Specific and global networks of gene regulation in Streptomyces coelicolor Ayad Hazim Hasan Submitted in accordance with the requirement for the degree of Doctor of Philosophy The University of Leeds, Faculty of Biological Sciences School of Molecular and Cellular Biology November 2015 Intellectual Property and Publication Statements The candidate confirms that the work submitted is his own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2015 The University of Leeds and Ayad Hazim Hasan. II Acknowledgments It is a pleasure to thank those who made this thesis possible. First of all, I would like to express my sincere appreciation for my honorific supervisor Dr. Kenneth McDowall for his continuous support, advice, patience, generosity and understanding. He always guided me in the right direction; without his suggestions and encouragement, this thesis would never have been written. Simply, I cannot imagine a better supervisor. My sincere thanks go to the sponsor of this project (KRG-HCDP Scholarship program). I also extend my sincere thanks to the Faculty of Biological Sciences and the Graduate School hardship program for their support during final year of my study. Special thanks to my colleagues Justin, Louise, David, Tom, Fayez, Jaskiran and Ayat for their help and wishes for the successful completion of the project. Their scientific chats and support inspirited me to solve many problems. I would also like to thank my lab mates, everyone who has been a member of Alex O’Neill’s group (especially my shisha mate Zeyad) and Peter Stockley’s group during my study. I would like to acknowledge the following individuals for their assist; Dr. Ryan Seipke for his time and valuable feedback on a preliminary version of this thesis, Dr. Joan Boyes, Dr Bin Hong, Professor Colin Smith, Dr. Iain Manfield and Mr Chris Jones. I am very thankful to Dr. Martin Huscroft and Ph.D. student Sara Narramore for their help with actinorhodin fractionation and giving me the opportunity to use their facilities in Chemistry G61 lab. I owe a lot to my spouse and partner Shelan and my little princes Awshar for their endless love, encouragement, sacrifice and motivation. I am in greatly in debt to my parents, brothers and sisters for their love and support throughout my life. Without their support, I would not be at this stage. Above all, I thank Almighty God for giving me the strength, patience and wisdom to overcome every obstacle and difficulty I encountered. Finally, I would like to show my gratitude to all those who supported me in any respect during the completion of the project. III Abstract Streptomycetes produce a plethora of secondary metabolites, including antibiotics, and undergo a complex developmental cycle. The GC-rich species, Streptomyces coelicolor is used by many laboratories as a model for studying gene regulation, morphological development, cellular physiology and microbial signalling. A genome-wide view of many factors that control S. coelicolor gene expression at the level of transcription initiation and beyond was obtained successfully from a combination of RNA-sequencing approaches. For instance, sites of transcription initiation, vegetative promoters, leaderless mRNAs, sites involved in the processing and degradation of rRNA, tRNA and mRNA, and small RNAs, including those that may be involved in attenuation-like switching mechanisms, were successfully detected. Many of the small RNAs identified in this study are novel. Overall, our approaches show the ability to identify new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor. Here we were able to show that AtrA activates the transcription of ssgR, the gene product of which is in turn required for the transcription of ssgA, the best-studied SALP, which has a crucial role in septation and the morphology aerial hyphae. AtrA also binds to the promoter region of leuA2, which encodes α-isopropylmalate that directly utilise acetyl- CoA. Interestingly, crude extract from M1146 strain (Δact, Δred, Δcpk, and Δcda) was found to inhibit the DNA-binding activity of AtrA; however, the specificity of the small molecule(s) interaction with AtrA should be investigated. Addition of the 3 x FLAG tag™ to the N- terminus of AtrA does not hinder its ability to substitute functionally for untagged AtrA in S. coelicolor and can be used for the mapping of AtrA binding sites by ChIP-sequencing. Taken together the above suggest that AtrA has a direct role in morphological development and coordinating the utilisation of acetyl-CoA for primary and secondary metabolism. IV Table of Contents Intellectual Property and Publication Statements .................................................................... II Acknowledgments .................................................................................................................... III Abstract ..................................................................................................................................... IV Table of Contents ....................................................................................................................... V List of Figures ............................................................................................................................ IX List of Tables ............................................................................................................................. XI List of Abbreviations ................................................................................................................ XII Chapter 1 ................................................................................................................................... 1 1 Introduction ........................................................................................................................ 1 1.1 Streptomyces ........................................................................................................... 1 1.1.1 Aerial hyphyae formation regulatory networks ..................................................... 2 1.1.2 Sopre maturation regulatory networks .................................................................. 3 1.2 Antibiotic production in Streptomyces ................................................................... 5 1.2.1 Undecylprodigiosin (RED) ....................................................................................... 7 1.2.2 Calcium Dependent Antibiotics (CDAs) ................................................................... 7 1.2.3 Cryptic Polyketide (CPK) .......................................................................................... 8 1.2.4 Methylenomycins (Mm).......................................................................................... 8 1.2.5 Actinorhodin biosynthesis overview in S. coelicolor .............................................. 9 1.3 ActII-ORF4, its regulon and regulation ................................................................. 15 1.4 The regulon of atrA - unpublished work leading to this project .......................... 21 1.5 General and Specific aims ..................................................................................... 23 Chapter 2 ................................................................................................................................. 25 2 Material and Methods ...................................................................................................... 25 2.1 Bacterial strains and culture conditions ............................................................... 25 2.2 Media and chemicals ............................................................................................ 26 2.2.1 R5 (Kieser et al., 2000) .......................................................................................... 26 2.2.2 Minimal medium (MM) (Hopwood, 1967) ........................................................... 27 2.2.3 Mannitol soya flour medium (MSF) (Hobbs et al., 1989) ..................................... 27 2.2.4 Yeast extract-malt extract medium (YEME) (Kieser et al., 2000) ......................... 27 2.2.5 2X YT medium (Kieser et al., 2000) ....................................................................... 27 2.2.6 Tryptone Soya Agar (TSA) ..................................................................................... 27 2.3 Antibiotics ............................................................................................................. 27 2.4 Preparation of S. coelicolor spore stocks .............................................................. 28 V 2.5 Nucleic acid methods ............................................................................................ 28 2.5.1 Extraction of chromosomal DNA from S. coelicolor and E. coli for use in PCR ..... 28 2.5.2 Streptomyces RNA extraction from broth culture ................................................ 29 2.5.3 DNase I Treatment ................................................................................................ 30 2.5.4 Quantify the Nucleic Acid Concentration ............................................................. 30 2.6 Preparation of mycelial patches ..........................................................................