University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations May 2016 Deciphering the Multi-tiered Regulatory Network That Links Cyclic-di-GMP Signaling to Virulence and Bacterial Behaviors Xiaochen Yuan University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Biology Commons, Microbiology Commons, and the Plant Pathology Commons Recommended Citation Yuan, Xiaochen, "Deciphering the Multi-tiered Regulatory Network That Links Cyclic-di-GMP Signaling to Virulence and Bacterial Behaviors" (2016). Theses and Dissertations. 1234. https://dc.uwm.edu/etd/1234 This Dissertation is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. DECIPHERING THE MULTI-TIERED REGULATORY NETWORK THAT LINKS CYCLIC-DI-GMP SIGNALING TO VIRULENCE AND BACTERIAL BEHAVIORS by Xiaochen Yuan A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Sciences at The University of Wisconsin–Milwaukee May 2016 ABSTRACT DECIPHERING THE MULTI-TIERED REGULATORY NETWORK THAT LINKS CYCLIC-DI-GMP SIGNALING TO VIRULENCE AND BACTERIAL BEHAVIORS by Xiaochen Yuan The University of Wisconsin-Milwaukee, 2016 Under the Supervision of Ching-Hong Yang, Ph.D. Bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that regulates multiple cellular behaviors in most major bacterial phyla. C-di-GMP signaling in bacterial often includes enzymes that are responsible for the synthesis and degradation of c-di-GMP, effector proteins or molecules that bind c-di-GMP, and targets that interact with effectors. However, little is known about the specificity of c-di-GMP signaling in controlling virulence and bacterial behaviors. In this work, we have investigated the c-di-GMP signaling network using the model plant pathogen Dickeya dadantii 3937. In Chapter 2, we characterized two PilZ domain proteins that regulate biofilm formation, swimming motility, Type III secretion system (T3SS) gene expression, and pectate lyase production in high c-di-GMP level conditions. YcgR3937 binds c-di-GMP both in vivo and in vitro. Next, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signaling, and flagellar master regulator FlhDC. We proposed FlhDC regulates T3SS through three distinct pathways, including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Genetic analysis showed that EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression. In chapter 3, we constructed a panel of single-deletion mutants, in which each GGDEF ii and/or EAL domain protein coding gene was individually either deleted or inactivated. Various cellular outputs were investigated using these mutants. We showed that GGDEF domain protein GcpA negatively regulates swimming motility, pectate lyase production, and T3SS gene expression. GcpD and GcpL only negatively regulate the expression of T3SS and swimming motility but not the pectate lyase production. iii TABLE OF CONTENTS Page Abstract……………………………………………………………………………… ii List of Figures………………………………………………………………………. v List of Tables………………………………………………………………………… vii List of Abbreviations………………………………………………………………... viii Acknowledgements…………………………………………………………………. ix CHAPTER 1. INTRODUCTION……………………………………………….. 1 1.1 Dickeya dadantii 3937……………………………………………………….... 2 1.1.1 Background and Significance of Dickeya dadantii 3937……………….. 2 1.1.2 Virulence mechanisms of D. dadantii 3937……………………………... 3 1.1.2.1 Type II secretion system…………………………………………... 4 1.1.2.2 Type III secretion system and its regulatory mechanism………… 6 1.1.2.3 Role and regulatory mechanism of chemotaxis and motility… 7 1.1.3 A bacterial second messenger c-di-GMP………………………………… 9 1.2 References……………………………………………………………….….…. 12 CHAPTER 2. CROSS-TALK BETWEEN A REGULATORY SMALL RNA, CYCLIC-DI-GMP SIGNALING, AND FLAGELLAR REGULATOR FLHDC FOR VIRULENCE AND BACTERIAL BEHAVORS………………………......…… 22 Abstract………………………………………………………………………….. 23 Introduction……………………………………………………………………… 23 Experimental procedures…………………………………………………………... 27 Results…………………………………………………………………………… 36 Discussion……………………………………..……………………………….. 46 References……………………………………………………………………….. 54 Tables and Figures…………………………………………………………………. 59 CHAPTER 3. STUDY OF GGDEF AND EAL DOMAIN PROTEINS IN C-DI-GMP SIGNALING IN D. dadantii 3937……………………………………………... 78 Abstract………………………………………………………………………….. 79 Introduction……………………………………………………………………… 79 Experimental procedures……………………………………………………….. 81 Results…………………………………………………………………………… 85 Discussion……………………………………………………………………….. 90 References……………………………………………………………………….. 94 Tables and Figures…………………………………………………………………. 97 CURRICULUM VITAE…………………………………………………………. 