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Analysis of Cyclodipeptide Biosynthetic Genes in Nocardiopsis Alba ATCC BAA-2165

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Analysis of Cyclodipeptide Biosynthetic Genes in Nocardiopsis Alba ATCC BAA-2165 Analysis of Cyclodipeptide Biosynthetic Genes in Nocardiopsis alba ATCC BAA-2165 A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Yongli Li May 2014 © 2014 Yongli Li. All Rights Reserved. 2 This thesis titled Analysis of Cyclodipeptide Biosynthetic Genes in Nocardiopsis alba ATCC BAA-2165 by YONGLI LI has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Shawn Chen Assistant Professor of Biological Sciences Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT LI, YONGLI., M.S., May 2014, Biological Sciences Analysis of Cyclodipeptide Biosynthetic Genes in Nocardiopsis alba ATCC BAA-2165 Director of Thesis: Shawn Chen Nocardiopsis alba ATCC BAA-2165 is an actinobacterium isolated from honeybee guts in Southern Ohio. It was reported N. alba showed antibiotic activity against several Gram-positive microorganisms, including two honeybee pathogens. Bioactivity-guided compound isolation led to an identification of two cyclodipeptides, albonoursin (cyclo(ΔPhe-ΔLeu)) and its analog (cyclo(mΔTyr-ΔLeu)), as the bioactive metabolites produced by N. alba. Despite its important environmental presence, characterization of the Nocardiopsis genus was limited due to the lack of genetic tools. In this project, we focused on the cyclodipeptides production of N. alba to establish a system for genetic analysis of Nocardiopsis. An albonoursin cyclodipeptide biosynthetic gene cluster, albABC, was identified in the N. alba genome. A PCR-targeting strategy was developed to generate an albABC deletion mutant of N. alba; the mutant, YL001, was shown to have lost the production of cyclodipeptides. Cyclodipeptides production in YL001 was restored by genetic complementation with the albABC cloned in a replicative native plasmid of Nocardiopsis. These results indicate that the albABC biosynthetic genes are responsible for the production of antibiotic cyclodipeptides of N. alba. Two additional mutants, in which a reporter gene gusA encoding β-Glucuronidase (GUS) is fused to albABC promoter in chromosome, were created to measure the transcriptional activity of the albABC genetic operon. With the GUS reporter assay, albABC gene expression was 4 found to be possibly controlled in response to osmolarity of the growth environment. A series of molecular microbiological methods have been developed for N. alba, which lays the foundation for further characterization of Nocardiopsis actinobacteria on a genetic basis. 5 ACKNOWLEDGMENTS I owe my advisor Dr. Shawn Chen a debt of gratitude for all he has done for my project design and thesis writing. His academic advices are of tremendous value and will be cherished for a long time. It has been a great pleasure to work with Shawn as a colleague. And it is my honor to graduate under his guidance. My acknowledgement also goes to our collaborators: Prof. Weiwen Zhang from Tianjin University, China; and Prof. Yu-Liang Yang from Agricultural Biotechnology Research Center, Taiwan. Prof. Weiwen Zhang showed great generosity and provided funding for sequencing the N. alba genome. Prof. Yu-Liang Yang and his group performed the chemistry isolations and analyses in this study. I could not have finished my study without their efforts. I would like to thank Ohio University for supporting this project with Ohio University Research Committee Award. I am so very glad to have Dr. Erin Murphy and Dr. Soichi Tanda on my committee. I herein give them my respectful thanks for their helpful suggestions and kind understanding. I will have to thank my lab mates, Dr. Yu Zeng, Dr. Wei Zhou, Aditya S. Kulkarni, Yahan Wei, Preeti B. Patil, Xinhao Liu, Yajun Lin, and Zifeng Deng for being such great 6 companions along the way. They have shown me the way to become a smart and humble human being. My special thanks go to Preeti B. Patil who originally isolated the N. alba pure culture from honeybee guts. It was her painstaking work that gave rise to the start of this project. She deserves the credit. Last but not least, I honestly appreciate the help and advice from our faculty members of the teaching labs: Stephanie Miller, Dr. Joan Cunningham, Molly Gurien, Karen Mammone, Dr. Donald Holzschu, just to name a few. I am truly grateful to have the chance of teaching for which has prepared me for public speaking. However, I don’t see teaching as a performance to be evaluated, but an opportunity to communicate and learn. Thus, I want to offer thanks to all my students and coworkers. Bruce Lee once said, “Under the heavens, there is but one family. It just so happens that people are different.” I totally agree with that after my four-year study at OU. