MOLECULAR BIOLOGY AND BIOCHEMISTRY OF THE REGULATION OF HRP/TYPE III SECRETION GENES IN THE CORN PATHOGEN PANTOEA STEWARTII SUBSP. STEWARTII DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Massimo Merighi, B. S., M. S. ***** The Ohio State University 2003 Dissertation Committee: Professor David L. Coplin, Adviser Approved by Professor Brian Ahmer ______________________ Professor Dietz W. Bauer Adviser Professor Terrence L. Graham Plant Pathology Copyright by Massimo Merighi 2003 ii ABSTRACT Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the expression of a Hrp/type III protein secretion/translocation system. The regulatory region of the hrp gene cluster consists of three adjacent operons: hrpXY encodes a two- component regulatory system, consisting of the response regulator HrpY and sensor PAS-kinase HrpX; hrpS encodes an NtrC-like enhancer-binding protein; and hrpL encodes an ECF sigma factor. In this study, we used genetic and biochemical approaches to delineate the following regulatory cascade: 1) HrpY activates hrpS; 2) HrpS activates hrpL; and 3) HrpL activates secretion and effector genes with ‘Hrp-box’ promoters. This pathway responds to environmental signals and global regulators. Mutant analysis showed that HrpX is required for full virulence. Deletion of its individual PAS-sensory domains revealed that they are not redundant and each may have a different role in modulating kinase activity. HrpX probably senses an intracellular signal, possibly related to nitrogen metabolism. pH, osmolarity and nicotinic acid controlled expression of hrpS independently of HrpX/HrpY. hrpY mutants were completely avirulent. Site-directed mutagenesis of the conserved D57 residue in the HrpY receiver domain showed that it is ii required for virulence and activation of hrpS and hrpXY in vivo. In vitro studies using purified His6 -HrpY indicated that D57 is the only phosphorylation site and that His6 -HrpY~P has increased affinity for PhrpS. Primer extension and deletion analyses located the PhrpS upstream of an IS-like element and showed that transcription of hrpL from a σ 54 promoter requires an upstream activating sequence. We also discovered that the absence of transcriptional terminators downstream of hrpL and hrpY creates a novel autoregulatory loop, wherein activation of hrpL by HrpS results in read-through transcription into hrpY and hrpS. Finally, the EsaR/EsaI quorum sensing system was shown to modulate the expression of hrp genes and virulence. Expression of hrpS was reduced in an esaI background and restored in an esaIR strain. iii “...Fatti non foste per viver come bruti, ma per seguir virtute e canoscenza’.” (“…(Men) were not created to live like wild animals, but to pursue virtue and knowledge.”) (Dante’s Inferno, Canto XXVI, 119-120) Dedicated to my parents, Graziella and Camillo iv ACKNOWLEDGMENTS First of all, I would like to thank my family, dad, mom, and sister, for their unconditional support and love throughout all these years so far away from my motherland Italy. I know how much sacrifice that has meant for you and I hope I made you proud in the end. Secondly, thanks to my lovely wife, Serena. Simply put, nothing of this could have ever happened without you. I will never forget your patience and encouragement during these last six years we have been living together here in America. Serena also helped me with many of the figures in Chapter 1. Obviously, I want to thank Dr. David Coplin, my adviser, for his mentorship, and the intellectual freedom and support I have enjoyed in the years spent in his lab. There I fully discovered my vocation as a molecular bacteriologist and I found the courage to explore many new areas of research. Secondly, I also have to thank him for the extensive editing of this dissertation. I also want to express my gratitude to the members of my SAC, Dr. Brian Ahmer, Dr. Dietz W. Bauer, and Dr. Terrence L. Graham, for many stimulating discussions and for their unconditional availability whenever I bugged them for using equipment or v discussing ideas. In particular, a special thanks goes to Dr. Bauer, for teaching me many things about academic life and beyond, and for trying, unsuccessfully, to make a decent pool player out of me (Sorry Dietz!). A special acknowledgement goes to current members of the lab, Doris R. Majerczak and Dr. Jong Hyun Ham. Doris has been a good friend and a wonderful co-worker, always available and extremely helpful. Many of the experiments in this dissertation are a result of the strict cooperation between the two of us. I will always carry with me a good memory of the time spent in the lab together. Jong Hyun has been a good friend with whom I shared many nights in the lab and many discussions about science and our futures. A particular thanks goes to Dr. Max Teplitski, who has been a dear friend of my wife and me. He has been