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Zeeb RA Ann Arbor, MI 48106 MOLECULAR AND BIOCHEMICAL STUDIES ON THIOPEPTIDE ANTIBIOTIC BIOSYNTHESIS A DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Robert Harvey Woodman, B.S. ******** The Ohio State University 1991 Dissertation Committee: Approved by Heinz G. Floss Edward J. Behrman rXirvo ^ Adviser George M. Milo The Ohio State Biochemistry Program Richard T . Sayre William R. Strohl Copyright by Robert Harvey Woodman 1 9 9 1 To Dr. Stella Elakovich, Dr. William R. Eure, Mr. Blanchard Hinton, Mr. Dean Hubbard, Mr. and Mrs. Thomas H. Smith III, and my parents, Robert C. and Carolyn B. Woodman for all they have done through the years. 11 ACKNOWLEDGEMENTS I thank Drs. Heinz G. Floss and William R. Strohl — my official and unofficial advisors, respectively — for allowing me to work on, and learn from, this project. I especially thank Dr. Strohl for his generous allowances of time, lab space, guidance, and advice in the course of my research. I express thanks to all the members of the Strohl lab, past and present, for their advice, help, humor, and tolerance. I especially thank Tracey Miller and Li Yun for their advice and assistance and Don Dosch for his friendship and several good pieces of advice. I also thank Mark and Cheryl Thompson for their friendship and gracious hospitality in the last days of this work; their help cannot be too highly praised. Thanks go also to all of the members of the Reeve lab, especially Diane Stroup and Vanessa Steigerwald, for friendship and splendid advice during my research. Finally, I thank all of the people to whom this work is dedicated. Each of these, in some significant way, has influenced my life and my career choices. (I am especially appreciative of my mother's frequent reminder "This, too, shall pass.") To them, I offer my heartfelt thanks for their friendship, help, and encouragement in my life. Ill VITA .March 8, 1962...................Born - Smithville, Missouri August 8, 1985..................B. S., cum laude, University of Southern Mississippi, Hattiesburg, Mississippi Sept. 1985 - Dec. 1986.......... Graduate Teaching Associate The Ohio State Biochemistry Program The Ohio State University Columbus, Ohio Jan. 1987 - present............ Graduate Research Associate Department of Microbiology The Ohio State University Columbus, Ohio PUBLICATION Woodman, R.H. 1985. Study of a precipitate from Brasenia schreberi. Bachelor of Science Thesis. Hattiesburg, Mississippi: Honors College, University of Southern Mississippi. PUBLISHED ABSTRACTS 1. Li, Y., R.H. Woodman, D.C. Dosch, W.R. Strohl, and H.G. Floss. 1989. Nucleotide sequence of a nosiheptide resistance gene from Streptomyces actuosus. Abstr. Ann. Meet. Am. Soc. Microbiol. 014, p. 306. 2. Li, Y., D. Dosch, R. Woodman, and W.R. Strohl. 1990. Regulation of nosiheptide production and resistance in Streptomyces actuosus. Ann. Meet. Soc. Industr. Microbiol. Abstract SlOO, p. 82. IV FIELDS OF STUDY Major field of study: The Ohio State Biochemistry Program Studies in the biochemistry of microorganisms. TABLE OF CONTENTS ACKNOWLEDGEMENTS....................................... i ü VITA................................................... iv LIST OF TABLES......................................... ix LIST OF FIGURES........................................ xi LIST OF ABBREVIATIONS.................................. xiv CHAPTER I: INTRODUCTION............................... 1 CHAPTER II: REVIEW OF THE LITERATURE................. 7 Streptomyces and Secondary Metabolites........... 7 Antibiotic Synthesis Regulation: Introduction.... 8 Antibiotic Synthesis Regulation: Classes of Regulatory Genes...................... 9 Antibiotic Synthesis Regulation: Pleiotropic Regulators................................... 12 Antibiotic Synthesis Regulation: Pathway- Specific Regulators.......................... 