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Acknowledgments............................................................................................................... 5 List of Tables .................................................................................................................... 11 List of Figures ................................................................................................................... 12 Chapter 1: Introduction ..................................................................................................... 15 1.1 General Feature of Actinobacteria .......................................................................... 16 1.2 Mycelial Actinobacteria—Actinomycetes .............................................................. 19 1.2.1 Classical Studies of Streptomyces Models ....................................................... 20 1.2.1.1 The Life Cycle of Streptomyces ................................................................ 20 1.2.1.2 Molecular Mechanisms of Streptomyces Development ............................ 21 1.2.3 Microbial Secondary Metabolism .................................................................... 23 1.2.4 Secondary Metabolites of Streptomyces .......................................................... 25 1.2.4.1 Introduction to the Biosynthesis of Secondary Metabolites ..................... 27 1.2.4.1.1 Polyketides ......................................................................................... 27 1.2.4.1.2 Nonribosomal Peptides ...................................................................... 28 1.2.4.1.3 Hybrid Peptide-polyketides ............................................................... 29 1.2.4.1.4 Cyclodipeptides ................................................................................. 29 1.2.4.2 Control of Secondary Metabolism in Streptomyces .................................. 31 1.2.4.2.1 Microbial Hormones .......................................................................... 32 1.2.4.2.2 Pathway-specific Regulators and Transcriptional Regulation ........... 32 1.2.4.2.3 Stringent Response ............................................................................ 33 1.2.4.2.4 Elemental Nutrients ........................................................................... 34 1.2.4.3 Recent Progress in Streptomyces Synthetic Biology ................................ 35 1.2.4.3.1 Synthetic Biology as a Discovery Tool ............................................. 35 1.2.4.3.2 Genetic Strategies for Refactoring Biosynthetic Clusters ................. 36 1.3 Development of Streptomyces Genetics and the Beyond ....................................... 38 8 1.3.1 Streptomyces Genetics ..................................................................................... 39 1.3.1.1 Restriction-modification System of Streptomyces .................................... 40 1.3.1.2 Plasmids and Introduction of DNA into Streptomyces ............................. 40 1.3.1.3 PCR-targeting System in Streptomyces coelicolor A3(2) ........................ 42 1.3.1.4 Transposon-based Mutagenesis in Streptomyces ...................................... 43 1.3.1.5 Reporter Systems ...................................................................................... 44 1.4 Introduction to Nocardiopsis Biology .................................................................... 45 1.4.1 The Nocardiopsis Genus .................................................................................. 45 1.4.2 The Ecology of Nocardiopsis .......................................................................... 46 1.4.3 Bioactive Metabolites Produced by Nocardiopsis Species .............................. 47 1.4.4 Recent Progress of Nocardiopsis Genomics and Genetics .............................. 48 Chapter 2: Preliminary Genome Mining and Hypotheses ................................................ 51 2.1 Genome Mining ...................................................................................................... 51 2.1.1 Background ...................................................................................................... 51 2.1.2 The Genome of Nocardiopsis alba ATCC BAA- 2165 ................................... 52 2.1.3 Genome Annotation ......................................................................................... 54 2.1.4 Bioinformatic Tools ......................................................................................... 54 2.1.5 Putative Biosynthetic Clusters in N. alba ........................................................ 54 2.1.6 A Putative Cyclodipeptides Biosynthetic Gene Cluster in
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