very close to us both in times of bad and good luck and we will never forget that. He also provided me with samples of purified Salmonella enterica BarA198 before publication for the experiments described in Chapter 4. Many other friends have helped me to endure the hardships of the Graduate School, some by discussing science with me, others by giving me different perspectives on the facts of life, some others just by being there when I needed it the most. My dear friends, Maria, Mensheng, Ricardo, and Dr. Trudy Torto, I will never forget you all and I hope the Fate will bring great happiness to your lives. vi I thank also the Ohio Agricultural Research Development Center for supporting me with a Director’s Fellowship for the first three years, the Graduate School of the Ohio State University for granting me a Presidential Fellowship and the Department of Plant Pathology for its support over one year and a half of my Ph.D. program. Some of the work presented in this dissertation was completed with the support of many people at various levels. In particular, most of the data in Chapter 2 was published in a peer-reviewed journal before the defense of this dissertation (Merighi et al., 2003, Mol. Plant Microbe Interact 16: 238-248). For that part, I acknowledge practical contributions from E. H. Stover (limited to the construction of strains DM701, DM729 and DM733 carrying the plasmid pRF205), who also provided the initial observations on the linear model of regulation and on the hrpS autoregulation several years before I came in the lab; N. Smith, for the data in Fig. 2.7; D. R. Majerczak, for her help in performing replicates of enzyme assays, for her independent construction of strains DM786 and plasmids pDM1296, pDM2560 and for her practical support in various steps leading to the construction of several plasmids and strains used in that work. I also acknowledge intellectual contributions by D. L. Coplin in the Discussion section of Chapter 2. Furthermore I have to acknowledge also the contribution by D. R. Majerczak of the two Ω interposon mutants used in Chapter 3, which Doris constructed under my supervision, and her help in running replicates of the enzyme assays described in Chapters 2 and 6. I vii finally thank Dr. Jyang Chuyn Jang for his friendship, mentorship and for having me in his lab as radiation user for all the experiments in Chapters 3, 4 and 5 involving radioactive isotopes. viii VITA 1971 Born, Italy, European Union 1990-1996: B.S. (“Laurea”), Department of Plant Pathology, University of Bologna, Italy, Graduated 110/110, Summa cum Laude. Experimental thesis: “Genetic fingerprinting by PCR and PFGE of Erwinia amylovora strains from the Mediterranean basin”. Field of Study: Plant Pathology, Molecular Phytobacteriology. Summer 1996: Research Intern, Max Plank Institute für Zellbiologie, Klaus Geider’s Bacterial Molecular Genetics Laboratory, Heidelberg, Germany. 1997-1998: Research Assistant (MIRAAF/National Plan of Plant Biotechnology), Institute of Plant Pathology, University of Bologna, Italy. 2001: M.S.(non-thesis), The Ohio State University 1998-present: Graduate Student, Graduate School of The Ohio State University, Department of Plant Pathology and Plant Molecular Biology/Biotechnology Program: -O.A.R.D.C. Graduate Fellow (1998-2001); ix -Graduate Research Associate (2001-2002 and 2003- present); -Presidential Fellow of the Ohio State University (2002-2003) PUBLICATIONS Refereed publications 1. Teplitski, M., Chen, H., Rajamani, S., Gao, M., Merighi, M., Sayre, R. T., Robinson, J. B., Rolfe, B. G., Bauer, W. D. (2004). Chlamydomonas reinhardtii secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria. Plant Physiol. 134:1-10. 2. Merighi, M., Majerczak, D. R., Stover, H. E. and Coplin, D. L. (2003). The HrpX/HrpY two-component system activates hrpS expression, the first step in the regulatory cascade controlling the Hrp regulon in Pantoea stewartii subsp. stewartii. Mol. Plant-Microbe Interact. 16:238- 248. 3. Merighi, M., Sandrini, A., Landini, S., Girotti, S., Ghini, S., Malaguti, S., Bazzi, C. (2000). Chemiluminescent and colorimetric x detection of Erwinia amylovora by immunoenzymatic determination of PCR amplicons from plasmid pEA29. Plant Dis. 84:49-54. 4. Zhang, Y., Merighi, M., Bazzi, C., K. Geider (1998). Genomic analysis by pulsed-field gel electrophoresis of Erwinia amylovora strains from the Mediterranean region including Italy. J. Plant Path. 80:225-232. Book Chapters 1. Merighi, M., Majerczak, D. R., and Coplin, D. L. “The hrp genes of Pantoea stewartii are regulated by a complex system that senses environmental signals”. In: “Plant Pathogenic Bacteria” p. 201-204, S. H. De Boer Editor, Kluwer Academic Publishers, The Netherlands, 2001. 2. Merighi, M., Sandrini, A., Landini, S., Malaguti, S., Porrini, C., Sabatini, A.G., Girotti, S., Ghini, S., and Bazzi, C.