and enjoy the pleasure that research brings to me although it does not always go smoothly. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 15 Citrus Canker .......................................................................................................... 15 Secretion Systems of Xanthomonas ....................................................................... 17 Type 2 Secretion System ................................................................................. 17 Type 3 Secretion System ................................................................................. 18 Type 4 Secretion System ................................................................................. 19 Other Virulence Traits of Xanthomonas .................................................................. 20 Extracellular Polysaccharides ........................................................................... 20 Lipopolysaccharides ......................................................................................... 21 Type IV Pilus .................................................................................................... 21 Flagellum .......................................................................................................... 22 Stringent Response Regulatory System ................................................................. 22 (p)ppGpp Synthesis and Its Regulation ............................................................ 23 Transcriptional Regulation by (p)ppGpp and DksA in E. coli ............................ 25 Transcriptional Regulation by (p)ppGpp in B.subtilis ........................................ 27 Stringent Response Regulators Conjure Bacterial Virulence ........................... 27 Hypothesis and Rationale ....................................................................................... 29 2 TRANSCRIPTOME ANALYSES OF ∆dksA, ∆spoT∆relA AND Xcc306 .................. 33 Introduction ............................................................................................................. 33 Results .................................................................................................................... 36 Revision of the Annotation of dksA ................................................................... 36 Sequence Analysis of dksA, relA and spoT ...................................................... 37 RNA-seq and Functional Enrichment Analysis ................................................. 38 DksA and (p)ppGpp Repress tRNA and Ribosome Protein Biosynthesis and Activate Histidine Metabolism ....................................................................... 39 Discussion and Conclusion ..................................................................................... 40 Materials and Methods............................................................................................ 42 Bacterial Strain, Growth Conditions .................................................................. 42 Generation of Mutant Strains and Complemented Strains ............................... 42 6 Site-directed Mutagenesis and Western Blot .................................................... 43 5’ RACE ............................................................................................................ 44 RNA Extraction, Sequencing and Data Analysis .............................................. 44 3 EXPERIMENTAL ASSAYS OF VIRULENCE-ASSOCIATED TRAITS REGULATED BY (p)ppGpp AND DksA .................................................................. 64 Introduction ............................................................................................................. 64 Results .................................................................................................................... 66 DksA and SpoT Are Required for Full Virulence .............................................. 66 (p)ppGpp and DksA Positively Regulate T3SS and T2SS................................ 67 (p)ppGpp and DksA Repress the Expression of Motility-related Genes ........... 69 (p)ppGpp and DksA Positively Regulate TonB-dependent Transporter Genes ........................................................................................................... 70 Differential Regulation of Xss Gene Cluster by (p)ppGpp and DksA ................ 71 Discussion and Conclusion ..................................................................................... 72 Materials and Methods............................................................................................ 75 Pathogenicity and HR Test ............................................................................... 75 Bacterial Growth Assay .................................................................................... 76 Extracellular Enzyme Activity Assay ................................................................. 76 RNA Extraction and RT-qPCR ......................................................................... 77 Motility Assay ................................................................................................... 78 GUS Activity Assay .......................................................................................... 78 Siderophore Production Assay ......................................................................... 79 Strains, Plasmids and Primers ......................................................................... 79 4 CONCLUDING REMARKS AND FUTURE DIRECTIONS .................................... 103 LIST OF REFERENCES ............................................................................................. 106 BIOGRAPHICAL SKETCH .......................................................................................... 120 7 LIST OF TABLES Table page 2-1 Data quality summary of RNA-seq ..................................................................... 58 2-2 Functional enrichment analysis based on the COG database ............................ 59 2-3 Gene expression of tRNA-coding genes ............................................................ 60 2-4 Gene expression of ribosome protein genes ...................................................... 62 3-1 Gene expression profile of T3SS- and T2SS-related genes in Xcc .................... 92 3-2 Downregulated T3SS-related genes