107 iv LIST OF FIGURES Figure Description Page CHAPTER 1 1 Regulation of pectate lyase production during D. dadantii 3937 pathogenesis... 18 2 Regulatory mechanism of T3SS in D. dadantii 3937.…………………….. 19 3 Flagellar transcriptional hierarchy in bacteria…………………………… 20 4 Modulation of c-di-GMP and its regulatory effects on diverse cellular behaviors.................................................................................................... 21 CHAPTER 2 5 Measurement of intracellular levels of c-di-GMP in wild-type Dickeya dadantii, ΔegcpB, ΔecpC, and ΔegcpBΔecpC ………...…………………. 64 6 Analysis of PilZ-domain proteins. (A) PilZ-domain proteins YcgR3937 and BcsA3937 in Dickeya dadantii 3937………………………………... 65 7 The impact of mutation of bcsA3937 and ycgR3937 on various virulence phenotypes were examined…………………………………………….. 66 8 The impact of mutation of bcsA3937 and ycgR3937 on hrpA promoter activity was examined…………………………………………………………… 67 9 Isothermal titration calorimetric analysis of c-di-GMP binding to wild-type YcgR3937........................................................................................... 68 10 Swimming motility was measured in D. dadantii…………………………….. 69 11 The impact of mutation of flhDC and fliA on the T3SS gene expression in D. dadantii 3937 was examined……………………………….... 70 12 FlhDC, independently of FliA, regulates the T3SS master regulator HrpL at transcriptional level through EcpC-RpoN-HrpL pathway in D. dadantii 3937…………………………………………………. 71 13 FlhDC and FliA inversely regulate RsmB at a post-transcriptional level………………………………………………………………………….. 72 14 Promoter activity of hrpA in different D. dadantii 3937 strains was examined………………………………………………………………... 73 15 FlhDC and FliA positively regulate the virulence of D. dadantii 3937 on Chinese cabbage (Brassica campestris)……………………………. 74 16 Measurement of D. dadantii virulence to African violet (Saintpaulia ionantha)…………………………………………………......... 75 17 Pectate lyase production and swimming motility were measured in D. dadantii 3937………………………………………………. 76 18 Model for the type III secretion system (T3SS) regulatory v network in D. dadantii……………………………………………………… 77 CHAPTER 3 19 GGDEF and/or EAL domain proteins in Dickeya dadantii 3937 ……......... 100 20 Analysis of of GGDEF and EAL domains in D. dadantii ………………… 101 21 Biofilm formation and swimming motility in wild-type D. dadantii, GGDEF and/or EAL single deletion mutants and complementation strains……………………………………………………. 102 22 TEM pictures of the wild-type D. dadantii and mutant strains...………….. 103 23 Swimming motility in D. dadantii 3937............................................... 104 24 Pectate lyase production, gene promoter activities and RsmA protein levels in D. dadantii3937…………………………………………………….... 105 25 T3SS gene hrpA (A and B) and hrpL (C) promoter activities in wild-type D. dadantii, GGDEF and/or EAL single deletion mutants and their complementation stains…………………………………. 106 vi LIST OF TABLES Table Description Page CHAPTER 2 1 Mean ± SEM (standard errors of the mean) of apparent FRET efficiency for wild-type and ΔegcpBΔecpC cells expressing the c-di-GMP sensor YFP-YcgR3937-CFP……………………… 59 2 Bacterial strains and plasmids……………………………………. 60 3 Primers used in this study………………..………………… 62 CHAPTER 3 4 Strains, plasmids, and primers used in this study ……… 97 vii LIST OF ABBREVIATIONS Ap Ampicillin C-di-GMP Cyclic diguanosine monophosphate CFP Cyan fluorescent protein Cm Chloramphenicol DGC Diguanylate cyclase Ecp EAL-domain containing protein FRET Förster resonance energy transfer Gcp GGDEF-domain containing protein GFP Green florescent protein Gm Gentamycin Hrp Hypersensitive response and pathogenicity IPTG Isopropyl-beta-D-thiogalactopyranoside ITC Isothermal tritration colorimetry Km Kanamycin LB Luria-Bertani media MM Minimal medium OD Optical density PCR Polymerase chain reaction PDE Phosphodiesterase Pel Pectate lyase Real-time RT-PCR Real-time reverse transcription polymerase chain reaction Sp Spectinomycine T2SS Type II secretion system T3SS Type III secretion system YFP Yellow florescent protein viii ACKNOWLEDEMENTS I would like to acknowledge the wonderful people who helped make this thesis a reality. Strat with special thanks to my advisor Dr. Ching-Hong Yang for giving me the opportunity to work in his lab. Without this support, advice, and encouragement, this thesis would not be possible. I would also like to thank the scientific training that he offered during my Ph.D. program, letting me know that what to be expected from a true scientist. I would like to thank my committee members Dr.