16 Antibiotic Synthesis Regulation; Antibiotic Resistance................................... 18 Peptide Antibiotics: General..................... 21 Thiopeptide Antibiotics: Thiostrepton and Nosiheptide.................................. 26 Molecular Biology of Thiostrepton and Nosiheptide Biosynthesis.................... 33 CHAPTER III: SPECIFIC AIMS OF THE RESEARCH........... 36 CHAPTER IV: METHODOLOGY.................. ,............ 39 Chemicals and Biochemicals........................ 39 Media Used in This Work........................... 41 Bacterial Strains and Plasmids................... 46 Antibiotics Usage................................. 46 Culturing of Organisms............................ 55 Preparation of Streptomyces Spores............... 58 N-methyl-N'-nitro-N-nitrosoguanidine Mutagenesis of Streptomyces Spores....................... 58 vi Nitrous Acid Mutagenesis of Streptomyces Spores... 59 Isolation of Mutant Strains of Streptomyces...... 60 Protoplast Fusion of Mutant Organisms............ 62 Preparation of Chromosomal DNA.................... 62 Plasmid DNA Isolation Methods..................... 65 Cesium Chloride Purification of Plasmids......... 70 Preparation of E. coli Competent Cells........... 71 Preparation of Protoplasts of Streptomyces Strains for Cloning.......................... 71 Transformation of E. coli with Plasmids.......... 73 Transformation of Streptomyces Protoplasts....... 74 Restriction Endonuclease Digestions.............. 74 Construction of Standards for Autoradiography 75 DNA Probes for Hybridization...................... 75 Isolation of Specific DNA Fragments.............. 78 DNA Ligations with Plasmids....................... 78 Radioactive Labelling of DNA...................... 79 Southern Hybridization............................ 81 Hybridization of DNA Probes to Colonies of Recombinant E . c o h .......................... 82 Autoradiography and Scintillation Counting....... 84 Fermentation of S. azur eus........................ 84 Isolation of High-Purity Thiostrepton............ 85 Isolation of High-Purity Nosiheptide............. 86 Quick Extractions of Thiostrepton and Nosiheptide. 88 Reduction of the Specific Activity of ^®S-L-cysteine............................... 89 Protocol for the First Synthesis Study of Thiopeptide Antibiotics...................... 89 Protocol for the Second Synthesis Study of Thiopeptide Antibiotics...................... 91 CHAPTER V: RESULTS AND DISCUSSION..................... 96 Mutants of Thiopeptide Producers.................. 96 Southern Hybridization Analysis of Thiopeptide Producers and Thiostrepton Non-producing Mutants...................................... 99 Transformation of S. azureus Mutants............... 133 Protoplast Fusion of Two Thiopeptide Antibiotic Non-producing Mutants and the Resulting Restoration of Production..................... 134 Discovery of Plasmid-Borne Thiostrepton Resistance in Streptomyces azureus....................... 136 Cloning of the nsM-Equivalent Gene from S. azureus............................... 181 Analysis of the Function of NshA................... 184 Time-course Fermentation: Analysis of S. azureus DNA................................ 194 Study of the Mode of Biosynthesis of Thiostrepton and Nosiheptide by Chloramphenicol Inhibition of Antibiotic Production................ 197 vii CHAPTER VI: SUMMARY AND CONCLUSIONS................... 227 LITERATURE CITED....................................... 231 vixi LIST OF TABLES Table 1: Predicted Number of Genes for the Biosynthesis of Thiostrepton and Nosiheptide............. 32 Table 2: Bacterial Strains Used in This Research..... 47 Table 3: Mutant Strains of Thiopeptide Producers Used m This S t u d y 51 Table 4: Plasmids Used in This Project............... 53 Table 5: Autoradiography Standards for DNA Hybrid­ ization to Thiostrepton Resistance.......... 76 Table 6: