Regulation of coronafacoyl phytotoxin production in the potato common scab pathogen Streptomyces scabies by © Zhenlong Cheng A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland December 2018 St. John’s Newfoundland and Labrador I Abstract The genus Streptomyces consists of hundreds of species of Gram-positive, filamentous bacteria, which have a complex developmental life cycle. Many specialized metabolites of clinical, agricultural and biotechnological value are produced by Streptomyces. Very few Streptomyces species have been shown to be pathogenic to plants. The best characterized pathogenic species is Streptomyces scabies, which is the main causative agent of potato common scab (CS) disease. CS is characterized by the formation of lesions on the potato tuber surface, which negatively impact the market value of the affected potatoes, leading to significant losses for growers. S. scabies and other CS-causing pathogens produce thaxtomin A, the key virulence factor involved in CS disease. S. scabies also produces N-coronafacoyl-L-isoleucine (CFA- L-Ile), which is a member of the coronafacoyl family of phytotoxins. The coronafacoyl phytotoxin biosynthetic gene cluster in S. scabies consists of 15 genes, of which 13 are enzyme-encoding genes and are co-transcribed. The remaining two genes are divergently co-transcribed from the biosynthetic genes. The first gene, scab79591/cfaR, encodes a PAS-LuxR family regulator that activates the transcription of the enzyme-coding genes. The second gene, scab79581/orf1, encodes a ThiF family protein of unknown function. This thesis examines the regulation of CFA-L-Ile biosynthesis in S. scabies and the role of the CfaR and ORF1 proteins. Our results show that CfaR is the principle regulator controlling expression of the coronafacoyl phytotoxin biosynthetic genes and CFA-L-Ile production, while ORF1 augments phytotoxin production in a CfaR dependent manner and may function as a “helper” of CfaR. Bioinformatics analysis suggests that ORF1 may II catalyze AMPylation of an unknown target molecule. In addition, this thesis addresses the effects of the plant-derived molecules cellobiose and suberin on CFA-L-Ile production as well as the role of CFA-L-Ile in controlling its own production. While cellobiose and suberin both induce thaxtomin A production, our results suggest that these molecules inhibit CFA-L-Ile production in S. scabies. In vitro binding assays showed that CFA-L-Ile inhibits the binding of CfaR to its target site in the coronafacoyl phytotoxin biosynthetic gene cluster. This suggests that CFA-L-Ile production is subjected to negative feedback regulation in S. scabies. III Acknowledgements First of all, I would like to thank my PhD supervisor, Dr. Dawn Bignell, for her unconditionally full support for my project. I wish to thank Dr. Kapil Tahlan for his kind help, especially for his unique and creative way of problem solving. It works, all the time. Thanks to Dr. Andrew Lang. You are always there whenever I need you. I thank my lab mates: Dr. Joanna Fyans (周安娜) for being kind and helpful and being a good friend. Luke Bown for doing HPLC analysis and keeping lab tidy. Phoebe Li and Jingyu Liu, your presence makes the lab vivid. Keep doing it and good luck! Brandon Piercey for the help with his expertise in bioinformatics. Nader F. AbuSara. We always have nice conversations. Dr. Santosh Srivastava. You are inspiring as a lab member and a future business partner. I thank Dr. Carole Beaulieu from Université de Sherbrooke for providing suberin. … Plus so many with whom I had the honor to work. IV Thanks to the School of Graduate Studies and the Department of Biology at Memorial University of Newfoundland for providing me the opportunity to pursue my research caeer. Special thanks to my wife, Maggie. You mean more than the rest of the world to me. I would like to take this opportunity to thank two little ones, LB (“Panda”) Cheng and Vega Liu. You keep me from depression. Lil’ AI, thanks for all the trails we hiked, which kept both of us fit. Last but not least, thank you, Barbara Yung! V Table of Contents Abstract…………………………………………………………………………………...II Acknowledgements………………………………………………………………………IV Table of Contents………………………………………………………………………...VI List of Figures………………………………………………………………………….XIV List of Tables…………………………………………………………………………XVIII List of Abbreviations and Symbols…………………………………………………….XIX List of Appendices……………………………………………………………………XXIII Chapter 1: Introduction and Overview…………………………………………………….1 1.1 General Features of Streptomyces……………………………………………………..1 1.2 Specialized Metabolism in Streptomyces Species……………………………………..3 1.3 Regulation of Specialized Metabolism in Streptomyces…………………….…………5 1.3.1 CSRs of specialized metabolism……………………………………..............5 1.3.1.1 ActII-ORF4: the CSR for actinorhodin production……………………...…6 1.3.1.2 A cascade of CSRs controlling tylosin production…………………………7 1.3.1.3 RedZ: a multi-functional CSR……………………………………………..9 1.3.2 Pleiotropic regulators of specialized metabolism…………………………..10 1.3.2.1 Two component systems (TCSs)…………………………………………10 VI 1.3.2.1.1 The AfsQ1/Q2 regulatory system………………………………………11 1.3.2.1.2 The AfsK-AfsR-AfsS regulatory System……………………………...12 1.3.2.1.3 The PhoR-PhoP system sensing phosphate starvation………………….12 1.3.2.2 Global regulators of specialized metabolism and morphological differentiation: bld genes……...………………………………………………….13 1.3.3 Regulation of specialized metabolism by small molecules…………………15 1.3.3.1 GBL regulatory cascades…………………………………………………15 1.3.3.2 PI factor…………………………………………………………………..17 1.3.3.3 Crosstalk and feedback control of specialized metabolite biosynthesis…..18 1.3.4 The stringent response to nitrogen, phosphate and carbon limitation……….19 1.3.5 Carbon catabolite repression of specialized metabolism in Streptomyces….20 1.4 Plant Pathogenicity in the Genus Streptomyces………………………………………21 1.5 Known or Predicted Virulence Factors Produced by S. scabies………………………23 1.5.1 Thaxtomins…………………………………………………………………23 1.5.2 Nec1………………………………………………………………………..26 1.5.3 Coronafacoyl phytotoxins………………………………………………….26 1.5.3.1 Coronafacoyl phytotoxin biosynthesis in S. scabies……………………...27 1.5.3.2 Regulation of coronafacoyl phytotoxin production in S. scabies…………28 VII 1.5.3.3 Role of coronafacoyl phytotoxins in S. scabies pathogenicity……………30 1.6 Thesis Objectives and Outline………………………………………………………..31 1.7 References……………………………………………………………………………34 1.8 Figures and Tables……………………………………………………………………67 Co-Authorship Statement………………………………………………………………...76 Chapter 2: Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies ……………………78 2.1 Abstract………………………………………………………………………………78 2.2 Introduction…………………………………………………………………………..79 2.3 Materials and Methods……………………………………………………………….82 2.3.1 Bacterial strains, cultivation and maintenance……………………………...82 2.3.2 Plasmids, primers and DNA manipulation………………………………….83 2.3.3 Construction of protein expression plasmids………...…………………….84 2.3.4 Protein overexpression and purification……………………………………84 2.3.5 Total RNA isolation………………………………………………………...85 2.3.6 Reverse transcription PCR………………………………………………….86 2.3.7 Primer extension analysis…………………………………………………..86 2.3.8 Electrophoretic mobility shift assay (EMSA)………………………………87 VIII 2.3.9 Glutaraldehyde cross-linking of CfaR proteins……………………………..88 2.3.10 Construction of cfaR, orf1 and cfaR+orf1 overexpression plasmids………88 2.3.11 Extraction and analysis of CFA-L-Ile production…………………………89 2.3.12 Bioinformatics analysis…………………………………………………...89 2.4 Results and Discussion…………………………….…………………………………90 2.4.1 CfaR binds to a single site located in the cfa1 promoter region……………..90 2.4.2 The CfaR PAS domain is required for DNA binding and protein dimerization in vitro……………………………………………………………………………92 2.4.3 Activation of coronafacoyl phytotoxin production by CfaR is enhanced by ORF1……………………………………………………………………………..94 2.4.4 Phylogenetic analysis indicates that CfaR is a novel member of the PAS- LuxR protein family……………………………………..……………………….95 2.5 Concluding Remarks…………………………………………………………………97 2.6 References……………………………………………………………………………98 2.7 Figures and Tables…………………………………………………………………..106 2.8 Supplementary Materials……………………………………………………………118 Chapter 3: ORF1 is a ThiF-family protein that promotes coronafacoyl phytotoxin production and pathogenicity in the common scab pathogen Streptomyces scabies…….122 3.1 Abstract……………………………………………………………………………..122 IX 3.2 Introduction…………………………………………………………………………123 3.3 Materials and Methods……………………………………………………………...126 3.3.1 Bacterial strains, cultivation and maintenance…………………………….126 3.3.2 Plasmids, primers and DNA manipulation………………………………...128 3.3.3 Bioinformatics analyses…………………………………………………..128 3.3.4 RNA isolation and analysis of gene expression…………………………...129 3.3.5 Construction of the S. scabies ΔtxtA/orf1, ΔtxtA/cfaR and ΔtxtA/cfaR/orf1 mutant strains…………………………………………………………………...129 3.3.6 Genetic complementation of the orf1 deletion mutant…………………….130 3.3.7 Construction of Streptomyces gene overexpression plasmids harbouring the ermEp*, kasOp* and SP44 promoters…………………………………………..130 3.3.8 eGFP fluorescence assay………………………………………………….132 3.3.9 Purification of HIS6-CfaR from S. scabies………………………………..133 3.3.10 Western blot analysis…………………………………………………….134 3.3.11 Mass spectrometry (MS) analysis of the purified HIS6-CfaR……………134 3.3.12